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Sample records for aldo-keto reductase superfamily

  1. Expression, purification, crystallization and preliminary X-ray analysis of perakine reductase, a new member of the aldo-keto reductase enzyme superfamily from higher plants

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    Rosenthal, Cindy [Department of Pharmaceutical Biology, Institute of Pharmacy, Johannes Gutenberg-University Mainz, Staudinger Weg 5, D-55099 Mainz (Germany); Mueller, Uwe [Berliner Elektronenspeicherring-Gesellschaft für Synchrotronstrahlung mbH, Albert-Einstein-Strasse 15, D-12489 Berlin (Germany); Panjikar, Santosh [European Molecular Biology Laboratory Hamburg, Outstation Deutsches Elektronen-Synchrotron, Notkestrasse 85, D-22603 Hamburg (Germany); Sun, Lianli [Department of Pharmaceutical Biology, Institute of Pharmacy, Johannes Gutenberg-University Mainz, Staudinger Weg 5, D-55099 Mainz (Germany); Department of TCM and Natural Drug Research, College of Pharmaceutical Sciences, 513 Zijingang Campus, Zhejiang University, 310058 Hangzhou (China); Ruppert, Martin [Department of Pharmaceutical Biology, Institute of Pharmacy, Johannes Gutenberg-University Mainz, Staudinger Weg 5, D-55099 Mainz (Germany); Zhao, Yu [Department of TCM and Natural Drug Research, College of Pharmaceutical Sciences, 513 Zijingang Campus, Zhejiang University, 310058 Hangzhou (China); Stöckigt, Joachim, E-mail: stoeckig@mail.uni-mainz.de [Department of Pharmaceutical Biology, Institute of Pharmacy, Johannes Gutenberg-University Mainz, Staudinger Weg 5, D-55099 Mainz (Germany); Department of TCM and Natural Drug Research, College of Pharmaceutical Sciences, 513 Zijingang Campus, Zhejiang University, 310058 Hangzhou (China)

    2006-12-01

    Perakine reductase, a novel member of the aldo-keto reductase enzyme superfamily of higher plants, is involved in the biosynthesis of monoterpenoid indole alkaloids in the Indian medicinal plant Rauvolfia serpentina. The enzyme has been crystallized in C-centered orthorhombic space group and diffracts to 2.0 Å resolution. Perakine reductase (PR) is a novel member of the aldo-keto reductase enzyme superfamily from higher plants. PR from the plant Rauvolfia serpentina is involved in the biosynthesis of monoterpenoid indole alkaloids by performing NADPH-dependent reduction of perakine, yielding raucaffrinoline. However, PR can also reduce cinnamic aldehyde and some of its derivatives. After heterologous expression of a triple mutant of PR in Escherichia coli, crystals of the purified and methylated enzyme were obtained by the hanging-drop vapour-diffusion technique at 293 K with 100 mM sodium citrate pH 5.6 and 27% PEG 4000 as precipitant. Crystals belong to space group C222{sub 1} and diffract to 2.0 Å, with unit-cell parameters a = 58.9, b = 93.0, c = 143.4 Å.

  2. Babesia microti Aldo-keto Reductase-Like Protein Involved in Antioxidant and Anti-parasite Response

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    Qiang Huang

    2017-10-01

    Full Text Available The intraerythrocytic apicomplexan Babesia microti is the primary causative agent of human babesiosis, which is an infectious disease that occurs in various regions around the world. Although the aldo-keto reductases (AKRs of this parasite have been sequenced and annotated, their biological properties remain unknown. AKRs are a superfamily of enzymes with diverse functions in the reduction of aldehydes and ketones. In the present study, we cloned the full-length cDNA of a B. microti aldo-keto reductase-like protein (BmAKR and analyzed the deduced amino acid sequence of the BmAKR protein. This protein has a conserved AKR domain with an N-terminal signal sequence. Bmakr was upregulated on the 8th day after infection, whereas it was downregulated during the later stages. The recombinant protein of BmAKR was expressed in a glutathione S-transferase-fused soluble form in Escherichia coli. Western blot analysis showed that the mouse anti-BmAKR antibody recognized native BmAKR from a parasite lysate. Immunofluorescence microscopy localized BmAKR to the cytoplasm of B. microti merozoites in mouse RBCs in this study. Bmakr expression was significantly upregulated in the presence of oxidant stress. Atovaquone, a known anti-babesiosis drug, and robenidine, a known anti-coccidiosis drug, induced upregulation of Bmakr mRNA, thereby suggesting that Bmakr may be involved in anti-parasite drug response.

  3. Inhibition of aldo-keto reductase family 1 member B10 by unsaturated fatty acids.

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    Hara, Akira; Endo, Satoshi; Matsunaga, Toshiyuki; Soda, Midori; El-Kabbani, Ossama; Yashiro, Koji

    2016-11-01

    A human member of the aldo-keto reductase (AKR) superfamily, AKR1B10, is a cytosolic NADPH-dependent reductase toward various carbonyl compounds including reactive aldehydes, and is normally expressed in intestines. The enzyme is overexpressed in several extraintestinal cancers, and suggested as a potential target for cancer treatment. We found that saturated and cis-unsaturated fatty acids inhibit AKR1B10. Among the saturated fatty acids, myristic acid was the most potent, showing the IC 50 value of 4.2 μM cis-Unsaturated fatty acids inhibited AKR1B10 more potently, and linoleic, arachidonic, and docosahexaenoic acids showed the lowest IC 50 values of 1.1 μM. The inhibition by these fatty acids was reversible and kinetically competitive with respect to the substrate, showing the K i values of 0.24-1.1 μM. These fatty acids, except for α-linoleic acid, were much less inhibitory to structurally similar aldose reductase. Site-directed mutagenesis study suggested that the fatty acids interact with several active site residues of AKR1B10, of which Gln114, Val301 and Gln303 are responsible for the inhibitory selectivity. Linoleic and arachidonic acids also effectively inhibited AKR1B10-mediated 4-oxo-2-nonenal metabolism in HCT-15 cells. Thus, the cis-unsaturated fatty acids may be used as an adjuvant therapy for treatment of cancers that up-regulate AKR1B10. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Stereospecific reduction of 5β-reduced steroids by human ketosteroid reductases of the AKR (aldo-keto reductase) superfamily: role of AKR1C1-AKR1C4 in the metabolism of testosterone and progesterone via the 5β-reductase pathway.

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    Jin, Yi; Mesaros, A Clementina; Blair, Ian A; Penning, Trevor M

    2011-07-01

    Active sex hormones such as testosterone and progesterone are metabolized to tetrahydrosteroids in the liver to terminate hormone action. One main metabolic pathway, the 5β-pathway, involves 5β-steroid reductase (AKR1D1, where AKR refers to the aldo-keto reductase superfamily), which catalyses the reduction of the 4-ene structure, and ketosteroid reductases (AKR1C1-AKR1C4), which catalyse the subsequent reduction of the 3-oxo group. The activities of the four human AKR1C enzymes on 5β-dihydrotestosterone, 5β-pregnane-3,20-dione and 20α-hydroxy-5β-pregnan-3-one, the intermediate 5β-dihydrosteroids on the 5β-pathway of testosterone and progesterone metabolism, were investigated. Product characterization by liquid chromatography-MS revealed that the reduction of the 3-oxo group of the three steroids predominantly favoured the formation of the corresponding 3α-hydroxy steroids. The stereochemistry was explained by molecular docking. Kinetic properties of the enzymes identified AKR1C4 as the major enzyme responsible for the hepatic formation of 5β-tetrahydrosteroid of testosterone, but indicated differential routes and roles of human AKR1C for the hepatic formation of 5β-tetrahydrosteroids of progesterone. Comparison of the kinetics of the AKR1C1-AKR1C4-catalysed reactions with those of AKR1D1 suggested that the three intermediate 5β-dihydrosteroids derived from testosterone and progesterone are unlikely to accumulate in liver, and that the identities and levels of 5β-reduced metabolites formed in peripheral tissues will be governed by the local expression of AKR1D1 and AKR1C1-AKR1C3.

  5. Lignases and aldo-keto reductases for conversion of lignin-containing materials to fermentable products

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    Scharf, Michael; Sethi, Amit

    2016-09-13

    Termites have specialized digestive systems that overcome the lignin barrier in wood to release fermentable simple sugars. Using the termite Reticulitermes flavipes and its gut symbionts, high-throughput titanium pyrosequencing and proteomics approaches experimentally compared the effects of lignin-containing diets on host-symbiont digestome composition. Proteomic investigations and functional digestive studies with recombinant lignocellulases conducted in parallel provided strong evidence of congruence at the transcription and translational levels and provide enzymatic strategies for overcoming recalcitrant lignin barriers in biofuel feedstocks. Briefly described, therefore, the disclosure provides a system for generating a fermentable product from a lignified plant material, the system comprising a cooperating series of at least two catalytically active polypeptides, where said catalytically active polypeptides are selected from the group consisting of: cellulase Cell-1, .beta.-glu cellulase, an aldo-keto-reductase, a catalase, a laccase, and an endo-xylanase.

  6. BIOLOGICAL ROLE OF ALDO-KETO REDUCTASES IN RETINOIC ACID BIOSYNTHESIS AND SIGNALING

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    F. Xavier eRuiz

    2012-04-01

    Full Text Available Several aldo-keto reductase (AKR enzymes from subfamilies 1B and 1C show retinaldehyde reductase activity, having low Km and kcat values. Only AKR1B10 and 1B12, with all-trans-retinaldehyde, and AKR1C3, with 9-cis-retinaldehyde, display high catalytic efficiency. Major structural determinants for retinaldehyde isomer specificity are located in the external loops (A and C for AKR1B10, and B for AKR1C3, as assessed by site-directed mutagenesis and molecular dynamics. Cellular models have shown that AKR1B and 1C enzymes are well suited to work in vivo as retinaldehyde reductases and to regulate retinoic acid (RA biosynthesis at hormone pre-receptor level. An additional physiological role for the retinaldehyde reductase activity of these enzymes, consistent with their tissue localization, is their participation in β-carotene absorption. Retinaldehyde metabolism may be subjected to subcellular compartmentalization, based on enzyme localization. While retinaldehyde oxidation to RA takes place in the cytosol, reduction to retinol could take place in the cytosol by AKRs or in the membranes of endoplasmic reticulum by microsomal retinaldehyde reductases. Upregulation of some AKR1 enzymes in different cancer types may be linked to their induction by oxidative stress and to their participation in different signaling pathways related to cell proliferation. AKR1B10 and AKR1C3, through their retinaldehyde reductase activity, trigger a decrease in the RA biosynthesis flow, resulting in RA deprivation and consequently lower differentiation, with an increased cancer risk in target tissues. Rational design of selective AKR inhibitors could lead to development of novel drugs for cancer treatment as well as reduction of chemotherapeutic drug resistance.

  7. Aldo-keto reductase enzymes detoxify glyphosate and improve herbicide resistance in plants.

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    Vemanna, Ramu S; Vennapusa, Amaranatha Reddy; Easwaran, Murugesh; Chandrashekar, Babitha K; Rao, Hanumantha; Ghanti, Kirankumar; Sudhakar, Chinta; Mysore, Kirankumar S; Makarla, Udayakumar

    2017-07-01

    In recent years, concerns about the use of glyphosate-resistant crops have increased because of glyphosate residual levels in plants and development of herbicide-resistant weeds. In spite of identifying glyphosate-detoxifying genes from microorganisms, the plant mechanism to detoxify glyphosate has not been studied. We characterized an aldo-keto reductase gene from Pseudomonas (PsAKR1) and rice (OsAKR1) and showed, by docking studies, both PsAKR1 and OsAKR1 can efficiently bind to glyphosate. Silencing AKR1 homologues in rice and Nicotiana benthamiana or mutation of AKR1 in yeast and Arabidopsis showed increased sensitivity to glyphosate. External application of AKR proteins rescued glyphosate-mediated cucumber seedling growth inhibition. Regeneration of tobacco transgenic lines expressing PsAKR1 or OsAKRI on glyphosate suggests that AKR can be used as selectable marker to develop transgenic crops. PsAKR1- or OsAKRI-expressing tobacco and rice transgenic plants showed improved tolerance to glyphosate with reduced accumulation of shikimic acid without affecting the normal photosynthetic rates. These results suggested that AKR1 when overexpressed detoxifies glyphosate in planta. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  8. 9,10-Phenanthrenequinone promotes secretion of pulmonary aldo-keto reductases with surfactant.

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    Matsunaga, Toshiyuki; Haga, Mariko; Watanabe, Gou; Shinoda, Yuhki; Endo, Satoshi; Kajiwara, Yu; Tanaka, Hiroyuki; Inagaki, Naoki; El-Kabbani, Ossama; Hara, Akira

    2012-02-01

    9,10-Phenanthrenequinone (9,10-PQ), a major quinone in diesel exhaust particles, induces apoptosis via the generation of reactive oxygen species (ROS) because of 9,10-PQ redox cycling. We have found that intratracheal infusion of 9,10-PQ facilitates the secretion of surfactant into rat alveolus. In the cultured rat lung, treatment with 9,10-PQ results in an increase in a lower-density surfactant by ROS generation through redox cycling of the quinone. The surfactant contains aldo-keto reductase (AKR) 1C15, which reduces 9,10-PQ and the enzyme level in the surfactant increases on treatment with 9,10-PQ suggesting an involvement of AKR1C15 in the redox cycling of the quinone. In six human cell types (A549, MKN45, Caco2, Hela, Molt4 and U937) only type II epithelial A549 cells secrete three human AKR1C subfamily members (AKR1C1, AKR1C2 and AKR1C3) with the surfactant into the medium; this secretion is highly increased by 9,10-PQ treatment. Using in vitro enzyme inhibition analysis, we have identified AKR1C3 as the most abundantly secreted AKR1C member. The AKR1C enzymes in the medium efficiently reduce 9,10-PQ and initiate its redox cycling accompanied by ROS production. The exposure of A549 cells to 9,10-PQ provokes viability loss, which is significantly protected by the addition of the AKR1C3 inhibitor and antioxidant enzyme and by the removal of the surfactants from the culture medium. Thus, the AKR1C enzymes secreted in pulmonary surfactants probably participate in the toxic mechanism triggered by 9,10-PQ.

  9. Structural and mutational studies on an aldo-keto reductase AKR5C3 from Gluconobacter oxydans

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    Liu, Xu; Wang, Chao; Zhang, Lujia; Yao, Zhiqiang; Cui, Dongbing; Wu, Liang; Lin, Jinping; Yuan, Yu-Ren Adam; Wei, Dongzhi

    2014-01-01

    An aldo-keto reductase AKR5C3 from Gluconobacter oxydans (designated as Gox0644) is a useful enzyme with various substrates, including aldehydes, diacetyl, keto esters, and α-ketocarbonyl compounds. The crystal structures of AKR5C3 in apoform in complex with NADPH and the D53A mutant (AKR5C3-D53A) in complex with NADPH are presented herein. Structure comparison and site-directed mutagenesis combined with biochemical kinetics analysis reveal that the conserved Asp53 in the AKR5C3 catalytic tetrad has a crucial role in securing active pocket conformation. The gain-of-function Asp53 to Ala mutation triggers conformational changes on the Trp30 and Trp191 side chains, improving NADPH affinity to AKR5C3, which helps increase catalytic efficiency. The highly conserved Trp30 and Trp191 residues interact with the nicotinamide moiety of NADPH and help form the NADPH-binding pocket. The AKR5C3-W30A and AKR5C3-W191Y mutants show decreased activities, confirming that both residues facilitate catalysis. Residue Trp191 is in the loop structure, and the AKR5C3-W191Y mutant does not react with benzaldehyde, which might also determine substrate recognition. Arg192, which is involved in the substrate binding, is another important residue. The introduction of R192G increases substrate-binding affinity by improving hydrophobicity in the substrate-binding pocket. These results not only supplement the AKRs superfamily with crystal structures but also provide useful information for understanding the catalytic properties of AKR5C3 and guiding further engineering of this enzyme. PMID:25131535

  10. Long-chain fatty acids inhibit human members of the aldo-keto reductase 1C subfamily.

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    Hara, Akira; Endo, Satoshi; Matsunaga, Toshiyuki; Soda, Midori; Yashiro, Koji; El-Kabbani, Ossama

    2017-11-01

    Four human hydroxysteroid dehydrogenases in the aldo-keto reductase (AKR) superfamily, AKR1C1-AKR1C4, are involved in the metabolism of steroids and other carbonyl compounds including drugs, and altered expression of AKRs (1C1, 1C2 and/or 1C3) is related to the pathogenesis of several extrahepatic cancers. Here, we report that unsaturated fatty acids (FAs) are potent competitive inhibitors of the AKR enzymes. The sensitivities to the FAs were different among the enzymes, especially between AKR1C1 and AKR1C2. The most potent inhibitors for AKR1C1, AKR1C2 and AKR1C4 were docosahexaenoic acid (Ki 0.77 µM), palmitoleic acid (Ki 0.41 µM) and linoleic acid (Ki 0.33 µM), respectively. AKR1C3 was the most sensitive to FA inhibition, showing low Ki values (0.23-0.29 µM) for oleic, linoleic, eicosapentaenoic and docosahexaenoic acids. Linoleic and oleic acids also inhibited AKR1C3-mediated metabolism of 9,10-phenanthrenequinone in colon DLD1 cells. Molecular docking and site-directed mutagenesis studies suggested upon FA binding to AKR1C1 and AKR1C3: (i) the carboxyl group of the FA binds to the oxyanion-binding site in the active site; (ii) the difference in FA sensitivity between AKR1C1 and AKR1C2 is due to their residue difference at position 54; (iii) Ser118, Phe306 and Phe311 of AKR1C3 are important for determining the inhibitory potency of FAs. © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  11. Aldo-Keto Reductases as Early Biomarkers of Hepatocellular Carcinoma: A Comparison Between Animal Models and Human HCC.

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    Torres-Mena, Julia Esperanza; Salazar-Villegas, Karla Noemí; Sánchez-Rodríguez, Ricardo; López-Gabiño, Belém; Del Pozo-Yauner, Luis; Arellanes-Robledo, Jaime; Villa-Treviño, Saúl; Gutiérrez-Nava, María Angélica; Pérez-Carreón, Julio Isael

    2018-04-01

    The intrinsic heterogeneity of hepatocellular carcinoma (HCC) represents a great challenge for its molecular classification and for detecting predictive biomarkers. Aldo-keto reductase (Akr) family members have shown differential expression in human HCC, while AKR1B10 overexpression is considered a biomarker; AKR7A3 expression is frequently reduced in HCC. To investigate the time-course expression of Akr members in the experimental hepatocarcinogenesis. Using DNA-microarray data, we analyzed the time-course gene expression profile from nodules to tumors (4-17 months) of 17 Akr members induced by the resistant hepatocyte carcinogenesis model in the rat. The expression of six members (Akr1c19, Akr1b10, Akr7a3, Akr1b1, Akr1cl1, and Akr1b8) was increased, comparable to that of Ggt and Gstp1, two well-known liver cancer markers. In particular, Akr7a3 and Akr1b10 expression also showed a time-dependent increment at mRNA and protein levels in a second hepatocarcinogenesis model induced with diethylnitrosamine. We confirmed that aldo-keto reductases 7A3 and 1B10 were co-expressed in nine biopsies of human HCC, independently from the presence of glypican-3 and cytokeratin-19, two well-known HCC biomarkers. Because it has been suggested that expression of Akr members is regulated through NRF2 activity at the antioxidant response element (ARE) sequences, we searched and identified at least two ARE sites in Akr1b1, Akr1b10, and Akr7a3 from rat and human gene sequences. Moreover, we observed higher NRF2 nuclear translocation in tumors as compared with non-tumor tissues. Our results demonstrate that Akr7a3 mRNA and protein levels are consistently co-expressed along with Akr1b10, in both experimental liver carcinogenesis and some human HCC samples. These results highlight the presence of AKR7A3 and AKR1B10 from early stages of the experimental HCC and introduce them as a potential application for early diagnosis, staging, and prognosis in human cancer.

  12. Overexpression of Aldo-Keto-Reductase in Azole-resistant Clinical Isolates of Candida Glabrata Determined by cDNA-AFLP

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    Mansour Heidari

    2013-01-01

    Full Text Available Background: Candida glabrata causes significant medical problems in immunocompromised patients. Many strains of this yeast are intrinsically resistant to azole antifungal agents, and treatment is problematic, leading to high morbidity and mortality rates in immunosuppressed individuals. The primary goal of this study was to investigate the genes involved in the drug resistance of clinical isolates of C. glabrata.Methods: The clinical isolates of C. glabrata were collected in an epidemiological survey of candidal infection inimmunocompromised patients and consisted of four fluconazole and itraconazole resistant isolates, two fluconazoleand itraconazole sensitive isolates, and C. glabrata CBS 138 as reference strain. Antifungal susceptibility patterns ofthe organisms were determined beforehand by the Clinical and Laboratory Standards Institute (CLSI. The potentialgene(s implicated in antifungal resistance were investigated using complementary DNA- Amplified Fragment Length Polymorphism (cDNA-AFLP. Semi-quantitative RT-PCR was carried out to evaluate the expression of gene(s in resistant isolates as compared to sensitive and reference strains.Results and conclusions: The aldo-keto-reductase superfamily (AKR gene was upregulated in the resistant clinicalisolates as assessed by cDNA-AFLP. Semi-quantitative RT-PCR revealed AKR mRNA expression approximately twice that seen in the sensitive isolates. Overexpression of the AKR gene was associated with increased fluconazole and itraconazole resistance in C. glabrata. The data suggest that upregulation of the AKR gene might give a new insight into the mechanism of azole resistance.

  13. Expression of the Aldo-Keto Reductases AKR1B1 and AKR1B10 in Human Cancers.

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    Brian eLaffin

    2012-06-01

    Full Text Available The American Cancer Society estimates that there will be more than 1.5 million new cases of cancer in 2011, underscoring the need for identification of new therapeutic targets and development of novel cancer therapies. Previous studies have implicated the human aldo-keto reductases AKR1B1 and AKR1B10 in cancer, and therefore we examined AKR1B1 and AKR1B10 expression across all major human cancer types using the Oncomine cancer gene expression database (Compendia Biosciences, www.oncomine.com. Using this database, we found that expression of of AKR1B1 and AKR1B10 varies greatly by cancer type and tissue of origin, including agreement with previous reports that AKR1B10 is significantly over-expressed in cancers of the lungs and liver. AKR1B1 is more broadly over-expressed in human cancers than AKR1B10, albeit at a generally lower magnitude. AKR1B1 over-expression was found to be associated with shortened patient survival in acute myelogenous leukemias and multiple myelomas. High AKR1B10 expression tends to predict less aggressive clinical course generally, notably within lung cancers, where it tends to be highly over-expressed compared to normal tissue. These findings suggest that AKR1B1 inhibitors in particular hold great potential as novel cancer therapeutics.

  14. Efficient Preparation of (S)-N-Boc-3-Hydroxylpiperidine Through Bioreduction by a Thermostable Aldo-KetoReductase.

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    He, Mengyan; Zhou, Shuo; Cui, Maolin; Jin, Xiaolu; Lai, Dunyue; Zhang, Shuangling; Wang, Zhiguo; Chen, Zhenming

    2017-04-01

    (S)-N-Boc-3-hydroxypiperidine ((S)-NBHP) is a key pharmaceutical intermediate and the chiral source in synthesizing Imbruvica, which is a newly approved drug in lymphoma therapy by targeting Bruton's tyrosine kinase (BTK). Current chemical synthesis of (S)-NBHP suffered from the need of noble metal catalyst and low yield. The single reported bioconversion of (S)-NBHP was achieved by using recombinant ketoreductase, but enzyme sequence was kept confidential and the catalytic process suffered from the thermodeactivation and substrate inhibition. In the current study, we presented a thermostable aldo-keto reductase (AKR)-AKR-43-which showed high activity toward N-Boc-3-piperidone (NBP) to produce (S)-NBHP, high enantioselectivity, and no substrate inhibition. The molecular simulations demonstrated the structural rationale for the enantioselectivity of AKR-43 toward NBP and supported the classic ordered two-step catalytic mechanism. The catalytic process was achieved by using glucose dehydrogenase (GDH) for cofactor recycling, and the optimal reaction conditions were determined to be 30 °C and pH 7.5. Within a reaction time of 16 h, the 16 % substrate concentration (w/w), over 99 % ee and under 3.5 % of enzyme loading (w/w) characterized a high efficiency process with promising industrial values.

  15. The catalytic mechanism of NADH-dependent reduction of 9,10-phenanthrenequinone by Candida tenuis xylose reductase reveals plasticity in an aldo-keto reductase active site.

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    Pival, Simone L; Klimacek, Mario; Nidetzky, Bernd

    2009-06-12

    Despite their widely varying physiological functions in carbonyl metabolism, AKR2B5 (Candida tenuis xylose reductase) and many related enzymes of the aldo-keto reductase protein superfamily utilise PQ (9,10-phenanthrenequinone) as a common in vitro substrate for NAD(P)H-dependent reduction. The catalytic roles of the conserved active-site residues (Tyr51, Lys80 and His113) of AKR2B5 in the conversion of the reactive alpha-dicarbonyl moiety of PQ are not well understood. Using wild-type and mutated (Tyr51, Lys80 and His113 individually replaced by alanine) forms of AKR2B5, we have conducted steady-state and transient kinetic studies of the effects of varied pH and deuterium isotopic substitutions in coenzyme and solvent on the enzymatic rates of PQ reduction. Each mutation caused a 10(3)-10(4)-fold decrease in the rate constant for hydride transfer from NADH to PQ, whose value in the wild-type enzyme was determined as approximately 8 x 10(2) s(-1). The data presented support an enzymic mechanism in which a catalytic proton bridge from the protonated side chain of Lys80 (pK=8.6+/-0.1) to the carbonyl group adjacent to the hydride acceptor carbonyl facilitates the chemical reaction step. His113 contributes to positioning of the PQ substrate for catalysis. Contrasting its role as catalytic general acid for conversion of the physiological substrate xylose, Tyr51 controls release of the hydroquinone product. The proposed chemistry of AKR2B5 action involves delivery of both hydrogens required for reduction of the alpha-dicarbonyl substrate to the carbonyl group undergoing (stereoselective) transformation. Hydride transfer from NADH probably precedes the transfer of a proton from Tyr51 whose pK of 7.3+/-0.3 in the NAD+-bound enzyme appears suitable for protonation of a hydroquinone anion (pK=8.8). These results show that the mechanism of AKR2B5 is unusually plastic in the exploitation of the active-site residues, for the catalytic assistance provided to carbonyl group

  16. Role of aldo-keto reductases and other doxorubicin pharmacokinetic genes in doxorubicin resistance, DNA binding, and subcellular localization

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    Heibein, Allan D; Guo, Baoqing; Sprowl, Jason A; MacLean, David A; Parissenti, Amadeo M

    2012-01-01

    Since proteins involved in chemotherapy drug pharmacokinetics and pharmacodynamics have a strong impact on the uptake, metabolism, and efflux of such drugs, they likely play critical roles in resistance to chemotherapy drugs in cancer patients. To investigate this hypothesis, we conducted a whole genome microarray study to identify difference in the expression of genes between isogenic doxorubicin-sensitive and doxorubicin-resistant MCF-7 breast tumour cells. We then assessed the degree of over-representation of doxorubicin pharmacokinetic and pharmacodynamic genes in the dataset of doxorubicin resistance genes. Of 27,958 Entrez genes on the array, 7.4 per cent or 2,063 genes were differentially expressed by ≥ 2-fold between wildtype and doxorubicin-resistant cells. The false discovery rate was set at 0.01 and the minimum p value for significance for any gene within the “hit list” was 0.01. Seventeen and 43 per cent of doxorubicin pharmacokinetic genes were over-represented in the hit list, depending upon whether the gene name was identical or within the same gene family, respectively. The most over-represented genes were within the 1C and 1B families of aldo-keto reductases (AKRs), which convert doxorubicin to doxorubicinol. Other genes convert doxorubicin to other metabolites or affect the influx, efflux, or cytotoxicity of the drug. In further support of the role of AKRs in doxorubicin resistance, we observed that, in comparison to doxorubicin, doxorubincol exhibited dramatically reduced cytotoxicity, reduced DNA-binding activity, and strong localization to extra nuclear lysosomes. Pharmacologic inhibition of the above AKRs in doxorubicin-resistant cells increased cellular doxorubicin levels, restored doxorubicin cytotoxicity and re-established doxorubicin localization to the nucleus. The properties of doxorubicinol were unaffected. These findings demonstrate the utility of using curated pharmacokinetic and pharmacodynamic knowledge bases to identify

  17. Anthracycline resistance mediated by reductive metabolism in cancer cells: The role of aldo-keto reductase 1C3

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    Hofman, Jakub; Malcekova, Beata; Skarka, Adam; Novotna, Eva; Wsol, Vladimir, E-mail: wsol@faf.cuni.cz

    2014-08-01

    Pharmacokinetic drug resistance is a serious obstacle that emerges during cancer chemotherapy. In this study, we investigated the possible role of aldo-keto reductase 1C3 (AKR1C3) in the resistance of cancer cells to anthracyclines. First, the reducing activity of AKR1C3 toward anthracyclines was tested using incubations with a purified recombinant enzyme. Furthermore, the intracellular reduction of daunorubicin and idarubicin was examined by employing the transfection of A549, HeLa, MCF7 and HCT 116 cancer cells with an AKR1C3 encoding vector. To investigate the participation of AKR1C3 in anthracycline resistance, we conducted MTT cytotoxicity assays with these cells, and observed that AKR1C3 significantly contributes to the resistance of cancer cells to daunorubicin and idarubicin, whereas this resistance was reversible by the simultaneous administration of 2′-hydroxyflavanone, a specific AKR1C3 inhibitor. In the final part of our work, we tracked the changes in AKR1C3 expression after anthracycline exposure. Interestingly, a reciprocal correlation between the extent of induction and endogenous levels of AKR1C3 was recorded in particular cell lines. Therefore, we suggest that the induction of AKR1C3 following exposure to daunorubicin and idarubicin, which seems to be dependent on endogenous AKR1C3 expression, eventually might potentiate an intrinsic resistance given by the normal expression of AKR1C3. In conclusion, our data suggest a substantial impact of AKR1C3 on the metabolism of daunorubicin and idarubicin, which affects their pharmacokinetic and pharmacodynamic behavior. In addition, we demonstrate that the reduction of daunorubicin and idarubicin, which is catalyzed by AKR1C3, contributes to the resistance of cancer cells to anthracycline treatment. - Highlights: • Metabolism of anthracyclines by AKR1C3 was studied at enzyme and cellular levels. • Anthracycline resistance mediated by AKR1C3 was demonstrated in cancer cells. • Induction of AKR1C3

  18. Ruthenium complexes as inhibitors of the aldo-keto reductases AKR1C1-1C3.

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    Traven, Katja; Sinreih, Maša; Stojan, Jure; Seršen, Sara; Kljun, Jakob; Bezenšek, Jure; Stanovnik, Branko; Turel, Iztok; Rižner, Tea Lanišnik

    2015-06-05

    The human aldo-keto reductases (AKRs) from the 1C subfamily are important targets for the development of new drugs. In this study, we have investigated the possible interactions between the recombinant AKR1C enzymes AKR1C1-AKR1C3 and ruthenium(II) complexes; in particular, we were interested in the potential inhibitory actions. Five novel ruthenium complexes (1a, 1b, 2a, 2b, 2c), two precursor ruthenium compounds (P1, P2), and three ligands (a, b, c) were prepared and included in this study. Two different types of novel ruthenium(II) complexes were synthesized. First, bearing the sulphur macrocycle [9]aneS3, S-bonded dimethylsulphoxide (dmso-S), and an N,N-donor ligand, with the general formula of [Ru([9]aneS3)(dmso)(N,N-ligand)](PF6)2 (1a, 1b), and second, with the general formula of [(η(6)-p-cymene)RuCl(N,N-ligand)]Cl (2a, 2b, 2c). All of these synthesized compounds were characterized by high-resolution NMR spectroscopy, X-ray crystallography (compounds a, b, c, 1a, 1b) and other standard physicochemical methods. To evaluate the potential inhibitory actions of these compounds on the AKR1C enzymes, we followed enzymatically catalyzed oxidation of the substrate 1-acenaphthenol by NAD(+) in the absence and presence of various micromolar concentrations of the individual compounds. Among 10 compounds, one ruthenium complex (2b) and two precursor ruthenium compounds (P1, P2) inhibited all three AKR1C enzymes, and one ruthenium complex (2a) inhibited only AKR1C3. Ligands a, b and c revealed no inhibition of the AKR1C enzymes. All four of the active compounds showed multiple binding with the AKR1C enzymes that was characterized by an initial instantaneous inhibition followed by a slow quasi-irreversible step. To the best of our knowledge, this is the first study that has examined interactions between these AKR1C enzymes and ruthenium(II) complexes. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  19. Characterization of the aldo-keto reductase 1C gene cluster on pig chromosome 10: possible associations with reproductive traits

    Directory of Open Access Journals (Sweden)

    Nonneman Dan J

    2006-09-01

    Full Text Available Abstract Background The rate of pubertal development and weaning to estrus interval are correlated and affect reproductive efficiency of swine. Quantitative trait loci (QTL for age of puberty, nipple number and ovulation rate have been identified in Meishan crosses on pig chromosome 10q (SSC10 near the telomere, which is homologous to human chromosome 10p15 and contains an aldo-keto reductase (AKR gene cluster with at least six family members. AKRs are tissue-specific hydroxysteroid dehydrogenases that interconvert weak steroid hormones to their more potent counterparts and regulate processes involved in development, homeostasis and reproduction. Because of their location in the swine genome and their implication in reproductive physiology, this gene cluster was characterized and evaluated for effects on reproductive traits in swine. Results Screening the porcine CHORI-242 BAC library with a full-length AKR1C4 cDNA identified 7 positive clones and sample sequencing of 5 BAC clones revealed 5 distinct AKR1C genes (AKR1CL2 and AKR1C1 through 4, which mapped to 126–128 cM on SSC10. Using the IMpRH7000rad and IMNpRH212000rad radiation hybrid panels, these 5 genes mapped between microsatellite markers SWR67 and SW2067. Comparison of sequence data with the porcine BAC fingerprint map show that the cluster of genes resides in a 300 kb region. Twelve SNPs were genotyped in gilts observed for age at first estrus and ovulation rate from the F8 and F10 generations of one-quarter Meishan descendants of the USMARC resource population. Age at puberty, nipple number and ovulation rate data were analyzed for association with genotypes by MTDFREML using an animal model. One SNP, a phenylalanine to isoleucine substitution in AKR1C2, was associated with age of puberty (p = 0.07 and possibly ovulation rate (p = 0.102. Two SNP in AKR1C4 were significantly associated with nipple number (p ≤ 0.03 and another possibly associated with age at puberty (p = 0

  20. Novel Aldo-Keto Reductases for the Biocatalytic Conversion of 3-Hydroxybutanal to 1,3-Butanediol: Structural and Biochemical Studies

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Taeho; Flick, Robert; Brunzelle, Joseph; Singer, Alex; Evdokimova, Elena; Brown, Greg; Joo, Jeong Chan; Minasov, George A.; Anderson, Wayne F.; Mahadevan, Radhakrishnan; Savchenko, Alexei; Yakunin, Alexander F. (KRICT); (Toronto); (NWU)

    2017-01-27

    The nonnatural alcohol 1,3-butanediol (1,3-BDO) is a valuable building block for the synthesis of various polymers. One of the potential pathways for the biosynthesis of 1,3-BDO includes the biotransformation of acetaldehyde to 1,3-BDO via 3-hydroxybutanal (3-HB) using aldolases and aldo-keto reductases (AKRs). This pathway requires an AKR selective for 3-HB, but inactive toward acetaldehyde, so it can be used for one-pot synthesis. In this work, we screened more than 20 purified uncharacterized AKRs for 3-HB reduction and identified 10 enzymes with significant activity and nine proteins with detectable activity. PA1127 fromPseudomonas aeruginosashowed the highest activity and was selected for comparative studies with STM2406 fromSalmonella entericaserovar Typhimurium, for which we have determined the crystal structure. Both AKRs used NADPH as a cofactor, reduced a broad range of aldehydes, and showed low activities toward acetaldehyde. The crystal structures of STM2406 in complex with cacodylate or NADPH revealed the active site with bound molecules of a substrate mimic or cofactor. Site-directed mutagenesis of STM2406 and PA1127 identified the key residues important for the activity against 3-HB and aromatic aldehydes, which include the residues of the substrate-binding pocket and C-terminal loop. Our results revealed that the replacement of the STM2406 Asn65 by Met enhanced the activity and the affinity of this protein toward 3-HB, resulting in a 7-fold increase inkcat/Km. Our work provides further insights into the molecular mechanisms of the substrate selectivity of AKRs and for the rational design of these enzymes toward new substrates.

    IMPORTANCEIn this study, we identified several aldo-keto reductases with significant activity in reducing 3

  1. Substrate Specificity, Inhibitor Selectivity and Structure-Function Relationships of Aldo-Keto Reductase 1B15: A Novel Human Retinaldehyde Reductase.

    Directory of Open Access Journals (Sweden)

    Joan Giménez-Dejoz

    Full Text Available Human aldo-keto reductase 1B15 (AKR1B15 is a newly discovered enzyme which shares 92% amino acid sequence identity with AKR1B10. While AKR1B10 is a well characterized enzyme with high retinaldehyde reductase activity, involved in the development of several cancer types, the enzymatic activity and physiological role of AKR1B15 are still poorly known. Here, the purified recombinant enzyme has been subjected to substrate specificity characterization, kinetic analysis and inhibitor screening, combined with structural modeling. AKR1B15 is active towards a variety of carbonyl substrates, including retinoids, with lower kcat and Km values than AKR1B10. In contrast to AKR1B10, which strongly prefers all-trans-retinaldehyde, AKR1B15 exhibits superior catalytic efficiency with 9-cis-retinaldehyde, the best substrate found for this enzyme. With ketone and dicarbonyl substrates, AKR1B15 also shows higher catalytic activity than AKR1B10. Several typical AKR inhibitors do not significantly affect AKR1B15 activity. Amino acid substitutions clustered in loops A and C result in a smaller, more hydrophobic and more rigid active site in AKR1B15 compared with the AKR1B10 pocket, consistent with distinct substrate specificity and narrower inhibitor selectivity for AKR1B15.

  2. A Novel Aldo-Keto Reductase, HdRed, from the Pacific Abalone Haliotis discus hannai, Which Reduces Alginate-derived 4-Deoxy-l-erythro-5-hexoseulose Uronic Acid to 2-Keto-3-deoxy-d-gluconate*

    Science.gov (United States)

    Mochizuki, Shogo; Nishiyama, Ryuji; Inoue, Akira; Ojima, Takao

    2015-01-01

    Abalone feeds on brown seaweeds and digests seaweeds' alginate with alginate lyases (EC 4.2.2.3). However, it has been unclear whether the end product of alginate lyases (i.e. unsaturated monouronate-derived 4-deoxy-l-erythro-5-hexoseulose uronic acid (DEH)) is assimilated by abalone itself, because DEH cannot be metabolized via the Embden-Meyerhof pathway of animals. Under these circumstances, we recently noticed the occurrence of an NADPH-dependent reductase, which reduced DEH to 2-keto-3-deoxy-d-gluconate, in hepatopancreas extract of the pacific abalone Haliotis discus hannai. In the present study, we characterized this enzyme to some extent. The DEH reductase, named HdRed in the present study, could be purified from the acetone-dried powder of hepatopancreas by ammonium sulfate fractionation followed by conventional column chromatographies. HdRed showed a single band of ∼40 kDa on SDS-PAGE and reduced DEH to 2-keto-3-deoxy-d-gluconate with an optimal temperature and pH at around 50 °C and 7.0, respectively. HdRed exhibited no appreciable activity toward 28 authentic compounds, including aldehyde, aldose, ketose, α-keto-acid, uronic acid, deoxy sugar, sugar alcohol, carboxylic acid, ketone, and ester. The amino acid sequence of 371 residues of HdRed deduced from the cDNA showed 18–60% identities to those of aldo-keto reductase (AKR) superfamily enzymes, such as human aldose reductase, halophilic bacterium reductase, and sea hare norsolorinic acid (a polyketide derivative) reductase-like protein. Catalytic residues and cofactor binding residues known in AKR superfamily enzymes were fairly well conserved in HdRed. Phylogenetic analysis for HdRed and AKR superfamily enzymes indicated that HdRed is an AKR belonging to a novel family. PMID:26555267

  3. A Novel Aldo-Keto Reductase, HdRed, from the Pacific Abalone Haliotis discus hannai, Which Reduces Alginate-derived 4-Deoxy-L-erythro-5-hexoseulose Uronic Acid to 2-Keto-3-deoxy-D-gluconate.

    Science.gov (United States)

    Mochizuki, Shogo; Nishiyama, Ryuji; Inoue, Akira; Ojima, Takao

    2015-12-25

    Abalone feeds on brown seaweeds and digests seaweeds' alginate with alginate lyases (EC 4.2.2.3). However, it has been unclear whether the end product of alginate lyases (i.e. unsaturated monouronate-derived 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH)) is assimilated by abalone itself, because DEH cannot be metabolized via the Embden-Meyerhof pathway of animals. Under these circumstances, we recently noticed the occurrence of an NADPH-dependent reductase, which reduced DEH to 2-keto-3-deoxy-D-gluconate, in hepatopancreas extract of the pacific abalone Haliotis discus hannai. In the present study, we characterized this enzyme to some extent. The DEH reductase, named HdRed in the present study, could be purified from the acetone-dried powder of hepatopancreas by ammonium sulfate fractionation followed by conventional column chromatographies. HdRed showed a single band of ∼ 40 kDa on SDS-PAGE and reduced DEH to 2-keto-3-deoxy-D-gluconate with an optimal temperature and pH at around 50 °C and 7.0, respectively. HdRed exhibited no appreciable activity toward 28 authentic compounds, including aldehyde, aldose, ketose, α-keto-acid, uronic acid, deoxy sugar, sugar alcohol, carboxylic acid, ketone, and ester. The amino acid sequence of 371 residues of HdRed deduced from the cDNA showed 18-60% identities to those of aldo-keto reductase (AKR) superfamily enzymes, such as human aldose reductase, halophilic bacterium reductase, and sea hare norsolorinic acid (a polyketide derivative) reductase-like protein. Catalytic residues and cofactor binding residues known in AKR superfamily enzymes were fairly well conserved in HdRed. Phylogenetic analysis for HdRed and AKR superfamily enzymes indicated that HdRed is an AKR belonging to a novel family. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Aldo-keto reductase 1C15 as a quinone reductase in rat endothelial cell: its involvement in redox cycling of 9,10-phenanthrenequinone.

    Science.gov (United States)

    Matsunaga, Toshiyuki; Shinoda, Yuhki; Inoue, Yukari; Shimizu, Yuki; Haga, Mariko; Endo, Satoshi; El-Kabbani, Ossama; Hara, Akira

    2011-07-01

    9,10-Phenanthrenequinone (9,10-PQ), a redox-active quinone in diesel exhausts, triggers cellular apoptosis via reactive oxygen species (ROS) generation in its redox cycling. This study found that induction of CCAAT/enhancer-binding protein-homologous protein (CHOP), a pro-apoptotic factor derived from endoplasmic reticulum stress, participates in the mechanism of rat endothelial cell damage. The 9,10-PQ-mediated CHOP induction was strengthened by a proteasome inhibitor (MG132) and the MG132-induced cell sensitization to the 9,10-PQ toxicity was abolished by a ROS inhibitor, suggesting that ROS generation and consequent proteasomal dysfunction are responsible for the CHOP up-regulation caused by 9,10-PQ. Aldo-keto reductase (AKR) 1C15 expressed in rat endothelial cells reduced 9,10-PQ into 9,10-dihydroxyphenanthrene concomitantly with superoxide anion formation, implying its participation in evoking the 9,10-PQ-redox cycling. The 9,10-PQ-induced damage was augmented by AKR1C15 over-expression. 9,10-PQ also provoked the AKR1C15 up-regulation, which sensitized against the quinone toxicity. These results suggest the presence of a negative feedback loop exacerbating the quinone toxicity in rat endothelial cells. © 2011 Informa UK, Ltd.

  5. A novel aldo-keto reductase from Jatropha curcas L. (JcAKR) plays a crucial role in the detoxification of methylglyoxal, a potent electrophile.

    Science.gov (United States)

    Mudalkar, Shalini; Sreeharsha, Rachapudi Venkata; Reddy, Attipalli Ramachandra

    2016-05-20

    Abiotic stress leads to the generation of reactive oxygen species (ROS) which further results in the production of reactive carbonyls (RCs) including methylglyoxal (MG). MG, an α, β-dicarbonyl aldehyde, is highly toxic to plants and the mechanism behind its detoxification is not well understood. Aldo-keto reductases (AKRs) play a role in detoxification of reactive aldehydes and ketones. In the present study, we cloned and characterised a putative AKR from Jatropha curcas (JcAKR). Phylogenetically, it forms a small clade with AKRs of Glycine max and Rauwolfia serpentina. JcAKR was heterologously expressed in Escherichia coli BL-21(DE3) cells and the identity of the purified protein was confirmed through MALDI-TOF analysis. The recombinant protein had high enzyme activity and catalytic efficiency in assays containing MG as the substrate. Protein modelling and docking studies revealed MG was efficiently bound to JcAKR. Under progressive drought and salinity stress, the enzyme and transcript levels of JcAKR were higher in leaves compared to roots. Further, the bacterial and yeast cells expressing JcAKR showed more tolerance towards PEG (5%), NaCl (200mM) and MG (5mM) treatments compared to controls. In conclusion, our results project JcAKR as a possible and potential target in crop improvement for abiotic stress tolerance. Copyright © 2016 Elsevier GmbH. All rights reserved.

  6. Aldo-keto reductase family 1 B10 protein detoxifies dietary and lipid-derived alpha, beta-unsaturated carbonyls at physiological levels

    Energy Technology Data Exchange (ETDEWEB)

    Zhong, Linlin [Department of Medical Microbiology, Immunology, and Cell Biology, SimmonsCooper Cancer Institute, Southern Illinois University School of Medicine, 913 N. Rutledge Street, Springfield, IL 62794-9626 (United States); Department of Neurobiology and Anatomy, China Medical University, Shenyang 110001 (China); Liu, Ziwen [Department of Medical Microbiology, Immunology, and Cell Biology, SimmonsCooper Cancer Institute, Southern Illinois University School of Medicine, 913 N. Rutledge Street, Springfield, IL 62794-9626 (United States); Department of Surgery, Peking Union Medical College Hospital, Beijing 100730 (China); Yan, Ruilan [Department of Medical Microbiology, Immunology, and Cell Biology, SimmonsCooper Cancer Institute, Southern Illinois University School of Medicine, 913 N. Rutledge Street, Springfield, IL 62794-9626 (United States); Johnson, Stephen [Carbon Dynamics Institute, LLC, 2835 via Verde Drive, Springfield, IL 62703-4325 (United States); Zhao, Yupei [Department of Surgery, Peking Union Medical College Hospital, Beijing 100730 (China); Fang, Xiubin [Department of Neurobiology and Anatomy, China Medical University, Shenyang 110001 (China); Cao, Deliang, E-mail: dcao@siumed.edu [Department of Medical Microbiology, Immunology, and Cell Biology, SimmonsCooper Cancer Institute, Southern Illinois University School of Medicine, 913 N. Rutledge Street, Springfield, IL 62794-9626 (United States)

    2009-09-18

    Alpha, beta-unsaturated carbonyls are highly reactive mutagens and carcinogens to which humans are exposed on a daily basis. This study demonstrates that aldo-keto reductase family 1 member B10 (AKR1B10) is a critical protein in detoxifying dietary and lipid-derived unsaturated carbonyls. Purified AKR1B10 recombinant protein efficiently catalyzed the reduction to less toxic alcohol forms of crotonaldehyde at 0.90 {mu}M, 4-hydroxynonenal (HNE) at 0.10 {mu}M, trans-2-hexanal at 0.10 {mu}M, and trans-2,4-hexadienal at 0.05 {mu}M, the concentrations at or lower than physiological exposures. Ectopically expressed AKR1B10 in 293T cells eliminated immediately HNE at 1 (subtoxic) or 5 {mu}M (toxic) by converting to 1,4-dihydroxynonene, protecting the cells from HNE toxicity. AKR1B10 protein also showed strong enzymatic activity toward glutathione-conjugated carbonyls. Taken together, our study results suggest that AKR1B10 specifically expressed in the intestine is physiologically important in protecting the host cell against dietary and lipid-derived cytotoxic carbonyls.

  7. Involvement of an aldo-keto reductase (AKR1C3) in redox cycling of 9,10-phenanthrenequinone leading to apoptosis in human endothelial cells.

    Science.gov (United States)

    Matsunaga, Toshiyuki; Arakaki, Marina; Kamiya, Tetsuro; Endo, Satoshi; El-Kabbani, Ossama; Hara, Akira

    2009-09-14

    9,10-Phenanthrenequinone (9,10-PQ), a major quinone found in diesel exhaust particles, is considered to generate reactive oxygen species (ROS) through its redox cycling. Here, we show that 9,10-PQ evokes apoptosis in human aortic endothelial cells (HAECs) and its apoptotic signaling includes ROS generation and caspase activation. The 9,10-PQ-induced cytotoxicity was inhibited by ROS scavengers, indicating that intracellular ROS generation is responsible for the 9,10-PQ-induced apoptosis. Comparison of mRNA expression levels and kinetic constants in the 9,10-PQ reduction among 10 human reductases suggests that aldo-keto reductase 1C3 (AKR1C3) is a 9,10-PQ reductase in HAECs. In in vitro 9,10-PQ reduction by AKR1C3, the reduced product 9,10-dihydroxyphenanthrene and superoxide anions were formed, suggesting the enzymatic two-electron reduction of 9,10-PQ that thereby causes oxidative stress through its redox cycling. In addition, the participation of AKR1C3 in 9,10-PQ-redox cycling was confirmed by the data that AKR1C3 overexpression in endothelial cells augmented the ROS generation and cytotoxicity by 9,10-PQ, and the ROS scavengers inhibited the toxic effects. Pretreatment of the overexpressing cells with AKR1C3 inhibitors, flufenamic acid and indomethacin, suppressed the 9,10-PQ-induced GSH depletion. These results suggest that AKR1C3 is a key enzyme in the initial step of 9,10-PQ-induced cytotoxicity in HAECs.

  8. Expression of human aldo-keto reductase 1C2 in cell lines of peritoneal endometriosis: potential implications in metabolism of progesterone and dydrogesterone and inhibition by progestins.

    Science.gov (United States)

    Beranič, Nataša; Brožič, Petra; Brus, Boris; Sosič, Izidor; Gobec, Stanislav; Lanišnik Rižner, Tea

    2012-05-01

    The human aldo-keto reductase AKR1C2 converts 5α-dihydrotestosterone to the less active 3α-androstanediol and has a minor 20-ketosteroid reductase activity that metabolises progesterone to 20α-hydroxyprogesterone. AKR1C2 is expressed in different peripheral tissues, but its role in uterine diseases like endometriosis has not been studied in detail. Some progestins used for treatment of endometriosis inhibit AKR1C1 and AKR1C3, with unknown effects on AKR1C2. In this study we investigated expression of AKR1C2 in the model cell lines of peritoneal endometriosis, and examined the ability of recombinant AKR1C2 to metabolise progesterone and progestin dydrogesterone, as well as its potential inhibition by progestins. AKR1C2 is expressed in epithelial and stromal endometriotic cell lines at the mRNA level. The recombinant enzyme catalyses reduction of progesterone to 20α-hydroxyprogesterone with a 10-fold lower catalytic efficiency than the major 20-ketosteroid reductase, AKR1C1. AKR1C2 also metabolises progestin dydrogesterone to its 20α-dihydrodydrogesterone, with 8.6-fold higher catalytic efficiency than 5α-dihydrotestosterone. Among the progestins that are currently used for treatment of endometriosis, dydrogesterone, medroxyprogesterone acetate and 20α-dihydrodydrogesterone act as AKR1C2 inhibitors with low μM K(i) values in vitro. Their potential in vivo effects should be further studied. Copyright © 2011 Elsevier Ltd. All rights reserved.

  9. 9,10-phenanthrenequinone induces monocytic differentiation of U937 cells through regulating expression of aldo-keto reductase 1C3.

    Science.gov (United States)

    Matsunaga, Toshiyuki; Hosogai, Mika; Arakaki, Marina; Endo, Satoshi; El-Kabbani, Ossama; Hara, Akira

    2012-01-01

    Persistent inhalation of diesel exhaust particles results in damaged lung cells through formation of reactive oxygen species (ROS), but the details of the toxicity mechanism against monocytes are poorly understood. In this study, we used human promyelomonocytic U937 cells as surrogates of monocytes and investigated the toxicity mechanism initiated by exposure to 9,10-phenanthrenequinone (9,10-PQ), a major quinone component in diesel exhaust particles. A 24-h incubation with 9,10-PQ provoked apoptotic cell death, which was due to signaling through the enhanced ROS generation and concomitant caspase activation. Flow cytometric analyses of U937 cells after long-term exposure to 9,10-PQ revealed induction of differentiation that was evidenced by increasing expression of CD11b/CD18, a cell-surface marker for monocytic differentiation into macrophages. The 9,10-PQ-induced differentiation was significantly abolished by ROS inhibitors, suggesting that ROS generation contributes to cell differentiation. The 9,10-PQ treatment increased the expression of aldo-keto reductase (AKR) 1C3, which reached a peak at 1 to 2 d post-treatment and then declined. The bell-shaped curve of the AKR1C3 expression by 9,10-PQ resembled that caused by phorbol 12-myristate 13-acetate, a differentiation inducer. Additionally, the concomitant treatment with tolfenamic acid, a selective AKR1C3 inhibitor, sensitized the differentiation induced by 9,10-PQ. These results suggest that ROS formation during 9,10-PQ treatment acutely leads to apoptosis of U937 cells and the initiation of monocytic differentiation, which proceeds after the provisional overexpression of AKR1C3.

  10. Exposure to 9,10-phenanthrenequinone accelerates malignant progression of lung cancer cells through up-regulation of aldo-keto reductase 1B10.

    Science.gov (United States)

    Matsunaga, Toshiyuki; Morikawa, Yoshifumi; Haga, Mariko; Endo, Satoshi; Soda, Midori; Yamamura, Keiko; El-Kabbani, Ossama; Tajima, Kazuo; Ikari, Akira; Hara, Akira

    2014-07-15

    Inhalation of 9,10-phenanthrenequinone (9,10-PQ), a major quinone in diesel exhaust, exerts fatal damage against a variety of cells involved in respiratory function. Here, we show that treatment with high concentrations of 9,10-PQ evokes apoptosis of lung cancer A549 cells through production of reactive oxygen species (ROS). In contrast, 9,10-PQ at its concentrations of 2 and 5 μM elevated the potentials for proliferation, invasion, metastasis and tumorigenesis, all of which were almost completely inhibited by addition of an antioxidant N-acetyl-l-cysteine, inferring a crucial role of ROS in the overgrowth and malignant progression of lung cancer cells. Comparison of mRNA expression levels of six aldo-keto reductases (AKRs) in the 9,10-PQ-treated cells advocated up-regulation of AKR1B10 as a major cause contributing to the lung cancer malignancy. In support of this, the elevation of invasive, metastatic and tumorigenic activities in the 9,10-PQ-treated cells was significantly abolished by the addition of a selective AKR1B10 inhibitor oleanolic acid. Intriguingly, zymographic and real-time PCR analyses revealed remarkable increases in secretion and expression, respectively, of matrix metalloproteinase 2 during the 9,10-PQ treatment, and suggested that the AKR1B10 up-regulation and resultant activation of mitogen-activated protein kinase cascade are predominant mechanisms underlying the metalloproteinase induction. In addition, HPLC analysis and cytochrome c reduction assay in in vitro 9,10-PQ reduction by AKR1B10 demonstrated that the enzyme catalyzes redox-cycling of this quinone, by which ROS are produced. Collectively, these results suggest that AKR1B10 is a key regulator involved in overgrowth and malignant progression of the lung cancer cells through ROS production due to 9,10-PQ redox-cycling. Copyright © 2014 Elsevier Inc. All rights reserved.

  11. Fluoxetine elevates allopregnanolone in female rat brain but inhibits a steroid microsomal dehydrogenase rather than activating an aldo-keto reductase.

    Science.gov (United States)

    Fry, J P; Li, K Y; Devall, A J; Cockcroft, S; Honour, J W; Lovick, T A

    2014-12-01

    Fluoxetine, a selective serotonin reuptake inhibitor, elevates brain concentrations of the neuroactive progesterone metabolite allopregnanolone, an effect suggested to underlie its use in the treatment of premenstrual dysphoria. One report showed fluoxetine to activate the aldo-keto reductase (AKR) component of 3α-hydroxysteroid dehydrogenase (3α-HSD), which catalyses production of allopregnanolone from 5α-dihydroprogesterone. However, this action was not observed by others. The present study sought to clarify the site of action for fluoxetine in elevating brain allopregnanolone. Adult male rats and female rats in dioestrus were treated with fluoxetine and their brains assayed for allopregnanolone and its precursors, progesterone and 5α-dihydroprogesterone. Subcellular fractions of rat brain were also used to investigate the actions of fluoxetine on 3α-HSD activity in both the reductive direction, producing allopregnanolone from 5α-dihydroprogesterone, and the reverse oxidative direction. Fluoxetine was also tested on these recombinant enzyme activities expressed in HEK cells. Short-term treatment with fluoxetine increased brain allopregnanolone concentrations in female, but not male, rats. Enzyme assays on native rat brain fractions and on activities expressed in HEK cells showed fluoxetine did not affect the AKR producing allopregnanolone from 5α-dihydroprogesterone but did inhibit the microsomal dehydrogenase oxidizing allopregnanolone to 5α-dihydroprogesterone. Fluoxetine elevated allopregnanolone in female rat brain by inhibiting its oxidation to 5α-dihydroprogesterone by a microsomal dehydrogenase. This is a novel site of action for fluoxetine, with implications for the development of new agents and/or dosing regimens to raise brain allopregnanolone. © 2014 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of The British Pharmacological Society.

  12. The Aldo-Keto Reductase AKR1B10 Is Up-Regulated in Keloid Epidermis, Implicating Retinoic Acid Pathway Dysregulation in the Pathogenesis of Keloid Disease.

    Science.gov (United States)

    Jumper, Natalie; Hodgkinson, Tom; Arscott, Guyan; Har-Shai, Yaron; Paus, Ralf; Bayat, Ardeshir

    2016-07-01

    Keloid disease is a recurrent fibroproliferative cutaneous tumor of unknown pathogenesis for which clinical management remains unsatisfactory. To obtain new insights into hitherto underappreciated aspects of keloid pathobiology, we took a laser capture microdissection-based, whole-genome microarray analysis approach to identify distinct keloid disease-associated gene expression patterns within defined keloid regions. Identification of the aldo-keto reductase enzyme AKR1B10 as highly up-regulated in keloid epidermis suggested that an imbalance of retinoic acid metabolism is likely associated with keloid disease. Here, we show that AKR1B10 transfection into normal human keratinocytes reproduced the abnormal retinoic acid pathway expression pattern we had identified in keloid epidermis. Cotransfection of AKR1B10 with a luciferase reporter plasmid showed reduced retinoic acid response element activity, supporting the hypothesis of retinoic acid synthesis deficiency in keloid epidermis. Paracrine signals released by AKR1B10-overexpressing keratinocytes into conditioned medium resulted in up-regulation of transforming growth factor-β1, transforming growth factor-β2, and collagens I and III in both keloid and normal skin fibroblasts, mimicking the typical profibrotic keloid profile. Our study results suggest that insufficient retinoic acid synthesis by keloid epidermal keratinocytes may contribute to the pathogenesis of keloid disease. We refocus attention on the role of injured epithelium in keloid disease and identify AKR1B10 as a potential new target in future management of keloid disease. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  13. Characterization of AKR4C15, a Novel Member of Aldo-Keto Reductase, in Comparison with Other Rice AKR(s).

    Science.gov (United States)

    Auiyawong, Budsakorn; Narawongsanont, Rawint; Tantitadapitak, Chonticha

    2017-08-01

    Environmental stresses often cause a rapid and excessive accumulation of reactive oxygen species (ROS), the toxicity of which is further amplified by downstream aldehyde production. Aldo-keto reductase (AKR) is a group of enzymes metabolizing aldehyde/ketone to the corresponding alcohol using NADPH as the cofactor. In this study, OsI_20197 (AKR4C15), a novel member of AKR4 subfamily C, was isolated and biochemically characterized. Kinetic studies on bacterially-expressed recombinant AKR4C15 revealed that the enzyme was capable of metabolizing a wide variety of aldehydes but clearly exhibited a preference for three carbon compounds, i.e. methylglyoxal, malondialdehyde and glyceraldehyde. In comparison with His-tagged proteins of AKR4C9 from Arabidopsis and several other rice AKR(s): OsI_04426, OsI_04428, OsI_04429, and OsI_15387, AKR4C15 was the one capable of most efficiently metabolizing MDA and had the highest value of catalytic efficiency, which was higher than the value of AKR4C9, approximately, by 30-fold; while its capability of metabolizing MG was on par with AKR4C9, OsI_04426 and OsI_04428 (AKR4C14); and was considerably higher than the activity of OsI_04429 and OsI_15387. In vivo research on transgenic Arabidopsis seedlings ectopically-expressing AKR4C15 showed that the levels of both MDA and MG were also significantly lower than the levels in wild-type seedlings under both normal and stress conditions, emphasizing the role of AKR4C15 in MG and MDA metabolism. In conclusion, AKR4C15, together with OsI_04426 and AKR4C14, may play protective roles against small reactive aldehydes and medium-chain aldehydes.

  14. Major differences exist in the function and tissue-specific expression of human aflatoxin B1 aldehyde reductase and the principal human aldo-keto reductase AKR1 family members.

    Science.gov (United States)

    O'connor, T; Ireland, L S; Harrison, D J; Hayes, J D

    1999-01-01

    Complementary DNA clones encoding human aflatoxin B(1) aldehyde reductase (AKR7A2), aldehyde reductase (AKR1A1), aldose reductase (AKR1B1), dihydrodiol dehydrogenase 1 (AKR1C1) and chlordecone reductase (AKR1C4) have been expressed in Escherichia coli. These members of the aldo-keto reductase (AKR) superfamily have been purified from E. coli as recombinant proteins. The recently identified AKR7A2 was shown to differ from the AKR1 isoenzymes in being able to catalyse the reduction of 2-carboxybenzaldehyde. Also, AKR7A2 was found to exhibit a narrow substrate specificity, with activity being restricted to succinic semialdehyde (SSA), 2-nitrobenzaldehyde, pyridine-2-aldehyde, isatin, 1,2-naphthoquinone (1,2-NQ) and 9,10-phenanthrenequinone. In contrast, AKR1A1 reduces a broad spectrum of carbonyl-containing compounds, displaying highest specific activity for SSA, 4-carboxybenzaldehyde, 4-nitrobenzaldehyde, pyridine-3-aldehyde, pyridine-4-aldehyde, 4-hydroxynonenal, phenylglyoxal, methylglyoxal, 2,3-hexanedione, 1, 2-NQ, 16-ketoestrone and d-glucuronic acid. Comparison between the kinetic properties of AKR7A2 and AKR1A1 showed that both recombinant enzymes exhibited roughly similar k(cat)/K(m) values for SSA, 1,2-NQ and 16-ketoestrone. Many of the compounds which are substrates for AKR1A1 also serve as substrates for AKR1B1, though the latter enzyme was shown to display a specific activity significantly less than that of AKR1A1 for most of the aromatic and aliphatic aldehydes studied. Neither AKR1C1 nor AKR1C4 was found to possess high reductase activity towards aliphatic aldehydes, aromatic aldehydes, aldoses or dicarbonyls. However, unlike AKR1A1 and AKR1B1, both AKR1C1 and AKR1C4 were able to catalyse the oxidation of 1-acenaphthenol and, in addition, AKR1C4 could oxidize di- and tri-hydroxylated bile acids. Specific antibodies raised against AKR7A2, AKR1A1, AKR1B1, AKR1C1 and AKR1C4 have been used to show the presence of all of the reductases in human hepatic

  15. Exposure to 9,10-phenanthrenequinone accelerates malignant progression of lung cancer cells through up-regulation of aldo-keto reductase 1B10

    International Nuclear Information System (INIS)

    Matsunaga, Toshiyuki; Morikawa, Yoshifumi; Haga, Mariko; Endo, Satoshi; Soda, Midori; Yamamura, Keiko; El-Kabbani, Ossama; Tajima, Kazuo; Ikari, Akira; Hara, Akira

    2014-01-01

    Inhalation of 9,10-phenanthrenequinone (9,10-PQ), a major quinone in diesel exhaust, exerts fatal damage against a variety of cells involved in respiratory function. Here, we show that treatment with high concentrations of 9,10-PQ evokes apoptosis of lung cancer A549 cells through production of reactive oxygen species (ROS). In contrast, 9,10-PQ at its concentrations of 2 and 5 μM elevated the potentials for proliferation, invasion, metastasis and tumorigenesis, all of which were almost completely inhibited by addition of an antioxidant N-acetyl-L-cysteine, inferring a crucial role of ROS in the overgrowth and malignant progression of lung cancer cells. Comparison of mRNA expression levels of six aldo-keto reductases (AKRs) in the 9,10-PQ-treated cells advocated up-regulation of AKR1B10 as a major cause contributing to the lung cancer malignancy. In support of this, the elevation of invasive, metastatic and tumorigenic activities in the 9,10-PQ-treated cells was significantly abolished by the addition of a selective AKR1B10 inhibitor oleanolic acid. Intriguingly, zymographic and real-time PCR analyses revealed remarkable increases in secretion and expression, respectively, of matrix metalloproteinase 2 during the 9,10-PQ treatment, and suggested that the AKR1B10 up-regulation and resultant activation of mitogen-activated protein kinase cascade are predominant mechanisms underlying the metalloproteinase induction. In addition, HPLC analysis and cytochrome c reduction assay in in vitro 9,10-PQ reduction by AKR1B10 demonstrated that the enzyme catalyzes redox-cycling of this quinone, by which ROS are produced. Collectively, these results suggest that AKR1B10 is a key regulator involved in overgrowth and malignant progression of the lung cancer cells through ROS production due to 9,10-PQ redox-cycling. - Highlights: • 9,10-PQ promotes invasion, metastasis and tumorigenicity in lung cancer cells. • The 9,10-PQ-elicited promotion is possibly due to AKR1B10 up

  16. Exposure to 9,10-phenanthrenequinone accelerates malignant progression of lung cancer cells through up-regulation of aldo-keto reductase 1B10

    Energy Technology Data Exchange (ETDEWEB)

    Matsunaga, Toshiyuki, E-mail: matsunagat@gifu-pu.ac.jp [Laboratory of Biochemistry, Gifu Pharmaceutical University, Gifu 501-1196 (Japan); Morikawa, Yoshifumi; Haga, Mariko; Endo, Satoshi [Laboratory of Biochemistry, Gifu Pharmaceutical University, Gifu 501-1196 (Japan); Soda, Midori; Yamamura, Keiko [Laboratory of Clinical Pharmacy, School of Pharmacy, Aichi Gakuin University, Nagoya 464-8650 (Japan); El-Kabbani, Ossama [Monash Institute of Pharmaceutical Sciences, Monash University, Victoria 3052 (Australia); Tajima, Kazuo [Faculty of Pharmaceutical Sciences, Hokuriku University, Kanazawa 920-1181 (Japan); Ikari, Akira [Laboratory of Biochemistry, Gifu Pharmaceutical University, Gifu 501-1196 (Japan); Hara, Akira [Faculty of Engineering, Gifu University, Gifu 501-1193 (Japan)

    2014-07-15

    Inhalation of 9,10-phenanthrenequinone (9,10-PQ), a major quinone in diesel exhaust, exerts fatal damage against a variety of cells involved in respiratory function. Here, we show that treatment with high concentrations of 9,10-PQ evokes apoptosis of lung cancer A549 cells through production of reactive oxygen species (ROS). In contrast, 9,10-PQ at its concentrations of 2 and 5 μM elevated the potentials for proliferation, invasion, metastasis and tumorigenesis, all of which were almost completely inhibited by addition of an antioxidant N-acetyl-L-cysteine, inferring a crucial role of ROS in the overgrowth and malignant progression of lung cancer cells. Comparison of mRNA expression levels of six aldo-keto reductases (AKRs) in the 9,10-PQ-treated cells advocated up-regulation of AKR1B10 as a major cause contributing to the lung cancer malignancy. In support of this, the elevation of invasive, metastatic and tumorigenic activities in the 9,10-PQ-treated cells was significantly abolished by the addition of a selective AKR1B10 inhibitor oleanolic acid. Intriguingly, zymographic and real-time PCR analyses revealed remarkable increases in secretion and expression, respectively, of matrix metalloproteinase 2 during the 9,10-PQ treatment, and suggested that the AKR1B10 up-regulation and resultant activation of mitogen-activated protein kinase cascade are predominant mechanisms underlying the metalloproteinase induction. In addition, HPLC analysis and cytochrome c reduction assay in in vitro 9,10-PQ reduction by AKR1B10 demonstrated that the enzyme catalyzes redox-cycling of this quinone, by which ROS are produced. Collectively, these results suggest that AKR1B10 is a key regulator involved in overgrowth and malignant progression of the lung cancer cells through ROS production due to 9,10-PQ redox-cycling. - Highlights: • 9,10-PQ promotes invasion, metastasis and tumorigenicity in lung cancer cells. • The 9,10-PQ-elicited promotion is possibly due to AKR1B10 up

  17. Lack of functional and expression homology between human and mouse aldo-keto reductase 1C enzymes: implications for modelling human cancers

    Directory of Open Access Journals (Sweden)

    Schrewe Heinrich

    2009-12-01

    Full Text Available Abstract Background Over recent years, enzymes of the aldo-keto reductase (AKR 1C subfamily have been implicated in the progression of prostate, breast, endometrial and leukemic cancers. This is due to the ability of AKR1C enzymes to modify androgens, estrogens, progesterone and prostaglandins (PGs in a tissue-specific manner, regulating the activity of nuclear receptors and other downstream effects. Evidence supporting a role for AKR1C enzymes in cancer derives mostly from studies with isolated primary cells from patients or immortalized cell lines. Mice are ideal organisms for in vivo studies, using knock-out or over-expression strains. However, the functional conservation of AKR1C enzymes between human and mice has yet to be described. Results In this study, we have characterized and compared the four human (AKR1C1,-1C2, -1C3 and -1C4 and the eight murine (AKR1C6, -1C12, -1C13, -1C14, -1C18, -1C19, -1C20 and -1C21 isoforms in their phylogeny, substrate preference and tissue distribution. We have found divergent evolution between human and murine AKR1C enzymes that was reflected by differing substrate preference. Murine enzymes did not perform the 11β-ketoreduction of prostaglandin (PG D2, an activity specific to human AKR1C3 and important in promoting leukemic cell survival. Instead, murine AKR1C6 was able to perform the 9-ketoreduction of PGE2, an activity absent amongst human isoforms. Nevertheless, reduction of the key steroids androstenedione, 5α-dihydrotestosterone, progesterone and estrone was found in murine isoforms. However, unlike humans, no AKR1C isoforms were detected in murine prostate, testes, uterus and haemopoietic progenitors. Conclusions This study exposes significant lack of phylogenetic and functional homology between human and murine AKR1C enzymes. Therefore, we conclude that mice are not suitable to model the role of AKR1C in human cancers and leukemia.

  18. Crystal structures of three classes of non-steroidal anti-inflammatory drugs in complex with aldo-keto reductase 1C3.

    Directory of Open Access Journals (Sweden)

    Jack U Flanagan

    Full Text Available Aldo-keto reductase 1C3 (AKR1C3 catalyses the NADPH dependent reduction of carbonyl groups in a number of important steroid and prostanoid molecules. The enzyme is also over-expressed in prostate and breast cancer and its expression is correlated with the aggressiveness of the disease. The steroid products of AKR1C3 catalysis are important in proliferative signalling of hormone-responsive cells, while the prostanoid products promote prostaglandin-dependent proliferative pathways. In these ways, AKR1C3 contributes to tumour development and maintenance, and suggest that inhibition of AKR1C3 activity is an attractive target for the development of new anti-cancer therapies. Non-steroidal anti-inflammatory drugs (NSAIDs are one well-known class of compounds that inhibits AKR1C3, yet crystal structures have only been determined for this enzyme with flufenamic acid, indomethacin, and closely related analogues bound. While the flufenamic acid and indomethacin structures have been used to design novel inhibitors, they provide only limited coverage of the NSAIDs that inhibit AKR1C3 and that may be used for the development of new AKR1C3 targeted drugs. To understand how other NSAIDs bind to AKR1C3, we have determined ten crystal structures of AKR1C3 complexes that cover three different classes of NSAID, N-phenylanthranilic acids (meclofenamic acid, mefenamic acid, arylpropionic acids (flurbiprofen, ibuprofen, naproxen, and indomethacin analogues (indomethacin, sulindac, zomepirac. The N-phenylanthranilic and arylpropionic acids bind to common sites including the enzyme catalytic centre and a constitutive active site pocket, with the arylpropionic acids probing the constitutive pocket more effectively. By contrast, indomethacin and the indomethacin analogues sulindac and zomepirac, display three distinctly different binding modes that explain their relative inhibition of the AKR1C family members. This new data from ten crystal structures greatly broadens

  19. The Role of Human Aldo-Keto Reductases (AKRs in the Metabolic Activation and Detoxication of Polycyclic Aromatic Hydrocarbons: Interconversion of PAH-catechols and PAH o-Quinones

    Directory of Open Access Journals (Sweden)

    Li eZhang

    2012-11-01

    Full Text Available Polycyclic aromatic hydrocarbons (PAH are ubiquitous environmental pollutants. They are procarcinogens requiring metabolic activation to elicit their deleterious effects. Aldo-keto reductases (AKR catalyze the oxidation of proximate carcinogenic PAH trans-dihydrodiols to yield electrophilic and redox-active PAH o-quiniones. AKRs are also found to be capable of reducing PAH o-quinones to form PAH catechols. The interconversion of o-quinones and catechols results in the redox cycling of PAH o-quinones to give rise to the generation of reactive oxygen species and subsequent oxidative DNA damage. On the other hand, PAH catechols can be intercepted through phase II metabolism by which PAH o-quinones could be detoxified and eliminated. The aim of the present review is to summarize the role of human AKRs in the metabolic activation/detoxication of PAH and the relevance of phase II conjugation reactions to human lung carcinogenesis.

  20. Sex hormones reduce NNK detoxification through inhibition of short-chain dehydrogenases/reductases and aldo-keto reductases in vitro.

    Science.gov (United States)

    Stapelfeld, Claudia; Maser, Edmund

    2017-10-01

    Carbonyl reduction is an important metabolic pathway for endogenous and xenobiotic substances. The tobacco specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK, nicotine-derived nitrosamine ketone) is classified as carcinogenic to humans (IARC, Group 1) and considered to play the most important role in tobacco-related lung carcinogenesis. Detoxification of NNK through carbonyl reduction is catalyzed by members of the AKR- and the SDR-superfamilies which include AKR1B10, AKR1C1, AKR1C2, AKR1C4, 11β-HSD1 and CBR1. Because some reductases are also involved in steroid metabolism, five different hormones were tested for their inhibitory effect on NNK carbonyl reduction. Two of those hormones were estrogens (estradiol and ethinylestradiol), another two hormones belong to the gestagen group (progesterone and drospirenone) and the last tested hormone was an androgen (testosterone). Furthermore, one of the estrogens (ethinylestradiol) and one of the gestagens (drospirenone) are synthetic hormones, used as hormonal contraceptives. Five of six NNK reducing enzymes (AKR1B10, AKR1C1, AKR1C2, AKR1C4 and 11β-HSD1) were significantly inhibited by the tested sex hormones. Only NNK reduction catalyzed by CBR1 was not significantly impaired. In the case of the other five reductases, gestagens had remarkably stronger inhibitory effects at a concentration of 25 μM (progesterone: 66-88% inhibition; drospirenone: 26-87% inhibition) in comparison to estrogens (estradiol: 17-51% inhibition; ethinylestradiol: 14-79% inhibition) and androgens (14-78% inhibition). Moreover, in most cases the synthetic hormones showed a greater ability to inhibit NNK reduction than the physiologic derivatives. These results demonstrate that male and female sex hormones have different inhibitory potentials, thus indicating that there is a varying detoxification capacity of NNK in men and women which could result in a different risk for developing lung cancer. Copyright © 2017 Elsevier B

  1. Trypanosoma cruzi: death phenotypes induced by ortho-naphthoquinone substrates of the aldo-keto reductase (TcAKR). Role of this enzyme in the mechanism of action of β-lapachone.

    Science.gov (United States)

    Garavaglia, Patricia Andrea; Rubio, María Fernanda; Laverrière, Marc; Tasso, Laura Mónica; Fichera, Laura Edith; Cannata, Joaquín J B; García, Gabriela Andrea

    2018-02-05

    Several ortho-naphthoquinones (o-NQs) have trypanocidal activity against Trypanosoma cruzi, the aetiological agent of Chagas disease. Previously, we demonstrated that the aldo-keto reductase from this parasite (TcAKR) reduces o-NQs, such as β-lapachone (β-Lap) and 9,10-phenanthrenequinone (9,10-PQ), with concomitant reactive oxygen species (ROS) production. Recent characterization of TcAKR activity and expression in two T. cruzi strains, CL Brener and Nicaragua, showed that TcAKR expression is 2.2-fold higher in CL Brener than in Nicaragua. Here, we studied the trypanocidal effect and induction of several death phenotypes by β-Lap and 9,10-PQ in epimastigotes of these two strains. The CL Brener strain was more resistant to both o-NQs than Nicaragua, indicating that greater TcAKR activity is unlikely to be a major influence on o-NQ toxicity. Evaluation of changes in ROS production, mitochondrial membrane potential, phosphatidylserine exposure and monodansylcadaverine labelling evidenced that β-Lap and 9,10-PQ induce different death phenotypes depending on the combination of drug and T. cruzi strain analysed. To study whether TcAKR participates in o-NQ activation in intact parasites, β-Lap and 9,10-PQ trypanocidal effect was next evaluated in TcAKR-overexpressing parasites. Only β-Lap was more effective and induced greater ROS production in TcAKR-overexpressing epimastigotes than in controls, suggesting that TcAKR may participate in β-Lap activation.

  2. Overexpression of aldo-keto reductase 1C3 (AKR1C3) in LNCaP cells diverts androgen metabolism towards testosterone resulting in resistance to the 5α-reductase inhibitor finasteride.

    Science.gov (United States)

    Byrns, Michael C; Mindnich, Rebekka; Duan, Ling; Penning, Trevor M

    2012-05-01

    Type 5 17β-hydroxysteroid dehydrogenase (AKR1C3) is the major enzyme in the prostate that reduces 4-androstene-3,17-dione (Δ(4)-Adione) to the androgen receptor (AR) ligand testosterone. AKR1C3 is upregulated in prostate cancer (PCa) and castrate resistant prostate cancer (CRPC) that develops after androgen deprivation therapy. PCa and CRPC often depend on intratumoral androgen biosynthesis and upregulation of AKR1C3 could contribute to intracellular synthesis of AR ligands and stimulation of proliferation through AR signaling. To test this hypothesis, we developed an LNCaP prostate cancer cell line overexpressing AKR1C3 (LNCaP-AKR1C3) and compared its metabolic and proliferative responses to Δ(4)-Adione treatment with that of the parental, AKR1C3 negative LNCaP cells. In LNCaP and LNCaP-AKR1C3 cells, metabolism proceeded via 5α-reduction to form 5α-androstane-3,17-dione and then (epi)androsterone-3-glucuronide. LNCaP-AKR1C3 cells made significantly higher amounts of testosterone-17β-glucuronide. When 5α-reductase was inhibited by finasteride, the production of testosterone-17β-glucuronide was further elevated in LNCaP-AKR1C3 cells. When AKR1C3 activity was inhibited with indomethacin the production of testosterone-17β-glucuronide was significantly decreased. Δ(4)-Adione treatment stimulated cell proliferation in both cell lines. Finasteride inhibited LNCaP cell proliferation, consistent with 5α-androstane-3,17-dione acting as the major metabolite that stimulates growth by binding to the mutated AR. However, LNCaP-AKR1C3 cells were resistant to the growth inhibitory properties of finasteride, consistent with the diversion of Δ(4)-Adione metabolism from 5α-reduced androgens to increased formation of testosterone. Indomethacin did not result in differences in Δ(4)-Adione induced proliferation since this treatment led to the same metabolic profile in LNCaP and LNCaP-AKR1C3 cells. We conclude that AKR1C3 overexpression diverts androgen metabolism to

  3. An Indomethacin Analogue, N-(4-Chlorobenzoyl)-melatonin, is a Selective Inhibitor of Aldo-keto Reductase 1C3 (Type 2 3α-HSD, Type 5 17β-HSD, and Prostaglandin F Synthase), a Potential Target for the Treatment of Hormone Dependent and Hormone Independent Malignancies

    Science.gov (United States)

    Byrns, Michael C.; Steckelbroeck, Stephan; Penning, Trevor M.

    2008-01-01

    Aldo-keto reductase (AKR) 1C3 (type 2 3α-HSD, type 5 17β-HSD, and prostaglandin F synthase) regulates ligand access to steroid hormone and prostaglandin receptors and may stimulate proliferation of prostate and breast cancer cells. NSAIDs are known inhibitors of AKR1C enzymes. An NSAID analogue that inhibits AKR1C3 but is inactive against the cyclooxygenases and the other AKR1C family members would provide an important tool to examine the role of AKR1C3 in proliferative signaling. We tested NSAIDs and NSAID analogues for inhibition of the reduction of 9,10-phenanthrenequinone (PQ) catalyzed by AKR1C3 and the closely related isoforms AKR1C1 and AKR1C2. Two of the compounds initially screened, indomethacin and its methyl ester, were specific for AKR1C3 versus the other AKR1C isoforms. Based on these results and the crystal structure of AKR1C3, we predicted that N-(4-chlorobenzoyl)-melatonin (CBM), an indomethacin analogue that does not inhibit the cyclooxygenases, would selectively inhibit AKR1C3. CBM inhibited the reduction of PQ by AKR1C3, but did not significantly inhibit AKR1C1 or AKR1C2. Indomethacin and CBM also inhibited the AKR1C3-catalyzed reduction of Δ4-androstene-3,17-dione but did not significantly inhibit the reduction of steroid hormones catalyzed by AKR1C1 or AKR1C2. The pattern of inhibition of AKR1C3 by indomethacin and CBM was uncompetitive versus PQ, but competitive versus Δ4-androstene-3,17-dione, indicating that two different inhibitory complexes form during the ordered bi-bi reactions. The identification of CBM as a specific inhibitor of AKR1C3 will aid the investigation of its roles in steroid hormone and prostaglandin signaling and the resultant effects on cancer development. PMID:17950253

  4. An indomethacin analogue, N-(4-chlorobenzoyl)-melatonin, is a selective inhibitor of aldo-keto reductase 1C3 (type 2 3alpha-HSD, type 5 17beta-HSD, and prostaglandin F synthase), a potential target for the treatment of hormone dependent and hormone independent malignancies.

    Science.gov (United States)

    Byrns, Michael C; Steckelbroeck, Stephan; Penning, Trevor M

    2008-01-15

    Aldo-keto reductase (AKR) 1C3 (type 2 3alpha-HSD, type 5 17beta-HSD, and prostaglandin F synthase) regulates ligand access to steroid hormone and prostaglandin receptors and may stimulate proliferation of prostate and breast cancer cells. NSAIDs are known inhibitors of AKR1C enzymes. An NSAID analogue that inhibits AKR1C3 but is inactive against the cyclooxygenases and the other AKR1C family members would provide an important tool to examine the role of AKR1C3 in proliferative signaling. We tested NSAIDs and NSAID analogues for inhibition of the reduction of 9,10-phenanthrenequinone (PQ) catalyzed by AKR1C3 and the closely related isoforms AKR1C1 and AKR1C2. Two of the compounds initially screened, indomethacin and its methyl ester, were specific for AKR1C3 versus the other AKR1C isoforms. Based on these results and the crystal structure of AKR1C3, we predicted that N-(4-chlorobenzoyl)-melatonin (CBM), an indomethacin analogue that does not inhibit the cyclooxygenases, would selectively inhibit AKR1C3. CBM inhibited the reduction of PQ by AKR1C3, but did not significantly inhibit AKR1C1 or AKR1C2. Indomethacin and CBM also inhibited the AKR1C3-catalyzed reduction of Delta(4)-androstene-3,17-dione but did not significantly inhibit the reduction of steroid hormones catalyzed by AKR1C1 or AKR1C2. The pattern of inhibition of AKR1C3 by indomethacin and CBM was uncompetitive versus PQ, but competitive versus Delta(4)-androstene-3,17-dione, indicating that two different inhibitory complexes form during the ordered bi bi reactions. The identification of CBM as a specific inhibitor of AKR1C3 will aid the investigation of its roles in steroid hormone and prostaglandin signaling and the resultant effects on cancer development.

  5. Aldo-keto Reductase Family 1 B10 as a Novel Target for Breast Cancer Treatment

    Science.gov (United States)

    2010-08-01

    University, Beijing 100084,People’s Republic of China . ¥To whom requests reprints: Deliang Cao, Department of Medical Microbiology, Immunology, & Cell...acids, phospholipids, triglycerides, and cholesterol, was analyzed by TLC . Lipid extracts and appropriate lipid standards were spotted on silica gel

  6. Crystallization and preliminary X-ray diffraction analysis of maize aldose reductase

    Energy Technology Data Exchange (ETDEWEB)

    Kiyota, Eduardo [Laboratório de Biologia Estrutural, Instituto de Química, Universidade Estadual de Campinas, CP 6154, 13083-970 Campinas-SP (Brazil); Centro de Biologia Molecular e Engenharia Genética, Universidade Estadual de Campinas, Campinas-SP (Brazil); Sousa, Sylvia Morais de [Centro de Biologia Molecular e Engenharia Genética, Universidade Estadual de Campinas, Campinas-SP (Brazil); Santos, Marcelo Leite dos; Costa Lima, Aline da [Laboratório de Biologia Estrutural, Instituto de Química, Universidade Estadual de Campinas, CP 6154, 13083-970 Campinas-SP (Brazil); Menossi, Marcelo [Departamento de Genética e Evolução, Instituto de Biologia, Universidade Estadual de Campinas, Campinas-SP (Brazil); Yunes, José Andrés [Laboratório de Biologia Molecular, Centro Infantil Boldrini, Campinas-SP (Brazil); Aparicio, Ricardo, E-mail: aparicio@iqm.unicamp.br [Laboratório de Biologia Estrutural, Instituto de Química, Universidade Estadual de Campinas, CP 6154, 13083-970 Campinas-SP (Brazil)

    2007-11-01

    Preliminary X-ray diffraction studies of apo maize aldose reductase at 2.0 Å resolution are reported. Maize aldose reductase (AR) is a member of the aldo-keto reductase superfamily. In contrast to human AR, maize AR seems to prefer the conversion of sorbitol into glucose. The apoenzyme was crystallized in space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 47.2, b = 54.5, c = 100.6 Å and one molecule in the asymmetric unit. Synchrotron X-ray diffraction data were collected and a final resolution limit of 2.0 Å was obtained after data reduction. Phasing was carried out by an automated molecular-replacement procedure and structural refinement is currently in progress. The refined structure is expected to shed light on the functional/enzymatic mechanism and the unusual activities of maize AR.

  7. Crystallization and preliminary X-ray diffraction analysis of maize aldose reductase

    International Nuclear Information System (INIS)

    Kiyota, Eduardo; Sousa, Sylvia Morais de; Santos, Marcelo Leite dos; Costa Lima, Aline da; Menossi, Marcelo; Yunes, José Andrés; Aparicio, Ricardo

    2007-01-01

    Preliminary X-ray diffraction studies of apo maize aldose reductase at 2.0 Å resolution are reported. Maize aldose reductase (AR) is a member of the aldo-keto reductase superfamily. In contrast to human AR, maize AR seems to prefer the conversion of sorbitol into glucose. The apoenzyme was crystallized in space group P2 1 2 1 2 1 , with unit-cell parameters a = 47.2, b = 54.5, c = 100.6 Å and one molecule in the asymmetric unit. Synchrotron X-ray diffraction data were collected and a final resolution limit of 2.0 Å was obtained after data reduction. Phasing was carried out by an automated molecular-replacement procedure and structural refinement is currently in progress. The refined structure is expected to shed light on the functional/enzymatic mechanism and the unusual activities of maize AR

  8. Pharmacological Characterization of a Novel Bifunctional Aldo-Keto Reductase 1C3 Inhibitor and Androgen Receptor Antagonist

    Science.gov (United States)

    2013-10-01

    metastatic form of the disease known as advanced prostate cancer (APC) or castrate resistant prostate cancer (CRPC). APC develops as a result of...Engl J Med 2011, 364, 1995-2005. 8. O’Donnell, A.; Judson, I.; Dowsett, M.; Raynaud , F.; Dearnaley, D.; Mason, M.; Harland, S.; Robbins, A

  9. The human short-chain dehydrogenase/reductase (SDR) superfamily: a bioinformatics summary.

    Science.gov (United States)

    Bray, James E; Marsden, Brian D; Oppermann, Udo

    2009-03-16

    The short-chain dehydrogenase/reductase (SDR) superfamily represents one of the largest protein superfamilies known to date. Enzymes of this family usually catalyse NAD(P)(H) dependent reactions with a substrate spectrum ranging from polyols, retinoids, steroids and fatty acid derivatives to xenobiotics. We have currently identified 73 SDR superfamily members within the human genome. A status report of the human SDR superfamily is provided in terms of 3D structure determination, co-factor preferences, subcellular localisation and functional annotation. A simple scoring system for measuring structural and functional information (SFS score) has also been introduced to monitor the status of 5 key metrics. Currently there are 17 SDR members with an SFS score of zero indicating that almost a quarter of the human SDR superfamily lacks substantial functional annotation.

  10. Substrate specificity of an aflatoxin-metabolizing aldehyde reductase.

    OpenAIRE

    Ellis, E M; Hayes, J D

    1995-01-01

    The enzyme from rat liver that reduces aflatoxin B1-dialdehyde exhibits a unique catalytic specificity distinct from that of other aldo-keto reductases. This enzyme, designated AFAR, displays high activity towards dicarbonyl-containing compounds with ketone groups on adjacent carbon atoms; 9,10-phenanthrenequinone, acenaphthenequinone and camphorquinone were found to be good substrates. Although AFAR can also reduce aromatic and aliphatic aldehydes such as succinic semialdehyde, it is inactiv...

  11. Reductive detoxification of acrolein as a potential role for aldehyde reductase (AKR1A) in mammals.

    Science.gov (United States)

    Kurahashi, Toshihiro; Kwon, Myoungsu; Homma, Takujiro; Saito, Yuka; Lee, Jaeyong; Takahashi, Motoko; Yamada, Ken-Ichi; Miyata, Satoshi; Fujii, Junichi

    2014-09-12

    Aldehyde reductase (AKR1A), a member of the aldo-keto reductase superfamily, suppresses diabetic complications via a reduction in metabolic intermediates; it also plays a role in ascorbic acid biosynthesis in mice. Because primates cannot synthesize ascorbic acid, a principle role of AKR1A appears to be the reductive detoxification of aldehydes. In this study, we isolated and immortalized mouse embryonic fibroblasts (MEFs) from wild-type (WT) and human Akr1a-transgenic (Tg) mice and used them to investigate the potential roles of AKR1A under culture conditions. Tg MEFs showed higher methylglyoxal- and acrolein-reducing activities than WT MEFs and also were more resistant to cytotoxicity. Enzymatic analyses of purified rat AKR1A showed that the efficiency of the acrolein reduction was about 20% that of glyceraldehyde. Ascorbic acid levels were quite low in the MEFs, and while the administration of ascorbic acid to the cells increased the intracellular levels of ascorbic acid, it had no affect on the resistance to acrolein. Endoplasmic reticulum stress and protein carbonylation induced by acrolein treatment were less evident in Tg MEFs than in WT MEFs. These data collectively indicate that one of the principle roles of AKR1A in primates is the reductive detoxification of aldehydes, notably acrolein, and protection from its detrimental effects. Copyright © 2014 Elsevier Inc. All rights reserved.

  12. Substrate Specificity, Inhibitor Selectivity and Structure-Function Relationships of Aldo-Keto Reductase 1B15: A Novel Human Retinaldehyde Reductase

    Czech Academy of Sciences Publication Activity Database

    Giménez-Dejoz, J.; Kolář, Michal H.; Ruiz, F. X.; Crespo, I.; Cousido-Siah, A.; Podjarny, A.; Barski, O. A.; Fanfrlík, Jindřich; Parés, X.; Farrés, J.; Porté, S.

    2015-01-01

    Roč. 10, č. 7 (2015), e0134506/1-e0134506/19 E-ISSN 1932-6203 Institutional support: RVO:61388963 Keywords : retinoic acid biosynthesis * site-directed mutagenesis * tumor marker AKR1B15 Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 3.057, year: 2015 http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0134506

  13. Genetic variation of Aflatoxin B(1) aldehyde reductase genes (AFAR) in human tumour cells

    DEFF Research Database (Denmark)

    Praml, Christian; Schulz, Wolfgang; Claas, Andreas

    2008-01-01

    AFAR genes play a key role in the detoxification of the carcinogen Aflatoxin B(1) (AFB(1)). In the rat, Afar1 induction can prevent AFB(1)-induced liver cancer. It has been proposed that AFAR enzymes can metabolise endogenous diketones and dialdehydes that may be cytotoxic and/or genotoxic. Furth...... many aldo-keto reductases. This polarity change may have an effect on the proposed substrate binding amino acids nearby (Met(47), Tyr(48), Asp(50)). Further population analyses and functional studies of the nine variants detected may show if these variants are disease-related....

  14. Dimerization and enzymatic activity of fungal 17β-hydroxysteroid dehydrogenase from the short-chain dehydrogenase/reductase superfamily

    Directory of Open Access Journals (Sweden)

    Kristan Katja

    2005-12-01

    Full Text Available Abstract Background 17β-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus (17β-HSDcl is a member of the short-chain dehydrogenase/reductase (SDR superfamily. SDR proteins usually function as dimers or tetramers and 17β-HSDcl is also a homodimer under native conditions. Results We have investigated here which secondary structure elements are involved in the dimerization of 17β-HSDcl and examined the importance of dimerization for the enzyme activity. Sequence similarity with trihydroxynaphthalene reductase from Magnaporthe grisea indicated that Arg129 and His111 from the αE-helices interact with the Asp121, Glu117 and Asp187 residues from the αE and αF-helices of the neighbouring subunit. The Arg129Asp and His111Leu mutations both rendered 17β-HSDcl monomeric, while the mutant 17β-HSDcl-His111Ala was dimeric. Circular dichroism spectroscopy analysis confirmed the conservation of the secondary structure in both monomers. The three mutant proteins all bound coenzyme, as shown by fluorescence quenching in the presence of NADP+, but both monomers showed no enzymatic activity. Conclusion We have shown by site-directed mutagenesis and structure/function analysis that 17β-HSDcl dimerization involves the αE and αF helices of both subunits. Neighbouring subunits are connected through hydrophobic interactions, H-bonds and salt bridges involving amino acid residues His111 and Arg129. Since the substitutions of these two amino acid residues lead to inactive monomers with conserved secondary structure, we suggest dimerization is a prerequisite for catalysis. A detailed understanding of this dimerization could lead to the development of compounds that will specifically prevent dimerization, thereby serving as a new type of inhibitor.

  15. Molecular cloning and biochemical characterization of a novel erythrose reductase from Candida magnoliae JH110

    Directory of Open Access Journals (Sweden)

    Ryu Yeon-Woo

    2010-06-01

    Full Text Available Abstract Background Erythrose reductase (ER catalyzes the final step of erythritol production, which is reducing erythrose to erythritol using NAD(PH as a cofactor. ER has gained interest because of its importance in the production of erythritol, which has extremely low digestibility and approved safety for diabetics. Although ERs were purified and characterized from microbial sources, the entire primary structure and the corresponding DNA for ER still remain unknown in most of erythritol-producing yeasts. Candida magnoliae JH110 isolated from honeycombs produces a significant amount of erythritol, suggesting the presence of erythrose metabolizing enzymes. Here we provide the genetic sequence and functional characteristics of a novel NADPH-dependent ER from C. magnoliae JH110. Results The gene encoding a novel ER was isolated from an osmophilic yeast C. magnoliae JH110. The ER gene composed of 849 nucleotides encodes a polypeptide with a calculated molecular mass of 31.4 kDa. The deduced amino acid sequence of ER showed a high degree of similarity to other members of the aldo-keto reductase superfamily including three ER isozymes from Trichosporonoides megachiliensis SNG-42. The intact coding region of ER from C. magnoliae JH110 was cloned, functionally expressed in Escherichia coli using a combined approach of gene fusion and molecular chaperone co-expression, and subsequently purified to homogeneity. The enzyme displayed a temperature and pH optimum at 42°C and 5.5, respectively. Among various aldoses, the C. magnoliae JH110 ER showed high specific activity for reduction of erythrose to the corresponding alcohol, erythritol. To explore the molecular basis of the catalysis of erythrose reduction with NADPH, homology structural modeling was performed. The result suggested that NADPH binding partners are completely conserved in the C. magnoliae JH110 ER. Furthermore, NADPH interacts with the side chains Lys252, Thr255, and Arg258, which could

  16. Substrate specificity of an aflatoxin-metabolizing aldehyde reductase.

    Science.gov (United States)

    Ellis, E M; Hayes, J D

    1995-01-01

    The enzyme from rat liver that reduces aflatoxin B1-dialdehyde exhibits a unique catalytic specificity distinct from that of other aldo-keto reductases. This enzyme, designated AFAR, displays high activity towards dicarbonyl-containing compounds with ketone groups on adjacent carbon atoms; 9,10-phenanthrenequinone, acenaphthenequinone and camphorquinone were found to be good substrates. Although AFAR can also reduce aromatic and aliphatic aldehydes such as succinic semialdehyde, it is inactive with glucose, galactose and xylose. The enzyme also exhibits low activity towards alpha,beta-unsaturated carbonyl-containing compounds. Determination of the apparent Km reveals that AFAR has highest affinity for 9,10-phenanthrenequinone and succinic semialdehyde, and low affinity for glyoxal and DL-glyceraldehyde. PMID:8526867

  17. Long-term In Vitro Treatment of Human Glioblastoma Cells with Temozolomide Increases Resistance In Vivo through Up-regulation of GLUT Transporter and Aldo-Keto Reductase Enzyme AKR1C Expression

    Directory of Open Access Journals (Sweden)

    Benjamin Le Calvé

    2010-09-01

    Full Text Available Glioblastoma (GBM is the most frequent malignant glioma. Treatment of GBM patients is multimodal with maximum surgical resection, followed by concurrent radiation and chemotherapy with the alkylating drug temozolomide (TMZ. The present study aims to identify genes implicated in the acquired resistance of two human GBM cells of astrocytic origin, T98G and U373, to TMZ. Resistance to TMZ was induced by culturing these cells in vitro for months with incremental TMZ concentrations up to 1 mM. Only partial resistance to TMZ has been achieved and was demonstrated in vivo in immunocompromised mice bearing orthotopic U373 and T98G xenografts. Our data show that long-term treatment of human astroglioma cells with TMZ induces increased expression of facilitative glucose transporter/solute carrier GLUT/SLC2A family members, mainly GLUT-3, and of the AKR1C family of proteins. The latter proteins are phase 1 drug-metabolizing enzymes involved in the maintenance of steroid homeostasis, prostaglandin metabolism, and metabolic activation of polycyclic aromatic hydrocarbons. GLUT-3 has been previously suggested to exert roles in GBM neovascularization processes, and TMZ was found to exert antiangiogenic effects in experimental gliomas. AKR1C1 was previously shown to be associated with oncogenic potential, with proproliferative effects similar to AKR1C3 in the latter case. Both AKR1C1 and AKR1C2 proteins are involved in cancer pro-proliferative cell chemoresistance. Selective targeting of GLUT-3 in GBM and/or AKR1C proteins (by means of jasmonates, for example could thus delay the acquisition of resistance to TMZ of astroglioma cells in the context of prolonged treatment with this drug.

  18. Role reduktas v nádorovém onemocnění.

    OpenAIRE

    Škarydová, Lucie

    2009-01-01

    Only a small attention was paid for long time to reducing enzymes, but today it is clear that these are an important part of the endogenous metabolism and also the phase I biotransformation of xenobiotics. The significant group of reducing enzymes are carbonyl reductases that belong to two superfamilies - short chain dehydrogenases/reductases (SDR) and aldo-keto reductases (AKR). Their role in cancer is now intensively studied and their functions in cancer it is possible to divide into two ma...

  19. Expansion of the mammalian 3 beta-hydroxysteroid dehydrogenase/plant dihydroflavonol reductase superfamily to include a bacterial cholesterol dehydrogenase, a bacterial UDP-galactose-4-epimerase, and open reading frames in vaccinia virus and fish lymphocystis disease virus.

    Science.gov (United States)

    Baker, M E; Blasco, R

    1992-04-13

    Mammalian 3 beta-hydroxysteroid dehydrogenase and plant dihydroflavonol reductases are descended from a common ancestor. Here we present evidence that Nocardia cholesterol dehydrogenase, E. coli UDP-galactose-4 epimerase, and open reading frames in vaccinia virus and fish lymphocystis disease virus are homologous to 3 beta-hydroxysteroid dehydrogenase and dihydroflavonol reductase. Analysis of a multiple alignment of these sequences indicates that viral ORFs are most closely related to the mammalian 3 beta-hydroxysteroid dehydrogenases. The ancestral protein of this superfamily is likely to be one that metabolized sugar nucleotides. The sequence similarity between 3 beta-hydroxysteroid dehydrogenase and the viral ORFs is sufficient to suggest that these ORFs have an activity that is similar to 3 beta-hydroxysteroid dehydrogenase or cholesterol dehydrogenase, although the putative substrates are not yet known.

  20. Structural and Biochemical Investigation of PglF from Campylobacter jejuni Reveals a New Mechanism for a Member of the Short Chain Dehydrogenase/Reductase Superfamily

    Energy Technology Data Exchange (ETDEWEB)

    Riegert, Alexander S. [Department; Thoden, James B. [Department; Schoenhofen, Ian C. [National; Watson, David C. [National; Young, N. Martin [National; Tipton, Peter A. [Department; Holden, Hazel M. [Department

    2017-11-03

    Within recent years it has become apparent that protein glycosylation is not limited to eukaryotes. Indeed, in Campylobacter jejuni, a Gram-negative bacterium, more than 60 of its proteins are known to be glycosylated. One of the sugars found in such glycosylated proteins is 2,4-diacetamido-2,4,6-trideoxy-α-d-glucopyranose, hereafter referred to as QuiNAc4NAc. The pathway for its biosynthesis, initiating with UDP-GlcNAc, requires three enzymes referred to as PglF, PglE, and PlgD. The focus of this investigation is on PglF, an NAD+-dependent sugar 4,6-dehydratase known to belong to the short chain dehydrogenase/reductase (SDR) superfamily. Specifically, PglF catalyzes the first step in the pathway, namely, the dehydration of UDP-GlcNAc to UDP-2-acetamido-2,6-dideoxy-α-d-xylo-hexos-4-ulose. Most members of the SDR superfamily contain a characteristic signature sequence of YXXXK where the conserved tyrosine functions as a catalytic acid or a base. Strikingly, in PglF, this residue is a methionine. Here we describe a detailed structural and functional investigation of PglF from C. jejuni. For this investigation five X-ray structures were determined to resolutions of 2.0 Å or better. In addition, kinetic analyses of the wild-type and site-directed variants were performed. On the basis of the data reported herein, a new catalytic mechanism for a SDR superfamily member is proposed that does not require the typically conserved tyrosine residue.

  1. Medium- and short-chain dehydrogenase/reductase gene and protein families : the SDR superfamily: functional and structural diversity within a family of metabolic and regulatory enzymes.

    Science.gov (United States)

    Kavanagh, K L; Jörnvall, H; Persson, B; Oppermann, U

    2008-12-01

    Short-chain dehydrogenases/reductases (SDRs) constitute a large family of NAD(P)(H)-dependent oxidoreductases, sharing sequence motifs and displaying similar mechanisms. SDR enzymes have critical roles in lipid, amino acid, carbohydrate, cofactor, hormone and xenobiotic metabolism as well as in redox sensor mechanisms. Sequence identities are low, and the most conserved feature is an alpha/beta folding pattern with a central beta sheet flanked by 2 - 3 alpha-helices from each side, thus a classical Rossmannfold motif for nucleotide binding. The conservation of this element and an active site, often with an Asn-Ser-Tyr-Lys tetrad, provides a platform for enzymatic activities encompassing several EC classes, including oxidoreductases, epimerases and lyases. The common mechanism is an underlying hydride and proton transfer involving the nicotinamide and typically an active site tyrosine residue, whereas substrate specificity is determined by a variable C-terminal segment. Relationships exist with bacterial haloalcohol dehalogenases, which lack cofactor binding but have the active site architecture, emphasizing the versatility of the basic fold in also generating hydride transfer-independent lyases. The conserved fold and nucleotide binding emphasize the role of SDRs as scaffolds for an NAD(P)(H) redox sensor system, of importance to control metabolic routes, transcription and signalling.

  2. Cloning, expression, and characterization of a novel xylose reductase from Rhizopus oryzae.

    Science.gov (United States)

    Zhang, Min; Jiang, Shao-tong; Zheng, Zhi; Li, Xing-jiang; Luo, Shui-zhong; Wu, Xue-feng

    2015-07-01

    Rhizopus oryzae is valuable as a producer of organic acids via lignocellulose catalysis. R. oryzae metabolizes xylose, which is one component of lignocellulose hydrolysate. In this study, a novel NADPH-dependent xylose reductase gene from R. oryzae AS 3.819 (Roxr) was cloned and expressed in Pichia pastoris GS115. Homology alignment suggested that the 320-residue protein contained domains and active sites belonging to the aldo/keto reductase family. SDS-PAGE demonstrated that the recombinant xylose reductase has a molecular weight of approximately 37 kDa. The optimal catalytic pH and temperature of the purified recombinant protein were 5.8 and 50 °C, respectively. The recombinant protein was stable from pH 4.4 to 6.5 and at temperatures below 42 °C. The recombinant enzyme has bias for D-xylose and L-arabinose as substrates and NADPH as its coenzyme. Real-time quantitative reverse transcription PCR tests suggested that native Roxr expression is regulated by a carbon catabolite repression mechanism. Site-directed mutagenesis at two possible key sites involved in coenzyme binding, Thr(226)  → Glu(226) and Val(274)  → Asn(274), were performed, respectively. The coenzyme specificity constants of the resulted RoXR(T226E) and RoXR(V274N) for NADH increased 18.2-fold and 2.4-fold, which suggested possibility to improve the NADH preference of this enzyme through genetic modification. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Identification and functional evaluation of the reductases and dehydrogenases from Saccharomyces cerevisiae involved in vanillin resistance.

    Science.gov (United States)

    Wang, Xinning; Liang, Zhenzhen; Hou, Jin; Bao, Xiaoming; Shen, Yu

    2016-04-01

    Vanillin, a type of phenolic released during the pre-treatment of lignocellulosic materials, is toxic to microorganisms and therefore its presence inhibits the fermentation. The vanillin can be reduced to vanillyl alcohol, which is much less toxic, by the ethanol producer Saccharomyces cerevisiae. The reducing capacity of S. cerevisiae and its vanillin resistance are strongly correlated. However, the specific enzymes and their contribution to the vanillin reduction are not extensively studied. In our previous work, an evolved vanillin-resistant strain showed an increased vanillin reduction capacity compared with its parent strain. The transcriptome analysis suggested the reductases and dehydrogenases of this vanillin resistant strain were up-regulated. Using this as a starting point, 11 significantly regulated reductases and dehydrogenases were selected in the present work for further study. The roles of these reductases and dehydrogenases in the vanillin tolerance and detoxification abilities of S. cerevisiae are described. Among the candidate genes, the overexpression of the alcohol dehydrogenase gene ADH6, acetaldehyde dehydrogenase gene ALD6, glucose-6-phosphate 1-dehydrogenase gene ZWF1, NADH-dependent aldehyde reductase gene YNL134C, and aldo-keto reductase gene YJR096W increased 177, 25, 6, 15, and 18 % of the strain μmax in the medium containing 1 g L(-1) vanillin. The in vitro detected vanillin reductase activities of strain overexpressing ADH6, YNL134C and YJR096W were notably higher than control. The vanillin specific reduction rate increased by 8 times in ADH6 overexpressed strain but not in YNL134C and YJR096W overexpressed strain. This suggested that the enzymes encoded by YNL134C and YJR096W might prefer other substrate and/or could not show their effects on vanillin on the high background of Adh6p in vivo. Overexpressing ALD6 and ZWF1 mainly increased the [NADPH]/[NADP(+)] and [GSH]/[GSSG] ratios but not the vanillin reductase activities. Their

  4. Molecular cloning of goat 20alpha-hydroxysteroid dehydrogenase cDNA.

    Science.gov (United States)

    Jayasekara, Walimuni Samantha Nilanthi; Yonezawa, Tomohiro; Ishida, Maho; Yamanouchi, Keitaro; Nishihara, Masugi

    2004-06-01

    20Alpha-hydroxysteroid dehydrogenase (20alpha-HSD), which catalyzes the conversion of progesterone to its inactive form 20alpha-dihydroprogesterone, is expressed in murine placenta and has been suggested to play roles in maintaining pregnancy. To understand the role of 20alpha-HSD during pregnancy in the goat, as a first step, cloning and sequencing of 20alpha-HSD cDNA were performed. The full nucleotide sequence of 20alpha-HSD cDNA was determined on samples obtained from the corpus luteum at the luteal phase of the estrous cycle and the placenta in late pregnancy by RT-PCR and 3' and 5' RACE systems. Cloned 20alpha-HSD cDNA consisted of 1124 bp and belonged to the aldo-keto reductase superfamily. From the start codon to stop codon there were 323 amino acids, the same as in other species. To verify whether the protein derived from goat 20alpha-HSD cDNA had 20alpha-HSD activity, the cDNA was expressed by bacteria. Bacterially expressed goat 20alpha-HSD protein showed 20alpha-HSD enzyme activity. A tissue distribution study demonstrated that 20alpha-HSD was expressed in the placenta, but not in the adrenal gland, liver and spleen during pregnancy. The present study suggests that goat 20alpha-HSD is another member of the aldo-keto reductase superfamily and that it plays a role in the placenta during pregnancy.

  5. A spontaneously immortalized Schwann cell line from aldose reductase-deficient mice as a useful tool for studying polyol pathway and aldehyde metabolism.

    Science.gov (United States)

    Niimi, Naoko; Yako, Hideji; Takaku, Shizuka; Kato, Hiroshi; Matsumoto, Takafumi; Nishito, Yasumasa; Watabe, Kazuhiko; Ogasawara, Saori; Mizukami, Hiroki; Yagihashi, Soroku; Chung, Sookja K; Sango, Kazunori

    2018-03-01

    The increased glucose flux into the polyol pathway via aldose reductase (AR) is recognized as a major contributing factor for the pathogenesis of diabetic neuropathy, whereas little is known about the functional significance of AR in the peripheral nervous system. Spontaneously immortalized Schwann cell lines established from long-term cultures of AR-deficient and normal C57BL/6 mouse dorsal root ganglia and peripheral nerves can be useful tools for studying the physiological and pathological roles of AR. These cell lines, designated as immortalized knockout AR Schwann cells 1 (IKARS1) and 1970C3, respectively, demonstrated distinctive Schwann cell phenotypes, such as spindle-shaped morphology and immunoreactivity to S100, p75 neurotrophin receptor, and vimentin, and extracellular release of neurotrophic factors. Conditioned media obtained from these cells promoted neuronal survival and neurite outgrowth of cultured adult mouse dorsal root ganglia neurons. Microarray and real-time RT-PCR analyses revealed significantly down-regulated mRNA expression of polyol pathway-related enzymes, sorbitol dehydrogenase and ketohexokinase, in IKARS1 cells compared with those in 1970C3 cells. In contrast, significantly up-regulated mRNA expression of aldo-keto reductases (AKR1B7 and AKR1B8) and aldehyde dehydrogenases (ALDH1L2, ALDH5A1, and ALDH7A1) was detected in IKARS1 cells compared with 1970C3 cells. Exposure to reactive aldehydes (3-deoxyglucosone, methylglyoxal, and 4-hydroxynonenal) significantly up-regulated the mRNA expression of AKR1B7 and AKR1B8 in IKARS1 cells, but not in 1970C3 cells. Because no significant differences in viability between these two cell lines after exposure to these aldehydes were observed, it can be assumed that the aldehyde detoxification is taken over by AKR1B7 and AKR1B8 in the absence of AR. © 2017 International Society for Neurochemistry.

  6. Conversion of Human Steroid 5[beta]-Reductase (AKR1D1) into 3[beta]-Hydroxysteroid Dehydrogenase by Single Point Mutation E120H: Example of Perfect Enzyme Engineering

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Mo; Drury, Jason E.; Christianson, David W.; Penning, Trevor M. (UPENN)

    2012-10-10

    Human aldo-keto reductase 1D1 (AKR1D1) and AKR1C enzymes are essential for bile acid biosynthesis and steroid hormone metabolism. AKR1D1 catalyzes the 5{beta}-reduction of {Delta}{sup 4}-3-ketosteroids, whereas AKR1C enzymes are hydroxysteroid dehydrogenases (HSDs). These enzymes share high sequence identity and catalyze 4-pro-(R)-hydride transfer from NADPH to an electrophilic carbon but differ in that one residue in the conserved AKR catalytic tetrad, His120 (AKR1D1 numbering), is substituted by a glutamate in AKR1D1. We find that the AKR1D1 E120H mutant abolishes 5{beta}-reductase activity and introduces HSD activity. However, the E120H mutant unexpectedly favors dihydrosteroids with the 5{alpha}-configuration and, unlike most of the AKR1C enzymes, shows a dominant stereochemical preference to act as a 3{beta}-HSD as opposed to a 3{alpha}-HSD. The catalytic efficiency achieved for 3{beta}-HSD activity is higher than that observed for any AKR to date. High resolution crystal structures of the E120H mutant in complex with epiandrosterone, 5{beta}-dihydrotestosterone, and {Delta}{sup 4}-androstene-3,17-dione elucidated the structural basis for this functional change. The glutamate-histidine substitution prevents a 3-ketosteroid from penetrating the active site so that hydride transfer is directed toward the C3 carbonyl group rather than the {Delta}{sup 4}-double bond and confers 3{beta}-HSD activity on the 5{beta}-reductase. Structures indicate that stereospecificity of HSD activity is achieved because the steroid flips over to present its {alpha}-face to the A-face of NADPH. This is in contrast to the AKR1C enzymes, which can invert stereochemistry when the steroid swings across the binding pocket. These studies show how a single point mutation in AKR1D1 can introduce HSD activity with unexpected configurational and stereochemical preference.

  7. CHARACTERIZATION OF STABLE BENZO(A)PYRENE-7,8-QUINONE-DNA ADDUCTS IN CALF THYMUS DNA

    Science.gov (United States)

    Benzo[alpyrene-7,8-dione (BPQ) is a reactive aldo-keto reductase-mediated product of B[a]P-7,8-diol, a major P450/epoxide hydrolase metabolite of the multi-species carcinogen, B[a]P. The role of BPQ in B[a]P's genotoxicity and carcinogenesis is evolving. Toxicity pathways involvi...

  8. CHARACTERIZATION OF STABLE BENZOLALPYRENE-7,8-QUINONE-DNA ADDUCTS IN CALF THYMUS DNA AND POLYDEOXYNUCLEOTIDES

    Science.gov (United States)

    Bcnzo[a]pyrene-7,8-dione (BPQ) is a reactive aldo-keto reductase-mediated product of B[a]P-7,8-diol, a major P450/epoxide hydrolase metabolite of the multi-species carcinogen, B[a]P. The role of BPQ in B[a]P's genotoxicity and carcinogenesis is evolving. Toxicity pathways involvi...

  9. Antibodies Targeting EMT

    Science.gov (United States)

    2017-10-01

    determine their targets on the cell. The newly discovered antibodies will then be engineered for utility as new highly specific drugs and diagnostics in...are from the aldo-keto reductase family (AKRs). Remarkably, 3 of the top 10 genes with induction in the mesenchymal TES2b cells Figure 1. Amino

  10. The role of aryl hydrocarbon receptor in regulation of enzymes involved in metabolic activation of polycyclic aromatic hydrocarbons in a model of rat liver progenitor cells

    Czech Academy of Sciences Publication Activity Database

    Vondráček, Jan; Krčmář, P.; Procházková, Jiřina; Trilecová, L.; Gavelová, M.; Skálová, L.; Szotáková, B.; Bunček, M.; Radilová, H.; Kozubík, Alois; Machala, M.

    2009-01-01

    Roč. 180, č. 2 (2009), s. 226-237 ISSN 0009-2797 R&D Projects: GA ČR(CZ) GA524/06/0517 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : cytochromes P450 * PAHs * aldo-keto reductases Subject RIV: BO - Biophysics Impact factor: 2.457, year: 2009

  11. The evolving doublecortin (DCX superfamily

    Directory of Open Access Journals (Sweden)

    Sapir Tamar

    2006-07-01

    Full Text Available Abstract Background Doublecortin (DCX domains serve as protein-interaction platforms. Mutations in members of this protein superfamily are linked to several genetic diseases. Mutations in the human DCX gene result in abnormal neuronal migration, epilepsy, and mental retardation; mutations in RP1 are associated with a form of inherited blindness, and DCDC2 has been associated with dyslectic reading disabilities. Results The DCX-repeat gene family is composed of eleven paralogs in human and in mouse. Its evolution was followed across vertebrates, invertebrates, and was traced to unicellular organisms, thus enabling following evolutionary additions and losses of genes or domains. The N-terminal and C-terminal DCX domains have undergone sub-specialization and divergence. Developmental in situ hybridization data for nine genes was generated. In addition, a novel co-expression analysis for most human and mouse DCX superfamily-genes was performed using high-throughput expression data extracted from Unigene. We performed an in-depth study of a complete gene superfamily using several complimentary methods. Conclusion This study reveals the existence and conservation of multiple members of the DCX superfamily in different species. Sequence analysis combined with expression analysis is likely to be a useful tool to predict correlations between human disease and mouse models. The sub-specialization of some members due to restricted expression patterns and sequence divergence may explain the successful addition of genes to this family throughout evolution.

  12. Two differentially regulated Arabidopsis genes define a new branch of the DFR superfamily

    DEFF Research Database (Denmark)

    Østergaard, L; Lauvergeat, V; Naested, H

    2001-01-01

    are highly divergent, suggesting differential regulation of the CRL genes. Phylogenetic analysis placed CRL1 and CRL2 in a separate branch of the DFR superfamily. Northern blotting showed strong AtCRL1 induction by abscisic acid (ABA), drought, and heat shock, and high expression level in seeds, thus......Two tandem genes were identified on Arabidopsis chromosome II (AtCRL1 and AtCRL2) encoding proteins with homology to members of the dihydroflavonol-4-reductase (DFR) superfamily. The encoded CRL1 and CRL2 proteins share 87% mutual amino acid sequence identity whereas their promoter regions...

  13. Direct enzyme assay evidence confirms aldehyde reductase function of Ydr541cp and Ygl039wp from Saccharomyces cerevisiae

    Science.gov (United States)

    Aldehyde reductase gene ARI1 is a recently characterized member of intermediate subfamily under SDR (short-chain dehydrogenase/reductase) superfamily that revealed mechanisms of in situ detoxification of furfural and HMF for tolerance of Saccharomyces cerevisiae. Uncharacterized open reading frames ...

  14. The Ras superfamily G-proteins.

    Science.gov (United States)

    Tetlow, Ashley L; Tamanoi, Fuyuhiko

    2013-01-01

    The Ras superfamily G-proteins are monomeric proteins of approximately 21kDa that act as a molecular switch to regulate a variety of cellular processes. The structure of the Ras superfamily G-proteins, their regulators as well as posttranslational modification of these proteins leading to their membrane association have been elucidated. The Ras superfamily G-proteins interact at their effector domains with their downstream effectors via protein-protein interactions. Mutational activation or overexpression of the Ras superfamily G-proteins has been observed in a number of human cancer cases. Over the years, a variety of approaches to inhibit the Ras superfamily G-proteins have been developed. These different approaches are discussed in this volume. © 2013 Elsevier Inc. All rights reserved.

  15. Two differentially regulated Arabidopsis genes define a new branch of the DFR superfamily

    DEFF Research Database (Denmark)

    Østergaard, L; Lauvergeat, V; Naested, H

    2001-01-01

    Two tandem genes were identified on Arabidopsis chromosome II (AtCRL1 and AtCRL2) encoding proteins with homology to members of the dihydroflavonol-4-reductase (DFR) superfamily. The encoded CRL1 and CRL2 proteins share 87% mutual amino acid sequence identity whereas their promoter regions...... are highly divergent, suggesting differential regulation of the CRL genes. Phylogenetic analysis placed CRL1 and CRL2 in a separate branch of the DFR superfamily. Northern blotting showed strong AtCRL1 induction by abscisic acid (ABA), drought, and heat shock, and high expression level in seeds, thus...... resembling the expression pattern of late embryogenic abundant ABA-responsive genes. Differential expression of the two genes during plant development was confirmed in plants expressing transcriptional fusions between the two promoters and the Escherichia coli beta-glucuronidase reporter gene. This showed...

  16. Structure and mechanism of dimethylsulfoxide reductase, a molybdopterin-containing enzyme of DMSO reductase family

    International Nuclear Information System (INIS)

    McEwan, A.G.; Ridge, J.P.; McDevitt, C.A.; Hanson, G.R.

    2001-01-01

    Full text: Apart from nitrogenase, enzymes containing molybdenum are members of a superfamily, the molybdopterin-containing enzymes. Most of these enzymes catalyse an oxygen atom transfer and two electron transfer reaction. During catalysis the Mo at the active site cycles between the Mo(VI) and Mo(IV) states. The DMSO reductase family of molybdopterin-containing enzymes all contain a bis(molybdopterin guanine dinucleotide)Mo cofactor and over thirty examples have now been described. Over the last five years crystal structures of dimethylsulfoxide (DMSO) reductase and four other enzymes of the DMSO reductase family have revealed that enzymes of this family have a similar tertiary structure. The Mo atom at the active site is coordinated by four thiolate ligands provided by the dithiolene side chains of the two MGD molecules of the bis(MGD)Mo cofactor as well as a ligand provided by an amino acid side chain. In addition, an oxygen atom in the form of an oxo, hydroxo or aqua group is also coordinated to the Mo atom. In the case of dimethylsulfoxide reductase X-ray crystallography of the product-reduced species and Raman spectroscopy has demonstrated that the enzyme contains a single exchangeable oxo group that is H-bonded to W116

  17. The genome, proteome and phylogenetic analysis of Sinorhizobium meliloti phage ΦM12, the founder of a new group of T4-superfamily phages.

    Science.gov (United States)

    Brewer, Tess E; Stroupe, M Elizabeth; Jones, Kathryn M

    2014-02-01

    Phage ΦM12 is an important transducing phage of the nitrogen-fixing rhizobial bacterium Sinorhizobium meliloti. Here we report the genome, phylogenetic analysis, and proteome of ΦM12, the first report of the genome and proteome of a rhizobium-infecting T4-superfamily phage. The structural genes of ΦM12 are most similar to T4-superfamily phages of cyanobacteria. ΦM12 is the first reported T4-superfamily phage to lack genes encoding class I ribonucleotide reductase (RNR) and exonuclease dexA, and to possess a class II coenzyme B12-dependent RNR. ΦM12's novel collection of genes establishes it as the founder of a new group of T4-superfamily phages, fusing features of cyanophages and phages of enteric bacteria. © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

  18. The T-superfamily of conotoxins.

    Science.gov (United States)

    Walker, C S; Steel, D; Jacobsen, R B; Lirazan, M B; Cruz, L J; Hooper, D; Shetty, R; DelaCruz, R C; Nielsen, J S; Zhou, L M; Bandyopadhyay, P; Craig, A G; Olivera, B M

    1999-10-22

    We report the discovery and initial characterization of the T-superfamily of conotoxins. Eight different T-superfamily peptides from five Conus species were identified; they share a consensus signal sequence, and a conserved arrangement of cysteine residues (- -CC- -CC-). T-superfamily peptides were found expressed in venom ducts of all major feeding types of Conus; the results suggest that the T-superfamily will be a large and diverse group of peptides, widely distributed in the 500 different Conus species. These peptides are likely to be functionally diverse; although the peptides are small (11-17 amino acids), their sequences are strikingly divergent, with different peptides of the superfamily exhibiting varying extents of post-translational modification. Of the three peptides tested for in vivo biological activity, only one was active on mice but all three had effects on fish. The peptides that have been extensively characterized are as follows: p5a, GCCPKQMRCCTL*; tx5a, gammaCCgammaDGW(+)CCT( section sign)AAO; and au5a, FCCPFIRYCCW (where gamma = gamma-carboxyglutamate, W(+) = bromotryptophan, O = hydroxyproline, T( section sign) = glycosylated threonine, and * = COOH-terminal amidation). We also demonstrate that the precursor of tx5a contains a functional gamma-carboxylation recognition signal in the -1 to -20 propeptide region, consistent with the presence of gamma-carboxyglutamate residues in this peptide.

  19. The nitrilase superfamily: classification, structure and function

    Science.gov (United States)

    Pace, Helen C; Brenner, Charles

    2001-01-01

    The nitrilase superfamily consists of thiol enzymes involved in natural product biosynthesis and post-translational modification in plants, animals, fungi and certain prokaryotes. On the basis of sequence similarity and the presence of additional domains, the superfamily can be classified into 13 branches, nine of which have known or deduced specificity for specific nitrile- or amide-hydrolysis or amide-condensation reactions. Genetic and biochemical analysis of the family members and their associated domains assists in predicting the localization, specificity and cell biology of hundreds of uncharacterized protein sequences. PMID:11380987

  20. The SUPERFAMILY database in 2007: families and functions

    OpenAIRE

    Wilson, Derek; Madera, Martin; Vogel, Christine; Chothia, Cyrus; Gough, Julian

    2006-01-01

    The SUPERFAMILY database provides protein domain assignments, at the SCOP ?superfamily? level, for the predicted protein sequences in over 400 completed genomes. A superfamily groups together domains of different families which have a common evolutionary ancestor based on structural, functional and sequence data. SUPERFAMILY domain assignments are generated using an expert curated set of profile hidden Markov models. All models and structural assignments are available for browsing and downloa...

  1. Relationship between Apolipoprotein Superfamily and Parkinson's Disease

    Directory of Open Access Journals (Sweden)

    Lin Li

    2017-01-01

    Conclusions: The Apo superfamily has been proved to be closely involved in the initiation, progression, and prognosis of PD. Apos and their genes are of great value in predicting the susceptibility of PD and hopeful to become the target of medical intervention to prevent the onset of PD or slow down the progress. Therefore, further large-scale studies are warranted to elucidate the precise mechanisms of Apos in PD.

  2. Designer TGFβ superfamily ligands with diversified functionality.

    Directory of Open Access Journals (Sweden)

    George P Allendorph

    Full Text Available Transforming Growth Factor--beta (TGFβ superfamily ligands, including Activins, Growth and Differentiation Factors (GDFs, and Bone Morphogenetic Proteins (BMPs, are excellent targets for protein-based therapeutics because of their pervasiveness in numerous developmental and cellular processes. We developed a strategy termed RASCH (Random Assembly of Segmental Chimera and Heteromer, to engineer chemically-refoldable TGFβ superfamily ligands with unique signaling properties. One of these engineered ligands, AB208, created from Activin-βA and BMP-2 sequences, exhibits the refolding characteristics of BMP-2 while possessing Activin-like signaling attributes. Further, we find several additional ligands, AB204, AB211, and AB215, which initiate the intracellular Smad1-mediated signaling pathways more strongly than BMP-2 but show no sensitivity to the natural BMP antagonist Noggin unlike natural BMP-2. In another design, incorporation of a short N-terminal segment from BMP-2 was sufficient to enable chemical refolding of BMP-9, without which was never produced nor refolded. Our studies show that the RASCH strategy enables us to expand the functional repertoire of TGFβ superfamily ligands through development of novel chimeric TGFβ ligands with diverse biological and clinical values.

  3. Origin and evolution of TNF and TNF receptor superfamilies

    Science.gov (United States)

    The tumor necrosis factor superfamily (TNFSF) and the TNF receptor superfamily (TNFRSF) have an ancient evolutionary origin that can be traced back to single copy genes within Arthropods. In humans, 18 TNFSF and 29 TNFRSF genes have been identified. Evolutionary models account for the increase in g...

  4. The plant short-chain dehydrogenase (SDR) superfamily: genome-wide inventory and diversification patterns.

    Science.gov (United States)

    Moummou, Hanane; Kallberg, Yvonne; Tonfack, Libert Brice; Persson, Bengt; van der Rest, Benoît

    2012-11-20

    Short-chain dehydrogenases/reductases (SDRs) form one of the largest and oldest NAD(P)(H) dependent oxidoreductase families. Despite a conserved 'Rossmann-fold' structure, members of the SDR superfamily exhibit low sequence similarities, which constituted a bottleneck in terms of identification. Recent classification methods, relying on hidden-Markov models (HMMs), improved identification and enabled the construction of a nomenclature. However, functional annotations of plant SDRs remain scarce. Wide-scale analyses were performed on ten plant genomes. The combination of hidden Markov model (HMM) based analyses and similarity searches led to the construction of an exhaustive inventory of plant SDR. With 68 to 315 members found in each analysed genome, the inventory confirmed the over-representation of SDRs in plants compared to animals, fungi and prokaryotes. The plant SDRs were first classified into three major types - 'classical', 'extended' and 'divergent' - but a minority (10% of the predicted SDRs) could not be classified into these general types ('unknown' or 'atypical' types). In a second step, we could categorize the vast majority of land plant SDRs into a set of 49 families. Out of these 49 families, 35 appeared early during evolution since they are commonly found through all the Green Lineage. Yet, some SDR families - tropinone reductase-like proteins (SDR65C), 'ABA2-like'-NAD dehydrogenase (SDR110C), 'salutaridine/menthone-reductase-like' proteins (SDR114C), 'dihydroflavonol 4-reductase'-like proteins (SDR108E) and 'isoflavone-reductase-like' (SDR460A) proteins - have undergone significant functional diversification within vascular plants since they diverged from Bryophytes. Interestingly, these diversified families are either involved in the secondary metabolism routes (terpenoids, alkaloids, phenolics) or participate in developmental processes (hormone biosynthesis or catabolism, flower development), in opposition to SDR families involved in primary

  5. Radiation of the Tnt1 retrotransposon superfamily in three Solanaceae genera

    Science.gov (United States)

    Manetti, Maria E; Rossi, Magdalena; Costa, Ana PP; Clausen, Andrea M; Van Sluys, Marie-Anne

    2007-01-01

    Background Tnt1 was the first active plant retrotransposon identified in tobacco after nitrate reductase gene disruption. The Tnt1 superfamily comprises elements from Nicotiana (Tnt1 and Tto1) and Lycopersicon (Retrolyc1 and Tlc1) species. The study presented here was conducted to characterise Tnt1-related sequences in 20 wild species of Solanum and five cultivars of Solanum tuberosum. Results Tnt1-related sequences were amplified from total genomic DNA using a PCR-based approach. Purified fragments were cloned and sequenced, and clustering analysis revealed three groups that differ in their U3 region. Using a network approach with a total of 453 non-redundant sequences isolated from Solanum (197), Nicotiana (140) and Lycopersicon (116) species, it is demonstrated that the Tnt1 superfamily can be treated as a population to resolve previous phylogenetic multifurcations. The resulting RNAseH network revealed that sequences group according to the Solanaceae genus, supporting a strong association with the host genome, whereas tracing the U3 region sequence association characterises the modular evolutionary pattern within the Tnt1 superfamily. Within each genus, and irrespective of species, nearly 20% of Tnt1 sequences analysed are identical, indicative of being part of an active copy. The network approach enabled the identification of putative "master" sequences and provided evidence that within a genus these master sequences are associated with distinct U3 regions. Conclusion The results presented here support the hypothesis that the Tnt1 superfamily was present early in the evolution of Solanaceae. The evidence also suggests that the RNAseH region of Tnt1 became fixed at the host genus level whereas, within each genus, propagation was ensured by the diversification of the U3 region. Different selection pressures seemed to have acted on the U3 and RNAseH modules of ancestral Tnt1 elements, probably due to the distinct functions of these regions in the retrotransposon

  6. Novel Methods for the Chemical Synthesis of Insulin Superfamily Peptides and of Analogues Containing Disulfide Isosteres.

    Science.gov (United States)

    Hossain, Mohammed Akhter; Wade, John D

    2017-09-19

    The insulin superfamily of peptides is ubiquitous within vertebrates and invertebrates and is characterized by the presence of a set of three disulfide bonds in a unique disposition. With the exception of insulin-like growth factors I and II, which are single chain peptides, the remaining 8 members of the human insulin superfamily are two-chain peptides containing one intramolecular and two intermolecular disulfide bridges. These structural features have long made the chemical synthesis of the peptides a considerable challenge, in particular, including their correct disulfide bond pairing and formation. However, they have also afforded the opportunity to develop modern solid phase synthesis methods for the preparation of such peptides that incorporate novel or improved chemical methods for the controlled introduction of both disulfide bonds and their surrogates, both during and after peptide chain assembly. In turn, this has enabled a detailed probing of the structure and function relationship of this small but complex superfamily of peptides. After initially using and subsequently identifying significant limitations of the approach of simultaneous random chain combination and oxidative folding, our laboratory undertook to develop robust chemical synthesis strategies in concert with orthogonal cysteine S-protecting groups and corresponding regioselective disulfide bond formation. These have included the separate synthesis of each of the two chains or of the two chains linked by an artificial C-peptide that is removed following postoxidative folding. These, in turn, have enabled an increased ease of acquisition in a good yield of not only members of human insulin superfamily but other insulin-like peptides. Importantly, these successful methods have enabled, for the first time, a detailed analysis of the role that the disulfide bonds play in the structure and function of such peptides. This was achieved by selective removal of the disulfide bonds or by the judicious

  7. CAP protein superfamily members in Toxocara canis.

    Science.gov (United States)

    Stroehlein, Andreas J; Young, Neil D; Hall, Ross S; Korhonen, Pasi K; Hofmann, Andreas; Sternberg, Paul W; Jabbar, Abdul; Gasser, Robin B

    2016-06-24

    Proteins of the cysteine-rich secretory proteins, antigen 5 and pathogenesis-related 1 (CAP) superfamily are recognized or proposed to play roles in parasite development and reproduction, and in modulating host immune attack and infection processes. However, little is known about these proteins for most parasites. In the present study, we explored CAP proteins of Toxocara canis, a socioeconomically important zoonotic roundworm. To do this, we mined and curated transcriptomic and genomic data, predicted and curated full-length protein sequences (n = 28), conducted analyses of these data and studied the transcription of respective genes in different developmental stages of T. canis. In addition, based on information available for Caenorhabditis elegans, we inferred that selected genes (including lon-1, vap-1, vap-2, scl-1, scl-8 and scl-11 orthologs) of T. canis and their interaction partners likely play central roles in this parasite's development and/or reproduction via TGF-beta and/or insulin-like signaling pathways, or via host interactions. In conclusion, this study could provide a foundation to guide future studies of CAP proteins of T. canis and related parasites, and might assist in finding new interventions against diseases caused by these parasites.

  8. Aldehyde dehydrogenase protein superfamily in maize.

    Science.gov (United States)

    Zhou, Mei-Liang; Zhang, Qian; Zhou, Ming; Qi, Lei-Peng; Yang, Xiong-Bang; Zhang, Kai-Xuan; Pang, Jun-Feng; Zhu, Xue-Mei; Shao, Ji-Rong; Tang, Yi-Xiong; Wu, Yan-Min

    2012-11-01

    Maize (Zea mays ssp. mays L.) is an important model organism for fundamental research in the agro-biotechnology field. Aldehydes were generated in response to a suite of environmental stresses that perturb metabolism including salinity, dehydration, desiccation, and cold and heat shock. Many biologically important aldehydes are metabolized by the superfamily of NAD(P)(+)-dependent aldehyde dehydrogenases. Here, starting from the database of Z. mays, we identified 28 aldehyde dehydrogenase (ALDH) genes and 48 transcripts by the in silico cloning method using the ALDH-conserved domain amino acid sequence of Arabidopsis and rice as a probe. Phylogenetic analysis shows that all 28 members of the ALDH gene families were classified to ten distinct subfamilies. Microarray data and quantitative real-time PCR analysis reveal that ZmALDH9, ZmALDH13, and ZmALDH17 genes involve the function of drought stress, acid tolerance, and pathogens infection. These results suggested that these three ZmALDH genes might be potentially useful in maize genetic improvement.

  9. Update on the olfactory receptor (OR gene superfamily

    Directory of Open Access Journals (Sweden)

    Olender Tsviya

    2008-09-01

    Full Text Available Abstract The olfactory receptor gene (OR superfamily is the largest in the human genome. The superfamily contains 390 putatively functional genes and 465 pseudogenes arranged into 18 gene families and 300 subfamilies. Even members within the same subfamily are often located on different chromosomes. OR genes are located on all autosomes except chromosome 20, plus the X chromosome but not the Y chromosome. The gene:pseudogene ratio is lowest in human, higher in chimpanzee and highest in rat and mouse -- most likely reflecting the greater need of olfaction for survival in the rodent than in the human. The OR genes undergo allelic exclusion, each sensory neurone expressing usually only one odourant receptor allele; the mechanism by which this phenomenon is regulated is not yet understood. The nomenclature system (based on evolutionary divergence of genes into families and subfamilies of the OR gene superfamily has been designed similarly to that originally used for the CYP gene superfamily.

  10. Ascorbic acid reverses the prolonged anesthetic action of pentobarbital in Akr1a-knockout mice.

    Science.gov (United States)

    Ito, Junitsu; Otsuki, Noriyuki; Zhang, Xuhong; Konno, Tasuku; Kurahashi, Toshihiro; Takahashi, Motoko; Yamato, Mayumi; Matsuoka, Yuta; Yamada, Ken-ichi; Miyata, Satoshi; Fujii, Junichi

    2014-01-24

    Aldehyde reductase (AKR1A), a member of the aldo-keto reductase superfamily, is highly expressed in the liver and is involved in both the detoxification of carbonyl compounds and ascorbic acid biosynthesis. By comparison with wild-type mice, Akr1a-knockout (Akr1a(-/-)) mice and human Akrla-transgenic (Akr1a(tg/+)) mice experience different anesthetic actions from pentobarbital-prolonged in Akr1a-knockout (Akr1a(-/-)) mice and shortened in human Akrla-transgenic (Akr1a(tg/+)) mice. We investigated this alteration in the anesthetic efficacy of pentobarbital in Akr1a genetically modified mice. Neither the cytosolic protein of wild-type mouse liver nor purified rat AKR1A directly reduced pentobarbital. Ascorbic acid administration neutralized the prolonged duration of the loss of the righting reflex (LORR) in Akr1a(-/-) mice, but preincubation of pentobarbital with ascorbic acid prior to administration did not change the anesthetic effect. Those results indicated that ascorbic acid does not directly reduce pentobarbital. Enzymatic activities and levels of the proteins of some cytochrome P450s that make up a potent detoxification system for pentobarbital showed no changes in the genetically modified mice examined. Thus, ascorbic acid also had no effect on the detoxification system in the liver. The prolonged duration of LORR in the Akr1a(-/-) mice caused by pentobarbital and the neutralization of the anesthetic effect by ascorbic acid together with other results imply that ascorbic acid alters the responses of the neuronal system to anesthetics. Pentobarbital action is increased under conditions of ascorbic acid deficiency, and this may have to be taken into account when anesthetizing malnourished patients. Copyright © 2013. Published by Elsevier Inc.

  11. Phylogenomic analysis of the cystatin superfamily in eukaryotes and prokaryotes

    Directory of Open Access Journals (Sweden)

    Turk Vito

    2009-11-01

    Full Text Available Abstract Background The cystatin superfamily comprises cysteine protease inhibitors that play key regulatory roles in protein degradation processes. Although they have been the subject of many studies, little is known about their genesis, evolution and functional diversification. Our aim has been to obtain a comprehensive insight into their origin, distribution, diversity, evolution and classification in Eukaryota, Bacteria and Archaea. Results We have identified in silico the full complement of the cystatin superfamily in more than 2100 prokaryotic and eukaryotic genomes. The analysis of numerous eukaryotic genomes has provided strong evidence for the emergence of this superfamily in the ancestor of eukaryotes. The progenitor of this superfamily was most probably intracellular and lacked a signal peptide and disulfide bridges, much like the extant Giardia cystatin. A primordial gene duplication produced two ancestral eukaryotic lineages, cystatins and stefins. While stefins remain encoded by a single or a small number of genes throughout the eukaryotes, the cystatins have undergone a more complex and dynamic evolution through numerous gene and domain duplications. In the cystatin superfamily we discovered twenty vertebrate-specific and three angiosperm-specific orthologous families, indicating that functional diversification has occurred only in multicellular eukaryotes. In vertebrate orthologous families, the prevailing trends were loss of the ancestral inhibitory activity and acquisition of novel functions in innate immunity. Bacterial cystatins and stefins may be emergency inhibitors that enable survival of bacteria in the host, defending them from the host's proteolytic activity. Conclusion This study challenges the current view on the classification, origin and evolution of the cystatin superfamily and provides valuable insights into their functional diversification. The findings of this comprehensive study provide guides for future

  12. Phylogenomic analysis of the GIY-YIG nuclease superfamily

    Directory of Open Access Journals (Sweden)

    Bujnicki Janusz M

    2006-04-01

    Full Text Available Abstract Background The GIY-YIG domain was initially identified in homing endonucleases and later in other selfish mobile genetic elements (including restriction enzymes and non-LTR retrotransposons and in enzymes involved in DNA repair and recombination. However, to date no systematic search for novel members of the GIY-YIG superfamily or comparative analysis of these enzymes has been reported. Results We carried out database searches to identify all members of known GIY-YIG nuclease families. Multiple sequence alignments together with predicted secondary structures of identified families were represented as Hidden Markov Models (HMM and compared by the HHsearch method to the uncharacterized protein families gathered in the COG, KOG, and PFAM databases. This analysis allowed for extending the GIY-YIG superfamily to include members of COG3680 and a number of proteins not classified in COGs and to predict that these proteins may function as nucleases, potentially involved in DNA recombination and/or repair. Finally, all old and new members of the GIY-YIG superfamily were compared and analyzed to infer the phylogenetic tree. Conclusion An evolutionary classification of the GIY-YIG superfamily is presented for the very first time, along with the structural annotation of all (subfamilies. It provides a comprehensive picture of sequence-structure-function relationships in this superfamily of nucleases, which will help to design experiments to study the mechanism of action of known members (especially the uncharacterized ones and will facilitate the prediction of function for the newly discovered ones.

  13. Structure and Function of the LmbE-like Superfamily

    Directory of Open Access Journals (Sweden)

    Shane Viars

    2014-05-01

    Full Text Available The LmbE-like superfamily is comprised of a series of enzymes that use a single catalytic metal ion to catalyze the hydrolysis of various substrates. These substrates are often key metabolites for eukaryotes and prokaryotes, which makes the LmbE-like enzymes important targets for drug development. Herein we review the structure and function of the LmbE-like proteins identified to date. While this is the newest superfamily of metallohydrolases, a growing number of functionally interesting proteins from this superfamily have been characterized. Available crystal structures of LmbE-like proteins reveal a Rossmann fold similar to lactate dehydrogenase, which represented a novel fold for (zinc metallohydrolases at the time the initial structure was solved. The structural diversity of the N-acetylglucosamine containing substrates affords functional diversity for the LmbE-like enzyme superfamily. The majority of enzymes identified to date are metal-dependent deacetylases that catalyze the hydrolysis of a N-acetylglucosamine moiety on substrate using a combination of amino acid side chains and a single bound metal ion, predominantly zinc. The catalytic zinc is coordinated to proteins via His2-Asp-solvent binding site. Additionally, studies indicate that protein dynamics play important roles in regulating access to the active site and facilitating catalysis for at least two members of this protein superfamily.

  14. Iron reductases from Pseudomonas aeruginosa.

    Science.gov (United States)

    Cox, C D

    1980-01-01

    Cell-free extracts of Pseudomonas aeruginosa contain enzyme activities which reduce Fe(III) to Fe(II) when iron is provided in certain chelates, but not when the iron is uncomplexed. Iron reductase activities for two substrates, ferripyochelin and ferric citrate, appear to be separate enzymes because of differences in heat stabilities, in locations in fractions of cell-free extracts, in reductant specificity, and in apparent sizes during gel filtration chromatography. Ferric citrate iron reductase is an extremely labile activity found in the cytoplasmic fraction, and ferripyochelin iron reductase is a more stable activity found in the periplasmic as well as cytoplasmic fraction of extracts. A small amount of activity detectable in the membrane fraction seemed to be loosely associated with the membranes. Although both enzymes have highest activity reduced nicotinamide adenine dinucleotide, reduced glutathione also worked with ferripyochelin iron reductase. In addition, oxygen caused an irreversible loss of a percentage of the ferripyochelin iron reductase following sparge of reaction mixtures, whereas the reductase for ferric citrate was not appreciably affected by oxygen. PMID:6766439

  15. A ferric-chelate reductase for iron uptake from soils.

    Science.gov (United States)

    Robinson, N J; Procter, C M; Connolly, E L; Guerinot, M L

    1999-02-25

    Iron deficiency afflicts more than three billion people worldwide, and plants are the principal source of iron in most diets. Low availability of iron often limits plant growth because iron forms insoluble ferric oxides, leaving only a small, organically complexed fraction in soil solutions. The enzyme ferric-chelate reductase is required for most plants to acquire soluble iron. Here we report the isolation of the FRO2 gene, which is expressed in iron-deficient roots of Arabidopsis. FRO2 belongs to a superfamily of flavocytochromes that transport electrons across membranes. It possesses intramembranous binding sites for haem and cytoplasmic binding sites for nucleotide cofactors that donate and transfer electrons. We show that FRO2 is allelic to the frd1 mutations that impair the activity of ferric-chelate reductase. There is a nonsense mutation within the first exon of FRO2 in frd1-1 and a missense mutation within FRO2 in frd1-3. Introduction of functional FRO2 complements the frd1-1 phenotype in transgenic plants. The isolation of FRO2 has implications for the generation of crops with improved nutritional quality and increased growth in iron-deficient soils.

  16. Phylogenomic evolutionary surveys of subtilase superfamily genes in fungi.

    Science.gov (United States)

    Li, Juan; Gu, Fei; Wu, Runian; Yang, JinKui; Zhang, Ke-Qin

    2017-03-30

    Subtilases belong to a superfamily of serine proteases which are ubiquitous in fungi and are suspected to have developed distinct functional properties to help fungi adapt to different ecological niches. In this study, we conducted a large-scale phylogenomic survey of subtilase protease genes in 83 whole genome sequenced fungal species in order to identify the evolutionary patterns and subsequent functional divergences of different subtilase families among the main lineages of the fungal kingdom. Our comparative genomic analyses of the subtilase superfamily indicated that extensive gene duplications, losses and functional diversifications have occurred in fungi, and that the four families of subtilase enzymes in fungi, including proteinase K-like, Pyrolisin, kexin and S53, have distinct evolutionary histories which may have facilitated the adaptation of fungi to a broad array of life strategies. Our study provides new insights into the evolution of the subtilase superfamily in fungi and expands our understanding of the evolution of fungi with different lifestyles.

  17. Isolated menthone reductase and nucleic acid molecules encoding same

    Science.gov (United States)

    Croteau, Rodney B; Davis, Edward M; Ringer, Kerry L

    2013-04-23

    The present invention provides isolated menthone reductase proteins, isolated nucleic acid molecules encoding menthone reductase proteins, methods for expressing and isolating menthone reductase proteins, and transgenic plants expressing elevated levels of menthone reductase protein.

  18. Superfamilies SDR and MDR: from early ancestry to present forms. Emergence of three lines, a Zn-metalloenzyme, and distinct variabilities.

    Science.gov (United States)

    Jörnvall, Hans; Hedlund, Joel; Bergman, Tomas; Oppermann, Udo; Persson, Bengt

    2010-05-21

    Two large gene and protein superfamilies, SDR and MDR (short- and medium-chain dehydrogenases/reductases), were originally defined from analysis of alcohol and polyol dehydrogenases. The superfamilies contain minimally 82 and 25 genes, respectively, in humans, minimally 324 and 86 enzyme families when known lines in other organisms are also included, and over 47,000 and 15,000 variants in existing sequence data bank entries. SDR enzymes have one-domain subunits without metal and MDR two-domain subunits without or with zinc, and these three lines appear to have emerged in that order from the universal cellular ancestor. This is compatible with their molecular architectures, present multiplicity, and overall distribution in the kingdoms of life, with SDR also of viral occurrence. An MDR-zinc, when present, is often, but not always, catalytic. It appears also to have a structural role in inter-domain interactions, coenzyme binding and substrate pocket formation, as supported by domain variability ratios and ligand positions. Differences among structural and catalytic zinc ions may be relative and involve several states. Combined, the comparisons trace evolutionary properties of huge superfamilies, with partially redundant enzymes in cellular redox functions. 2010 Elsevier Inc. All rights reserved.

  19. VITAL (Vanguard Investigations of Therapeutic Approaches to Lung Cancer)

    Science.gov (United States)

    2009-01-01

    organotypic culture environment mimics lung stratified epithelium, complete with basal cells, ciliated columnar cells, and mucus - producing goblet...Yang SC, Luo J, et al. Cyclooxygenase-2-dependent regulation of E- cadherin: prostaglandin E(2) induces transcriptional repressors ZEB1 and snail in...5AC, oligomeric mucus /gel-forming XM_001130382 214385_s_at AKR1C2 3.3 4.2 3.5 3.5 0.0000 aldo-keto reductase family 1, member C2 NM_001354 209699_x_at

  20. Differential nitrate accumulation, nitrate reduction, nitrate reductase ...

    African Journals Online (AJOL)

    use

    2011-12-07

    Dec 7, 2011 ... reductase activity and nitrite accumulation depend on the exogenous nitrate. Nitrite itself is reduced to ammonium by palstidic nitrite reductase. Nitrite reductase is activated by both nitrate and nitrite ions by positive feed forward, whereas nitrate metabolites, most likely ammonium and glutamine; down.

  1. Structural and evolutionary bioinformatics of the SPOUT superfamily of methyltransferases

    Directory of Open Access Journals (Sweden)

    Purta Elzbieta

    2007-03-01

    Full Text Available Abstract Background SPOUT methyltransferases (MTases are a large class of S-adenosyl-L-methionine-dependent enzymes that exhibit an unusual alpha/beta fold with a very deep topological knot. In 2001, when no crystal structures were available for any of these proteins, Anantharaman, Koonin, and Aravind identified homology between SpoU and TrmD MTases and defined the SPOUT superfamily. Since then, multiple crystal structures of knotted MTases have been solved and numerous new homologous sequences appeared in the databases. However, no comprehensive comparative analysis of these proteins has been carried out to classify them based on structural and evolutionary criteria and to guide functional predictions. Results We carried out extensive searches of databases of protein structures and sequences to collect all members of previously identified SPOUT MTases, and to identify previously unknown homologs. Based on sequence clustering, characterization of domain architecture, structure predictions and sequence/structure comparisons, we re-defined families within the SPOUT superfamily and predicted putative active sites and biochemical functions for the so far uncharacterized members. We have also delineated the common core of SPOUT MTases and inferred a multiple sequence alignment for the conserved knot region, from which we calculated the phylogenetic tree of the superfamily. We have also studied phylogenetic distribution of different families, and used this information to infer the evolutionary history of the SPOUT superfamily. Conclusion We present the first phylogenetic tree of the SPOUT superfamily since it was defined, together with a new scheme for its classification, and discussion about conservation of sequence and structure in different families, and their functional implications. We identified four protein families as new members of the SPOUT superfamily. Three of these families are functionally uncharacterized (COG1772, COG1901, and COG4080

  2. Methylenetetrahydrofolate reductase (MTHFR) gene polymorphism ...

    African Journals Online (AJOL)

    Administrator

    2011-10-19

    Oct 19, 2011 ... Polymorphisms of the methylenetetrahydrofolate reductase (MTHFR) gene are associated with abortion, early embryo loss and recurrent spontaneous abortion in human. However, information on the association between MTHFR polymorphism and cow abortion is scarce. In the present study, the effects.

  3. Association between methylenetetrahydrofolate reductase (MTHFR ...

    African Journals Online (AJOL)

    Association between methylenetetrahydrofolate reductase (MTHFR) C677T gene polymorphism and risk of ischemic stroke in North Indian population: A hospital based case–control study. Amit Kumar, Shubham Misra, Anjali Hazarika, Pradeep Kumar, Ram Sagar, Abhishek Pathak, Kamalesh Chakravarty, Kameshwar ...

  4. Methylenetetrahydrofolate reductase (MTHFR) gene polymorphism ...

    African Journals Online (AJOL)

    Polymorphisms of the methylenetetrahydrofolate reductase (MTHFR) gene are associated with abortion, early embryo loss and recurrent spontaneous abortion in human. However, information on the association between MTHFR polymorphism and cow abortion is scarce. In the present study, the effects of MTHFR ...

  5. Trametes versicolor carboxylate reductase uncovered.

    Science.gov (United States)

    Winkler, Margit; Winkler, Christoph K

    The first carboxylate reductase from Trametes versicolor was identified, cloned, and expressed in Escherichia coli . The enzyme reduces aromatic acids such as benzoic acid and derivatives, cinnamic acid, and 3-phenylpropanoic acid, but also aliphatic acids such as octanoic acid are reduced.

  6. The SDR (short-chain dehydrogenase/reductase and related enzymes) nomenclature initiative.

    Science.gov (United States)

    Persson, Bengt; Kallberg, Yvonne; Bray, James E; Bruford, Elspeth; Dellaporta, Stephen L; Favia, Angelo D; Duarte, Roser Gonzalez; Jörnvall, Hans; Kavanagh, Kathryn L; Kedishvili, Natalia; Kisiela, Michael; Maser, Edmund; Mindnich, Rebekka; Orchard, Sandra; Penning, Trevor M; Thornton, Janet M; Adamski, Jerzy; Oppermann, Udo

    2009-03-16

    Short-chain dehydrogenases/reductases (SDR) constitute one of the largest enzyme superfamilies with presently over 46,000 members. In phylogenetic comparisons, members of this superfamily show early divergence where the majority have only low pairwise sequence identity, although sharing common structural properties. The SDR enzymes are present in virtually all genomes investigated, and in humans over 70 SDR genes have been identified. In humans, these enzymes are involved in the metabolism of a large variety of compounds, including steroid hormones, prostaglandins, retinoids, lipids and xenobiotics. It is now clear that SDRs represent one of the oldest protein families and contribute to essential functions and interactions of all forms of life. As this field continues to grow rapidly, a systematic nomenclature is essential for future annotation and reference purposes. A functional subdivision of the SDR superfamily into at least 200 SDR families based upon hidden Markov models forms a suitable foundation for such a nomenclature system, which we present in this paper using human SDRs as examples.

  7. Phylogenomic evolutionary surveys of subtilase superfamily genes in fungi

    OpenAIRE

    Juan Li; Fei Gu; Runian Wu; JinKui Yang; Ke-Qin Zhang

    2017-01-01

    Subtilases belong to a superfamily of serine proteases which are ubiquitous in fungi and are suspected to have developed distinct functional properties to help fungi adapt to different ecological niches. In this study, we conducted a large-scale phylogenomic survey of subtilase protease genes in 83 whole genome sequenced fungal species in order to identify the evolutionary patterns and subsequent functional divergences of different subtilase families among the main lineages of the fungal king...

  8. Systematic classification of the His-Me finger superfamily.

    Science.gov (United States)

    Jablonska, Jagoda; Matelska, Dorota; Steczkiewicz, Kamil; Ginalski, Krzysztof

    2017-11-16

    The His-Me finger endonucleases, also known as HNH or ββα-metal endonucleases, form a large and diverse protein superfamily. The His-Me finger domain can be found in proteins that play an essential role in cells, including genome maintenance, intron homing, host defense and target offense. Its overall structural compactness and non-specificity make it a perfectly-tailored pathogenic module that participates on both sides of inter- and intra-organismal competition. An extremely low sequence similarity across the superfamily makes it difficult to identify and classify new His-Me fingers. Using state-of-the-art distant homology detection methods, we provide an updated and systematic classification of His-Me finger proteins. In this work, we identified over 100 000 proteins and clustered them into 38 groups, of which three groups are new and cannot be found in any existing public domain database of protein families. Based on an analysis of sequences, structures, domain architectures, and genomic contexts, we provide a careful functional annotation of the poorly characterized members of this superfamily. Our results may inspire further experimental investigations that should address the predicted activity and clarify the potential substrates, to provide more detailed insights into the fundamental biological roles of these proteins. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  9. The Evolution of the Actin Binding NET Superfamily

    Directory of Open Access Journals (Sweden)

    Tim eHawkins

    2014-06-01

    Full Text Available The arabidopsis Networked protein superfamily are plant-specific actin binding proteins which specifically label different membrane compartments and identify specialized sites of interaction between actin and membranes unique to plants. There are 13 members of the superfamily in arabidopsis which group into 4 distinct clades or subfamilies. NET homologues are absent from the genomes of metazoa and fungi, furthermore in Plantae NET sequences are also absent from the genome of mosses and more ancient extant plant clades. A single subfamily of the NET proteins are found encoded in the club moss genome; an extant species of the earliest vascular plants. Gymnosperms have examples from subfamilies 4 and 3 with a hybrid form of NET1 and 2 which shows characteristics of both NET1 and NET2. In addition to NET3 and 4 subfamilies, the NET1 and pollen-expressed NET2 subfamilies are only found as independent sequences in angiosperms. This is consistent with the divergence of reproductive actin. The four subfamilies are conserved across monocots and eudicots with the numbers of members of each clade expanding at this point due in part to regions of genome duplication. Since the emergence of the NET superfamily at the dawn of vascular plants they have continued to develop and diversify in a manner which has mirrored the divergence and complexity of plant species through evolution in the ‘March of Progress’.

  10. YLL056C from Saccharomyces cerevisiae encodes a novel protein with aldehyde reductase activity.

    Science.gov (United States)

    Wang, Han-Yu; Xiao, Di-Fan; Zhou, Chang; Wang, Lin-Lu; Wu, Lan; Lu, Ya-Ting; Xiang, Quan-Ju; Zhao, Ke; Li, Xi; Ma, Meng -Gen

    2017-06-01

    The short-chain dehydrogenase/reductase (SDR) family, the largest family in dehydrogenase/reductase superfamily, is divided into "classical," "extended," "intermediate," "divergent," "complex," and "atypical" groups. Recently, several open reading frames (ORFs) were characterized as intermediate SDR aldehyde reductase genes in Saccharomyces cerevisiae. However, no functional protein in the atypical group has been characterized in S. cerevisiae till now. Herein, we report that an uncharacterized ORF YLL056C from S. cerevisiae was significantly upregulated under high furfural (2-furaldehyde) or 5-(hydroxymethyl)-2-furaldehyde concentrations, and transcription factors Yap1p, Hsf1p, Pdr1/3p, Yrr1p, and Stb5p likely controlled its upregulated transcription. This ORF indeed encoded a protein (Yll056cp), which was grouped into the atypical subgroup 7 in the SDR family and localized to the cytoplasm. Enzyme activity assays showed that Yll056cp is not a quinone or ketone reductase but an NADH-dependent aldehyde reductase, which can reduce at least seven aldehyde compounds. This enzyme showed the best Vmax, Kcat, and Kcat/Km to glycolaldehyde, but the highest affinity (Km) to formaldehyde. The optimum pH and temperature of this enzyme was pH 6.5 for reduction of glycolaldehyde, furfural, formaldehyde, butyraldehyde, and propylaldehyde, and 30 °C for reduction of formaldehyde or 35 °C for reduction of glycolaldehyde, furfural, butyraldehyde, and propylaldehyde. Temperature and pH affected stability of this enzyme and this influence varied with aldehyde substrate. Metal ions, salts, and chemical protective additives, especially at high concentrations, had different influence on enzyme activities for reduction of different aldehydes. This research provided guidelines for study of more uncharacterized atypical SDR enzymes from S. cerevisiae and other organisms.

  11. Research progress on the roles of aldose reductase in diabetic retinopathy

    Directory of Open Access Journals (Sweden)

    Hong-Zhe Li

    2015-07-01

    Full Text Available Aldose reductase(ARbelonging to nicotinamide-adenine dinucleotide phosphate(NADPH-dependent aldehyde-keto reductase superfamily, is the key rate-limiting enzyme in the polyol pathway which plays an important role in the body's high-sugar metabolism. AR is widely present in the kidneys, blood vessels, lens, retina, heart, skeletal muscle and other tissues and organs, converts glucose to sorbitol which easy permeability of cell membranes, cause cell swelling, degeneration, necrosis, and have a close relationship with the development of chronic complications of diabetes mellitus. Diabetic retinopathy(DRis a multifactorial disease, the exact cause is currently unknown, but polyol pathway has been demonstrated to play an important role in the pathogenesis of DR. Clinical risk factors such as blood sugar control, blood pressure and other treatments for DR only play a part effect of remission or invalid, if we can find out DR genes associated with the disease, this will contribute to a better understanding of the pathological mechanisms and contribute to the development of new treatments and drugs. The current research progress of AR, AR gene polymorphism, Aldose reductase inhibitors to DR was reviewed in this article.

  12. Immunocytochemical localization of APS reductase and bisulfite reductase in three Desulfovibrio species

    NARCIS (Netherlands)

    Kremer, D.R.; Veenhuis, M.; Fauque, G.; Peck Jr., H.D.; LeGall, J.; Lampreia, J.; Moura, J.J.G.; Hansen, T.A.

    1988-01-01

    The localization of APS reductase and bisulfite reductase in Desulfovibrio gigas, D. vulgaris Hildenborough and D. thermophilus was studied by immunoelectron microscopy. Polyclonal antibodies were raised against the purified enzymes from each strain. Cells fixed with formaldehyde/glutaraldehyde were

  13. Fatty acyl-CoA reductase

    Energy Technology Data Exchange (ETDEWEB)

    Reiser, Steven E.; Somerville, Chris R.

    1998-12-01

    The present invention relates to bacterial enzymes, in particular to an acyl-CoA reductase and a gene encoding an acyl-CoA reductase, the amino acid and nucleic acid sequences corresponding to the reductase polypeptide and gene, respectively, and to methods of obtaining such enzymes, amino acid sequences and nucleic acid sequences. The invention also relates to the use of such sequences to provide transgenic host cells capable of producing fatty alcohols and fatty aldehydes.

  14. Evolution of substrate specificity and protein-protein interactions in three enzyme superfamilies

    OpenAIRE

    Plach, Maximilian

    2017-01-01

    Superfamilies are a classification system to combine proteins that are related through a common evolutionary origin, share similar sequences, structures, and core reaction mechanisms, but exert different functions. Today, for most superfamilies tens of thousands of sequences and hundreds of structures are known and most of the different functions of their members have been elucidated. Superfamilies thus provide a formal and biologically sensible framework to study evolutionary relationships be...

  15. Oxygen and xenobiotic reductase activities of cytochrome P450.

    NARCIS (Netherlands)

    Goeptar, A.R.; Scheerens, H.; Vermeulen, N.P.E.

    1995-01-01

    The oxygen reductase and xenobiotic reductase activities of cytochrome P450 (P450) are reviewed. During the oxygen reductase activity of P450, molecular oxygen is reduced to superoxide anion radicals (O

  16. Comparative analysis of cation/proton antiporter superfamily in plants

    Energy Technology Data Exchange (ETDEWEB)

    Ye, Chuyu [ORNL; Yang, Xiaohan [ORNL; Xia, Xinli [Beijing Forestry University, China; Yin, Weilun [Beijing Forestry University, China

    2013-01-01

    The cation/proton antiporter superfamily is associated with the transport of monovalent cations across membranes. This superfamily was annotated in the Arabidopsis genome and some members were functionally characterized. In the present study, a systematic analysis of the cation/proton antiporter genes in diverse plant specieswas reported.We identified 240 cation/proton antiporters in alga, moss, and angiosperm. A phylogenetic tree was constructed showing these 240members are separated into three families, i.e., Na+/H+ exchangers, K+ efflux antiporters, and cation/H+ exchangers. Our analysis revealed that tandem and/or segmental duplications contribute to the expansion of cation/H+ exchangers in the examined angiospermspecies. Sliding windowanalysis of the nonsynonymous/synonymous substitution ratios showed some differences in the evolutionary fate of cation/proton antiporter paralogs. Furthermore, we identified over-represented motifs among these 240 proteins and foundmostmotifs are family specific, demonstrating diverse evolution of the cation/proton antiporters among three families. In addition, we investigated the co-expressed genes of the cation/proton antiporters in Arabidopsis thaliana. The results showed some biological processes are enriched in the co-expressed genes, suggesting the cation/proton antiporters may be involved in these biological processes. Taken together, this study furthers our knowledge on cation/proton antiporters in plants.

  17. The Arabidopsis thaliana ABC protein superfamily, a complete inventory.

    Science.gov (United States)

    Sánchez-Fernández, R; Davies, T G; Coleman, J O; Rea, P A

    2001-08-10

    We describe the first complete inventory of ATP-binding cassette (ABC) proteins from a multicellular organism, the model plant Arabidopsis thaliana. By the application of several search criteria, Arabidopsis was found to contain a total of 129 open reading frames (ORFs) capable of encoding ABC proteins, of which 103 possessed contiguous transmembrane spans and were identified as putative intrinsic membrane proteins. Fifty-two of the putative intrinsic membrane proteins contained at least two transmembrane domains (TMDs) and two nucleotide-binding folds (NBFs) and could be classified as belonging to one of five subfamilies of full-molecule transporters. The other 51 putative membrane proteins, all of which were half-molecule transporters, fell into five subfamilies. Of the remaining ORFs identified, all of which encoded proteins lacking TMDs, 11 could be classified into three subfamilies. There were no obvious homologs in other organisms for 15 of the ORFs which encoded a heterogeneous group of non-intrinsic ABC proteins (NAPs). Unrooted phylogenetic analyses substantiated the subfamily designations. Notable features of the Arabidopsis ABC superfamily was the presence of a large yeast-like PDR subfamily, and the absence of genes encoding bona fide cystic fibrosis transmembrane conductance regulator (CFTR), sulfonylurea receptor (SUR), and heavy metal tolerance factor 1 (HMT1) homologs. Arabidopsis was unusual in its large allocation of ORFs (a minimum of 0.5%) to members of the ABC protein superfamily.

  18. Membrane and Protein Interactions of the Pleckstrin Homology Domain Superfamily

    Directory of Open Access Journals (Sweden)

    Marc Lenoir

    2015-10-01

    Full Text Available The human genome encodes about 285 proteins that contain at least one annotated pleckstrin homology (PH domain. As the first phosphoinositide binding module domain to be discovered, the PH domain recruits diverse protein architectures to cellular membranes. PH domains constitute one of the largest protein superfamilies, and have diverged to regulate many different signaling proteins and modules such as Dbl homology (DH and Tec homology (TH domains. The ligands of approximately 70 PH domains have been validated by binding assays and complexed structures, allowing meaningful extrapolation across the entire superfamily. Here the Membrane Optimal Docking Area (MODA program is used at a genome-wide level to identify all membrane docking PH structures and map their lipid-binding determinants. In addition to the linear sequence motifs which are employed for phosphoinositide recognition, the three dimensional structural features that allow peripheral membrane domains to approach and insert into the bilayer are pinpointed and can be predicted ab initio. The analysis shows that conserved structural surfaces distinguish which PH domains associate with membrane from those that do not. Moreover, the results indicate that lipid-binding PH domains can be classified into different functional subgroups based on the type of membrane insertion elements they project towards the bilayer.

  19. Protein superfamily classification using fuzzy rule-based classifier.

    Science.gov (United States)

    Mansoori, Eghbal G; Zolghadri, Mansoor J; Katebi, Seraj D

    2009-03-01

    In this paper, we have proposed a fuzzy rule-based classifier for assigning amino acid sequences into different superfamilies of proteins. While the most popular methods for protein classification rely on sequence alignment, our approach is alignment-free and so more human readable. It accounts for the distribution of contiguous patterns of n amino acids ( n-grams) in the sequences as features, alike other alignment-independent methods. Our approach, first extracts a plenty of features from a set of training sequences, then selects only some best of them, using a proposed feature ranking method. Thereafter, using these features, a novel steady-state genetic algorithm for extracting fuzzy classification rules from data is used to generate a compact set of interpretable fuzzy rules. The generated rules are simple and human understandable. So, the biologists can utilize them, for classification purposes, or incorporate their expertise to interpret or even modify them. To evaluate the performance of our fuzzy rule-based classifier, we have compared it with the conventional nonfuzzy C4.5 algorithm, beside some other fuzzy classifiers. This comparative study is conducted through classifying the protein sequences of five superfamily classes, downloaded from a public domain database. The obtained results show that the generated fuzzy rules are more interpretable, with acceptable improvement in the classification accuracy.

  20. Membrane and Protein Interactions of the Pleckstrin Homology Domain Superfamily.

    Science.gov (United States)

    Lenoir, Marc; Kufareva, Irina; Abagyan, Ruben; Overduin, Michael

    2015-10-23

    The human genome encodes about 285 proteins that contain at least one annotated pleckstrin homology (PH) domain. As the first phosphoinositide binding module domain to be discovered, the PH domain recruits diverse protein architectures to cellular membranes. PH domains constitute one of the largest protein superfamilies, and have diverged to regulate many different signaling proteins and modules such as Dbl homology (DH) and Tec homology (TH) domains. The ligands of approximately 70 PH domains have been validated by binding assays and complexed structures, allowing meaningful extrapolation across the entire superfamily. Here the Membrane Optimal Docking Area (MODA) program is used at a genome-wide level to identify all membrane docking PH structures and map their lipid-binding determinants. In addition to the linear sequence motifs which are employed for phosphoinositide recognition, the three dimensional structural features that allow peripheral membrane domains to approach and insert into the bilayer are pinpointed and can be predicted ab initio. The analysis shows that conserved structural surfaces distinguish which PH domains associate with membrane from those that do not. Moreover, the results indicate that lipid-binding PH domains can be classified into different functional subgroups based on the type of membrane insertion elements they project towards the bilayer.

  1. TNF Superfamily: A Growing Saga of Kidney Injury Modulators

    Directory of Open Access Journals (Sweden)

    Maria D. Sanchez-Niño

    2010-01-01

    Full Text Available Members of the TNF superfamily participate in kidney disease. Tumor necrosis factor (TNF and Fas ligand regulate renal cell survival and inflammation, and therapeutic targeting improves the outcome of experimental renal injury. TNF-related apoptosis-inducing ligand (TRAIL and its potential decoy receptor osteoprotegerin are the two most upregulated death-related genes in human diabetic nephropathy. TRAIL activates NF-kappaB in tubular cells and promotes apoptosis in tubular cells and podocytes, especially in a high-glucose environment. By contrast, osteoprotegerin plays a protective role against TRAIL-induced apoptosis. Another family member, TNF-like weak inducer of apoptosis (TWEAK induces inflammation and tubular cell death or proliferation, depending on the microenvironment. While TNF only activates canonical NF-kappaB signaling, TWEAK promotes both canonical and noncanonical NF-kappaB activation in tubular cells, regulating different inflammatory responses. TWEAK promotes the secretion of MCP-1 and RANTES through NF-kappaB RelA-containing complexes and upregulates CCl21 and CCL19 expression through NF-kappaB inducing kinase (NIK- dependent RelB/NF-kappaB2 complexes. In vivo TWEAK promotes postnephrectomy compensatory renal cell proliferation in a noninflammatory milieu. However, in the inflammatory milieu of acute kidney injury, TWEAK promotes tubular cell death and inflammation. Therapeutic targeting of TNF superfamily cytokines, including multipronged approaches targeting several cytokines should be further explored.

  2. Transient receptor potential (TRP gene superfamily encoding cation channels

    Directory of Open Access Journals (Sweden)

    Pan Zan

    2011-01-01

    Full Text Available Abstract Transient receptor potential (TRP non-selective cation channels constitute a superfamily, which contains 28 different genes. In mammals, this superfamily is divided into six subfamilies based on differences in amino acid sequence homology between the different gene products. Proteins within a subfamily aggregate to form heteromeric or homomeric tetrameric configurations. These different groupings have very variable permeability ratios for calcium versus sodium ions. TRP expression is widely distributed in neuronal tissues, as well as a host of other tissues, including epithelial and endothelial cells. They are activated by environmental stresses that include tissue injury, changes in temperature, pH and osmolarity, as well as volatile chemicals, cytokines and plant compounds. Their activation induces, via intracellular calcium signalling, a host of responses, including stimulation of cell proliferation, migration, regulatory volume behaviour and the release of a host of cytokines. Their activation is greatly potentiated by phospholipase C (PLC activation mediated by coupled GTP-binding proteins and tyrosine receptors. In addition to their importance in maintaining tissue homeostasis, some of these responses may involve various underlying diseases. Given the wealth of literature describing the multiple roles of TRP in physiology in a very wide range of different mammalian tissues, this review limits itself to the literature describing the multiple roles of TRP channels in different ocular tissues. Accordingly, their importance to the corneal, trabecular meshwork, lens, ciliary muscle, retinal, microglial and retinal pigment epithelial physiology and pathology is reviewed.

  3. Characterization of xylose reductase from Candida tropicalis ...

    African Journals Online (AJOL)

    USER

    2010-08-02

    Aug 2, 2010 ... Xylose reductase gene, enzyme cofactors and plasmids. E.coli BL21(DE3) was used as host strains for ... C. tropicalis xylose reductase gene was isolated from plasmid. pMD18-T (TaKaRa, Japan). Enzyme ..... the gels is instable, soft and even dissolve in the solution containing multivalent anions or high ...

  4. Radical SAM, a novel protein superfamily linking unresolved steps in familiar biosynthetic pathways with radical mechanisms: functional characterization using new analysis and information visualization methods

    Science.gov (United States)

    Sofia, Heidi J.; Chen, Guang; Hetzler, Beth G.; Reyes-Spindola, Jorge F.; Miller, Nancy E.

    2001-01-01

    A novel protein superfamily with over 600 members was discovered by iterative profile searches and analyzed with powerful bioinformatics and information visualization methods. Evidence exists that these proteins generate a radical species by reductive cleavage of S-adenosylmethionine (SAM) through an unusual Fe-S center. The superfamily (named here Radical SAM) provides evidence that radical-based catalysis is important in a number of previously well- studied but unresolved biochemical pathways and reflects an ancient conserved mechanistic approach to difficult chemistries. Radical SAM proteins catalyze diverse reactions, including unusual methylations, isomerization, sulfur insertion, ring formation, anaerobic oxidation and protein radical formation. They function in DNA precursor, vitamin, cofactor, antibiotic and herbicide biosynthesis and in biodegradation pathways. One eukaryotic member is interferon-inducible and is considered a candidate drug target for osteoporosis; another is observed to bind the neuronal Cdk5 activator protein. Five defining members not previously recognized as homologs are lysine 2,3-aminomutase, biotin synthase, lipoic acid synthase and the activating enzymes for pyruvate formate-lyase and anaerobic ribonucleotide reductase. Two functional predictions for unknown proteins are made based on integrating other data types such as motif, domain, operon and biochemical pathway into an organized view of similarity relationships. PMID:11222759

  5. Human microsomal carbonyl reducing enzymes in the metabolism of xenobiotics: well-known and promising members of the SDR superfamily.

    Science.gov (United States)

    Skarydová, Lucie; Wsól, Vladimír

    2012-05-01

    The best known, most widely studied enzyme system in phase I biotransformation is cytochrome P450 (CYP), which participates in the metabolism of roughly 9 of 10 drugs in use today. The main biotransformation isoforms of CYP are associated with the membrane of the endoplasmatic reticulum (ER). Other enzymes that are also active in phase I biotransformation are carbonyl reducing enzymes. Much is known about the role of cytosolic forms of carbonyl reducing enzymes in the metabolism of xenobiotics, but their microsomal forms have been mostly poorly studied. The only well-known microsomal carbonyl reducing enzyme taking part in the biotransformation of xenobiotics is 11β-hydroxysteroid dehydrogenase 1, a member of the short-chain dehydrogenase/reductase superfamily. Physiological roles of microsomal carbonyl reducing enzymes are better known than their participation in the metabolism of xenobiotics. This review is a summary of the fragmentary information known about the roles of the microsomal forms. Besides 11β-hydroxysteroid dehydrogenase 1, it has been reported, so far, that retinol dehydrogenase 12 participates only in the detoxification of unsaturated aldehydes formed upon oxidative stress. Another promising group of microsomal biotransformation carbonyl reducing enzymes are some members of 17β-hydroxysteroid dehydrogenases. Generally, it is clear that this area is, overall, quite unexplored, but carbonyl reducing enzymes located in the ER have proven very interesting. The study of these enzymes could shed new light on the metabolism of several clinically used drugs or they could become an important target in connection with some diseases.

  6. Promiscuity and electrostatic flexibility in the alkaline phosphatase superfamily.

    Science.gov (United States)

    Pabis, Anna; Kamerlin, Shina Caroline Lynn

    2016-04-01

    Catalytic promiscuity, that is, the ability of single enzymes to facilitate the turnover of multiple, chemically distinct substrates, is a widespread phenomenon that plays an important role in the evolution of enzyme function. Additionally, such pre-existing multifunctionality can be harnessed in artificial enzyme design. The members of the alkaline phosphatase superfamily have served extensively as both experimental and computational model systems for enhancing our understanding of catalytic promiscuity. In this Opinion, we present key recent computational studies into the catalytic activity of these highly promiscuous enzymes, highlighting the valuable insight they have provided into both the molecular basis for catalytic promiscuity in general, and its implications for the evolution of phosphatase activity. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  7. The cellulose synthase superfamily in fully sequenced plants and algae

    Directory of Open Access Journals (Sweden)

    Xu Ying

    2009-07-01

    Full Text Available Abstract Background The cellulose synthase superfamily has been classified into nine cellulose synthase-like (Csl families and one cellulose synthase (CesA family. The Csl families have been proposed to be involved in the synthesis of the backbones of hemicelluloses of plant cell walls. With 17 plant and algal genomes fully sequenced, we sought to conduct a genome-wide and systematic investigation of this superfamily through in-depth phylogenetic analyses. Results A single-copy gene is found in the six chlorophyte green algae, which is most closely related to the CslA and CslC families that are present in the seven land plants investigated in our analyses. Six proteins from poplar, grape and sorghum form a distinct family (CslJ, providing further support for the conclusions from two recent studies. CslB/E/G/H/J families have evolved significantly more rapidly than their widely distributed relatives, and tend to have intragenomic duplications, in particular in the grape genome. Conclusion Our data suggest that the CslA and CslC families originated through an ancient gene duplication event in land plants. We speculate that the single-copy Csl gene in green algae may encode a mannan synthase. We confirm that the rest of the Csl families have a different evolutionary origin than CslA and CslC, and have proposed a model for the divergence order among them. Our study provides new insights about the evolution of this important gene family in plants.

  8. Plant tropane alkaloid biosynthesis evolved independently in the Solanaceae and Erythroxylaceae.

    Science.gov (United States)

    Jirschitzka, Jan; Schmidt, Gregor W; Reichelt, Michael; Schneider, Bernd; Gershenzon, Jonathan; D'Auria, John Charles

    2012-06-26

    The pharmacologically important tropane alkaloids have a scattered distribution among angiosperm families, like many other groups of secondary metabolites. To determine whether tropane alkaloids have evolved repeatedly in different lineages or arise from an ancestral pathway that has been lost in most lines, we investigated the tropinone-reduction step of their biosynthesis. In species of the Solanaceae, which produce compounds such as atropine and scopolamine, this reaction is known to be catalyzed by enzymes of the short-chain dehydrogenase/reductase family. However, in Erythroxylum coca (Erythroxylaceae), which accumulates cocaine and other tropane alkaloids, no proteins of the short-chain dehydrogenase/reductase family were found that could catalyze this reaction. Instead, purification of E. coca tropinone-reduction activity and cloning of the corresponding gene revealed that a protein of the aldo-keto reductase family carries out this reaction in E. coca. This protein, designated methylecgonone reductase, converts methylecgonone to methylecgonine, the penultimate step in cocaine biosynthesis. The protein has highest sequence similarity to other aldo-keto reductases, such as chalcone reductase, an enzyme of flavonoid biosynthesis, and codeinone reductase, an enzyme of morphine alkaloid biosynthesis. Methylecgonone reductase reduces methylecgonone (2-carbomethoxy-3-tropinone) stereospecifically to 2-carbomethoxy-3β-tropine (methylecgonine), and has its highest activity, protein level, and gene transcript level in young, expanding leaves of E. coca. This enzyme is not found at all in root tissues, which are the site of tropane alkaloid biosynthesis in the Solanaceae. This evidence supports the theory that the ability to produce tropane alkaloids has arisen more than once during the evolution of the angiosperms.

  9. Biochemical properties of human dehydrogenase/reductase (SDR family) member 7.

    Science.gov (United States)

    Stambergova, Hana; Skarydova, Lucie; Dunford, James E; Wsol, Vladimir

    2014-01-25

    Dehydrogenase/reductase (SDR family) member 7 (DHRS7, retSDR4, SDR34C1) is a previously uncharacterized member of the short-chain dehydrogenase/reductase (SDR) superfamily. While human SDR members are known to play an important role in various (patho)biochemical pathways including intermediary metabolism and biotransformation of xenobiotics, only 20% of them are considered to be well characterized. Based on phylogenetic tree and SDR sequence clusters analysis DHRS7 is a close relative to well-known SDR member 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) that participates in metabolism of endogenous and xenobiotic substances with carbonyl group. The aim of present study is to determine the basic biochemical properties of DHRS7 and its possible involvement in metabolism of substrates with carbonyl group. For the first time the computational predictions of this membrane protein and membrane topology were experimentally confirmed. DHRS7 has been demonstrated to be an integral protein facing the lumen of the endoplasmic reticulum with lack of posttranscriptional glycosylation modification. Subsequently, NADP(H) cofactor preference and enzymatic reducing activity of DHRS7 was determined towards endogenous substrates with a steroid structure (cortisone, 4-androstene-3,17-dion) and also toward relevant exogenous substances bearing a carbonyl group harmful to human health (1,2-naphtoquinone, 9,10-phenantrenequinone). In addition to 11β-HSD1, DHRS7 is another enzyme from SDR superfamily that have been proved, at least in vitro, to contribute to the metabolism of xenobiotics with carbonyl group. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  10. Carboxylic acid reductase enzymes (CARs).

    Science.gov (United States)

    Winkler, Margit

    2018-04-01

    Carboxylate reductases (CARs) are emerging as valuable catalysts for the selective one-step reduction of carboxylic acids to their corresponding aldehydes. The substrate scope of CARs is exceptionally broad and offers potential for their application in diverse synthetic processes. Two major fields of application are the preparation of aldehydes as end products for the flavor and fragrance sector and the integration of CARs in cascade reactions with aldehydes as the key intermediates. The latest applications of CARs are dominated by in vivo cascades and chemo-enzymatic reaction sequences. The challenge to fully exploit product selectivity is discussed. Recent developments in the characterization of CARs are summarized, with a focus on aspects related to the domain architecture and protein sequences of CAR enzymes. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. Ferrisiderophore reductase activity in Bacillus megaterium.

    Science.gov (United States)

    Arceneaux, J E; Byers, B R

    1980-01-01

    The release of iron from ferrisiderophores (microbial ferric-chelating iron transport cofactors) by cell-free extracts of Bacillus megaterium was demonstrated. Reductive transfer of iron from ferrisiderophores to the ferrous-chelating agent ferrozine was measured spectrophotometrically. This ferrisiderophore reductase activity (reduced nicotinamide adenine dinucleotide phosphate:ferrisiderophore oxidoreductase) was associated primarily with the cell soluble rather than particulate (membrane) fraction. Ferrisiderophore reductase was inhibited by oxygen and required the addition of a reductant (reduced nicotinamide adenine dinucleotide phosphate was most effective) for maximal activity. The activity was destroyed by both heat and protease treatments and was inhibited by iodoacetamide treatment. Ferrisiderophore reductase activity for several microbial ferrisiderophores was measured; highest activity was displayed for ferrischizokinen, the ferrisiderophore produced by this organism. The Km and Vmax values of the reductase for ferrischizokinen were 2.5 x 10(-4) M and 35.7 nmol/min per mg of the ferrisiderophore reductase reaction. Preliminary fractionation of the cell soluble material by gel filtration chromatography resulted in the demonstration of ferrisiderophore reductase activity in three peaks of different molecular weight. Ferrisiderophore reductase probably mediates entrance of iron into cellular metabolism. PMID:6444944

  12. Tetrathionate reductase of Salmonella thyphimurium: a molybdenum containing enzyme

    International Nuclear Information System (INIS)

    Hinojosa-Leon, M.; Dubourdieu, M.; Sanchez-Crispin, J.A.; Chippaux, M.

    1986-01-01

    Use of radioactive molybdenum demonstrates that the tetrathionate reductase of Salmonella typhimurium is a molydenum containing enzyme. It is proposed that this enzyme shares with other molybdo-proteins, such as nitrate reductase, a common molybdenum containing cofactor the defect of which leads to the loss of the tetrathionate reductase and nitrate reductase activities

  13. Characterization of the Tetraspan Junctional Complex (4JC) superfamily.

    Science.gov (United States)

    Chou, Amy; Lee, Andre; Hendargo, Kevin J; Reddy, Vamsee S; Shlykov, Maksim A; Kuppusamykrishnan, Harikrishnan; Medrano-Soto, Arturo; Saier, Milton H

    2017-03-01

    Connexins or innexins form gap junctions, while claudins and occludins form tight junctions. In this study, statistical data, derived using novel software, indicate that these four junctional protein families and eleven other families of channel and channel auxiliary proteins are related by common descent and comprise the Tetraspan (4 TMS) Junctional Complex (4JC) Superfamily. These proteins all share similar 4 transmembrane α-helical (TMS) topologies. Evidence is presented that they arose via an intragenic duplication event, whereby a 2 TMS-encoding genetic element duplicated tandemly to give 4 TMS proteins. In cases where high resolution structural data were available, the conclusion of homology was supported by conducting structural comparisons. Phylogenetic trees reveal the probable relationships of these 15 families to each other. Long homologues containing fusions to other recognizable domains as well as internally duplicated or fused domains are reported. Large "fusion" proteins containing 4JC domains proved to fall predominantly into family-specific patterns as follows: (1) the 4JC domain was N-terminal; (2) the 4JC domain was C-terminal; (3) the 4JC domain was duplicated or occasionally triplicated and (4) mixed fusion types were present. Our observations provide insight into the evolutionary origins and subfunctions of these proteins as well as guides concerning their structural and functional relationships. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. Nuclear Receptors in atherosclerosis: a superfamily with many 'Goodfellas'.

    Science.gov (United States)

    Kurakula, Kondababu; Hamers, Anouk A J; de Waard, Vivian; de Vries, Carlie J M

    2013-04-10

    Nuclear Receptors form a superfamily of 48 transcription factors that exhibit a plethora of functions in steroid hormone signaling, regulation of metabolism, circadian rhythm and cellular differentiation. In this review, we describe our current knowledge on the role of Nuclear Receptors in atherosclerosis, which is a multifactorial disease of the vessel wall. Various cell types are involved in this chronic inflammatory pathology in which multiple cellular processes and numerous genes are dysregulated. Systemic risk factors for atherosclerosis are among others adverse blood lipid profiles, enhanced circulating cytokine levels, as well as increased blood pressure. Since many Nuclear Receptors modulate lipid profiles or regulate blood pressure they indirectly affect atherosclerosis. In the present review, we focus on the functional involvement of Nuclear Receptors within the atherosclerotic vessel wall, more specifically on their modulation of cellular functions in endothelial cells, smooth muscle cells and macrophages. Collectively, this overview shows that most of the Nuclear Receptors are athero-protective in atherosclerotic lesions. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  15. Phylogenetic relationships among superfamilies of Neritimorpha (Mollusca: Gastropoda).

    Science.gov (United States)

    Uribe, Juan E; Colgan, Don; Castro, Lyda R; Kano, Yasunori; Zardoya, Rafael

    2016-11-01

    Despite the extraordinary morphological and ecological diversity of Neritimorpha, few studies have focused on the phylogenetic relationships of this lineage of gastropods, which includes four extant superfamilies: Neritopsoidea, Hydrocenoidea, Helicinoidea, and Neritoidea. Here, the nucleotide sequences of the complete mitochondrial genomes of Georissa bangueyensis (Hydrocenoidea), Neritina usnea (Neritoidea), and Pleuropoma jana (Helicinoidea) and the nearly complete mt genomes of Titiscania sp. (Neritopsoidea) and Theodoxus fluviatilis (Neritoidea) were determined. Phylogenetic reconstructions using probabilistic methods were based on mitochondrial (13 protein coding genes and two ribosomal rRNA genes), nuclear (partial 28S rRNA, 18S rRNA, actin, and histone H3 genes) and combined sequence data sets. All phylogenetic analyses except one converged on a single, highly supported tree in which Neritopsoidea was recovered as the sister group of a clade including Helicinoidea as the sister group of Hydrocenoidea and Neritoidea. This topology agrees with the fossil record and supports at least three independent invasions of land by neritimorph snails. The mitochondrial genomes of Titiscania sp., G. bangueyensis, N. usnea, and T. fluviatilis share the same gene organization previously described for Nerita mt genomes whereas that of P. jana has undergone major rearrangements. We sequenced about half of the mitochondrial genome of another species of Helicinoidea, Viana regina, and confirmed that this species shares the highly derived gene order of P. jana. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. Inhibitors of Nucleotidyltransferase Superfamily Enzymes Suppress Herpes Simplex Virus Replication

    Science.gov (United States)

    Wang, Hong; Tollefson, Ann E.; Ying, Baoling; Korom, Maria; Cheng, Xiaohong; Cao, Feng; Davis, Katie L.; Wold, William S. M.

    2014-01-01

    Herpesviruses are large double-stranded DNA viruses that cause serious human diseases. Herpesvirus DNA replication depends on multiple processes typically catalyzed by nucleotidyltransferase superfamily (NTS) enzymes. Therefore, we investigated whether inhibitors of NTS enzymes would suppress replication of herpes simplex virus 1 (HSV-1) and HSV-2. Eight of 42 NTS inhibitors suppressed HSV-1 and/or HSV-2 replication by >10-fold at 5 μM, with suppression at 50 μM reaching ∼1 million-fold. Five compounds in two chemical families inhibited HSV replication in Vero and human foreskin fibroblast cells as well as the approved drug acyclovir did. The compounds had 50% effective concentration values as low as 0.22 μM with negligible cytotoxicity in the assays employed. The inhibitors suppressed accumulation of viral genomes and infectious particles and blocked events in the viral replication cycle before and during viral DNA replication. Acyclovir-resistant mutants of HSV-1 and HSV-2 remained highly sensitive to the NTS inhibitors. Five of six NTS inhibitors of the HSVs also blocked replication of another herpesvirus pathogen, human cytomegalovirus. Therefore, NTS enzyme inhibitors are promising candidates for new herpesvirus treatments that may have broad efficacy against members of the herpesvirus family. PMID:25267681

  17. Thioredoxin Reductase and its Inhibitors

    Science.gov (United States)

    Saccoccia, Fulvio; Angelucci, Francesco; Boumis, Giovanna; Carotti, Daniela; Desiato, Gianni; Miele, Adriana E; Bellelli, Andrea

    2014-01-01

    Thioredoxin plays a crucial role in a wide number of physiological processes, which span from reduction of nucleotides to deoxyriboucleotides to the detoxification from xenobiotics, oxidants and radicals. The redox function of Thioredoxin is critically dependent on the enzyme Thioredoxin NADPH Reductase (TrxR). In view of its indirect involvement in the above mentioned physio/pathological processes, inhibition of TrxR is an important clinical goal. As a general rule, the affinities and mechanisms of binding of TrxR inhibitors to the target enzyme are known with scarce precision and conflicting results abound in the literature. A relevant analysis of published results as well as the experimental procedures is therefore needed, also in view of the critical interest of TrxR inhibitors. We review the inhibitors of TrxR and related flavoreductases and the classical treatment of reversible, competitive, non competitive and uncompetitive inhibition with respect to TrxR, and in some cases we are able to reconcile contradictory results generated by oversimplified data analysis. PMID:24875642

  18. Enzymes of the AKR1B and AKR1C subfamilies and uterine diseases

    Directory of Open Access Journals (Sweden)

    Tea eLanisnik Rizner

    2012-03-01

    Full Text Available Endometrial and cervical cancers, uterine myoma, and endometriosis are very common uterine diseases. Worldwide, more than 800,000 women are affected annually by gynecological cancers, as a result of which, more than 360,000 die. During their reproductive age, about 70% of women develop uterine myomas, 10% to 15% suffer from endometriosis, and 35% to 50% from infertility associated with endometriosis. Uterine diseases are associated with aberrant inflammatory responses and concomitant increased production of prostaglandins (PG. They are also related to decreased differentiation, due to low levels of protective progesterone and retinoic acid, and to enhanced proliferation, due to high local concentrations of estrogens. The pathogenesis of these diseases can thus be attributed to disturbed PG, estrogen and retinoid metabolism and actions. Five human members of the aldo-keto reductase 1B (AKR1B and 1C (AKR1C superfamilies, i.e., AKR1B1, AKR1B10, AKR1C1, AKR1C2 and AKR1C3, have roles in these processes and can thus be implicated in uterine diseases. AKR1B1 and AKR1C3 catalyze the formation of PGF2alpha which stimulates cell proliferation. AKR1C3 converts PGD2 to 9alpha,11beta-PGF2, and thus counteracts the formation of 15deoxy-PGJ2, which can activate pro-apoptotic peroxisome-proliferator-activated receptor beta. AKR1B10 catalyzes the reduction of retinal to retinol, and in thus lessens the formation of retinoic acid, with potential pro-differentiating actions. The AKR1C1-AKR1C3 enzymes also act as 17-keto- and 20-ketosteroid reductases to varying extents, and are implicated in increased estradiol and decreased progesterone levels. This review comprises a short introduction to uterine diseases, followed by an overview of the current literature on the AKR1B and AKR1C expression in the uterus and in uterine diseases. The potential implications of the AKR1B and AKR1C enzymes and their pathophysiologies are then discussed, followed by conclusions and

  19. Diacetyl/l-Xylulose Reductase Mediates Chemical Redox Cycling in Lung Epithelial Cells.

    Science.gov (United States)

    Yang, Shaojun; Jan, Yi-Hua; Mishin, Vladimir; Heck, Diane E; Laskin, Debra L; Laskin, Jeffrey D

    2017-07-17

    Reactive carbonyls such as diacetyl (2,3-butanedione) and 2,3-pentanedione in tobacco and many food and consumer products are known to cause severe respiratory diseases. Many of these chemicals are detoxified by carbonyl reductases in the lung, in particular, dicarbonyl/l-xylulose reductase (DCXR), a multifunctional enzyme important in glucose metabolism. DCXR is a member of the short-chain dehydrogenase/reductase (SDR) superfamily. Using recombinant human enzyme, we discovered that DCXR mediates redox cycling of a variety of quinones generating superoxide anion, hydrogen peroxide, and, in the presence of transition metals, hydroxyl radicals. Redox cycling activity preferentially utilized NADH as a cosubstrate and was greatest for 9,10-phenanthrenequinone and 1,2-naphthoquinone, followed by 1,4-naphthoquinone and 2-methyl-1,4-naphthoquinone (menadione). Using 9,10-phenanthrenequinone as the substrate, quinone redox cycling was found to inhibit DCXR reduction of l-xylulose and diacetyl. Competitive inhibition of enzyme activity by the quinone was observed with respect to diacetyl (K i = 190 μM) and l-xylulose (K i = 940 μM). Abundant DCXR activity was identified in A549 lung epithelial cells when diacetyl was used as a substrate. Quinones inhibited reduction of this dicarbonyl, causing an accumulation of diacetyl in the cells and culture medium and a decrease in acetoin, the reduced product of diacetyl. The identification of DCXR as an enzyme activity mediating chemical redox cycling suggests that it may be important in generating cytotoxic reactive oxygen species in the lung. These activities, together with the inhibition of dicarbonyl/l-xylulose metabolism by redox-active chemicals, as well as consequent deficiencies in pentose metabolism, are likely to contribute to lung injury following exposure to dicarbonyls and quinones.

  20. Two-Stage Approach for Protein Superfamily Classification

    Directory of Open Access Journals (Sweden)

    Swati Vipsita

    2013-01-01

    Full Text Available We deal with the problem of protein superfamily classification in which the family membership of newly discovered amino acid sequence is predicted. Correct prediction is a matter of great concern for the researchers and drug analyst which helps them in discovery of new drugs. As this problem falls broadly under the category of pattern classification problem, we have made all efforts to optimize feature extraction in the first stage and classifier design in the second stage with an overall objective to maximize the performance accuracy of the classifier. In the feature extraction phase, Genetic Algorithm- (GA- based wrapper approach is used to select few eigenvectors from the principal component analysis (PCA space which are encoded as binary strings in the chromosome. On the basis of position of 1’s in the chromosome, the eigenvectors are selected to build the transformation matrix which then maps the original high-dimension feature space to lower dimension feature space. Using PCA-NSGA-II (non-dominated sorting GA, the nondominated solutions obtained from the Pareto front solve the trade-off problem by compromising between the number of eigenvectors selected and the accuracy obtained by the classifier. In the second stage, recursive orthogonal least square algorithm (ROLSA is used for training radial basis function network (RBFN to select optimal number of hidden centres as well as update the output layer weighting matrix. This approach can be applied to large data set with much lower requirements of computer memory. Thus, very small architectures having few number of hidden centres are obtained showing higher level of performance accuracy.

  1. Respiratory arsenate reductase as a bidirectional enzyme

    Science.gov (United States)

    Richey, C.; Chovanec, P.; Hoeft, S.E.; Oremland, R.S.; Basu, P.; Stolz, J.F.

    2009-01-01

    The haloalkaliphilic bacterium Alkalilimnicola ehrlichii is capable of anaerobic chemolithoautotrophic growth by coupling the oxidation of arsenite (As(III)) to the reduction of nitrate and carbon dioxide. Analysis of its complete genome indicates that it lacks a conventional arsenite oxidase (Aox), but instead possesses two operons that each encode a putative respiratory arsenate reductase (Arr). Here we show that one homolog is expressed under chemolithoautotrophic conditions and exhibits both arsenite oxidase and arsenate reductase activity. We also demonstrate that Arr from two arsenate respiring bacteria, Alkaliphilus oremlandii and Shewanella sp. strain ANA-3, is also biochemically reversible. Thus Arr can function as a reductase or oxidase. Its physiological role in a specific organism, however, may depend on the electron potentials of the molybdenum center and [Fe–S] clusters, additional subunits, or constitution of the electron transfer chain. This versatility further underscores the ubiquity and antiquity of microbial arsenic metabolism.

  2. Structural updates of alignment of protein domains and consequences on evolutionary models of domain superfamilies.

    Science.gov (United States)

    Mutt, Eshita; Rani, Sudha Sane; Sowdhamini, Ramanathan

    2013-11-15

    Influx of newly determined crystal structures into primary structural databases is increasing at a rapid pace. This leads to updation of primary and their dependent secondary databases which makes large scale analysis of structures even more challenging. Hence, it becomes essential to compare and appreciate replacement of data and inclusion of new data that is critical between two updates. PASS2 is a database that retains structure-based sequence alignments of protein domain superfamilies and relies on SCOP database for its hierarchy and definition of superfamily members. Since, accurate alignments of distantly related proteins are useful evolutionary models for depicting variations within protein superfamilies, this study aims to trace the changes in data in between PASS2 updates. In this study, differences in superfamily compositions, family constituents and length variations between different versions of PASS2 have been tracked. Studying length variations in protein domains, which have been introduced by indels (insertions/deletions), are important because theses indels act as evolutionary signatures in introducing variations in substrate specificity, domain interactions and sometimes even regulating protein stability. With this objective of classifying the nature and source of variations in the superfamilies during transitions (between the different versions of PASS2), increasing length-rigidity of the superfamilies in the recent version is observed. In order to study such length-variant superfamilies in detail, an improved classification approach is also presented, which divides the superfamilies into distinct groups based on their extent of length variation. An objective study in terms of transition between the database updates, detailed investigation of the new/old members and examination of their structural alignments is non-trivial and will help researchers in designing experiments on specific superfamilies, in various modelling studies, in linking

  3. Utility of the Amborella trichopoda expansin superfamily in elucidating the history of angiosperm expansins.

    Science.gov (United States)

    Seader, Victoria H; Thornsberry, Jennifer M; Carey, Robert E

    2016-03-01

    Expansins form a superfamily of plant proteins that assist in cell wall loosening during growth and development. The superfamily is divided into four families: EXPA, EXPB, EXLA, and EXLB (Sampedro and Cosgrove in Genome Biol 6:242, 2005. doi: 10.1186/gb-2005-6-12-242 ). Previous studies on Arabidopsis, rice, and Populus trichocarpa have clarified the evolutionary history of expansins in angiosperms (Sampedro et al. in Plant J 44:409-419, 2005. doi: 10.1111/j.1365-313X.2005.02540.x ). Amborella trichopoda is a flowering plant that diverged very early. Thus, it is a sister lineage to all other extant angiosperms (Amborella Genome Project in 342:1241089, 2013. doi: 10.1126/science.1241089 ). Because of this relationship, comparing the A. trichopoda expansin superfamily with those of other flowering plants may indicate which expansin genes were present in the last common ancestor of all angiosperms. The A. trichopoda expansin superfamily was assembled using BLAST searches with angiosperm expansin queries. The search results were analyzed and annotated to isolate the complete A. trichopoda expansin superfamily. This superfamily is similar to other angiosperm expansin superfamilies, but is somewhat smaller. This is likely because of a lack of genome duplication events (Amborella Genome Project 2013). Phylogenetic and syntenic analyses of A. trichopoda expansins have improved our understanding of the evolutionary history of expansins in angiosperms. Nearly all of the A. trichopoda expansins were placed into an existing Arabidopsis-rice expansin clade. Based on the results of phylogenetic and syntenic analyses, we estimate there were 12-13 EXPA genes, 2 EXPB genes, 1 EXLA gene, and 2 EXLB genes in the last common ancestor of all angiosperms.

  4. Towards a systematic analysis of human short-chain dehydrogenases/reductases (SDR): Ligand identification and structure-activity relationships.

    Science.gov (United States)

    Bhatia, Chitra; Oerum, Stephanie; Bray, James; Kavanagh, Kathryn L; Shafqat, Naeem; Yue, Wyatt; Oppermann, Udo

    2015-06-05

    Short-chain dehydrogenases/reductases (SDRs) constitute a large, functionally diverse branch of enzymes within the class of NAD(P)(H) dependent oxidoreductases. In humans, over 80 genes have been identified with distinct metabolic roles in carbohydrate, amino acid, lipid, retinoid and steroid hormone metabolism, frequently associated with inherited genetic defects. Besides metabolic functions, a subset of atypical SDR proteins appears to play critical roles in adapting to redox status or RNA processing, and thereby controlling metabolic pathways. Here we present an update on the human SDR superfamily and a ligand identification strategy using differential scanning fluorimetry (DSF) with a focused library of oxidoreductase and metabolic ligands to identify substrate classes and inhibitor chemotypes. This method is applicable to investigate structure-activity relationships of oxidoreductases and ultimately to better understand their physiological roles. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  5. Differential nitrate accumulation, nitrate reduction, nitrate reductase ...

    African Journals Online (AJOL)

    use

    2011-12-07

    Dec 7, 2011 ... nitrate salts supply on nitrate accumulation, amino acid biosynthesis, total protein production, nitrate reductase activity and carbohydrate biosynthesis in the roots and leaves of the plants. The results indicate that both sodium and potassium nitrate supplementation had stimulatory effects on all of the.

  6. Methylenetetrahydrofolate reductase A1298C polymorphism and ...

    African Journals Online (AJOL)

    Methylenetetrahydrofolate reductase A1298C polymorphism and breast cancer risk: A meta analysis of 33 studies. ... were searched for case‑control studies relating the association between MTHFR A1298C polymorphism and BC risk and estimated summary odds ratios (ORs) with confidence intervals (CIs) for assessment.

  7. Xylose reductase from the thermophilic fungus Talaromyces ...

    Indian Academy of Sciences (India)

    Given the potential application of xylose reductase enzymes that preferentially utilize the reduced form of nicotinamide adenine dinucleotide (NADH) rather than NADPH in the fermentation of five carbon sugars by genetically engineered microorganisms, the coenzyme selectivity of TeXR was altered by site-directed ...

  8. Methylenetetrahydrofolate reductase (MTHFR) C677T gene ...

    Indian Academy of Sciences (India)

    vitamin B12 and riboflavin that are required in Hcy metabolic pathway. Gene that encodes the methylenete- trahydrofolate reductase (MTHFR) enzyme that .... tors like climate, food habits, lifestyle and genetic makeup are common. Validation of the results of the present study in different ethnic groups with larger sample ...

  9. phenotype correlation of methylene tetrahydrofolate reductase ...

    African Journals Online (AJOL)

    Rabah M. Shawky

    2014-06-21

    Jun 21, 2014 ... ORIGINAL ARTICLE. Study of genotype–phenotype correlation of methylene tetrahydrofolate reductase (MTHFR) gene polymorphisms in a sample of Egyptian autistic children. Rabah M. Shawky a,. *, Farida El-baz b. , Tarek M. Kamal c. , Reham M. Elhossiny b. ,. Mona A. Ahmed b. , Ghada H. El Nady d.

  10. Spectroscopic Signature of a Ubiquitous Metal Binding Site in the Metallo-beta-lactamase Superfamily

    Energy Technology Data Exchange (ETDEWEB)

    V Campos-Bermudez; J Gonzalez; D Tierney; A Vila

    2011-12-31

    The metallo-{beta}-lactamase (M{beta}L) superfamily is a functionally diverse group of metalloproteins sharing a distinctive {alpha}{beta}/{alpha}{beta} fold and a characteristic metal binding motif. A large number of open reading frames identified in genomic sequencing efforts have been annotated as members of this superfamily through sequence comparisons. However, structural and functional studies performed on purified proteins are normally needed to unequivocally include a newly discovered protein in the M{beta}L superfamily. Here we report the spectroscopic characterization of recombinant YcbL, a gene product annotated as a member of the M{beta}L superfamily whose function in vivo remains unknown. By taking advantage of the structural features characterizing the M{beta}L superfamily metal binding motif, we performed spectroscopic studies on Zn(II)- and Co(II)-substituted YcbL to structurally interrogate the metal binding site. The dinuclear center in Co(II)-YcbL was shown to display characteristic electronic absorption features in the visible region, which were also observed in an engineered M{beta}L aimed at mimicking this metal site. Thus, the spectroscopic features reported herein can be employed as a signature to readily identify and characterize the presence of these ubiquitous metal binding sites.

  11. Human dehydrogenase/reductase (SDR family) member 8 (DHRS8): a description and evaluation of its biochemical properties.

    Science.gov (United States)

    Lundová, Tereza; Štambergová, Hana; Zemanová, Lucie; Svobodová, Markéta; Havránková, Jana; Šafr, Miroslav; Wsól, Vladimír

    2016-01-01

    Dehydrogenase/reductase (SDR family) member 8 (DHRS8, SDR16C2) belongs to the short-chain dehydrogenase/reductase (SDR) superfamily, one of the largest enzyme groups. In addition to the well-known members which participate in the metabolism of important eobiotics and xenobiotics, this superfamily contains many poorly characterized proteins. DHRS8 is a member of the Multisubstrate NADP(H)-dependent SDR16C family, which generally contains insufficiently described enzymes. Despite the limited knowledge about DHRS8, preliminary indicators have emerged regarding its significant function in the modulation of steroidal activity, at least in the case of 3α-adiol, lipid metabolism and detoxification. The aim of this study was to describe additional biochemical properties of DHRS8 and to unify knowledge about this enzyme. The DHRS8 was prepared in recombinant form and its membrane topology in the endoplasmic reticulum as an integral protein with cytosolic orientation was demonstrated. The enzyme participates in the NAD(+)-dependent oxidation of steroid hormones as β-estradiol and testosterone in vitro; apparent K m and V max values were 39.86 µM and 0.80 nmol × mg(-1) × min(-1) for β-estradiol and 1207.29 µM and 3.45 nmol × mg(-1) × min(-1) for testosterone. Moreover, synthetic steroids (methyltestosterone and nandrolone) used as anabolics as well as all-trans-retinol were for the first time identified as substrates of DHRS8. This knowledge of its in vitro activity together with a newly described expression pattern at the protein level in tissues involved in steroidogenesis (adrenal gland and testis) and detoxification (liver, lung, kidney and small intestine) could suggest a potential role of DHRS8 in vivo.

  12. Geometric Restraint Drives On- and Off-pathway Catalysis by the Escherichia coli Menaquinol:Fumarate Reductase

    Energy Technology Data Exchange (ETDEWEB)

    Tomasiak, Thomas M.; Archuleta, Tara L.; Andréll, Juni; Luna-Chávez, César; Davis, Tyler A.; Sarwar, Maruf; Ham, Amy J.; McDonald, W. Hayes; Yankovskaya, Victoria; Stern, Harry A.; Johnston, Jeffrey N.; Maklashina, Elena; Cecchini, Gary; Iverson, Tina M. (Rochester-Med); (VA); (Vanderbilt); (MRCLMB); (UCSF)

    2012-01-05

    Complex II superfamily members catalyze the kinetically difficult interconversion of succinate and fumarate. Due to the relative simplicity of complex II substrates and their similarity to other biologically abundant small molecules, substrate specificity presents a challenge in this system. In order to identify determinants for on-pathway catalysis, off-pathway catalysis, and enzyme inhibition, crystal structures of Escherichia coli menaquinol:fumarate reductase (QFR), a complex II superfamily member, were determined bound to the substrate, fumarate, and the inhibitors oxaloacetate, glutarate, and 3-nitropropionate. Optical difference spectroscopy and computational modeling support a model where QFR twists the dicarboxylate, activating it for catalysis. Orientation of the C2-C3 double bond of activated fumarate parallel to the C(4a)-N5 bond of FAD allows orbital overlap between the substrate and the cofactor, priming the substrate for nucleophilic attack. Off-pathway catalysis, such as the conversion of malate to oxaloacetate or the activation of the toxin 3-nitropropionate may occur when inhibitors bind with a similarly activated bond in the same position. Conversely, inhibitors that do not orient an activatable bond in this manner, such as glutarate and citrate, are excluded from catalysis and act as inhibitors of substrate binding. These results support a model where electronic interactions via geometric constraint and orbital steering underlie catalysis by QFR.

  13. A superfamily of archaeal, bacterial, and eukaryotic proteins homologous to animal transglutaminases.

    Science.gov (United States)

    Makarova, K S; Aravind, L; Koonin, E V

    1999-08-01

    Computer analysis using profiles generated by the PSI-BLAST program identified a superfamily of proteins homologous to eukaryotic transglutaminases. The members of the new protein superfamily are found in all archaea, show a sporadic distribution among bacteria, and were detected also in eukaryotes, such as two yeast species and the nematode Caenorhabditis elegans. Sequence conservation in this superfamily primarily involves three motifs that center around conserved cysteine, histidine, and aspartate residues that form the catalytic triad in the structurally characterized transglutaminase, the human blood clotting factor XIIIa'. On the basis of the experimentally demonstrated activity of the Methanobacterium phage pseudomurein endoisopeptidase, it is proposed that many, if not all, microbial homologs of the transglutaminases are proteases and that the eukaryotic transglutaminases have evolved from an ancestral protease.

  14. Bacterial Multidrug Efflux Pumps of the Major Facilitator Superfamily as Targets for Modulation.

    Science.gov (United States)

    Kumar, Sanath; He, Guixin; Kakarla, Prathusha; Shrestha, Ugina; Ranjana, K C; Ranaweera, Indrika; Willmon, T Mark; Barr, Sharla R; Hernandez, Alberto J; Varela, Manuel F

    2016-01-01

    Causative agents of infectious disease that are multidrug resistant bacterial pathogens represent a serious public health concern due to the increasingly difficult nature of achieving efficacious clinical treatments. Of the various acquired and intrinsic antimicrobial agent resistance determinants, integral-membrane multidrug efflux pumps of the major facilitator superfamily constitute a major mechanism of bacterial resistance. The major facilitator superfamily (MFS) encompasses thousands of known related secondary active and passive solute transporters, including multidrug efflux pumps, from bacteria to humans. This review article addresses recent developments involving the targeting by various modulators of bacterial multidrug efflux pumps from the major facilitator superfamily. It is currently of tremendous interest to modulate bacterial multidrug efflux pumps in order to eventually restore the clinical efficacy of therapeutic agents against recalcitrant bacterial infections. Such MFS multidrug efflux pumps are good targets for modulation.

  15. Differential catalytic promiscuity of the alkaline phosphatase superfamily bimetallo core reveals mechanistic features underlying enzyme evolution.

    Science.gov (United States)

    Sunden, Fanny; AlSadhan, Ishraq; Lyubimov, Artem; Doukov, Tzanko; Swan, Jeffrey; Herschlag, Daniel

    2017-12-22

    Members of enzyme superfamilies specialize in different reactions but often exhibit catalytic promiscuity for one another's reactions, consistent with catalytic promiscuity as an important driver in the evolution of new enzymes. Wanting to understand how catalytic promiscuity and other factors may influence evolution across a superfamily, we turned to the well-studied alkaline phosphatase (AP) superfamily, comparing three of its members, two evolutionarily distinct phosphatases and a phosphodiesterase. We mutated distinguishing active-site residues to generate enzymes that had a common Zn 2+ bimetallo core but little sequence similarity and different auxiliary domains. We then tested the catalytic capabilities of these pruned enzymes with a series of substrates. A substantial rate enhancement of ∼10 11 -fold for both phosphate mono- and diester hydrolysis by each enzyme indicated that the Zn 2+ bimetallo core is an effective mono/di-esterase generalist and that the bimetallo cores were not evolutionarily tuned to prefer their cognate reactions. In contrast, our pruned enzymes were ineffective sulfatases, and this limited promiscuity may have provided a driving force for founding the distinct one-metal-ion branch that contains all known AP superfamily sulfatases. Finally, our pruned enzymes exhibited 10 7 -10 8 -fold phosphotriesterase rate enhancements, despite absence of such enzymes within the AP superfamily. We speculate that the superfamily active-site architecture involved in nucleophile positioning prevents accommodation of the additional triester substituent. Overall, we suggest that catalytic promiscuity, and the ease or difficulty of remodeling and building onto existing protein scaffolds, have greatly influenced the course of enzyme evolution. Uncovering principles and properties of enzyme function, promiscuity, and repurposing provides lessons for engineering new enzymes. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Keanekaragaman Jenis Kupu-Kupu Superfamili Papilionoidae di Banyuwindu, Limbangan Kendal

    Directory of Open Access Journals (Sweden)

    Ratna Oqtafiana

    2013-03-01

    Full Text Available Kupu-kupu turut memberi andil dalam mempertahankan keseimbangan ekosistem dan memperkaya keanekaragaman hayati. Tujuan dari penelitian ini adalah untuk mengetahui keanekaragaman jenis kupu-kupu superfamili Papilionoidae di Dukuh Banyuwindu Desa Limbangan Kecamatan Limbangan Kabupaten Kendal khususnya di habitat hutan sekunder, permukiman, Daerah Aliran Sungai (DAS dan persawahan.Populasi dalam penelitian ini adalah semua jenis kupu-kupu superfamili Papilionoidae yang ada di Banyuwindu, Limbangan Kendal. Sampel penelitian ini adalah jenis kupu-kupu superfamili Papilionoidae yang teramati di Banyuwindu Limbangan Kendal khususnya di habitat hutan sekunder, permukiman, DAS dan persawahan. Penelitian dilakukan dengan metode Indeks Point Abudance (IPA atau metode titik hitung.Hasil penelitian ditemukan sebanyak 62 jenis kupu-kupu superfamili Papilionoidae yang terdiri dari 737 individu yang tergolong kedalam empat famili yaitu Papilionidae, Pieridae, Lycaenidae dan Nymphalidae. Hasil analisis indeks keanekaragaman jenis berkisar antara 2,74-3,09, indeks kemerataan jenis berkisar antara 0,86-0,87 dan memiliki dominansi berkisar antara 0,07-0,09. Indeks keanekaragaman jenis dan indeks kemerataan jenis tertinggi tercatat pada habitat permukiman yaitu 3,09 dan 0,87 dan memiliki dominansi 0,07 sedangkan terendah tercatat pada habitat persawahan yaitu 2,74 dan 0,86 dan memiliki dominansi 0,07.Butterfly also contribute in maintaining the ecological balance and enrich biodiversity. The aim of this research was to determine the diversity of butterflies’ superfamily Papilionoidae in Banyuwindu Hamlet Limbangan Sub district Kendal Regency, especially in the secondary forest habitat, settlements, river flow area (RFA and rice field. The population in this research were all kinds of butterflies’ Papilionoidae superfamily in Banyuwindu, Limbangan Kendal. The sample was kind of butterfly superfamily Papilionoidae that observed in Banyuwindu Limbangan Kendal

  17. The nitric-oxide reductase from Paracoccus denitrificans uses a single specific proton pathway.

    Science.gov (United States)

    ter Beek, Josy; Krause, Nils; Reimann, Joachim; Lachmann, Peter; Ädelroth, Pia

    2013-10-18

    The NO reductase from Paracoccus denitrificans reduces NO to N2O (2NO + 2H(+) + 2e(-) → N2O + H2O) with electrons donated by periplasmic cytochrome c (cytochrome c-dependent NO reductase; cNOR). cNORs are members of the heme-copper oxidase superfamily of integral membrane proteins, comprising the O2-reducing, proton-pumping respiratory enzymes. In contrast, although NO reduction is as exergonic as O2 reduction, there are no protons pumped in cNOR, and in addition, protons needed for NO reduction are derived from the periplasmic solution (no contribution to the electrochemical gradient is made). cNOR thus only needs to transport protons from the periplasm into the active site without the requirement to control the timing of opening and closing (gating) of proton pathways as is needed in a proton pump. Based on the crystal structure of a closely related cNOR and molecular dynamics simulations, several proton transfer pathways were suggested, and in principle, these could all be functional. In this work, we show that residues in one of the suggested pathways (denoted pathway 1) are sensitive to site-directed mutation, whereas residues in the other proposed pathways (pathways 2 and 3) could be exchanged without severe effects on turnover activity with either NO or O2. We further show that electron transfer during single-turnover reduction of O2 is limited by proton transfer and can thus be used to study alterations in proton transfer rates. The exchange of residues along pathway 1 showed specific slowing of this proton-coupled electron transfer as well as changes in its pH dependence. Our results indicate that only pathway 1 is used to transfer protons in cNOR.

  18. Mycobacterial F420H2-Dependent Reductases Promiscuously Reduce Diverse Compounds through a Common Mechanism

    Directory of Open Access Journals (Sweden)

    Chris Greening

    2017-05-01

    Full Text Available An unusual aspect of actinobacterial metabolism is the use of the redox cofactor F420. Studies have shown that actinobacterial F420H2-dependent reductases promiscuously hydrogenate diverse organic compounds in biodegradative and biosynthetic processes. These enzymes therefore represent promising candidates for next-generation industrial biocatalysts. In this work, we undertook the first broad survey of these enzymes as potential industrial biocatalysts by exploring the extent, as well as mechanistic and structural bases, of their substrate promiscuity. We expressed and purified 11 enzymes from seven subgroups of the flavin/deazaflavin oxidoreductase (FDOR superfamily (A1, A2, A3, B1, B2, B3, B4 from the model soil actinobacterium Mycobacterium smegmatis. These enzymes reduced compounds from six chemical classes, including fundamental monocycles such as a cyclohexenone, a dihydropyran, and pyrones, as well as more complex quinone, coumarin, and arylmethane compounds. Substrate range and reduction rates varied between the enzymes, with the A1, A3, and B1 groups exhibiting greatest promiscuity. Molecular docking studies suggested that structurally diverse compounds are accommodated in the large substrate-binding pocket of the most promiscuous FDOR through hydrophobic interactions with conserved aromatic residues and the isoalloxazine headgroup of F420H2. Liquid chromatography-mass spectrometry (LC/MS and gas chromatography-mass spectrometry (GC/MS analysis of derivatized reaction products showed reduction occurred through a common mechanism involving hydride transfer from F420H- to the electron-deficient alkene groups of substrates. Reduction occurs when the hydride donor (C5 of F420H- is proximal to the acceptor (electrophilic alkene of the substrate. These findings suggest that engineered actinobacterial F420H2-dependent reductases are promising novel biocatalysts for the facile transformation of a wide range of α,β-unsaturated compounds.

  19. Lapachol inhibition of vitamin K epoxide reductase and vitamin K quinone reductase.

    Science.gov (United States)

    Preusch, P C; Suttie, J W

    1984-11-01

    Lapachol [2-hydroxy-3-(3-methyl-2-butenyl)-1,4-naphthoquinone] has been shown to be a potent inhibitor of both vitamin K epoxide reductase and the dithiothreitol-dependent vitamin K quinone reductase of rat liver microsomes in vitro. These observations explain the anticoagulant activity of lapachol previously observed in both rats and humans. Lapachol inhibition of the vitamin K epoxide and quinone reductases resembled coumarin anticoagulant inhibition, and was observed in normal strain but not in warfarin-resistant strain rat liver microsomes. This similarity of action suggests that the lactone functionality of the coumarins is not critical for their activity. The initial-velocity steady-state inhibition patterns for lapachol inhibition of the solubilized vitamin K epoxide reductase were consistent with tight binding of lapachol to the oxidized form of the enzyme, and somewhat lower affinity for the reduced form. It is proposed that lapachol assumes a 4-enol tautomeric structure similar to that of the 4-hydroxy coumarins. These structures are analogs of the postulated hydroxyvitamin K enolate intermediate bound to the oxidized form of the enzyme in the chemical reaction mechanism of vitamin K epoxide reductase, thus explaining their high affinity.

  20. Selaginella moellendorffii has a reduced and highly conserved expansin superfamily with genes more closely related to angiosperms than to bryophytes.

    Science.gov (United States)

    Carey, Robert E; Hepler, Nathan K; Cosgrove, Daniel J

    2013-01-03

    Expansins are plant cell wall loosening proteins encoded by a large superfamily of genes, consisting of four families named EXPA, EXPB, EXLA, and EXLB. The evolution of the expansin superfamily is well understood in angiosperms, thanks to synteny-based evolutionary studies of the gene superfamily in Arabidopsis, rice, and Populus. Analysis of the expansin superfamily in the moss Physcomitrella patens revealed a superfamily without EXLA or EXLB genes that has evolved considerably and independently of angiosperm expansins. The sequencing of the Selaginella moellendorffii genome has allowed us to extend these analyses into an early diverging vascular plant. The expansin superfamily in Selaginella moellendorffii has now been assembled from genomic scaffolds. A smaller (and less diverse) superfamily is revealed, consistent with studies of other gene families in Selaginella. Selaginella has an expansin superfamily, which, like Physcomitrella, lacks EXLA or EXLB genes, but does contain two EXPA genes that are related to a particular Arabidopsis-rice clade involved in root hair development. From sequence-based phylogenetic analysis, most Selaginella expansins lie outside the Arabidopsis-rice clades, leading us to estimate the minimum number of expansins present in the last common ancestor of Selaginella and angiosperms at 2 EXPA genes and 1 EXPB gene. These results confirm Selaginella as an important intermediary between bryophytes and angiosperms.

  1. Cell Adhesion Molecules of the Immunoglobulin Superfamily in the Nervous System

    DEFF Research Database (Denmark)

    Walmod, Peter Schledermann; Pedersen, Martin Volmer; Berezin, Vladimir

    2007-01-01

    Cell adhesion molecules (CAMs) are proteins mediating cell-cell or cell-extracellular matrix (ECM) interactions. CAMs are traditionally divided into four groups, the cadherins, the selectins, the integrins and CAMs belonging to the immunoglobulin superfamily (IgSF). The present chapter describes...

  2. Spatial and temporal expression of immunoglobulin superfamily member 1 in the rat

    NARCIS (Netherlands)

    Joustra, Sjoerd D.; Meijer, Onno C.; Heinen, Charlotte A.; Mol, Isabel M.; Laghmani, El Houari; Sengers, Rozemarijn M. A.; Carreno, Gabriela; van Trotsenburg, A. S. Paul; Biermasz, Nienke R.; Bernard, Daniel J.; Wit, Jan M.; Oostdijk, Wilma; van Pelt, Ans M. M.; Hamer, Geert; Wagenaar, Gerry T. M.

    2015-01-01

    Loss-of-function mutations in the immunoglobulin superfamily member 1 (IGSF1) gene cause an X-linked syndrome of central hypothyroidism, macroorchidism, variable prolactin and GH deficiency, delayed pubertal testosterone rise, and obesity. To understand the pathophysiology of this syndrome,

  3. Disease causing mutations in the TNF and TNFR superfamilies: Focus on molecular mechanisms driving disease

    NARCIS (Netherlands)

    Lobito, Adrian A.; Gabriel, Tanit L.; Medema, Jan Paul; Kimberley, Fiona C.

    2011-01-01

    The tumor necrosis factor (TNF) and TNF receptor (TNFR) superfamilies comprise multidomain proteins with diverse roles in cell activation, proliferation and cell death. These proteins play pivotal roles in the initiation, maintenance and termination of immune responses and have vital roles outside

  4. Fetal antigen 1 (FA1), a circulating member of the epidermal growth factor (EGF) superfamily

    DEFF Research Database (Denmark)

    Jensen, Charlotte Harken; Krogh, T N; Støving, René Klinkby

    1997-01-01

    We describe an ELISA technique for quantification of fetal antigen 1 (FA1), a glycoprotein belonging to the EGF-superfamily. The ELISA is based on immunospecifically purified polyclonal antibodies and has a dynamic range of 0.7-5.3 ng/ml, intra- and inter-assay C.V.s of less than 3.2% and an aver...

  5. NADH-Ferricyanide Reductase of Leaf Plasma Membranes : Partial Purification and Immunological Relation to Potato Tuber Microsomal NADH-Ferricyanide Reductase and Spinach Leaf NADH-Nitrate Reductase.

    Science.gov (United States)

    Askerlund, P; Laurent, P; Nakagawa, H; Kader, J C

    1991-01-01

    Plasma membranes obtained by two-phase partitioning of microsomal fractions from spinach (Spinacea oleracea L. cv Medania) and sugar beet leaves (Beta vulgaris L.) contained relatively high NADH-ferricyanide reductase and NADH-nitrate reductase (NR; EC 1.6.6.1) activities. Both of these activities were latent. To investigate whether these activities were due to the same enzyme, plasma membrane polypeptides were separated with SDS-PAGE and analyzed with immunoblotting methods. Antibodies raised against microsomal NADH-ferricyanide reductase (tentatively identified as NADH-cytochrome b(5) reductase, EC 1.6.2.2), purified from potato (Solanum tuberosum L. cv Bintje) tuber microsomes, displayed one single band at 43 kilodaltons when reacted with spinach plasma membranes, whereas lgG produced against NR from spinach leaves gave a major band at 110 kilodaltons together with a few fainter bands of lower molecular mass. Immunoblotting analysis using inside-out and right-side-out plasma membrane vesicles strongly indicated that NR was not an integral protein but probably trapped inside the plasma membrane vesicles during homogenization. Proteins from spinach plasma membranes were solubilized with the zwitterionic detergent 3-[(3-cholamidopropyl) dimethylammonio] 1-propane-sulfonate and separated on a Mono Q anion exchange column at pH 5.6 with fast protein liquid chromatography. One major peak of NADH-ferricyanide reductase activity was found after separation. The peak fraction was enriched about 70-fold in this activity compared to the plasma membrane. When the peak fractions were analyzed with SDS-PAGE the NADH-ferricyanide reductase activity strongly correlated with a 43 kilodalton polypeptide which reacted with the antibodies against potato microsomal NADH-ferricyanide reductase. Thus, our data indicate that most, if not all, of the truly membrane-bound NADH-ferricyanide reductase activity of leaf plasma membranes is due to an enzyme very similar to potato tuber

  6. Reduced mRNA expression of PTGDS in peripheral blood mononuclear cells of rapid-cycling bipolar disorder patients compared with healthy control subjects

    DEFF Research Database (Denmark)

    Munkholm, Klaus; Peijs, Lone; Kessing, Lars Vedel

    2015-01-01

    BACKGROUND: Disturbances related to the arachidonic acid cascade and prostaglandin metabolism may be involved in the pathophysiology of bipolar disorder, as supported by a recent genome-wide association study meta-analysis; however, evidence from clinical studies on a transcriptional level...... is lacking. Two enzymes in the arachidonic acid cascade are the prostaglandin D synthase (PTGDS), which catalyzes the conversion of prostaglandin H2 to prostaglandin D2 (PGD2), and the aldo-keto reductase family 1 member C3 (AKR1C3), which catalyzes the reduction of PGD2. We aimed to test the hypothesis...... that mRNA expression of PTGDS and AKR1C3 is deregulated in rapid-cycling disorder patients in a euthymic or current affective state compared with healthy control subjects, and that expression alters with affective states. METHODS: PTGDS and AKR1C3 mRNA expression in peripheral blood mononuclear cells...

  7. A comparative proteomic analysis of Bacillus coagulans in response to lactate stress during the production of L-lactic acid.

    Science.gov (United States)

    Wang, Xiuwen; Qin, Jiayang; Wang, Landong; Xu, Ping

    2014-12-01

    The growth rate and maximum biomass of Bacillus coagulans 2-6 were inhibited by lactate; inhibition by sodium lactate was stronger than by calcium lactate. The differences of protein expressions by B. coagulans 2-6 under the lactate stress were determined using two-dimensional electrophoresis coupled with mass spectrometric identification. Under the non-stress condition, calcium lactate stress and sodium lactate stress, the number of detected protein spots was 1,571 ± 117, 1,281 ± 231 and 904 ± 127, respectively. Four proteins with high expression under lactate stress were identified: lactate dehydrogenase, cysteine synthase A, aldo/keto reductase and ribosomal protein L7/L12. These proteins are thus potential targets for the reconstruction of B. coagulans to promote its resistance to lactate stress.

  8. Fundamental roles of reactive oxygen species and protective mechanisms in the female reproductive system

    Directory of Open Access Journals (Sweden)

    Okada Futoshi

    2005-09-01

    Full Text Available Abstract Controlled oxidation, such as disulfide bond formation in sperm nuclei and during ovulation, plays a fundamental role in mammalian reproduction. Excess oxidation, however, causes oxidative stress, resulting in the dysfunction of the reproductive process. Antioxidation reactions that reduce the levels of reactive oxygen species are of prime importance in reproductive systems in maintaining the quality of gametes and support reproduction. While anti-oxidative enzymes, such as superoxide dismutase and peroxidase, play a central role in eliminating oxidative stress, reduction-oxidation (redox systems, comprised of mainly glutathione and thioredoxin, function to reduce the levels of oxidized molecules. Aldo-keto reductase, using NADPH as an electron donor, detoxifies carbonyl compounds resulting from the oxidation of lipids and proteins. Thus, many antioxidative and redox enzyme genes are expressed and aggressively protect gametes and embryos in reproductive systems.

  9. Human carbonyl reductase 4 is a mitochondrial NADPH-dependent quinone reductase.

    Science.gov (United States)

    Endo, Satoshi; Matsunaga, Toshiyuki; Kitade, Yukio; Ohno, Satoshi; Tajima, Kazuo; El-Kabbani, Ossama; Hara, Akira

    2008-12-26

    A protein encoded in the gene Cbr4 on human chromosome 4q32.3 belongs to the short-chain dehydrogenase/reductase family. Contrary to the functional annotation as carbonyl reductase 4 (CBR4), we show that the recombinant tetrameric protein, composed of 25-kDa subunits, exhibits NADPH-dependent reductase activity for o- and p-quinones, but not for other aldehydes and ketones. The enzyme was insensitive to dicumarol and quercetin, potent inhibitors of cytosolic quinone reductases. The 25-kDa CBR4 was detected in human liver, kidney and cell lines on Western blotting using anti-CBR4 antibodies. The overexpression of CBR4 in bovine endothelial cells reveals that the enzyme has a non-cleavable mitochondrial targeting signal. We further demonstrate that the in vitro quinone reduction by CBR4 generates superoxide through the redox cycling, and suggest that the enzyme may be involved in the induction of apoptosis by cytotoxic 9,10-phenanthrenequinone.

  10. Nitroreductase reactions of Arabidopsis thaliana thioredoxin reductase.

    Science.gov (United States)

    Miskiniene, V; Sarlauskas, J; Jacquot, J P; Cenas, N

    1998-09-07

    Arabidopsis thaliana NADPH:thioredoxin reductase (TR, EC 1.6.4.5) catalyzed redox cycling of aromatic nitrocompounds, including the explosives 2,4,6-trinitrotoluene and tetryl, and the herbicide 3,5-dinitro-o-cresol. The yield of nitro anion radicals was equal to 70-90%. Redox cycling of tetryl was accompanied by formation of N-methylpicramide. Bimolecular rate constants of nitroaromatic reduction (kcat/Km) and reaction catalytic constants (kcat) increased upon an increase in oxidant single-electron reduction potential (E(1)7). Using compounds with an unknown E(1)7 value, the reactivity of TR increased parallelly to the increase in reactivity of ferredoxin:NADP+ reductase of Anabaena PCC 7119 (EC 1.18.1.2). This indicated that the main factor determining reactivity of nitroaromatics towards TR was their energetics of single-electron reduction. Incubation of reduced TR in the presence of tetryl or 2,4-dinitrochlorobenzene resulted in a loss of thioredoxin reductase activity, most probably due to modification of reduced catalytic disulfide, whereas nitroreductase reaction rates were unchanged. This means that on the analogy of quinone reduction by TR (D. Bironaite, Z. Anusevicius, J.-P. Jacquot, N. Cenas, Biochim. Biophys. Acta 1383 (1998) 82-92), FAD and not catalytic disulfide of TR was responsible for the reduction of nitroaromatics. Tetryl, 2,4,6-trinitrotoluene and thioredoxin increased the FAD fluorescence intensity of TR. This finding suggests that nitroaromatics may bind close to the thioredoxin-binding site at the catalytic disulfide domain of TR, and induce a conformational change of enzymes (S.B. Mulrooney, C.H. Williams Jr., Protein Sci. 6 (1997) 2188-2195). Our data indicate that certain nitroaromatic herbicides, explosives and other classes of xenobiotics may interfere with the reduction of thioredoxin by plant TR, and confer prooxidant properties to this antioxidant enzyme.

  11. Methemoglobin reductase activity in intact fish red blood cells

    DEFF Research Database (Denmark)

    Jensen, Frank B; Nielsen, Karsten

    2018-01-01

    Red blood cells (RBCs) possess methemoglobin reductase activity that counters the ongoing oxidation of hemoglobin (Hb) to methemoglobin (metHb), which in circulating blood is caused by Hb autoxidation or reactions with nitrite. We describe an assay for determining metHb reductase activity in intact...

  12. 21 CFR 864.7375 - Glutathione reductase assay.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Glutathione reductase assay. 864.7375 Section 864.7375 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED... reductase deficiency, or riboflavin deficiency. (b) Classification. Class II (performance standards). [45 FR...

  13. A Comprehensive Bioinformatics Analysis of the Nudix Superfamily in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    D. Gunawardana

    2009-01-01

    Full Text Available Nudix enzymes are a superfamily with a conserved common reaction mechanism that provides the capacity for the hydrolysis of a broad spectrum of metabolites. We used hidden Markov models based on Nudix sequences from the PFAM and PROSITE databases to identify Nudix hydrolases encoded by the Arabidopsis genome. 25 Nudix hydrolases were identified and classified into 11 individual families by pairwise sequence alignments. Intron phases were strikingly conserved in each family. Phylogenetic analysis showed that all multimember families formed monophyletic clusters. Conserved familial sequence motifs were identified with the MEME motif analysis algorithm. One motif (motif 4 was found in three diverse families. All proteins containing motif 4 demonstrated a degree of preference for substrates containing an ADP moiety. We conclude that HMM model-based genome scanning and MEME motif analysis, respectively, can significantly improve the identification and assignment of function of new members of this mechanistically-diverse protein superfamily.

  14. Evolutionary Pattern of N-Glycosylation Sequon Numbers in Eukaryotic ABC Protein Superfamilies

    DEFF Research Database (Denmark)

    Rao, R Shyama Prasad; Buus, Ole Thomsen; Wollenweber, Bernd

    2010-01-01

    and their underlying causes have largely been unexplored. We computed the actual and probabilistic occurrence of NXS/T sequons in ABC protein superfamilies from eight diverse eukaryotic organisms. The ABC proteins contained significantly higher NXS/T sequon numbers compared to respective genome-wide average......, but the sequon density was significantly lower owing to the increase in protein size and decrease in sequon specific amino acids. However, mammalian ABC proteins have significantly higher sequon density, and both serine and threonine containing sequons (NXS and NXT) have been positively selected...... higher than expected in plant ABC proteins which have the lowest number of NXS/T sequons. Accord- ingly, compared to overall proteins, N-glycosylation sequons in ABC protein superfamilies have a distinct pattern of occurrence, and the results are discussed in an evolutionary perspective...

  15. Regulation of TGF-β Superfamily Signaling by SMAD Mono-Ubiquitination

    Science.gov (United States)

    Xie, Feng; Zhang, Zhengkui; van Dam, Hans; Zhang, Long; Zhou, Fangfang

    2014-01-01

    TGF-β(transforming growth factor-β) superfamily signaling mediators are important regulators of diverse physiological and pathological events. TGF-β signals are transduced by transmembrane type I and type II serine/threonine kinase receptors and their downstream effectors, the SMAD (drosophila mothers against decapentaplegic protein) proteins. Numerous studies have already demonstrated crucial regulatory roles for modification of TGF-β pathway components by poly-ubiquitination. Recently, several studies also uncovered mono-ubiquitination of SMADs as a mechanism for SMAD activation or inactivation. Mono-ubiquitination and subsequent deubiquitination of SMAD proteins accordingly play important roles in the control of TGF-β superfamily signaling. This review highlights the major pathways regulated by SMAD mono-ubiquitination. PMID:25317929

  16. Superfamily assignments for the yeast proteome through integration of structure prediction with the gene ontology.

    Directory of Open Access Journals (Sweden)

    Lars Malmström

    2007-04-01

    Full Text Available Saccharomyces cerevisiae is one of the best-studied model organisms, yet the three-dimensional structure and molecular function of many yeast proteins remain unknown. Yeast proteins were parsed into 14,934 domains, and those lacking sequence similarity to proteins of known structure were folded using the Rosetta de novo structure prediction method on the World Community Grid. This structural data was integrated with process, component, and function annotations from the Saccharomyces Genome Database to assign yeast protein domains to SCOP superfamilies using a simple Bayesian approach. We have predicted the structure of 3,338 putative domains and assigned SCOP superfamily annotations to 581 of them. We have also assigned structural annotations to 7,094 predicted domains based on fold recognition and homology modeling methods. The domain predictions and structural information are available in an online database at http://rd.plos.org/10.1371_journal.pbio.0050076_01.

  17. A Tale from TGF-β Superfamily for Thymus Ontogeny and Function

    Science.gov (United States)

    Jurberg, Arnon Dias; Vasconcelos-Fontes, Larissa; Cotta-de-Almeida, Vinícius

    2015-01-01

    Multiple signaling pathways control every aspect of cell behavior, organ formation, and tissue homeostasis throughout the lifespan of any individual. This review takes an ontogenetic view focused on the large superfamily of TGF-β/bone morphogenetic protein ligands to address thymus morphogenesis and function in T cell differentiation. Recent findings on a role of GDF11 for reversing aging-related phenotypes are also discussed. PMID:26441956

  18. DUF538 protein superfamily is predicted to be chlorophyll hydrolyzing enzymes in plants

    OpenAIRE

    Gholizadeh, Ashraf

    2015-01-01

    The possible hydrolytic activity towards chlorophyll molecules was predicted for DUF538 protein superfamily in plants. It was examined by using computational as well as experimental tools including in vitro chlorophyll degradation, antioxidant compounds production and in vivo real-time gene expression tests. Comparison of the computational data with the experimental results indicated that DUF538 proteins might be chlorophyll hydrolyzing enzyme (most probably carboxyesterase) which degrade chl...

  19. Recent advances in the study of enzyme promiscuity in the tautomerase superfamily.

    Science.gov (United States)

    Baas, Bert-Jan; Zandvoort, Ellen; Geertsema, Edzard M; Poelarends, Gerrit J

    2013-05-27

    Catalytic promiscuity and evolution: Many enzymes exhibit catalytic promiscuity--the ability to catalyze reactions other than their biologically relevant one. These reactions can serve as starting points for both natural and laboratory evolution of new enzymatic functions. Recent advances in the study of enzyme promiscuity in the tautomerase superfamily are discussed. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Taxonomic distribution and origins of the extended LHC (light-harvesting complex antenna protein superfamily

    Directory of Open Access Journals (Sweden)

    Brinkmann Henner

    2010-07-01

    Full Text Available Abstract Background The extended light-harvesting complex (LHC protein superfamily is a centerpiece of eukaryotic photosynthesis, comprising the LHC family and several families involved in photoprotection, like the LHC-like and the photosystem II subunit S (PSBS. The evolution of this complex superfamily has long remained elusive, partially due to previously missing families. Results In this study we present a meticulous search for LHC-like sequences in public genome and expressed sequence tag databases covering twelve representative photosynthetic eukaryotes from the three primary lineages of plants (Plantae: glaucophytes, red algae and green plants (Viridiplantae. By introducing a coherent classification of the different protein families based on both, hidden Markov model analyses and structural predictions, numerous new LHC-like sequences were identified and several new families were described, including the red lineage chlorophyll a/b-binding-like protein (RedCAP family from red algae and diatoms. The test of alternative topologies of sequences of the highly conserved chlorophyll-binding core structure of LHC and PSBS proteins significantly supports the independent origins of LHC and PSBS families via two unrelated internal gene duplication events. This result was confirmed by the application of cluster likelihood mapping. Conclusions The independent evolution of LHC and PSBS families is supported by strong phylogenetic evidence. In addition, a possible origin of LHC and PSBS families from different homologous members of the stress-enhanced protein subfamily, a diverse and anciently paralogous group of two-helix proteins, seems likely. The new hypothesis for the evolution of the extended LHC protein superfamily proposed here is in agreement with the character evolution analysis that incorporates the distribution of families and subfamilies across taxonomic lineages. Intriguingly, stress-enhanced proteins, which are universally found in the

  1. BOC, an Ig superfamily member, associates with CDO to positively regulate myogenic differentiation

    OpenAIRE

    Kang, Jong-Sun; Mulieri, Philip J.; Hu, Yulan; Taliana, Lavinia; Krauss, Robert S.

    2002-01-01

    CDO is a cell surface receptor-like protein that positively regulates myogenic differentiation. Reported here is the identification of BOC, which, with CDO, defines a newly recognized subfamily within the immunoglobulin superfamily. cdo and boc are co-expressed in muscle precursors in the developing mouse embryo. Like CDO, BOC accelerates differentiation of cultured myoblast cell lines and participates in a positive feedback loop with the myogenic transcription factor, MyoD. CDO and BOC form ...

  2. A Comparative Analysis of Synonymous Codon Usage Bias Pattern in Human Albumin Superfamily

    Directory of Open Access Journals (Sweden)

    Hoda Mirsafian

    2014-01-01

    Full Text Available Synonymous codon usage bias is an inevitable phenomenon in organismic taxa across the three domains of life. Though the frequency of codon usage is not equal across species and within genome in the same species, the phenomenon is non random and is tissue-specific. Several factors such as GC content, nucleotide distribution, protein hydropathy, protein secondary structure, and translational selection are reported to contribute to codon usage preference. The synonymous codon usage patterns can be helpful in revealing the expression pattern of genes as well as the evolutionary relationship between the sequences. In this study, synonymous codon usage bias patterns were determined for the evolutionarily close proteins of albumin superfamily, namely, albumin, α-fetoprotein, afamin, and vitamin D-binding protein. Our study demonstrated that the genes of the four albumin superfamily members have low GC content and high values of effective number of codons (ENC suggesting high expressivity of these genes and less bias in codon usage preferences. This study also provided evidence that the albumin superfamily members are not subjected to mutational selection pressure.

  3. Lipid- and polyion complex-based micelles as agonist platforms for TNFR superfamily receptors.

    Science.gov (United States)

    Gilbreth, Ryan N; Novarra, Shabazz; Wetzel, Leslie; Florinas, Stelios; Cabral, Horacio; Kataoka, Kazunori; Rios-Doria, Jonathan; Christie, Ronald J; Baca, Manuel

    2016-07-28

    Receptor clustering is important for signaling among the therapeutically relevant TNFR superfamily of receptors. In nature, this clustering is driven by trimeric ligands often presented in large numbers as cell surface proteins. Molecules capable of driving similar levels of clustering could make good agonists and hold therapeutic value. However, recapitulating such extensive clustering using typical biotherapeutic formats, such as antibodies, is difficult. Consequently, generating effective agonists of TNFR superfamily receptors is challenging. Toward addressing this challenge we have used lipid- and polyion complex-based micelles as platforms for presenting receptor-binding biologics in a multivalent format that facilitates receptor clustering and imparts strong agonist activity. We show that receptor-binding scFvs or small antibody mimetics that have no agonist activity on their own can be transformed into potent agonists through multivalent presentation on a micelle surface and that the activity of already active multivalent agonists can be enhanced. Using this strategy, we generated potent agonists against two different TNFR superfamily receptors and mouse tumor model studies demonstrate that these micellar agonists have therapeutic efficacy in vivo. Due to its ease of implementation and applicability independent of agonist molecular format, we anticipate that this strategy could be useful for developing agonists to a variety of receptors that rely on clustering to signal. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Diversity of conotoxin gene superfamilies in the venomous snail, Conus victoriae.

    Directory of Open Access Journals (Sweden)

    Samuel D Robinson

    Full Text Available Animal venoms represent a vast library of bioactive peptides and proteins with proven potential, not only as research tools but also as drug leads and therapeutics. This is illustrated clearly by marine cone snails (genus Conus, whose venoms consist of mixtures of hundreds of peptides (conotoxins with a diverse array of molecular targets, including voltage- and ligand-gated ion channels, G-protein coupled receptors and neurotransmitter transporters. Several conotoxins have found applications as research tools, with some being used or developed as therapeutics. The primary objective of this study was the large-scale discovery of conotoxin sequences from the venom gland of an Australian cone snail species, Conus victoriae. Using cDNA library normalization, high-throughput 454 sequencing, de novo transcriptome assembly and annotation with BLASTX and profile hidden Markov models, we discovered over 100 unique conotoxin sequences from 20 gene superfamilies, the highest diversity of conotoxins so far reported in a single study. Many of the sequences identified are new members of known conotoxin superfamilies, some help to redefine these superfamilies and others represent altogether new classes of conotoxins. In addition, we have demonstrated an efficient combination of methods to mine an animal venom gland and generate a library of sequences encoding bioactive peptides.

  5. Conotoxin superfamily prediction using diffusion maps dimensionality reduction and subspace classifier.

    Science.gov (United States)

    Yin, Jiang-Bo; Fan, Yong-Xian; Shen, Hong-Bin

    2011-09-01

    Conotoxins are disulfide-rich small peptides that are invaluable channel-targeted peptides and target neuronal receptors, which have been demonstrated to be potent pharmaceuticals in the treatment of Alzheimer's disease, Parkinson's disease, and epilepsy. Accurate prediction of conotoxin superfamily would have many important applications towards the understanding of its biological and pharmacological functions. In this study, a novel method, named dHKNN, is developed to predict conotoxin superfamily. Firstly, we extract the protein's sequential features composed of physicochemical properties, evolutionary information, predicted secondary structures and amino acid composition. Secondly, we use the diffusion maps for dimensionality reduction, which interpret the eigenfunctions of Markov matrices as a system of coordinates on the original data set in order to obtain efficient representation of data geometric descriptions. Finally, an improved K-local hyperplane distance nearest neighbor subspace classifier method called dHKNN is proposed for predicting conotoxin superfamilies by considering the local density information in the diffusion space. The overall accuracy of 91.90% is obtained through the jackknife cross-validation test on a benchmark dataset, indicating the proposed dHKNN is promising.

  6. TNF and TNF Receptor Superfamily Members in HIV infection: New Cellular Targets for Therapy?

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    Amit Kumar

    2013-01-01

    Full Text Available Tumor necrosis factor (TNF and TNF receptors (TNFR superfamily members are engaged in diverse cellular phenomena such as cellular proliferation, morphogenesis, apoptosis, inflammation, and immune regulation. Their role in regulating viral infections has been well documented. Viruses have evolved with numerous strategies to interfere with TNF-mediated signaling indicating the importance of TNF and TNFR superfamily in viral pathogenesis. Recent research reports suggest that TNF and TNFRs play an important role in the pathogenesis of HIV. TNFR signaling modulates HIV replication and HIV proteins interfere with TNF/TNFR pathways. Since immune activation and inflammation are the hallmark of HIV infection, the use of TNF inhibitors can have significant impact on HIV disease progression. In this review, we will describe how HIV infection is modulated by signaling mediated through members of TNF and TNFR superfamily and in turn how these latter could be targeted by HIV proteins. Finally, we will discuss the emerging therapeutics options based on modulation of TNF activity that could ultimately lead to the cure of HIV-infected patients.

  7. Origination, expansion, evolutionary trajectory, and expression bias of AP2/ERF superfamily in Brassica napus

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    Xiaoming Song

    2016-08-01

    Full Text Available The AP2/ERF superfamily, one of the most important transcription factor families, plays crucial roles in response to biotic and abiotic stresses. So far, a comprehensive evolutionary inference of its origination and expansion has not been available. Here, we identified 515 AP2/ERF genes in B. napus, a neo-tetraploid forming ~7500 years ago, and found that 82.14% of them were duplicated in the tetraploidization. A prominent subgenome bias was revealed in gene expression, tissue-specific, and gene conversion. Moreover, a large-scale analysis across plants and alga suggested that this superfamily could have been originated from AP2 family, expanding to form other families (ERF, and RAV. This process was accompanied by duplicating and/or alternative deleting AP2 domain, intragenic domain sequence conversion, and/or by acquiring other domains, resulting in copy number variations, alternatively contributing to functional innovation. We found that significant positive selection occurred at certain critical nodes during the evolution of land plants, possibly responding to changing environment. In conclusion, the present research revealed origination, functional innovation, and evolutionary trajectory of the AP2/ERF superfamily, contributing to understanding their roles in plant stress tolerance.

  8. Identification of a 4-Deoxy-l-erythro-5-hexoseulose Uronic Acid Reductase, FlRed, in an Alginolytic Bacterium Flavobacterium sp. Strain UMI-01

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    Akira Inoue

    2015-01-01

    Full Text Available In alginate-assimilating bacteria, alginate is depolymerized to unsaturated monosaccharide by the actions of endolytic and exolytic alginate lyases (EC 4.2.2.3 and EC 4.2.2.11. The monosaccharide is non-enzymatically converted to 4-deoxy-l-ery thro-5-hexoseulose uronic acid (DEH, then reduced to 2-keto-3-deoxy-d-gluconate (KDG by a specific reductase, and metabolized through the Entner–Doudoroff pathway. Recently, the NADPH-dependent reductase A1-R that belongs to short-chain dehydrogenases/reductases (SDR superfamily was identified as the DEH-reductase in Sphingomonas sp. A1. We have subsequently noticed that an SDR-like enzyme gene, flred, occurred in the genome of an alginolytic bacterium Flavobacterium sp. strain UMI-01. In the present study, we report on the deduced amino-acid sequence of flred and DEH-reducing activity of recombinant FlRed. The deduced amino-acid sequence of flred comprised 254 residues and showed 34% amino-acid identities to that of A1-R from Sphingomonas sp. A1 and 80%–88% to those of SDR-like enzymes from several alginolytic bacteria. Common sequence motifs of SDR-superfamily enzymes, e.g., the catalytic tetrad Asn-Lys-Tyr-Ser and the cofactor-binding sequence Thr-Gly-x-x-x-Gly-x-Gly in Rossmann fold, were completely conserved in FlRed. On the other hand, an Arg residue that determined the NADPH-specificity of Sphingomonas A1-R was replaced by Glu in FlRed. Thus, we investigated cofactor-preference of FlRed using a recombinant enzyme. As a result, the recombinant FlRed (recFlRed was found to show high specificity to NADH. recFlRed exhibited practically no activity toward variety of aldehyde, ketone, keto ester, keto acid and aldose substrates except for DEH. On the basis of these results, we conclude that FlRed is the NADH-dependent DEH-specific SDR of Flavobacterium sp. strain UMI-01.

  9. Functional mechanism of C-terminal tail in the enzymatic role of porcine testicular carbonyl reductase: a combined experiment and molecular dynamics simulation study of the C-terminal tail in the enzymatic role of PTCR.

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    Minky Son

    Full Text Available Porcine testicular carbonyl reductase, PTCR which is one of the short chain dehydrogenases/reductases (SDR superfamily catalyzes the NADPH-dependent reduction of carbonyl compounds including steroids and prostaglandins. Previously we reported C-terminal tail of PTCR was deleted due to a nonsynonymous single nucleotide variation (nsSNV. Here we identified from kinetic studies that the enzymatic properties for 5α-dihydrotestosterone (5α-DHT were different between wild-type and C-terminal-deleted PTCRs. Compared to wild-type PTCR, C-terminal-deleted PTCR has much higher reduction rate. To investigate structural difference between wild-type and C-terminal-deleted PTCRs upon 5α-DHT binding, we performed molecular dynamics simulations for two complexes. Using trajectories, molecular interactions including hydrogen bonding patterns, distance between 5α-DHT and catalytic Tyr193, and interaction energies are analyzed and compared. During the MD simulation time, the dynamic behavior of C-terminal tail in wild-type PTCR is also examined using essential dynamics analysis. The results of our simulations reveal that the binding conformation of 5α-DHT in C-terminal-deleted PTCR is more favorable for reduction reaction in PTCR, which shows strong agreement with kinetic data. These structural findings provide valuable information to understand substrate specificity of PTCR and further kinetic properties of enzymes belonging to the SDR superfamily.

  10. Structure of a short-chain dehydrogenase/reductase (SDR) within a genomic island from a clinical strain of Acinetobacter baumannii.

    Science.gov (United States)

    Shah, Bhumika S; Tetu, Sasha G; Harrop, Stephen J; Paulsen, Ian T; Mabbutt, Bridget C

    2014-10-01

    Over 15% of the genome of an Australian clinical isolate of Acinetobacter baumannii occurs within genomic islands. An uncharacterized protein encoded within one island feature common to this and other International Clone II strains has been studied by X-ray crystallography. The 2.4 Å resolution structure of SDR-WM99c reveals it to be a new member of the classical short-chain dehydrogenase/reductase (SDR) superfamily. The enzyme contains a nucleotide-binding domain and, like many other SDRs, is tetrameric in form. The active site contains a catalytic tetrad (Asn117, Ser146, Tyr159 and Lys163) and water molecules occupying the presumed NADP cofactor-binding pocket. An adjacent cleft is capped by a relatively mobile helical subdomain, which is well positioned to control substrate access.

  11. Variant Cell Lines of Haplopappus gracilis with Disturbed Activities of Nitrate Reductase and Nitrite Reductase.

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    Gilissen, L J; Barneix, A J; van Staveren, M; Breteler, H

    1985-07-01

    Selected variant cell lines of Haplopappus gracilis (Nutt) Gray that showed disturbed growth after transfer from an alanine medium to NO(3) (-) medium were characterized. The in vivo NO(3) (-) reductase activity (NRA) was lower in these lines than in the wild type. In vitro NRA assays suggest that decreased in vivo NRA was not caused by a lower amount of active enzyme. Cells of the variant lines revealed up to 75% lower extractable activity of NO(2) (-) reductase as compared with the wild type. This coincided with higher accumulation of NO(2) (-) by the variant than by the wild type cells after transfer from alanine medium to NO(3) (-) medium. NO(2) (-) accumulation was transient or continuous, depending on cell line, metabolic state of the cells, and light conditions.

  12. Human carbonyl reductase 1 participating in intestinal first-pass drug metabolism is inhibited by fatty acids and acyl-CoAs.

    Science.gov (United States)

    Hara, Akira; Endo, Satoshi; Matsunaga, Toshiyuki; El-Kabbani, Ossama; Miura, Takeshi; Nishinaka, Toru; Terada, Tomoyuki

    2017-08-15

    Human carbonyl reductase 1 (CBR1), a member of the short-chain dehydrogenase/reductase (SDR) superfamily, reduces a variety of carbonyl compounds including endogenous isatin, prostaglandin E 2 and 4-oxo-2-nonenal. It is also a major non-cytochrome P450 enzyme in the phase I metabolism of carbonyl-containing drugs, and is highly expressed in the intestine. In this study, we found that long-chain fatty acids and their CoA ester derivatives inhibit CBR1. Among saturated fatty acids, myristic, palmitic and stearic acids were inhibitory, and stearic acid was the most potent (IC 50 9µM). Unsaturated fatty acids (oleic, elaidic, γ-linolenic and docosahexaenoic acids) and acyl-CoAs (palmitoyl-, stearoyl- and oleoyl-CoAs) were more potent inhibitors (IC 50 1.0-2.5µM), and showed high inhibitory selectivity to CBR1 over its isozyme CBR3 and other SDR superfamily enzymes (DCXR and DHRS4) with CBR activity. The inhibition by these fatty acids and acyl-CoAs was competitive with respect to the substrate, showing the K i values of 0.49-1.2µM. Site-directed mutagenesis of the substrate-binding residues of CBR1 suggested that the interactions between the fatty acyl chain and the enzyme's Met141 and Trp229 are important for the inhibitory selectivity. We also examined CBR1 inhibition by oleic acid in cellular levels: The fatty acid effectively inhibited CBR1-mediated 4-oxo-2-nonenal metabolism in colon cancer DLD1 cells and increased sensitivity to doxorubicin in the drug-resistant gastric cancer MKN45 cells that highly express CBR1. The results suggest a possible new food-drug interaction through inhibition of CBR1-mediated intestinal first-pass drug metabolism by dietary fatty acids. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Structure of Human B12 Trafficking Protein CblD Reveals Molecular Mimicry and Identifies a New Subfamily of Nitro-FMN Reductases.

    Science.gov (United States)

    Yamada, Kazuhiro; Gherasim, Carmen; Banerjee, Ruma; Koutmos, Markos

    2015-12-04

    In mammals, B12 (or cobalamin) is an essential cofactor required by methionine synthase and methylmalonyl-CoA mutase. A complex intracellular pathway supports the assimilation of cobalamin into its active cofactor forms and delivery to its target enzymes. MMADHC (the methylmalonic aciduria and homocystinuria type D protein), commonly referred to as CblD, is a key chaperone involved in intracellular cobalamin trafficking, and mutations in CblD cause methylmalonic aciduria and/or homocystinuria. Herein, we report the first crystal structure of the globular C-terminal domain of human CblD, which is sufficient for its interaction with MMADHC (the methylmalonic aciduria and homocystinuria type C protein), or CblC, and for supporting the cytoplasmic cobalamin trafficking pathway. CblD contains an α+β fold that is structurally reminiscent of the nitro-FMN reductase superfamily. Two of the closest structural relatives of CblD are CblC, a multifunctional enzyme important for cobalamin trafficking, and the activation domain of methionine synthase. CblD, CblC, and the activation domain of methionine synthase share several distinguishing features and, together with two recently described corrinoid-dependent reductive dehalogenases, constitute a new subclass within the nitro-FMN reductase superfamily. We demonstrate that CblD enhances oxidation of cob(II)alamin bound to CblC and that disease-causing mutations in CblD impair the kinetics of this reaction. The striking structural similarity of CblD to CblC, believed to be contiguous in the cobalamin trafficking pathway, suggests the co-option of molecular mimicry as a strategy for achieving its function. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Structures of mammalian cytosolic quinone reductases.

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    Foster, C E; Bianchet, M A; Talalay, P; Faig, M; Amzel, L M

    2000-08-01

    The metabolism of quinone compounds presents one source of oxidative stress in mammals, as many pathways proceed by mechanisms that generate reactive oxygen species as by-products. One defense against quinone toxicity is the enzyme NAD(P)H:quinone oxidoreductase type 1 (QR1), which metabolizes quinones by a two-electron reduction mechanism, thus averting production of radicals. QR1 is expressed in the cytoplasm of many tissues, and is highly inducible. A closely related homologue, quinone reductase type 2 (QR2), has been identified in several mammalian species. QR2 is also capable of reducing quinones to hydroquinones, but unlike QR1, cannot use NAD(P)H. X-ray crystallographic studies of QR1 and QR2 illustrate that despite their different biochemical properties, these enzymes have very similar three-dimensional structures. In particular, conserved features of the active sites point to the close relationship between these two enzymes.

  15. Hydroxylated naphthoquinones as substrates for Escherichia coli anaerobic reductases.

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    Rothery, R A; Chatterjee, I; Kiema, G; McDermott, M T; Weiner, J H

    1998-01-01

    We have used two hydroxylated naphthoquinol menaquinol analogues, reduced plumbagin (PBH2, 5-hydroxy-2-methyl-1,4-naphthoquinol) and reduced lapachol [LPCH2, 2-hydroxy-3-(3-methyl-2-butenyl)-1, 4-naphthoquinol], as substrates for Escherichia coli anaerobic reductases. These compounds have optical, solubility and redox properties that make them suitable for use in studies of the enzymology of menaquinol oxidation. Oxidized plumbagin and oxidized lapachol have well resolved absorbances at 419 nm (epsilon=3.95 mM-1. cm-1) and 481 nm (epsilon=2.66 mM-1.cm-1) respectively (in Mops/KOH buffer, pH 7.0). PBH2 is a good substrate for nitrate reductase A (Km=282+/-28 microM, kcat=120+/-6 s-1) and fumarate reductase (Km=155+/-24 microM, kcat=30+/-2 s-1), but not for DMSO reductase. LPCH2 is a good substrate for nitrate reductase A (Km=57+/-35 microM, kcat=68+/-13 s-1), fumarate reductase (Km=85+/-27 microM, kcat=74+/-6 s-1) and DMSO reductase (Km=238+/-30 microM, kcat=191+/-21 s-1). The sensitivity of enzymic LPCH2 and PBH2 oxidation to 2-n-heptyl-4-hydroxyquinoline N-oxide inhibition is consistent with their oxidation occurring at sites of physiological quinol binding. PMID:9576848

  16. The cytochrome bd respiratory oxygen reductases.

    Science.gov (United States)

    Borisov, Vitaliy B; Gennis, Robert B; Hemp, James; Verkhovsky, Michael I

    2011-11-01

    Cytochrome bd is a respiratory quinol: O₂ oxidoreductase found in many prokaryotes, including a number of pathogens. The main bioenergetic function of the enzyme is the production of a proton motive force by the vectorial charge transfer of protons. The sequences of cytochromes bd are not homologous to those of the other respiratory oxygen reductases, i.e., the heme-copper oxygen reductases or alternative oxidases (AOX). Generally, cytochromes bd are noteworthy for their high affinity for O₂ and resistance to inhibition by cyanide. In E. coli, for example, cytochrome bd (specifically, cytochrome bd-I) is expressed under O₂-limited conditions. Among the members of the bd-family are the so-called cyanide-insensitive quinol oxidases (CIO) which often have a low content of the eponymous heme d but, instead, have heme b in place of heme d in at least a majority of the enzyme population. However, at this point, no sequence motif has been identified to distinguish cytochrome bd (with a stoichiometric complement of heme d) from an enzyme designated as CIO. Members of the bd-family can be subdivided into those which contain either a long or a short hydrophilic connection between transmembrane helices 6 and 7 in subunit I, designated as the Q-loop. However, it is not clear whether there is a functional consequence of this difference. This review summarizes current knowledge on the physiological functions, genetics, structural and catalytic properties of cytochromes bd. Included in this review are descriptions of the intermediates of the catalytic cycle, the proposed site for the reduction of O₂, evidence for a proton channel connecting this active site to the bacterial cytoplasm, and the molecular mechanism by which a membrane potential is generated. 2011 Elsevier B.V. All rights reserved.

  17. On The Regulation of Spinach Nitrate Reductase 1

    Science.gov (United States)

    Sanchez, Juan; Heldt, Hans W.

    1990-01-01

    A coupled assay has been worked out to study spinach (Spinacea oleracea L.) nitrate reductase under low, more physiological concentrations of NADH. In this assay the reduction of nitrate is coupled to the oxidation of malate catalyzed by spinach NAD-malate dehydrogenase. The use of this coupled system allows the assay of nitrate reductase activity at steady-state concentrations of NADH below micromolar. We have used this coupled assay to study the kinetic parameters of spinach nitrate reductase and to reinvestigate the putative regulatory role of adenine nucleotides, inorganic phosphate, amino acids, and calcium and calmodulin. PMID:16667335

  18. Nitrite-Reductase and Peroxynitrite Isomerization Activities of Methanosarcina acetivorans Protoglobin

    Science.gov (United States)

    Ascenzi, Paolo; Leboffe, Loris; Pesce, Alessandra; Ciaccio, Chiara; Sbardella, Diego; Bolognesi, Martino; Coletta, Massimo

    2014-01-01

    Within the globin superfamily, protoglobins (Pgb) belong phylogenetically to the same cluster of two-domain globin-coupled sensors and single-domain sensor globins. Multiple functional roles have been postulated for Methanosarcina acetivorans Pgb (Ma-Pgb), since the detoxification of reactive nitrogen and oxygen species might co-exist with enzymatic activity(ies) to facilitate the conversion of CO to methane. Here, the nitrite-reductase and peroxynitrite isomerization activities of the CysE20Ser mutant of Ma-Pgb (Ma-Pgb*) are reported and analyzed in parallel with those of related heme-proteins. Kinetics of nitrite-reductase activity of ferrous Ma-Pgb* (Ma-Pgb*-Fe(II)) is biphasic and values of the second-order rate constant for the reduction of NO2– to NO and the concomitant formation of nitrosylated Ma-Pgb*-Fe(II) (Ma-Pgb*-Fe(II)-NO) are kapp1 = 9.6±0.2 M–1 s–1 and kapp2 = 1.2±0.1 M–1 s–1 (at pH 7.4 and 20°C). The kapp1 and kapp2 values increase by about one order of magnitude for each pH unit decrease, between pH 8.3 and 6.2, indicating that the reaction requires one proton. On the other hand, kinetics of peroxynitrite isomerization catalyzed by ferric Ma-Pgb* (Ma-Pgb*-Fe(III)) is monophasic and values of the second order rate constant for peroxynitrite isomerization by Ma-Pgb*-Fe(III) and of the first order rate constant for the spontaneous conversion of peroxynitrite to nitrate are happ = 3.8×104 M–1 s–1 and h0 = 2.8×10–1 s–1 (at pH 7.4 and 20°C). The pH-dependence of hon and h0 values reflects the acid-base equilibrium of peroxynitrite (pKa = 6.7 and 6.9, respectively; at 20°C), indicating that HOONO is the species that reacts preferentially with the heme-Fe(III) atom. These results highlight the potential role of Pgbs in the biosynthesis and scavenging of reactive nitrogen and oxygen species. PMID:24827820

  19. Nitrite-reductase and peroxynitrite isomerization activities of Methanosarcina acetivorans protoglobin.

    Directory of Open Access Journals (Sweden)

    Paolo Ascenzi

    Full Text Available Within the globin superfamily, protoglobins (Pgb belong phylogenetically to the same cluster of two-domain globin-coupled sensors and single-domain sensor globins. Multiple functional roles have been postulated for Methanosarcina acetivorans Pgb (Ma-Pgb, since the detoxification of reactive nitrogen and oxygen species might co-exist with enzymatic activity(ies to facilitate the conversion of CO to methane. Here, the nitrite-reductase and peroxynitrite isomerization activities of the CysE20Ser mutant of Ma-Pgb (Ma-Pgb* are reported and analyzed in parallel with those of related heme-proteins. Kinetics of nitrite-reductase activity of ferrous Ma-Pgb* (Ma-Pgb*-Fe(II is biphasic and values of the second-order rate constant for the reduction of NO2- to NO and the concomitant formation of nitrosylated Ma-Pgb*-Fe(II (Ma-Pgb*-Fe(II-NO are k(app1= 9.6 ± 0.2 M(-1 s(-1 and k(app2 = 1.2 ± 0.1 M(-1 s(-1 (at pH 7.4 and 20 °C. The k(app1 and k(app2 values increase by about one order of magnitude for each pH unit decrease, between pH 8.3 and 6.2, indicating that the reaction requires one proton. On the other hand, kinetics of peroxynitrite isomerization catalyzed by ferric Ma-Pgb* (Ma-Pgb*-Fe(III is monophasic and values of the second order rate constant for peroxynitrite isomerization by Ma-Pgb*-Fe(III and of the first order rate constant for the spontaneous conversion of peroxynitrite to nitrate are h(app = 3.8 × 10(4 M(-1 s(-1 and h0 = 2.8 × 10(-1 s(-1 (at pH 7.4 and 20 °C. The pH-dependence of hon and h0 values reflects the acid-base equilibrium of peroxynitrite (pKa = 6.7 and 6.9, respectively; at 20 °C, indicating that HOONO is the species that reacts preferentially with the heme-Fe(III atom. These results highlight the potential role of Pgbs in the biosynthesis and scavenging of reactive nitrogen and oxygen species.

  20. Investigating the Proton Donor in the NO Reductase from Paracoccus denitrificans.

    Science.gov (United States)

    ter Beek, Josy; Krause, Nils; Ädelroth, Pia

    2016-01-01

    Variant nomenclature: the variants were made in the NorB subunit if not indicated by the superscript c, which are variants in the NorC subunit (e.g. E122A = exchange of Glu-122 in NorB for an Ala, E71cD; exchange of Glu-71 in NorC for an Asp). Bacterial NO reductases (NORs) are integral membrane proteins from the heme-copper oxidase superfamily. Most heme-copper oxidases are proton-pumping enzymes that reduce O2 as the last step in the respiratory chain. With electrons from cytochrome c, NO reductase (cNOR) from Paracoccus (P.) denitrificans reduces NO to N2O via the following reaction: 2NO+2e-+2H+→N2O+H2O. Although this reaction is as exergonic as O2-reduction, cNOR does not contribute to the electrochemical gradient over the membrane. This means that cNOR does not pump protons and that the protons needed for the reaction are taken from the periplasmic side of the membrane (since the electrons are donated from this side). We previously showed that the P. denitrificans cNOR uses a single defined proton pathway with residues Glu-58 and Lys-54 from the NorC subunit at the entrance. Here we further strengthened the evidence in support of this pathway. Our further aim was to define the continuation of the pathway and the immediate proton donor for the active site. To this end, we investigated the region around the calcium-binding site and both propionates of heme b3 by site directed mutagenesis. Changing single amino acids in these areas often had severe effects on cNOR function, with many variants having a perturbed active site, making detailed analysis of proton transfer properties difficult. Our data does however indicate that the calcium ligation sphere and the region around the heme b3 propionates are important for proton transfer and presumably contain the proton donor. The possible evolutionary link between the area for the immediate donor in cNOR and the proton loading site (PLS) for pumped protons in oxygen-reducing heme-copper oxidases is discussed.

  1. Historical perspectives on tumor necrosis factor and its superfamily: 25 years later, a golden journey.

    Science.gov (United States)

    Aggarwal, Bharat B; Gupta, Subash C; Kim, Ji Hye

    2012-01-19

    Although activity that induced tumor regression was observed and termed tumor necrosis factor (TNF) as early as the 1960s, the true identity of TNF was not clear until 1984, when Aggarwal and coworkers reported, for the first time, the isolation of 2 cytotoxic factors: one, derived from macrophages (molecular mass 17 kDa), was named TNF, and the second, derived from lymphocytes (20 kDa), was named lymphotoxin. Because the 2 cytotoxic factors exhibited 50% amino acid sequence homology and bound to the same receptor, they came to be called TNF-α and TNF-β. Identification of the protein sequences led to cloning of their cDNA. Based on sequence homology to TNF-α, now a total of 19 members of the TNF superfamily have been identified, along with 29 interacting receptors, and several molecules that interact with the cytoplasmic domain of these receptors. The roles of the TNF superfamily in inflammation, apoptosis, proliferation, invasion, angiogenesis, metastasis, and morphogenesis have been documented. Their roles in immunologic, cardiovascular, neurologic, pulmonary, and metabolic diseases are becoming apparent. TNF superfamily members are active targets for drug development, as indicated by the recent approval and expanding market of TNF blockers used to treat rheumatoid arthritis, psoriasis, Crohns disease, and osteoporosis, with a total market of more than US $20 billion. As we learn more about this family, more therapeutics will probably emerge. In this review, we summarize the initial discovery of TNF-α, and the insights gained regarding the roles of this molecule and its related family members in normal physiology and disease.

  2. Lachesin: an immunoglobulin superfamily protein whose expression correlates with neurogenesis in grasshopper embryos.

    Science.gov (United States)

    Karlstrom, R O; Wilder, L P; Bastiani, M J

    1993-06-01

    We describe the developmental expression in grasshopper (Schistocerca americana) and molecular characterization in grasshopper and fruit fly (Drosophila melanogaster) of Lachesin, a novel immunoglobulin superfamily protein. Lachesin is expressed on the surfaces of differentiating neuronal cells from the onset of neurogenesis in both the central and peripheral nervous systems. Lachesin expression begins in some cells of the neurogenic ectoderm immediately after engrailed expression begins in the posterior cells of each future segment. All neurogenic cells express Lachesin early, but only those cells that become neuroblasts continue to express Lachesin. Ectodermal cells in the neurogenic region that adopt non-neuronal fates lose Lachesin at the time that they diverge from a potentially neurogenic pathway. Neuroblasts, ganglion mother cells and neurons all express Lachesin early in their lives, but expression becomes restricted to a subset of neurons as development progresses. Sensory neurons express Lachesin as they delaminate from the body wall ectoderm. Lachesin is also present on growing axons of the CNS and PNS and becomes restricted to a subset of axons later in development. This expression is unique among known insect neurogenic genes and suggests a role for Lachesin in early neuronal differentiation and axon outgrowth. Grasshopper Lachesin is a 38 x 10(3) M(r) protein linked to cell membranes through a glycosyl phosphatidylinositol anchor. We have cloned the Lachesin gene from both grasshopper and fly. The proteins are highly conserved (70% identical) between the two species. Lachesin is similar to Drosophila amalgam, bovine OBCAM and the human poliovirus receptor, putting it into a subgroup of the immunoglobulin superfamily containing one V- and two C2-type immunoglobulin domains. Lachesin is also similar to several other vertebrate immunoglobulin superfamily proteins (TAG-1, F11, L1 and NgCAM) known to function in neurite outgrowth and other cell surface

  3. Aldehyde Dehydrogenase Gene Superfamily in Populus: Organization and Expression Divergence between Paralogous Gene Pairs.

    Directory of Open Access Journals (Sweden)

    Feng-Xia Tian

    Full Text Available Aldehyde dehydrogenases (ALDHs constitute a superfamily of NAD(P+-dependent enzymes that catalyze the irreversible oxidation of a wide range of reactive aldehydes to their corresponding nontoxic carboxylic acids. ALDHs have been studied in many organisms from bacteria to mammals; however, no systematic analyses incorporating genome organization, gene structure, expression profiles, and cis-acting elements have been conducted in the model tree species Populus trichocarpa thus far. In this study, a comprehensive analysis of the Populus ALDH gene superfamily was performed. A total of 26 Populus ALDH genes were found to be distributed across 12 chromosomes. Genomic organization analysis indicated that purifying selection may have played a pivotal role in the retention and maintenance of PtALDH gene families. The exon-intron organizations of PtALDHs were highly conserved within the same family, suggesting that the members of the same family also may have conserved functionalities. Microarray data and qRT-PCR analysis indicated that most PtALDHs had distinct tissue-specific expression patterns. The specificity of cis-acting elements in the promoter regions of the PtALDHs and the divergence of expression patterns between nine paralogous PtALDH gene pairs suggested that gene duplications may have freed the duplicate genes from the functional constraints. The expression levels of some ALDHs were up- or down-regulated by various abiotic stresses, implying that the products of these genes may be involved in the adaptation of Populus to abiotic stresses. Overall, the data obtained from our investigation contribute to a better understanding of the complexity of the Populus ALDH gene superfamily and provide insights into the function and evolution of ALDH gene families in vascular plants.

  4. FLORA: a novel method to predict protein function from structure in diverse superfamilies.

    Directory of Open Access Journals (Sweden)

    Oliver C Redfern

    2009-08-01

    Full Text Available Predicting protein function from structure remains an active area of interest, particularly for the structural genomics initiatives where a substantial number of structures are initially solved with little or no functional characterisation. Although global structure comparison methods can be used to transfer functional annotations, the relationship between fold and function is complex, particularly in functionally diverse superfamilies that have evolved through different secondary structure embellishments to a common structural core. The majority of prediction algorithms employ local templates built on known or predicted functional residues. Here, we present a novel method (FLORA that automatically generates structural motifs associated with different functional sub-families (FSGs within functionally diverse domain superfamilies. Templates are created purely on the basis of their specificity for a given FSG, and the method makes no prior prediction of functional sites, nor assumes specific physico-chemical properties of residues. FLORA is able to accurately discriminate between homologous domains with different functions and substantially outperforms (a 2-3 fold increase in coverage at low error rates popular structure comparison methods and a leading function prediction method. We benchmark FLORA on a large data set of enzyme superfamilies from all three major protein classes (alpha, beta, alphabeta and demonstrate the functional relevance of the motifs it identifies. We also provide novel predictions of enzymatic activity for a large number of structures solved by the Protein Structure Initiative. Overall, we show that FLORA is able to effectively detect functionally similar protein domain structures by purely using patterns of structural conservation of all residues.

  5. Multiple gains of spliceosomal introns in a superfamily of vertebrate protease inhibitor genes

    Directory of Open Access Journals (Sweden)

    Frese Marc-André

    2009-08-01

    Full Text Available Abstract Background Intron gains reportedly are very rare during evolution of vertebrates, and the mechanisms underlying their creation are largely unknown. Previous investigations have shown that, during metazoan radiation, the exon-intron patterns of serpin superfamily genes were subject to massive changes, in contrast to many other genes. Results Here we investigated intron dynamics in the serpin superfamily in lineages pre- and postdating the split of vertebrates. Multiple intron gains were detected in a group of ray-finned fishes, once the canonical groups of vertebrate serpins had been established. In two genes, co-occurrence of non-standard introns was observed, implying that intron gains in vertebrates may even happen concomitantly or in a rapidly consecutive manner. DNA breakage/repair processes associated with genome compaction are introduced as a novel factor potentially favoring intron gain, since all non-canonical introns were found in a lineage of ray-finned fishes that experienced genomic downsizing. Conclusion Multiple intron acquisitions were identified in serpin genes of a lineage of ray-finned fishes, but not in any other vertebrates, suggesting that insertion rates for introns may be episodically increased. The co-occurrence of non-standard introns within the same gene discloses the possibility that introns may be gained simultaneously. The sequences flanking the intron insertion points correspond to the proto-splice site consensus sequence MAG↑N, previously proposed to serve as intron insertion site. The association of intron gains in the serpin superfamily with a group of fishes that underwent genome compaction may indicate that DNA breakage/repair processes might foster intron birth.

  6. Characterization of mitochondrial thioredoxin reductase from C. elegans

    International Nuclear Information System (INIS)

    Lacey, Brian M.; Hondal, Robert J.

    2006-01-01

    Thioredoxin reductase catalyzes the NADPH-dependent reduction of the catalytic disulfide bond of thioredoxin. In mammals and other higher eukaryotes, thioredoxin reductases contain the rare amino acid selenocysteine at the active site. The mitochondrial enzyme from Caenorhabditis elegans, however, contains a cysteine residue in place of selenocysteine. The mitochondrial C. elegans thioredoxin reductase was cloned from an expressed sequence tag and then produced in Escherichia coli as an intein-fusion protein. The purified recombinant enzyme has a k cat of 610 min -1 and a K m of 610 μM using E. coli thioredoxin as substrate. The reported k cat is 25% of the k cat of the mammalian enzyme and is 43-fold higher than a cysteine mutant of mammalian thioredoxin reductase. The enzyme would reduce selenocysteine, but not hydrogen peroxide or insulin. The flanking glycine residues of the GCCG motif were mutated to serine. The mutants improved substrate binding, but decreased the catalytic rate

  7. Regulation of ribonucleotide reductase by Spd1 involves multiple mechanisms

    DEFF Research Database (Denmark)

    Nestoras, Konstantinos; Mohammed, Asma Hadi; Schreurs, Ann-Sofie

    2010-01-01

    The correct levels of deoxyribonucleotide triphosphates and their relative abundance are important to maintain genomic integrity. Ribonucleotide reductase (RNR) regulation is complex and multifaceted. RNR is regulated allosterically by two nucleotide-binding sites, by transcriptional control, and...

  8. Reduction of Folate by Dihydrofolate Reductase from Thermotoga maritima

    NARCIS (Netherlands)

    Loveridge, E Joel; Hroch, Lukas; Hughes, Robert L; Williams, Thomas; Davies, Rhidian L; Angelastro, Antonio; Luk, Louis Y P; Maglia, Giovanni; Allemann, Rudolf K

    2017-01-01

    Mammalian dihydrofolate reductases (DHFR) catalyse the reduction of folate more efficiently than the equivalent bacterial enzymes, despite typically having similar efficiencies for the reduction of their natural substrate dihydrofolate. In contrast, we show here that DHFR from the hyperthermophilic

  9. Meeting report - TGF-β superfamily: signaling in development and disease.

    Science.gov (United States)

    Zhang, Ying E; Newfeld, Stuart J

    2013-11-01

    The latest advances on the transforming growth factor β (TGF-β) and bone morphogenetic protein (BMP) signaling pathways were reported at the July 2013 FASEB Summer Research Conference 'The TGF-β Superfamily: Development and Disease'. The meeting was held in Steamboat Springs, Colorado, USA at 6700 feet above sea level in the Rocky Mountains. This was the seventh biannual meeting in the series. In attendance were investigators from a broad range of disciplines with a common interest in the mechanics of TGF-β and BMP signaling pathways, their normal developmental and homeostatic functions, and the diseases associated with pathway misregulation.

  10. Cloning, functional expression and characterization of a bifunctional 3-hydroxybutanal dehydrogenase /reductase involved in acetone metabolism by Desulfococcus biacutus.

    Science.gov (United States)

    Frey, Jasmin; Rusche, Hendrik; Schink, Bernhard; Schleheck, David

    2016-11-25

    The strictly anaerobic, sulfate-reducing bacterium Desulfococcus biacutus can utilize acetone as sole carbon and energy source for growth. Whereas in aerobic and nitrate-reducing bacteria acetone is activated by carboxylation with CO 2 to acetoacetate, D. biacutus involves CO as a cosubstrate for acetone activation through a different, so far unknown pathway. Proteomic studies indicated that, among others, a predicted medium-chain dehydrogenase/reductase (MDR) superfamily, zinc-dependent alcohol dehydrogenase (locus tag DebiaDRAFT_04514) is specifically and highly produced during growth with acetone. The MDR gene DebiaDRAFT_04514 was cloned and overexpressed in E. coli. The purified recombinant protein required zinc as cofactor, and accepted NADH/NAD + but not NADPH/NADP + as electron donor/acceptor. The pH optimum was at pH 8, and the temperature optimum at 45 °C. Highest specific activities were observed for reduction of C 3 - C 5 -aldehydes with NADH, such as propanal to propanol (380 ± 15 mU mg -1 protein), butanal to butanol (300 ± 24 mU mg -1 ), and 3-hydroxybutanal to 1,3-butanediol (248 ± 60 mU mg -1 ), however, the enzyme also oxidized 3-hydroxybutanal with NAD + to acetoacetaldehyde (83 ± 18 mU mg -1 ). The enzyme might play a key role in acetone degradation by D. biacutus, for example as a bifunctional 3-hydroxybutanal dehydrogenase/reductase. Its recombinant production may represent an important step in the elucidation of the complete degradation pathway.

  11. Aldose reductase inhibitory activity and antioxidant capacity of pomegranate extracts

    OpenAIRE

    Karasu, Çimen; Cumaoğlu, Ahmet; Gürpinar, Ali Rifat; Kartal, Murat; Kovacikova, Lucia; Milackova, Ivana; Stefek, Milan

    2012-01-01

    The pomegranate, Punica granatum L., has been the subject of current interest as a medicinal agent with wide-ranging therapeutic indications. In the present study, pomegranate ethanolic seed and hull extracts were tested, in comparison with a commercial sample, for the inhibition of aldose reductase, an enzyme involved in the etiology of diabetic complications. In vitro inhibition of rat lens aldose reductase was determined by a conventional method. Pomegranate ethanolic hull extract and comm...

  12. Isethionate formation from taurine in Chromohalobacter salexigens: purification of sulfoacetaldehyde reductase.

    Science.gov (United States)

    Krejcík, Zdenek; Hollemeyer, Klaus; Smits, Theo H M; Cook, Alasdair M

    2010-05-01

    Bacterial generation of isethionate (2-hydroxyethanesulfonate) from taurine (2-aminoethanesulfonate) by anaerobic gut bacteria was established in 1980. That phenomenon in pure culture was recognized as a pathway of assimilation of taurine-nitrogen. Based on the latter work, we predicted from genome-sequence data that the marine gammaproteobacterium Chromohalobacter salexigens DSM 3043 would exhibit this trait. Quantitative conversion of taurine to isethionate, identified by mass spectrometry, was confirmed, and the taurine-nitrogen was recovered as cell material. An eight-gene cluster was predicted to encode the inducible vectorial, scalar and regulatory enzymes involved, some of which were known from other taurine pathways. The genes (Csal_0153-Csal_0156) encoding a putative ATP-binding-cassette (ABC) transporter for taurine (TauAB(1)B(2)C) were shown to be inducibly transcribed by reverse transcription (RT-) PCR. An inducible taurine : 2-oxoglutarate aminotransferase [EC 2.6.1.55] was found (Csal_0158); the reaction yielded glutamate and sulfoacetaldehyde. The sulfoacetaldehyde was reduced to isethionate by NADPH-dependent sulfoacetaldehyde reductase (IsfD), a member of the short-chain alcohol dehydrogenase superfamily. The 27 kDa protein (SDS-PAGE) was identified by peptide-mass fingerprinting as the gene product of Csal_0161. The putative exporter of isethionate (IsfE) is encoded by Csal_0160; isfE was inducibly transcribed (RT-PCR). The presumed transcriptional regulator, TauR (Csal_0157), may autoregulate its own expression, typical of GntR-type regulators. Similar gene clusters were found in several marine and terrestrial gammaproteobacteria, which, in the gut canal, could be the source of not only mammalian, but also arachnid and cephalopod isethionate.

  13. Engineering Styrene Monooxygenase for Biocatalysis: Reductase-Epoxidase Fusion Proteins.

    Science.gov (United States)

    Heine, Thomas; Tucker, Kathryn; Okonkwo, Nonye; Assefa, Berhanegebriel; Conrad, Catleen; Scholtissek, Anika; Schlömann, Michael; Gassner, George; Tischler, Dirk

    2017-04-01

    The enantioselective epoxidation of styrene and related compounds by two-component styrene monooxygenases (SMOs) has targeted these enzymes for development as biocatalysts. In the present work, we prepare genetically engineered fusion proteins that join the C-terminus of the epoxidase (StyA) to the N-terminus of the reductase (StyB) through a linker peptide and demonstrate their utility as biocatalysts in the synthesis of Tyrain purple and other indigoid dyes. A single-vector expression system offers a simplified platform for transformation and expansion of the catalytic function of styrene monooxygenases, and the resulting fusion proteins are self-regulated and couple efficiently NADH oxidation to styrene epoxidation. We find that the reductase domain proceeds through a sequential ternary-complex mechanism at low FAD concentration and a double-displacement mechanism at higher concentrations of FAD. Single-turnover studies indicate an observed rate constant for FAD-to-FAD hydride transfer of ~8 s -1 . This step is rate limiting in the styrene epoxidation reaction and helps to ensure that flavin reduction and styrene epoxidation reactions proceed without wasteful side reactions. Comparison of the reductase activity of the fusion proteins with the naturally occurring reductase, SMOB, and N-terminally histidine-tagged reductase, NSMOB, suggests that the observed changes in catalytic mechanism are due in part to an increase in flavin-binding affinity associated with the N-terminal extension of the reductase.

  14. Characterization of human DHRS4: an inducible short-chain dehydrogenase/reductase enzyme with 3beta-hydroxysteroid dehydrogenase activity.

    Science.gov (United States)

    Matsunaga, Toshiyuki; Endo, Satoshi; Maeda, Satoshi; Ishikura, Shuhei; Tajima, Kazuo; Tanaka, Nobutada; Nakamura, Kazuo T; Imamura, Yorishige; Hara, Akira

    2008-09-15

    Human DHRS4 is a peroxisomal member of the short-chain dehydrogenase/reductase superfamily, but its enzymatic properties, except for displaying NADP(H)-dependent retinol dehydrogenase/reductase activity, are unknown. We show that the human enzyme, a tetramer composed of 27kDa subunits, is inactivated at low temperature without dissociation into subunits. The cold inactivation was prevented by a mutation of Thr177 with the corresponding residue, Asn, in cold-stable pig DHRS4, where this residue is hydrogen-bonded to Asn165 in a substrate-binding loop of other subunit. Human DHRS4 reduced various aromatic ketones and alpha-dicarbonyl compounds including cytotoxic 9,10-phenanthrenequinone. The overexpression of the peroxisomal enzyme in cultured cells did not increase the cytotoxicity of 9,10-phenanthrenequinone. While its activity towards all-trans-retinal was low, human DHRS4 efficiently reduced 3-keto-C(19)/C(21)-steroids into 3beta-hydroxysteroids. The stereospecific conversion to 3beta-hydroxysteroids was observed in endothelial cells transfected with vectors expressing the enzyme. The mRNA for the enzyme was ubiquitously expressed in human tissues and several cancer cells, and the enzyme in HepG2 cells was induced by peroxisome-proliferator-activated receptor alpha ligands. The results suggest a novel mechanism of cold inactivation and role of the inducible human DHRS4 in 3beta-hydroxysteroid synthesis and xenobiotic carbonyl metabolism.

  15. Methylenetetrahydrofolate Reductase Activity and Folate Metabolism

    Directory of Open Access Journals (Sweden)

    Nursen Keser

    2014-04-01

    Full Text Available Folate is a vital B vitamin which is easily water-soluble. It is a natural source which is found in the herbal and animal foods. Folate has important duties in the human metabolism, one of them is the adjustment of the level of plasma homocysteine. Reduction in MTHFR (methylenetetrahydrofolate reductase,which is in charge of the metabolism of homocysteine activity affects the level of homocysteine. Therefore MTHFR is an important enzyme in folate metabolism. Some of the mutations occurring in the MTHFR gene is a risk factor for various diseases and may be caused the hyperhomocysteinemia or the homocystinuria, and they also may lead to metabolic problems. MTHFR is effective in the important pathways such as DNA synthesis, methylation reactions and synthesis of RNA. C677T and A1298C are the most commonly occurring polymorphisms in the gene of MTHFR. The frequency of these polymorphisms show differences in the populations. MTHFR, folate distribution, metabolism of homocysteine and S-adenosylmethionine, by the MTHFR methylation the genetic defects have the potential of affecting the risk of disease in the negative or positive way.

  16. Interactions between inhibitors of dihydrofolate reductase.

    Science.gov (United States)

    Bowden, K; Hall, A D; Birdsall, B; Feeney, J; Roberts, G C

    1989-03-01

    The binding of substrates and inhibitors to dihydrofolate reductase was studied by steady-state kinetics and high-field 1H-n.m.r. spectroscopy. A series of 5-substituted 2,4-diaminopyrimidines were examined and were found to be 'tightly binding' inhibitors of the enzyme (Ki less than 10(-9) M). Studies on the binding of 4-substituted benzenesulphonamides and benzenesulphonic acids also established the existence of a 'sulphonamide-binding site' on the enzyme. Subsequent n.m.r. experiments showed that there are two binding sites for the sulphonamides on the enzyme, one of which overlaps the coenzyme (NADPH) adenine-ring-binding site. An examination of the pH-dependence of the binding of sulphonamides to the enzyme indicated the influence of an ionizable group on the enzyme that was not directly involved in the sulphonamide binding. The change in pKa value from 6.7 to 7.2 observed on sulphonamide binding suggests the involvement of a histidine residue, which could be histidine-28.

  17. Aldose reductase mediates retinal microglia activation

    Energy Technology Data Exchange (ETDEWEB)

    Chang, Kun-Che; Shieh, Biehuoy; Petrash, J. Mark, E-mail: mark.petrash@ucdenver.edu

    2016-04-29

    Retinal microglia (RMG) are one of the major immune cells in charge of surveillance of inflammatory responses in the eye. In the absence of an inflammatory stimulus, RMG reside predominately in the ganglion layer and inner or outer plexiform layers. However, under stress RMG become activated and migrate into the inner nuclear layer (INL) or outer nuclear layer (ONL). Activated RMG in cell culture secrete pro-inflammatory cytokines in a manner sensitive to downregulation by aldose reductase inhibitors. In this study, we utilized CX3CR1{sup GFP} mice carrying AR mutant alleles to evaluate the role of AR on RMG activation and migration in vivo. When tested on an AR{sup WT} background, IP injection of LPS induced RMG activation and migration into the INL and ONL. However, this phenomenon was largely prevented by AR inhibitors or in AR null mice, or was exacerbated in transgenic mice that over-express AR. LPS-induced increases in ocular levels of TNF-α and CX3CL-1 in WT mice were substantially lower in AR null mice or were reduced by AR inhibitor treatment. These studies demonstrate that AR expression in RMG may contribute to the proinflammatory phenotypes common to various eye diseases such as uveitis and diabetic retinopathy. - Highlights: • AR inhibition prevents retinal microglial activation. • Endotoxin-induced ocular cytokine production is reduced in AR null mice. • Overexpression of AR spontaneously induces retinal microglial activation.

  18. Immunoglobulin superfamily members encoded by viruses and their multiple roles in immune evasion.

    Science.gov (United States)

    Farré, Domènec; Martínez-Vicente, Pablo; Engel, Pablo; Angulo, Ana

    2017-05-01

    Pathogens have developed a plethora of strategies to undermine host immune defenses in order to guarantee their survival. For large DNA viruses, these immune evasion mechanisms frequently rely on the expression of genes acquired from host genomes. Horizontally transferred genes include members of the immunoglobulin superfamily, whose products constitute the most diverse group of proteins of vertebrate genomes. Their promiscuous immunoglobulin domains, which comprise the building blocks of these molecules, are involved in a large variety of functions mediated by ligand-binding interactions. The flexible structural nature of the immunoglobulin domains makes them appealing targets for viral capture due to their capacity to generate high functional diversity. Here, we present an up-to-date review of immunoglobulin superfamily gene homologs encoded by herpesviruses, poxviruses, and adenoviruses, that include CD200, CD47, Fc receptors, interleukin-1 receptor 2, interleukin-18 binding protein, CD80, carcinoembryonic antigen-related cell adhesion molecules, and signaling lymphocyte activation molecules. We discuss their distinct structural attributes, binding properties, and functions, shaped by evolutionary pressures to disarm specific immune pathways. We include several novel genes identified from extensive genome database surveys. An understanding of the properties and modes of action of these viral proteins may guide the development of novel immune-modulatory therapeutic tools. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Nitrilase superfamily aryl acylamidase from the halotolerant mangrove Streptomyces sp. 211726.

    Science.gov (United States)

    Ma, Yanling; Xu, Wei; Zhang, Jun; Zhang, Sihong; Hong, Kui; Deng, Zixin; Sun, Yuhui

    2014-10-01

    A novel nitrilase superfamily amidase gene, designated azl13, was cloned from Streptomyces sp. 211726. Bioinformatic and biochemical analysis indicated that Azl13 belongs to a new subfamily in branch 13 of the nitrilase superfamily. His6-Azl13 was expressed in Escherichia coli BL21(DE3) and had the expected molecular mass of 31 kDa, and the enzymatic activity was best at 40 °C, pH 8.0. His6-Azl13 had amidase, aryl acylamidase, and acyl transferase activities, and it displayed an unusually wide substrate spectrum. His6-Azl13 was most active on 4-guanidinobutyramide, which is probably its natural substrate, moderately active on short-chain aliphatic amides and weakly active hydrolyzing aromatic and heterocyclic amides. His6-Azl13 also catalyzed acyl transfer to hydroxylamine from acetamide or the herbicide propanil. The substrate spectrum differs from that of the Pseudomonas amidase RamA, probably reflecting high salinity adaptation. The broad substrate spectrum of Azl13 is potentially useful for chemical synthesis and biodegradation.

  20. The Anabaena sensory rhodopsin transducer defines a novel superfamily of prokaryotic small-molecule binding domains

    Directory of Open Access Journals (Sweden)

    De Souza Robson F

    2009-08-01

    Full Text Available Abstract The Anabaena sensory rhodopsin transducer (ASRT is a small protein that has been claimed to function as a signaling molecule downstream of the cyanobacterial sensory rhodopsin. However, orthologs of ASRT have been detected in several bacteria that lack rhodopsin, raising questions about the generality of this function. Using sequence profile searches we show that ASRT defines a novel superfamily of β-sandwich fold domains. Through contextual inference based on domain architectures and predicted operons and structural analysis we present strong evidence that these domains bind small molecules, most probably sugars. We propose that the intracellular versions like ASRT probably participate as sensors that regulate a diverse range of sugar metabolism operons or even the light sensory behavior in Anabaena by binding sugars or related metabolites. We also show that one of the extracellular versions define a predicted sugar-binding structure in a novel cell-surface lipoprotein found across actinobacteria, including several pathogens such as Tropheryma, Actinomyces and Thermobifida. The analysis of this superfamily also provides new data to investigate the evolution of carbohydrate binding modes in β-sandwich domains with very different topologies. Reviewers: This article was reviewed by M. Madan Babu and Mark A. Ragan.

  1. Isolation of a novel LPS-induced component of the ML superfamily in Ciona intestinalis.

    Science.gov (United States)

    Vizzini, Aiti; Bonura, Angela; Longo, Valeria; Sanfratello, Maria Antonietta; Parrinello, Daniela; Cammarata, Matteo; Colombo, Paolo

    2015-11-01

    ML superfamily represents a group of proteins playing important roles in lipid metabolism and innate immune response. In this study, we report the identification of the first component of the ML superfamily in the invertebrate Ciona intestinalis by means of a subtractive hybridization strategy. Sequence homology and phylogenetic analysis showed that this protein forms a specific clade with vertebrate components of the Niemann-Pick type C2 protein and, for this reason, it has been named Ci-NPC2. The putative Ci-NPC2 is a 150 amino acids long protein with a short signal peptide, seven cysteine residues, three putative lipid binding site and a three-dimensional model showing a characteristic β-strand structure. Gene expression analysis demonstrated that the Ci-NPC2 protein is positively upregulated after LPS inoculum with a peak of expression 1 h after challenge. Finally, in-situ hybridization demonstrated that the Ci-NPC2 protein is preferentially expressed in hemocytes inside the vessel lumen. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. Functional analysis of Hsp70 superfamily proteins of rice (Oryza sativa).

    Science.gov (United States)

    Sarkar, Neelam K; Kundnani, Preeti; Grover, Anil

    2013-07-01

    Heat stress results in misfolding and aggregation of cellular proteins. Heat shock proteins (Hsp) enable the cells to maintain proper folding of proteins, both in unstressed as well as stressed conditions. Hsp70 genes encode for a group of highly conserved chaperone proteins across the living systems encompassing bacteria, plants, and animals. In the cellular chaperone network, Hsp70 family proteins interconnect other chaperones and play a dominant role in various cell processes. To assess the functionality of rice Hsp70 genes, rice genome database was analyzed. Rice genome contains 32 Hsp70 genes. Rice Hsp70 superfamily genes are represented by 24 Hsp70 family and 8 Hsp110 family members. Promoter and transcript expression analysis divulges that Hsp70 superfamily genes plays important role in heat stress. Ssc1 (mitochondrial Hsp70 protein in yeast) deleted yeast show compromised growth at 37 °C. Three mitochondrial rice Hsp70 sequences (i.e., mtHsp70-1, mtHsp70-2, and mtHsp70-3) complemented the Ssc1 mutation of yeast to differential extents. The information presented in this study provides detailed understanding of the Hsp70 protein family of rice, the crop species that is the major food for the world population.

  3. TGF-β superfamily signaling in testis formation and early male germline development.

    Science.gov (United States)

    Young, Julia C; Wakitani, Shoichi; Loveland, Kate L

    2015-09-01

    The TGF-β ligand superfamily contains at least 40 members, many of which are produced and act within the mammalian testis to facilitate formation of sperm. Their progressive expression at key stages and in specific cell types determines the fertility of adult males, influencing testis development and controlling germline differentiation. BMPs are essential for the interactive instructions between multiple cell types in the early embryo that drive initial specification of gamete precursors. In the nascent foetal testis, several ligands including Nodal, TGF-βs, Activins and BMPs, serve as key masculinizing switches by regulating male germline pluripotency, somatic and germline proliferation, and testicular vascularization and architecture. In postnatal life, local production of these factors determine adult testis size by regulating Sertoli cell multiplication and differentiation, in addition to specifying germline differentiation and multiplication. Because TGF-β superfamily signaling is integral to testis formation, it affects processes that underlie testicular pathologies, including testicular cancer, and its potential to contribute to subfertility is beginning to be understood. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. Evolutionary Pattern of N-Glycosylation Sequon Numbers  in Eukaryotic ABC Protein Superfamilies

    Directory of Open Access Journals (Sweden)

    R. Shyama Prasad Rao

    2010-02-01

    Full Text Available Many proteins contain a large number of NXS/T sequences (where X is any amino acid except proline which are the potential sites of asparagine (N linked glycosylation. However, the patterns of occurrence of these N-glycosylation sequons in related proteins or groups of proteins and their underlying causes have largely been unexplored. We computed the actual and probabilistic occurrence of NXS/T sequons in ABC protein superfamilies from eight diverse eukaryotic organisms. The ABC proteins contained significantly higher NXS/T sequon numbers compared to respective genome-wide average, but the sequon density was significantly lower owing to the increase in protein size and decrease in sequon specific amino acids. However, mammalian ABC proteins have significantly higher sequon density, and both serine and threonine containing sequons (NXS and NXT have been positively selected—against the recent findings of only threonine specific Darwinian selection of sequons in proteins. The occurrence of sequons was positively correlated with the frequency of sequon specific amino acids and negatively correlated with proline and the NPS/T sequences. Further, the NPS/T sequences were significantly higher than expected in plant ABC proteins which have the lowest number of NXS/T sequons. Accord- ingly, compared to overall proteins, N-glycosylation sequons in ABC protein superfamilies have a distinct pattern of occurrence, and the results are discussed in an evolutionary perspective.

  5. NADH-Ferricyanide Reductase of Leaf Plasma Membranes 1

    Science.gov (United States)

    Askerlund, Per; Laurent, Pascal; Nakagawa, Hiroki; Kader, Jean-Claude

    1991-01-01

    Plasma membranes obtained by two-phase partitioning of microsomal fractions from spinach (Spinacea oleracea L. cv Medania) and sugar beet leaves (Beta vulgaris L.) contained relatively high NADH-ferricyanide reductase and NADH-nitrate reductase (NR; EC 1.6.6.1) activities. Both of these activities were latent. To investigate whether these activities were due to the same enzyme, plasma membrane polypeptides were separated with SDS-PAGE and analyzed with immunoblotting methods. Antibodies raised against microsomal NADH-ferricyanide reductase (tentatively identified as NADH-cytochrome b5 reductase, EC 1.6.2.2), purified from potato (Solanum tuberosum L. cv Bintje) tuber microsomes, displayed one single band at 43 kilodaltons when reacted with spinach plasma membranes, whereas lgG produced against NR from spinach leaves gave a major band at 110 kilodaltons together with a few fainter bands of lower molecular mass. Immunoblotting analysis using inside-out and right-side-out plasma membrane vesicles strongly indicated that NR was not an integral protein but probably trapped inside the plasma membrane vesicles during homogenization. Proteins from spinach plasma membranes were solubilized with the zwitterionic detergent 3-[(3-cholamidopropyl) dimethylammonio] 1-propane-sulfonate and separated on a Mono Q anion exchange column at pH 5.6 with fast protein liquid chromatography. One major peak of NADH-ferricyanide reductase activity was found after separation. The peak fraction was enriched about 70-fold in this activity compared to the plasma membrane. When the peak fractions were analyzed with SDS-PAGE the NADH-ferricyanide reductase activity strongly correlated with a 43 kilodalton polypeptide which reacted with the antibodies against potato microsomal NADH-ferricyanide reductase. Thus, our data indicate that most, if not all, of the truly membrane-bound NADH-ferricyanide reductase activity of leaf plasma membranes is due to an enzyme very similar to potato tuber

  6. The ATPase of the phi29 DNA packaging motor is a member of the hexameric AAA+ superfamily.

    Science.gov (United States)

    Schwartz, Chad; De Donatis, Gian Marco; Fang, Huaming; Guo, Peixuan

    2013-08-15

    The AAA+ superfamily of proteins is a class of motor ATPases performing a wide range of functions that typically exist as hexamers. The ATPase of phi29 DNA packaging motor has long been a subject of debate in terms of stoichiometry and mechanism of action. Here, we confirmed the stoichiometry of phi29 motor ATPase to be a hexamer and provide data suggesting that the phi29 motor ATPase is a member of the classical hexameric AAA+ superfamily. Native PAGE, EMSA, capillary electrophoresis, ATP titration, and binomial distribution assay show that the ATPase is a hexamer. Mutations in the known Walker motifs of the ATPase validated our previous assumptions that the protein exists as another member of this AAA+ superfamily. Our data also supports the finding that the phi29 DNA packaging motor uses a revolution mechanism without rotation or coiling (Schwartz et al., this issue). Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

  7. Transcripts of Anthocyanidin Reductase and Leucoanthocyanidin Reductase and Measurement of Catechin and Epicatechin in Tartary Buckwheat

    Directory of Open Access Journals (Sweden)

    Yeon Bok Kim

    2014-01-01

    Full Text Available Anthocyanidin reductase (ANR and leucoanthocyanidin reductase (LAR play an important role in the monomeric units biosynthesis of proanthocyanidins (PAs such as catechin and epicatechin in several plants. The aim of this study was to clone ANR and LAR genes involved in PAs biosynthesis and examine the expression of these two genes in different organs under different growth conditions in two tartary buckwheat cultivars, Hokkai T8 and T10. Gene expression was carried out by quantitative real-time RT-PCR, and catechin and epicatechin content was analyzed by high performance liquid chromatography. The expression pattern of ANR and LAR did not match the accumulation pattern of PAs in different organs of two cultivars. Epicatechin content was the highest in the flowers of both cultivars and it was affected by light in only Hokkai T8 sprouts. ANR and LAR levels in tartary buckwheat might be regulated by different mechanisms for catechin and epicatechin biosynthesis under light and dark conditions.

  8. Isolation and characterization of cDNAs encoding leucoanthocyanidin reductase and anthocyanidin reductase from Populus trichocarpa.

    Directory of Open Access Journals (Sweden)

    Lijun Wang

    Full Text Available Proanthocyanidins (PAs contribute to poplar defense mechanisms against biotic and abiotic stresses. Transcripts of PA biosynthetic genes accumulated rapidly in response to infection by the fungus Marssonina brunnea f.sp. multigermtubi, treatments of salicylic acid (SA and wounding, resulting in PA accumulation in poplar leaves. Anthocyanidin reductase (ANR and leucoanthocyanidin reductase (LAR are two key enzymes of the PA biosynthesis that produce the main subunits: (+-catechin and (--epicatechin required for formation of PA polymers. In Populus, ANR and LAR are encoded by at least two and three highly related genes, respectively. In this study, we isolated and functionally characterized genes PtrANR1 and PtrLAR1 from P. trichocarpa. Phylogenetic analysis shows that Populus ANR1 and LAR1 occurr in two distinct phylogenetic lineages, but both genes have little difference in their tissue distribution, preferentially expressed in roots. Overexpression of PtrANR1 in poplar resulted in a significant increase in PA levels but no impact on catechin levels. Antisense down-regulation of PtrANR1 showed reduced PA accumulation in transgenic lines, but increased levels of anthocyanin content. Ectopic expression of PtrLAR1 in poplar positively regulated the biosynthesis of PAs, whereas the accumulation of anthocyanin and flavonol was significantly reduced (P<0.05 in all transgenic plants compared to the control plants. These results suggest that both PtrANR1 and PtrLAR1 contribute to PA biosynthesis in Populus.

  9. Purification and Properties of an NADPH-Aldose Reductase (Aldehyde Reductase) from Euonymus japonica Leaves

    Science.gov (United States)

    Negm, Fayek B.

    1986-01-01

    The enzyme aldose (aldehyde) reductase was partially purified (142-fold) and characterized from Euonymus japonica leaves. The reductase, a dimer, had an average molecular weight of 67,000 as determined by gel filtration on Sephadex G-100. The enzyme was NADPH specific and reduced a broad range of substrates including aldoses, aliphatic aldehydes, and aromatic aldehydes. Maximum activity was observed at pH 8 in phosphate and Tris-HCl buffers and at pH 8.6 to 9.0 in glycine-NaOH buffer using dl-glyceraldehyde or 3-pyridinecarboxaldehyde as substrate. NADP was a competitive inhibitor with respect to NADPH with a Ki of 60 micromolar. Glycerol was an uncompetitive inhibitor to dl-glyceraldehyde (K′i = 460 millimolar). The Euonymus enzyme was inhibited by sulfhydryl inhibitor, phenobarbital, and high concentrations of Li2SO4. Pyrazol and metal chelating agents inhibited the enzyme slightly. Enzyme activity was detected in the leaves and berries of Celastrus orbiculatus and several species of Euonymus. Probable function of this enzyme is to reduce d-galactose to galactitol, a characteristic metabolite in phloem sap of members of the Celastraceae family. Images Fig. 1 PMID:16664750

  10. A Unique Short-Chain Dehydrogenase/Reductase in Arabidopsis Glucose Signaling and Abscisic Acid Biosynthesis and Functions

    Science.gov (United States)

    Cheng, Wan-Hsing; Endo, Akira; Zhou, Li; Penney, Jessica; Chen, Huei-Chi; Arroyo, Analilia; Leon, Patricia; Nambara, Eiji; Asami, Tadao; Seo, Mitsunori; Koshiba, Tomokazu; Sheen, Jen

    2002-01-01

    Glc has hormone-like functions and controls many vital processes through mostly unknown mechanisms in plants. We report here on the molecular cloning of GLUCOSE INSENSITIVE1 (GIN1) and ABSCISIC ACID DEFICIENT2 (ABA2) which encodes a unique Arabidopsis short-chain dehydrogenase/reductase (SDR1) that functions as a molecular link between nutrient signaling and plant hormone biosynthesis. SDR1 is related to SDR superfamily members involved in retinoid and steroid hormone biosynthesis in mammals and sex determination in maize. Glc antagonizes ethylene signaling by activating ABA2/GIN1 and other abscisic acid (ABA) biosynthesis and signaling genes, which requires Glc and ABA synergistically. Analyses of aba2/gin1 null mutants define dual functions of endogenous ABA in inhibiting the postgermination developmental switch modulated by distinct Glc and osmotic signals and in promoting organ and body size and fertility in the absence of severe stress. SDR1 is sufficient for the multistep conversion of plastid- and carotenoid-derived xanthoxin to abscisic aldehyde in the cytosol. The surprisingly restricted spatial and temporal expression of SDR1 suggests the dynamic mobilization of ABA precursors and/or ABA. PMID:12417697

  11. Human dehydrogenase/reductase (SDR family) member 11 is a novel type of 17β-hydroxysteroid dehydrogenase.

    Science.gov (United States)

    Endo, Satoshi; Miyagi, Namiki; Matsunaga, Toshiyuki; Hara, Akira; Ikari, Akira

    2016-03-25

    We report characterization of a member of the short-chain dehydrogenase/reductase superfamily encoded in a human gene, DHRS11. The recombinant protein (DHRS11) efficiently catalyzed the conversion of the 17-keto group of estrone, 4- and 5-androstenes and 5α-androstanes into their 17β-hydroxyl metabolites with NADPH as a coenzyme. In contrast, it exhibited reductive 3β-hydroxysteroid dehydrogenase activity toward 5β-androstanes, 5β-pregnanes, 4-pregnenes and bile acids. Additionally, DHRS11 reduced α-dicarbonyls (such as diacetyl and methylglyoxal) and alicyclic ketones (such as 1-indanone and loxoprofen). The enzyme activity was inhibited in a mixed-type manner by flavonoids, and competitively by carbenoxolone, glycyrrhetinic acid, zearalenone, curcumin and flufenamic acid. The expression of DHRS11 mRNA was observed widely in human tissues, most abundantly in testis, small intestine, colon, kidney and cancer cell lines. Thus, DHRS11 represents a novel type of 17β-hydroxysteroid dehydrogenase with unique catalytic properties and tissue distribution. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. Short-chain dehydrogenase/reductase catalyzing the final step of noscapine biosynthesis is localized to laticifers in opium poppy.

    Science.gov (United States)

    Chen, Xue; Facchini, Peter J

    2014-01-01

    The final step in the biosynthesis of the phthalideisoquinoline alkaloid noscapine involves a purported dehydrogenation of the narcotinehemiacetal keto moiety. A short-chain dehydrogenase/reductase (SDR), designated noscapine synthase (NOS), that catalyzes dehydrogenation of narcotinehemiacetal to noscapine was identified in opium poppy and functionally characterized. The NOS gene was isolated using an integrated transcript and metabolite profiling strategy and subsequently expressed in Escherichia coli. Noscapine synthase is highly divergent from other characterized members of the NADPH-dependent SDR superfamily involved in benzylisoquinoline alkaloid metabolism, and it exhibits exclusive substrate specificity for narcotinehemiacetal. Kinetic analyses showed that NOS exhibits higher catalytic efficiency with NAD+ as the cofactor compared with NADP+. Suppression of NOS transcript levels in opium poppy plants subjected to virus-induced gene silencing resulted in a corresponding reduction in the accumulation of noscapine and an increase in narcotinehemiacetal levels in the latex. Noscapine and NOS transcripts were detected in all opium poppy organs, but both were most abundant in stems. Unlike other putative biosynthetic genes clustered in the opium poppy genome, and their corresponding proteins, NOS transcripts and the cognate enzyme were abundant in latex, indicating that noscapine metabolism is completed in a distinct cell type compared with the rest of the pathway.

  13. 5α-reductases in human physiology: an unfolding story.

    Science.gov (United States)

    Traish, Abdulmaged M

    2012-01-01

    5α-reductases are a family of isozymes expressed in a wide host of tissues including the central nervous system (CNS) and play a pivotal role in male sexual differentiation, development and physiology. A comprehensive literature search from 1970 to 2011 was made through PubMed and the relevant information was summarized. 5α reductases convert testosterone, progesterone, deoxycorticosterone, aldosterone and corticosterone into their respective 5α-dihydro-derivatives, which serve as substrates for 3α-hydroxysteroid dehydrogenase enzymes. The latter transforms these 5α-reduced metabolites into a subclass of neuroactive steroid hormones with distinct physiological functions. The neuroactive steroid hormones modulate a multitude of functions in human physiology encompassing regulation of sexual differentiation, neuroprotection, memory enhancement, anxiety, sleep and stress, among others. In addition, 5α -reductase type 3 is also implicated in the N-glycosylation of proteins via formation of dolichol phosphate. The family of 5α-reductases was targeted for drug development to treat pathophysiological conditions, such as benign prostatic hyperplasia and androgenetic alopecia. While the clinical use of 5α-reductase inhibitors was well established, the scope and the magnitude of the adverse side effects of such drugs, especially on the CNS, is still unrecognized due to lack of knowledge of the various physiological functions of this family of enzymes, especially in the CNS. There is an urgent need to better understand the function of 5α-reductases and the role of neuroactive steroids in human physiology in order to minimize the potential adverse side effects of inhibitors targeting 5α-reductases to treat benign prostatic hyperplasia and androgenic alopecia.

  14. Pancreaticobiliary cancers with deficient methylenetetrahydrofolate reductase genotypes.

    Science.gov (United States)

    Matsubayashi, Hiroyuki; Skinner, Halcyon G; Iacobuzio-Donahue, Christine; Abe, Tadayoshi; Sato, Norihiro; Riall, Taylor Sohn; Yeo, Charles J; Kern, Scott E; Goggins, Michael

    2005-08-01

    Methyl group deficiency might promote carcinogenesis by inducing DNA breaks and DNA hypomethylation. We hypothesized that deficient methylenetetrahydrofolate reductase (MTHFR) genotypes could promote pancreatic cancer development. First, we performed a case-control study of germline MTHFR polymorphisms (C677T, A1298C) in 303 patients with pancreatic cancer and 305 matched control subjects. Pancreatic neoplasms frequently lose an MTHFR allele during tumorigenesis; we hypothesized that such loss could promote carcinogenesis. We therefore evaluated the cancer MTHFR genotypes of 82 patients with pancreaticobiliary cancers and correlated them to genome-wide measures of chromosomal deletion by using 386 microsatellite markers. Finally, MTHFR genotypes were correlated with global DNA methylation in 68 cancer cell lines. Germline MTHFR polymorphisms were not associated with an increased likelihood of having pancreatic cancer. Fractional allelic loss (a measure of chromosomal loss) trended higher in cancers with 677T genotypes than in cancers with other genotypes (P = .055). Among cancers with loss of an MTHFR allele, cancers with 677T MTHFR alleles had more deletions at folate-sensitive fragile sites (36.9%) and at tumor suppressor gene loci (68.5%) than 677C cancers (28.7% and 47.8%, P = .079 and .014, respectively). LINE1 methylation was lower in cancers with less functional 677T/TT genotypes (24.4%) than in those with 677CT (26.0%) and CC/C genotypes (32.5%) (P = .014). Cancers with defective MTHFR genotypes have more DNA hypomethylation and more chromosomal losses. Deficient MTHFR function due to loss of an MTHFR allele by an evolving neoplasm might, by promoting chromosomal losses, accelerate cancer development.

  15. Sulfite reductase protects plants against sulfite toxicity.

    Science.gov (United States)

    Yarmolinsky, Dmitry; Brychkova, Galina; Fluhr, Robert; Sagi, Moshe

    2013-02-01

    Plant sulfite reductase (SiR; Enzyme Commission 1.8.7.1) catalyzes the reduction of sulfite to sulfide in the reductive sulfate assimilation pathway. Comparison of SiR expression in tomato (Solanum lycopersicum 'Rheinlands Ruhm') and Arabidopsis (Arabidopsis thaliana) plants revealed that SiR is expressed in a different tissue-dependent manner that likely reflects dissimilarity in sulfur metabolism between the plant species. Using Arabidopsis and tomato SiR mutants with modified SiR expression, we show here that resistance to ectopically applied sulfur dioxide/sulfite is a function of SiR expression levels and that plants with reduced SiR expression exhibit higher sensitivity than the wild type, as manifested in pronounced leaf necrosis and chlorophyll bleaching. The sulfite-sensitive mutants accumulate applied sulfite and show a decline in glutathione levels. In contrast, mutants that overexpress SiR are more tolerant to sulfite toxicity, exhibiting little or no damage. Resistance to high sulfite application is manifested by fast sulfite disappearance and an increase in glutathione levels. The notion that SiR plays a role in the protection of plants against sulfite is supported by the rapid up-regulation of SiR transcript and activity within 30 min of sulfite injection into Arabidopsis and tomato leaves. Peroxisomal sulfite oxidase transcripts and activity levels are likewise promoted by sulfite application as compared with water injection controls. These results indicate that, in addition to participating in the sulfate assimilation reductive pathway, SiR also plays a role in protecting leaves against the toxicity of sulfite accumulation.

  16. Evolution of Enzymatic Activities in the Enolase Superfamily: L-Fuconate Dehydratase from Xanthomonas campestris

    Energy Technology Data Exchange (ETDEWEB)

    Yew,W.; Fedorov, A.; Fedorov, E.; Rakus, J.; Pierce, R.; Almo, S.; Gerlt, J.

    2006-01-01

    Many members of the mechanistically diverse enolase superfamily have unknown functions. In this report the authors use both genome (operon) context and screening of a library of acid sugars to assign the L-fuconate dehydratase (FucD) function to a member of the mandelate racemase (MR) subgroup of the superfamily encoded by the Xanthomonas campestris pv. campestris str. ATCC 33913 genome (GI: 21233491). Orthologues of FucD are found in both bacteria and eukaryotes, the latter including the rTS beta protein in Homo sapiens that has been implicated in regulating thymidylate synthase activity. As suggested by sequence alignments and confirmed by high-resolution structures in the presence of active site ligands, FucD and MR share the same active site motif of functional groups: three carboxylate ligands for the essential Mg2+ located at the ends of th third, fourth, and fifth-strands in the (/)7-barrel domain (Asp 248, Glu 274, and Glu 301, respectively), a Lys-x-Lys motif at the end of the second-strand (Lys 218 and Lys 220), a His-Asp dyad at the end of the seventh and sixth-strands (His 351 and Asp 324, respectively), and a Glue at the end of the eighth-strand (Glu 382). The mechanism of the FucD reaction involves initial abstraction of the 2-proton by Lys 220, acid catalysis of the vinylogous-elimination of the 3-OH group by His 351, and stereospecific ketonization of the resulting 2-keto-3-deoxy-L-fuconate product. Screening of the library of acid sugars revealed substrate and functional promiscuity: In addition to L-fuconate, FucD also catalyzes the dehydration of L-galactonate, D-arabinonate, D-altronate, L-talonate, and D-ribonate. The dehydrations of L-fuconate, L-galactonate, and D-arabinonate are initiated by abstraction of the 2-protons by Lys 220. The dehydrations of L-talonate and D-ribonate are initiated by abstraction of the 2-protons by His 351; however, protonation of the enediolate intermediates by the conjugate acid of Lys 220 yields L

  17. RASOnD - A comprehensive resource and search tool for RAS superfamily oncogenes from various species

    Directory of Open Access Journals (Sweden)

    Singh Tej P

    2011-07-01

    Full Text Available Abstract Background The Ras superfamily plays an important role in the control of cell signalling and division. Mutations in the Ras genes convert them into active oncogenes. The Ras oncogenes form a major thrust of global cancer research as they are involved in the development and progression of tumors. This has resulted in the exponential growth of data on Ras superfamily across different public databases and in literature. However, no dedicated public resource is currently available for data mining and analysis on this family. The present database was developed to facilitate straightforward accession, retrieval and analysis of information available on Ras oncogenes from one particular site. Description We have developed the RAS Oncogene Database (RASOnD as a comprehensive knowledgebase that provides integrated and curated information on a single platform for oncogenes of Ras superfamily. RASOnD encompasses exhaustive genomics and proteomics data existing across diverse publicly accessible databases. This resource presently includes overall 199,046 entries from 101 different species. It provides a search tool to generate information about their nucleotide and amino acid sequences, single nucleotide polymorphisms, chromosome positions, orthologies, motifs, structures, related pathways and associated diseases. We have implemented a number of user-friendly search interfaces and sequence analysis tools. At present the user can (i browse the data (ii search any field through a simple or advance search interface and (iii perform a BLAST search and subsequently CLUSTALW multiple sequence alignment by selecting sequences of Ras oncogenes. The Generic gene browser, GBrowse, JMOL for structural visualization and TREEVIEW for phylograms have been integrated for clear perception of retrieved data. External links to related databases have been included in RASOnD. Conclusions This database is a resource and search tool dedicated to Ras oncogenes. It has

  18. Ensembler: Enabling High-Throughput Molecular Simulations at the Superfamily Scale.

    Directory of Open Access Journals (Sweden)

    Daniel L Parton

    2016-06-01

    Full Text Available The rapidly expanding body of available genomic and protein structural data provides a rich resource for understanding protein dynamics with biomolecular simulation. While computational infrastructure has grown rapidly, simulations on an omics scale are not yet widespread, primarily because software infrastructure to enable simulations at this scale has not kept pace. It should now be possible to study protein dynamics across entire (superfamilies, exploiting both available structural biology data and conformational similarities across homologous proteins. Here, we present a new tool for enabling high-throughput simulation in the genomics era. Ensembler takes any set of sequences-from a single sequence to an entire superfamily-and shepherds them through various stages of modeling and refinement to produce simulation-ready structures. This includes comparative modeling to all relevant PDB structures (which may span multiple conformational states of interest, reconstruction of missing loops, addition of missing atoms, culling of nearly identical structures, assignment of appropriate protonation states, solvation in explicit solvent, and refinement and filtering with molecular simulation to ensure stable simulation. The output of this pipeline is an ensemble of structures ready for subsequent molecular simulations using computer clusters, supercomputers, or distributed computing projects like Folding@home. Ensembler thus automates much of the time-consuming process of preparing protein models suitable for simulation, while allowing scalability up to entire superfamilies. A particular advantage of this approach can be found in the construction of kinetic models of conformational dynamics-such as Markov state models (MSMs-which benefit from a diverse array of initial configurations that span the accessible conformational states to aid sampling. We demonstrate the power of this approach by constructing models for all catalytic domains in the human

  19. Expression profiling and integrative analysis of the CESA/CSL superfamily in rice

    Directory of Open Access Journals (Sweden)

    Tu Yuanyuan

    2010-12-01

    Full Text Available Abstract Background The cellulose synthase and cellulose synthase-like gene superfamily (CESA/CSL is proposed to encode enzymes for cellulose and non-cellulosic matrix polysaccharide synthesis in plants. Although the rice (Oryza sativa L. genome has been sequenced for a few years, the global expression profiling patterns and functions of the OsCESA/CSL superfamily remain largely unknown. Results A total of 45 identified members of OsCESA/CSL were classified into two clusters based on phylogeny and motif constitution. Duplication events contributed largely to the expansion of this superfamily, with Cluster I and II mainly attributed to tandem and segmental duplication, respectively. With microarray data of 33 tissue samples covering the entire life cycle of rice, fairly high OsCESA gene expression and rather variable OsCSL expression were observed. While some members from each CSL family (A1, C9, D2, E1, F6 and H1 were expressed in all tissues examined, many of OsCSL genes were expressed in specific tissues (stamen and radicles. The expression pattern of OsCESA/CSL and OsBC1L which extensively co-expressed with OsCESA/CSL can be divided into three major groups with ten subgroups, each showing a distinct co-expression in tissues representing typically distinct cell wall constitutions. In particular, OsCESA1, -3 & -8 and OsCESA4, -7 & -9 were strongly co-expressed in tissues typical of primary and secondary cell walls, suggesting that they form as a cellulose synthase complex; these results are similar to the findings in Arabidopsis. OsCESA5/OsCESA6 is likely partially redundant with OsCESA3 for OsCESA complex organization in the specific tissues (plumule and radicle. Moreover, the phylogenetic comparison in rice, Arabidopsis and other species can provide clues for the prediction of orthologous gene expression patterns. Conclusions The study characterized the CESA/CSL of rice using an integrated approach comprised of phylogeny, transcriptional

  20. Thermolabile defect of methylenetetrahydrofolate reductase in coronary artery disease.

    Science.gov (United States)

    Kang, S S; Passen, E L; Ruggie, N; Wong, P W; Sora, H

    1993-10-01

    To determine whether or not a moderate genetic defect of homocysteine metabolism is associated with the development of coronary artery disease, we studied the prevalence of thermolabile methylenetetrahydrofolate reductase, which is probably the most common genetic defect of homocysteine metabolism. Three hundred thirty-nine subjects who underwent coronary angiography were classified into three groups: (1) patients with severe coronary artery stenosis (> or = 70% occlusion in one or more coronary arteries or > or = 50% occlusion in the left main coronary artery), (2) patients with mild to moderate coronary artery stenosis (< 70% occlusion in one or more coronary arteries or < 50% occlusion in the left main coronary artery), and (3) patients with non-coronary heart disease or noncardiac chest pain (nonstenotic coronary arteries). The thermolability of methylenetetrahydrofolate reductase was prospectively determined in all subjects. Plasma homocyst(e)ine levels were then measured in those with thermolabile methylenetetrahydrofolate reductase. The traditional risk factors for coronary artery disease were thereafter ascertained by chart review of all subjects. The prevalence of thermolabile methylenetetrahydrofolate reductase was 18.1% in group 1, 13.4% in group 2, and 7.9% in group 3. There was a significant difference between the prevalence of thermolabile methylenetetrahydrofolate reductase in groups 1 and 3 (P < .04). All individuals with thermolabile methylenetetrahydrofolate reductase irrespective of their clinical grouping had higher plasma homocyst(e)ine levels than normal (group 1, 14.86 +/- 5.85; group 2, 15.36 +/- 5.70; group 3, 13.39 +/- 3.80; normal, 8.50 +/- 2.8 nmol/mL). Nonetheless, there was no statistically significant difference in the plasma homocyst(e)ine concentrations of these patients with or without coronary artery stenosis. Using discriminant function analysis, thermolabile methylenetetrahydrofolate reductase was predictive of angiographically

  1. The role of biliverdin reductase in colorectal cancer

    International Nuclear Information System (INIS)

    Bauer, M.

    2010-01-01

    In recent years, the effects of biliverdin and bilirubin have been studied extensively, and an inhibitory effect of bile pigments in cancer progression has been proposed. In this study we focused on the effects of biliverdin reductase, the enzyme that converts biliverdin to bilirubin, in colorectal cancer. For in vitro experiments we used a human colorectal carcinoma cell line and transfected it with an expression construct of shRNA specific for biliverdin reductase, to create cells with stable knock-down of enzyme expression. Cell proliferation was analyzed using the CASY model TT cell counting device. Western blot protein analysis was performed to study intracellular signaling cascades. Samples of human colorectal cancer were analyzed using immunohistochemistry. We were able to confirm the antiproliferative effects of bile pigments on cancer cells in vitro. However, this effect was attenuated in biliverdin reductase knock down cells. ERK and Akt activation seen under biliverdin and bilirubin treatment was also reduced in biliverdin reductase deficient cells. Immunohistochemical analysis of tumor samples from patients with colorectal cancer showed elevated biliverdin reductase levels. High enzyme expression was associated with lower overall and disease free patient survival. We conclude that BVR is required for bile pigment mediated effects regarding cancer cell proliferation and modulation of intracellular signaling cascades. The role of BVR overexpression in vivo and its exact influence on cancer progression and patient survival need to be further investigated. (author) [de

  2. Phi Class of Glutathione S-transferase Gene Superfamily Widely Exists in Nonplant Taxonomic Groups.

    Science.gov (United States)

    Munyampundu, Jean-Pierre; Xu, You-Ping; Cai, Xin-Zhong

    2016-01-01

    Glutathione S-transferases (GSTs) constitute a superfamily of enzymes involved in detoxification of noxious compounds and protection against oxidative damage. GST class Phi (GSTF), one of the important classes of plant GSTs, has long been considered as plant specific but was recently found in basidiomycete fungi. However, the range of nonplant taxonomic groups containing GSTFs remains unknown. In this study, the distribution and phylogenetic relationships of nonplant GSTFs were investigated. We identified GSTFs in ascomycete fungi, myxobacteria, and protists Naegleria gruberi and Aureococcus anophagefferens. GSTF occurrence in these bacteria and protists correlated with their genome sizes and habitats. While this link was missing across ascomycetes, the distribution and abundance of GSTFs among ascomycete genomes could be associated with their lifestyles to some extent. Sequence comparison, gene structure, and phylogenetic analyses indicated divergence among nonplant GSTFs, suggesting polyphyletic origins during evolution. Furthermore, in silico prediction of functional partners suggested functional diversification among nonplant GSTFs.

  3. Oligonucleotide primers for specific detection of actinobacterial laccases from superfamilies I and K.

    Science.gov (United States)

    Fernandes, Tatiana Alves Rigamonte; da Silveira, Wendel Batista; Passos, Flávia Maria Lopes; Zucchi, Tiago Domingues

    2014-08-01

    Although many putative laccase-like genes have been assigned to members of the phylum Actinobacteria, few of the related enzymes have been characterized so far. It is noteworthy, however, that this small number of enzymes has presented properties with industrial relevance. This observation, combined with the recognized biotechnological potential and the capability of this phylum to degrade recalcitrant soil polymers, has attracted attention for bioprospective approaches. In the present work, we have designed and tested primers that were specific for detection of sub-groups of laccase-like genes within actinomycetes, which corresponded to the superfamilies I and K from the classification presented by the laccase and multicopper oxidase engineering database. The designed primers have amplified laccase-like gene fragments from actinomycete isolates that were undetectable by primers available from the literature. Furthermore, phylogenetic alignments suggest that some of these fragments may belong to new laccases-like proteins, and thus emphasize the benefits of designing subgroup-specific primers.

  4. Evolution of a protein superfamily: relationships between vertebrate lens crystallins and microorganism dormancy proteins.

    Science.gov (United States)

    Wistow, G

    1990-02-01

    A search of sequence databases shows that spherulin 3a, an encystment-specific protein of Physarum polycephalum, is probably structurally related to the beta- and gamma-crystallins, vertebrate ocular lens proteins, and to Protein S, a sporulation-specific protein of Myxococcus xanthus. The beta- and gamma-crystallins have two similar domains thought to have arisen by two successive gene duplication and fusion events. Molecular modeling confirms that spherulin 3a has all the characteristics required to adopt the tertiary structure of a single gamma-crystallin domain. The structure of spherulin 3a thus illustrates an earlier stage in the evolution of this protein superfamily. The relationship of beta- and gamma-crystallins to spherulin 3a and Protein S suggests that the lens proteins were derived from an ancestor with a role in stress-response, perhaps a response to osmotic stress.

  5. The Tumor Necrosis Factor Superfamily of Cytokines in the Inflammatory Myopathies: Potential Targets for Therapy

    Directory of Open Access Journals (Sweden)

    Boel De Paepe

    2012-01-01

    Full Text Available The idiopathic inflammatory myopathies (IM represent a heterogeneous group of autoimmune diseases, of which dermatomyositis (DM, polymyositis (PM, and sporadic inclusion body myositis (IBM are the most common. The crucial role played by tumor necrosis factor alpha (TNFα in the IM has long been recognized. However, so far, 18 other members of the TNF superfamily have been characterized, and many of these have not yet received the attention they deserve. In this paper, we summarize current findings for all TNF cytokines in IM, pinpointing what we know already and where current knowledge fails. For each TNF family member, possibilities for treating inflammatory diseases in general and the IM in particular are explored.

  6. Cell Adhesion Molecules of the Immunoglobulin Superfamily in the Nervous System

    DEFF Research Database (Denmark)

    Walmod, Peter Schledermann; Pedersen, Martin Volmer; Berezin, Vladimir

    2007-01-01

    CAMs belonging to IgSF, that exclusively or in part, are expressed in the nervous system. The chapter includes descriptions of myelin protein zero (P0), integrin-associated protein (CD47), neuroplastin, activated leukocyte-cell adhesion molecule (ALCAM), melanoma cell adhesion molecule (MCAM......Cell adhesion molecules (CAMs) are proteins mediating cell-cell or cell-extracellular matrix (ECM) interactions. CAMs are traditionally divided into four groups, the cadherins, the selectins, the integrins and CAMs belonging to the immunoglobulin superfamily (IgSF). The present chapter describes......), myelinassociated glycoprotein (MAG), the neural cell adhesion molecules 1 and 2 (NCAM, NCAM2), Down Syndrome cell adhesion molecule (DSCAM) and Down Syndrome cell adhesion molecule-like-1 (DSCAML1), sidekick 1 and 2 (SDK1, SDK2), signal-regulatory proteins (SIRPs), nectins, nectin-like proteins (necls...

  7. Neural Cell Adhesion Molecules of the Immunoglobulin Superfamily Regulate Synapse Formation, Maintenance, and Function.

    Science.gov (United States)

    Sytnyk, Vladimir; Leshchyns'ka, Iryna; Schachner, Melitta

    2017-05-01

    Immunoglobulin superfamily adhesion molecules are among the most abundant proteins in vertebrate and invertebrate nervous systems. Prominent family members are the neural cell adhesion molecules NCAM and L1, which were the first to be shown to be essential not only in development but also in synaptic function and as key regulators of synapse formation, synaptic activity, plasticity, and synaptic vesicle recycling at distinct developmental and activity stages. In addition to interacting with each other, adhesion molecules interact with ion channels and cytokine and neurotransmitter receptors. Mutations in their genes are linked to neurological disorders associated with abnormal development and synaptic functioning. This review presents an overview of recent studies on these molecules and their crucial impact on neurological disorders. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. Sequence-based protein superfamily classification using computational intelligence techniques: a review.

    Science.gov (United States)

    Vipsita, Swati; Rath, Santanu Kumar

    2015-01-01

    Protein superfamily classification deals with the problem of predicting the family membership of newly discovered amino acid sequence. Although many trivial alignment methods are already developed by previous researchers, but the present trend demands the application of computational intelligent techniques. As there is an exponential growth in size of biological database, retrieval and inference of essential knowledge in the biological domain become a very cumbersome task. This problem can be easily handled using intelligent techniques due to their ability of tolerance for imprecision, uncertainty, approximate reasoning, and partial truth. This paper discusses the various global and local features extracted from full length protein sequence which are used for the approximation and generalisation of the classifier. The various parameters used for evaluating the performance of the classifiers are also discussed. Therefore, this review article can show right directions to the present researchers to make an improvement over the existing methods.

  9. The CDI toxin of Yersinia kristensenii is a novel bacterial member of the RNase A superfamily

    Energy Technology Data Exchange (ETDEWEB)

    Batot, Gaëlle; Michalska, Karolina; Ekberg, Greg; Irimpan, Ervin M.; Joachimiak, Grazyna; Jedrzejczak, Robert; Babnigg, Gyorgy; Hayes, Christopher S.; Joachimiak, Andrzej; Goulding, Celia W.

    2017-04-10

    Contact-dependent growth inhibition (CDI) is an important mechanism of inter-bacterial competition found in many Gram-negative pathogens. CDI+ cells express cell-surface CdiA proteins that bind neighboring bacteria and deliver C-terminal toxin domains (CdiA-CT) to inhibit target-cell growth. CDI+ bacteria also produce CdiI immunity proteins, which specifically neutralize cognate CdiA-CT toxins to prevent self-inhibition. Here, we present the crystal structure of the CdiA-CT/CdiI(Ykris) complex from Yersinia kris-tensenii ATCC 33638. CdiA-CTYkris adopts the same fold as angiogenin and other RNase A paralogs, but the toxin does not share sequence similarity with these nucleases and lacks the characteristic disulfide bonds of the superfamily. Consistent with the structural homology, CdiA-CTYkris has potent RNase activity in vitro and in vivo. Structure-guided mutagenesis reveals that His175, Arg186, Thr276 and Tyr278 contribute to CdiA-CTYkris activity, suggesting that these residues participate in substrate binding and/or catalysis. CdiI(Ykris) binds directly over the putative active site and likely neutralizes toxicity by blocking access to RNA substrates. Significantly, CdiA-CTYkris is the first non-vertebrate protein found to possess the RNase A superfamily fold, and homologs of this toxin are associated with secretion systems in many Gram-negative and Gram-positive bacteria. These observations suggest that RNase Alike toxins are commonly deployed in inter-bacterial competition.

  10. Pulmonary artery hypertension in childhood: The transforming growth factor-β superfamily-related genes

    Directory of Open Access Journals (Sweden)

    Shi-Min Yuan

    2018-04-01

    Full Text Available Pulmonary artery hypertension (PAH is very rare in childhood, and it can be divided into heritable, idiopathic drug- and toxin-induced and other disease (connective tissue disease, human immunodeficiency virus infection, portal hypertension, congenital heart disease, or schistosomiasis-associated types. PAH could not be interpreted solely by pathophysiological theories. The impact of the transforming growth factor-β superfamily-related genes on the development of PAH in children remains to be clarified. Pertinent literature on the transforming growth factor-β superfamily-related genes in relation to PAH in children published after the year 2000 was reviewed and analyzed. Bone morphogenetic protein receptor type II gene mutation promotes cell division or prevents cell death, resulting in an overgrowth of cells in small arteries throughout the lungs. About 20% of individuals with a bone morphogenetic protein receptor type II gene mutation develop symptomatic PAH. In heritable PAH, bone morphogenetic protein receptor type II mutations may be absent; while mutations of other genes, such as type I receptor activin receptor-like kinase 1 and the type III receptor endoglin (both associated with hereditary hemorrhagic telangiectasia, caveolin-1 and KCNK3, the gene encoding potassium channel subfamily K, member 3, can be detected, instead. Gene mutations, environmental changes and acquired adjustment, etc. may explain the development of PAH. The researches on PAH rat model and familial PAH members may facilitate the elucidations of the mechanisms and further provide theories for prophylaxis and treatment of PAH. Key Words: bone morphogenetic proteins, mutation, pulmonary hypertension

  11. Intramolecular electron transfer in Pseudomonas aeruginosa cd(1) nitrite reductase

    DEFF Research Database (Denmark)

    Farver, Ole; Brunori, Maurizio; Cutruzzolà, Francesca

    2009-01-01

    The cd(1) nitrite reductases, which catalyze the reduction of nitrite to nitric oxide, are homodimers of 60 kDa subunits, each containing one heme-c and one heme-d(1). Heme-c is the electron entry site, whereas heme-d(1) constitutes the catalytic center. The 3D structure of Pseudomonas aeruginosa...... nitrite reductase has been determined in both fully oxidized and reduced states. Intramolecular electron transfer (ET), between c and d(1) hemes is an essential step in the catalytic cycle. In earlier studies of the Pseudomonas stutzeri enzyme, we observed that a marked negative cooperativity...... is controlling this internal ET step. In this study we have investigated the internal ET in the wild-type and His369Ala mutant of P. aeruginosa nitrite reductases and have observed similar cooperativity to that of the Pseudomonas stutzeri enzyme. Heme-c was initially reduced, in an essentially diffusion...

  12. Effect of ammonium and nitrate on ferric chelate reductase and nitrate reductase in Vaccinium species.

    Science.gov (United States)

    Poonnachit, U; Darnell, R

    2004-04-01

    Most Vaccinium species have strict soil requirements for optimal growth, requiring low pH, high iron availability and nitrogen primarily in the ammonium form. These soils are limited and are often located near wetlands. Vaccinium arboreum is a wild species adapted to a wide range of soils, including high pH, low iron, and nitrate-containing soils. This broader soil adaptation in V. arboreum may be related to increased efficiency of iron or nitrate uptake compared with the cultivated Vaccinium species. Nitrate, ammonium and iron uptake, and nitrate reductase (NR) and ferric chelate reductase (FCR) activities were compared in two Vaccinium species grown hydroponically in either nitrate or ammonia, with or without iron. The species studied were the wild V. arboreum and the cultivated V. corymbosum interspecific hybrid, which exhibits the strict soil requirements of most Vaccinium species. Ammonium uptake was significantly greater than nitrate uptake in both species, while nitrate uptake was greater in the wild species, V. arboreum, compared with the cultivated species, V. corymbosum. The increased nitrate uptake in V. arboreum was correlated with increased root NR activity compared with V. corymbosum. The lower nitrate uptake in V. corymbosum was reflected in decreased plant dry weight in this species compared with V. arboreum. Root FCR activity increased significantly in V. corymbosum grown under iron-deficient conditions, compared with the same species grown under iron-sufficient conditions or with V. arboreum grown under either iron condition. V. arboreum appears to be more efficient in acquiring nitrate compared with V. corymbosum, possibly due to increased NR activity and this may partially explain the wider soil adaptation of V. arboreum.

  13. Proanthocyanidin synthesis in Theobroma cacao: genes encoding anthocyanidin synthase, anthocyanidin reductase, and leucoanthocyanidin reductase.

    Science.gov (United States)

    Liu, Yi; Shi, Zi; Maximova, Siela; Payne, Mark J; Guiltinan, Mark J

    2013-12-05

    The proanthocyanidins (PAs), a subgroup of flavonoids, accumulate to levels of approximately 10% total dry weight of cacao seeds. PAs have been associated with human health benefits and also play important roles in pest and disease defense throughout the plant. To dissect the genetic basis of PA biosynthetic pathway in cacao (Theobroma cacao), we have isolated three genes encoding key PA synthesis enzymes, anthocyanidin synthase (ANS), anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR). We measured the expression levels of TcANR, TcANS and TcLAR and PA content in cacao leaves, flowers, pod exocarp and seeds. In all tissues examined, all three genes were abundantly expressed and well correlated with PA accumulation levels, suggesting their active roles in PA synthesis. Overexpression of TcANR in an Arabidopsis ban mutant complemented the PA deficient phenotype in seeds and resulted in reduced anthocyanidin levels in hypocotyls. Overexpression of TcANS in tobacco resulted in increased content of both anthocyanidins and PAs in flower petals. Overexpression of TcANS in an Arabidopsis ldox mutant complemented its PA deficient phenotype in seeds. Recombinant TcLAR protein converted leucoanthocyanidin to catechin in vitro. Transgenic tobacco overexpressing TcLAR had decreased amounts of anthocyanidins and increased PAs. Overexpressing TcLAR in Arabidopsis ldox mutant also resulted in elevated synthesis of not only catechin but also epicatechin. Our results confirm the in vivo function of cacao ANS and ANR predicted based on sequence homology to previously characterized enzymes from other species. In addition, our results provide a clear functional analysis of a LAR gene in vivo.

  14. Expression and site-directed mutagenesis of human dihydrofolate reductase

    Energy Technology Data Exchange (ETDEWEB)

    Prendergast, N.J.; Delcamp, T.J.; Smith, P.L.; Freisheim, J.H.

    1988-05-17

    A procaryotic high-level expression vector for human dihydrofolate reductase has been constructed and the protein characterized as a first step toward structure-function studies of this enzyme. A vector bearing the tac promoter, four synthetic oligodeoxynucleotides, and a restriction fragment from the dihydrofolate reductase cDNA were ligated in a manner which optimized the transcriptional and translational frequency of the enzyme mRNA. The reductase, comprising ca. 17% of the total soluble protein in the host bacteria, was purified to apparent homogeneity as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and characterized by amino acid composition, partial amino acid sequence, and steady-sate kinetic analysis. This expression vector has been used as a template for double-stranded plasmid DNA site-specific mutagenesis. Functional studies on a Cys-6 ..-->.. Ser-6 mutant enzyme support the contention that Cys-6 is obligatory for organomercurial activation of human dihydrofolate reductase. The Ser-6 mutant enzyme was not activated to any extent following a 24-h incubation with p-(hydroxymercuri)benzoate and nicotinamide adenine dinucleotide phosphate (reduced) (NADPH), whereas the k/sub cat/ for Cys-6 reductase increased 2-fold under identical conditions. The specific activities of the Cys-6 and Ser-6 enzymes were virtually identical as determined by methotrexate titration as were the K/sub m/ values for both dihydrofolate and NADPH. The Ser-6 mutant showed a decreased temperature stability and was more sensitive to inactivation by ..cap alpha..-chymotrypsin when compared to the wild-type enzyme. These results suggest that the Ser-6 mutant reductase is conformationally altered relative to the Cys-6 native enzyme.

  15. The Glutathione-S-Transferase, Cytochrome P450 and Carboxyl/Cholinesterase Gene Superfamilies in Predatory Mite Metaseiulus occidentalis.

    Directory of Open Access Journals (Sweden)

    Ke Wu

    Full Text Available Pesticide-resistant populations of the predatory mite Metaseiulus (= Typhlodromus or Galendromus occidentalis (Arthropoda: Chelicerata: Acari: Phytoseiidae have been used in the biological control of pest mites such as phytophagous Tetranychus urticae. However, the pesticide resistance mechanisms in M. occidentalis remain largely unknown. In other arthropods, members of the glutathione-S-transferase (GST, cytochrome P450 (CYP and carboxyl/cholinesterase (CCE gene superfamilies are involved in the diverse biological pathways such as the metabolism of xenobiotics (e.g. pesticides in addition to hormonal and chemosensory processes. In the current study, we report the identification and initial characterization of 123 genes in the GST, CYP and CCE superfamilies in the recently sequenced M. occidentalis genome. The gene count represents a reduction of 35% compared to T. urticae. The distribution of genes in the GST and CCE superfamilies in M. occidentalis differs significantly from those of insects and resembles that of T. urticae. Specifically, we report the presence of the Mu class GSTs, and the J' and J" clade CCEs that, within the Arthropoda, appear unique to Acari. Interestingly, the majority of CCEs in the J' and J" clades contain a catalytic triad, suggesting that they are catalytically active. They likely represent two Acari-specific CCE clades that may participate in detoxification of xenobiotics. The current study of genes in these superfamilies provides preliminary insights into the potential molecular components that may be involved in pesticide metabolism as well as hormonal/chemosensory processes in the agriculturally important M. occidentalis.

  16. HLA superfamily assignment is a predictor of immune response to cancer testis antigens and survival in ovarian cancer.

    Science.gov (United States)

    Szender, J Brian; Eng, Kevin H; Matsuzaki, Junko; Miliotto, Anthony; Gnjatic, Sacha; Tsuji, Takemasa; Odunsi, Kunle

    2016-07-01

    To characterize the association between major histocompatibility complex (MHC) types and spontaneous antibody development to the cancer testis (CT) antigen NY-ESO-1. Tumor expression of NY-ESO-1 and serum antibodies to NY-ESO-1 were characterized in addition to human leukocyte antigen (HLA) type for patients with epithelial ovarian cancer. HLA types were assigned to structure-based superfamilies and statistical associations were examined. HLA types were compared to existing reference libraries of HLA frequencies in a European-Caucasian American population. Out of 126 patients identified, 81% were expression positive and 48% had spontaneous antibody responses to NY-ESO-1. There was an association between HLA-B superfamily and seropositivity among patients with tumors expressing NY-ESO-1 (pHLA-B superfamily assignment were driven by HLA-B44. Among all patients, the B27 superfamily was over-represented compared with the general population (pHLA type appears to be associated with spontaneous anti-CT antigen antibodies, as well as with the overall risk of ovarian cancer. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. Oribatid Mites of the Superfamilies Gymnodamaeoidea and Plater emaeoidea (Acari: Oribatida fr om S teppe of Russia

    Directory of Open Access Journals (Sweden)

    Badamdorj Bayartogtokh

    2004-06-01

    Full Text Available Oribatid mites belonging to the superfamilies Gymnodamaeoidea and Plateremaeoidea collected from steppe soils of Russia are studied. Two new species, Pedrocortesella minuta sp. nov . and Pleodamaeus tuberculatus sp. nov . are described. In addition, three known species, Licnodamaeus pulcherrimus (Paoli, 1908 and Plesiodamaeus glaber Mihelèiè, 1957 are r edescribed, with notes on their distributions.

  18. The chemical versatility of the beta-alpha-beta fold : Catalytic promiscuity and divergent evolution in the tautomerase superfamily

    NARCIS (Netherlands)

    Poelarends, G. J.; Veetil, V. Puthan; Whitman, C. P.

    2008-01-01

    Tautomerase superfamily members have an amino-terminal proline and a beta-alpha-beta fold, and include 4-oxalocrotonate tautomerase (4-OT), 5-(carboxymethyl)-2-hydroxymuconate isomerase (CHMI), trans- and cis-3-chloroacrylic acid dehalogenase (CaaD and cis-CaaD, respectively), malonate semialdehyde

  19. Plasmid-encoded diacetyl (acetoin) reductase in Leuconostoc pseudomesenteroides

    DEFF Research Database (Denmark)

    Rattray, Fergal P; Myling-Petersen, Dorte; Larsen, Dianna

    2003-01-01

    A plasmid-borne diacetyl (acetoin) reductase (butA) from Leuconostoc pseudomesenteroides CHCC2114 was sequenced and cloned. Nucleotide sequence analysis revealed an open reading frame encoding a protein of 257 amino acids which had high identity at the amino acid level to diacetyl (acetoin...... diacetyl (acetoin) reductase activity with NADH as coenzyme, but not with NADPH as coenzyme, suggesting the presence of another diacetyl (acetoin)-reducing activity in L. pseudomesenteroides. Plasmid-curing experiments demonstrated that the butA gene is carried on a 20-kb plasmid in L. pseudomesenteroides....

  20. Compensatory periplasmic nitrate reductase activity supports anaerobic growth of Pseudomonas aeruginosa PAO1 in the absence of membrane nitrate reductase

    Science.gov (United States)

    Van Alst, Nadine E.; Sherrill, Lani A.; Iglewski, Barbara H.; Haidaris, Constantine G.

    2009-01-01

    Nitrate serves as a terminal electron acceptor under anaerobic conditions in Pseudomonas aeruginosa. Reduction of nitrate to nitrite generates a transmembrane proton motive force allowing ATP synthesis and anaerobic growth. Inner membrane-bound nitrate reductase NarGHI is encoded within the narK1K2GHJI operon and the periplasmic nitrate reductase NapAB is encoded within the napEFDABC operon. The role of the two dissimilatory nitrate reductases in anaerobic growth, and the regulation of their expression were examined by using a set of deletion mutants in P. aeruginosa PAO1. NarGHI mutants were unable to grow anaerobically, but plate cultures remained viable up to 120 hr. In contrast, nitrate sensor-response regulator mutant ΔnarXL displayed growth arrest initially, but resumed growth after 72 hr and reached early stationary phase in liquid culture after 120 hr. Genetic, transcriptional, and biochemical studies demonstrated that anaerobic growth recovery by the NarXL mutant was the result of NapAB periplasmic nitrate reductase expression. A novel transcriptional start site for napEFDABC expression was identified in the NarXL mutant grown anaerobically. Furthermore, mutagenesis of a consensus NarL-binding site monomer upstream of the novel transcriptional start site restored anaerobic growth recovery in the NarXL mutant. The data suggest that during anaerobic growth of wild type P. aeruginosa PAO1, nitrate response regulator NarL directly represses expression of periplasmic nitrate reductase, while inducing maximal expression of membrane nitrate reductase. PMID:19935885

  1. Optimum conditions for cotton nitrate reductase extraction and ...

    African Journals Online (AJOL)

    Conditions of nitrate reductase extraction and activity measurement should be adapted to plant species, and to the organs of the same plant, because of extreme weaknesses and instabilities of the enzyme. Different extraction and reaction media have been compared in order to define the best conditions for cotton callus ...

  2. Intraethnic variation in steroid-5-alpha-reductase polymorphisms in ...

    Indian Academy of Sciences (India)

    2015-06-01

    Jun 1, 2015 ... in prostate cancer patients: a potential factor implicated in 5-alpha-reductase inhibitor treatment. LUIS ALBERTO HENRÍQUEZ-HERNÁNDEZ1,2,3∗, ALMUDENA VALENCIANO2, PALMIRA FORO-ARNALOT4,. MARÍA JESÚS ÁLVAREZ-CUBERO5,6, JOSÉ MANUEL COZAR7, JOSÉ FRANCISCO ...

  3. Intraethnic variation in steroid-5-alpha-reductase polymorphisms in ...

    Indian Academy of Sciences (India)

    2015-06-01

    Jun 1, 2015 ... Herrera-Ramos E., Rodríguez-Gallego C. and Lara P. C. 2015 Intraethnic variation in steroid-5-alpha-reductase polymorphisms in prostate ... generation. This study was approved by the Research and. Ethics Committee of each institution participant in the study. DNA was isolated from 300 µL of ...

  4. Bioinformatic analysis of dihydrofolate reductase predicted in the ...

    African Journals Online (AJOL)

    ... 5 alpha helices and 8 beta-strands. Twelve binding site residues were predicted in KCA1_1610 relative to the template protein 2zzaA in protein database (PDB). The predicted structure of KCA1_1610 dihydrofolate reductase can serve as a new template as an addition to structural genomics and generation of models for ...

  5. Dizygotic twinning is not associated with methylenetetrahydrofolate reductase haplotypes

    NARCIS (Netherlands)

    Montgomery, GW; Zhao, Z.Z.; Morley, K.I.; Marsh, A.J.; Boomsma, D.I.; Martin, N.G.; Duffy, DL

    2003-01-01

    Background: Folate metabolism is critical to embryonic development, influencing neural tube defects (NTD) and recurrent early pregnancy loss. Polymorphisms in 5,10-methylenetetrahydrofolate reductase (MTHFR) have been associated with dizygotic (DZ) twinning through pregnancy loss. Methods: The C677T

  6. Optimum conditions for cotton nitrate reductase extraction and ...

    African Journals Online (AJOL)

    GREGO

    mM of glutamine in the extraction buffer stimulates significantly, in vitro, the reduction of nitrate. Enzyme activity is moreover optimal when 1 M of exogenous nitrate, as substrate, is added to the reaction medium. At these optimum conditions of nitrate reductase activity determination, the substrate was completely reduced ...

  7. Cloning and expression analysis of dihydroxyflavonol 4-reductase ...

    African Journals Online (AJOL)

    Southern blot analysis indicate that DFR is presented as a single copy in the Ascocenda spp. genome. The AscoDFR gene was highly expressed in the flower stages 2 and 3 of development as well as in the sepal and petal of the orchid flower. Keywords: Orchid, dihydroxyflavonol 4-reductase, anthocyanins, gene cloning ...

  8. Crystallographic analysis of tricolosan bound to enoyl reductase.

    NARCIS (Netherlands)

    Roujeinikova, A.; Levy, C.W.; Rowsell, S.; Sedelnikova, S.; Baker, P.J.; Minshull, C.A.; Mistry, A.; Colls, J.G.; Camble, R.; Stuitje, A.R.; Slabas, A.R.; Rafferty, J.B.; Pauptit, R.A.; Viner, A; Rice, D.W.

    1999-01-01

    Molecular genetic studies with strains of Escherichia coli resistant to triclosan, an ingredient of many anti-bacterial household goods, have suggested that this compound works by acting as an inhibitor of enoyl reductase (ENR) and thereby blocking lipid biosynthesis. We present structural analyses

  9. Molecular Cloning and Expression of Bacterial Mercuric Reductase ...

    African Journals Online (AJOL)

    USER

    2010-06-21

    Jun 21, 2010 ... In order to characterize the bacterial mercuric reductase (merA) gene, mercury resistant (Hgr). Escherichia coli strains have been isolated from various mercury contaminated sites of India. Their minimum inhibitory concentration (MIC) for Hg and zone of inhibition for different antibiotics were measured, and ...

  10. Aldose reductase inhibitory activity and antioxidant capacity of pomegranate extracts.

    Science.gov (United States)

    Karasu, Cimen; Cumaoğlu, Ahmet; Gürpinar, Ali Rifat; Kartal, Murat; Kovacikova, Lucia; Milackova, Ivana; Stefek, Milan

    2012-03-01

    The pomegranate, Punica granatum L., has been the subject of current interest as a medicinal agent with wide-ranging therapeutic indications. In the present study, pomegranate ethanolic seed and hull extracts were tested, in comparison with a commercial sample, for the inhibition of aldose reductase, an enzyme involved in the etiology of diabetic complications. In vitro inhibition of rat lens aldose reductase was determined by a conventional method. Pomegranate ethanolic hull extract and commercial pomegranate hull extract exhibited similar aldose reductase inhibitory activity characterized by IC(50) values ranging from 3 to 33.3 μg/ml. They were more effective than pomegranate ethanolic seed extract with IC(50) ranging from 33.3 to 333 μg/ml. Antioxidant action of the novel compounds was documented in a DPPH test and in a liposomal membrane model, oxidatively stressed by peroxyl radicals. All the plant extracts showed considerable antioxidant potential in the DPPH assay. Pomegranate ethanolic hull extract and commercial pomegranate hull extract executed similar protective effects on peroxidatively damaged liposomal membranes characterized by 10ethanolic seed extract showed significantly lower antioxidant activity compared to both hull extracts studied. Pomegranate extracts are thus presented as bifunctional agents combining aldose reductase inhibitory action with antioxidant activity and with potential therapeutic use in prevention of diabetic complications.

  11. Transcriptional modulation of genes encoding nitrate reductase in ...

    African Journals Online (AJOL)

    2016-10-26

    Oct 26, 2016 ... The free aluminum (Al) content in soil can reach levels that are toxic to plants, and this has frequently limited increased productivity of cultures. Four genes encoding nitrate reductase (NR) were identified, named ZmNR1–4. With the aim of evaluating NR activity and the transcriptional modulation of the.

  12. Transcriptional modulation of genes encoding nitrate reductase in ...

    African Journals Online (AJOL)

    The free aluminum (Al) content in soil can reach levels that are toxic to plants, and this has frequently limited increased productivity of cultures. Four genes encoding nitrate reductase (NR) were identified, named ZmNR1–4. With the aim of evaluating NR activity and the transcriptional modulation of the ZmNR1, ZmNR2, ...

  13. Evaluation of the conserve flavin reductase gene from three ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-12-15

    Dec 15, 2009 ... means of PCR technique. The nucleic acid sequences of the PCR primers were designed using conserved nucleic acid sequences of the flavin reductase enzyme from. Rhodococcus sp. strain IGTS8. The oligonucleotide primers were as follows: 5'-GAA TTC ATG TCT GAC. AAG CCG AAT GCC-3' (forward) ...

  14. Dihydrofolate reductase: A potential drug target in trypanosomes and leishmania

    Science.gov (United States)

    Zuccotto, Fabio; Martin, Andrew C. R.; Laskowski, Roman A.; Thornton, Janet M.; Gilbert, Ian H.

    1998-05-01

    Dihydrofolate reductase has successfully been used as a drug target in the area of anti-cancer, anti-bacterial and anti-malarial chemotherapy. Little has been done to evaluate it as a drug target for treatment of the trypanosomiases and leishmaniasis. A crystal structure of Leishmania major dihydrofolate reductase has been published. In this paper, we describe the modelling of Trypanosoma cruzi and Trypanosoma brucei dihydrofolate reductases based on this crystal structure. These structures and models have been used in the comparison of protozoan, bacterial and human enzymes in order to highlight the different features that can be used in the design of selective anti-protozoan agents. Comparison has been made between residues present in the active site, the accessibility of these residues, charge distribution in the active site, and the shape and size of the active sites. Whilst there is a high degree of similarity between protozoan, human and bacterial dihydrofolate reductase active sites, there are differences that provide potential for selective drug design. In particular, we have identified a set of residues which may be important for selective drug design and identified a larger binding pocket in the protozoan than the human and bacterial enzymes.

  15. The intramolecular electron transfer between copper sites of nitrite reductase

    DEFF Research Database (Denmark)

    Farver, O; Eady, R R; Abraham, Z H

    1998-01-01

    The intramolecular electron transfer (ET) between the type 1 Cu(I) and the type 2 Cu(II) sites of Alcaligenes xylosoxidans dissimilatory nitrite reductase (AxNiR) has been studied in order to compare it with the analogous process taking place in ascorbate oxidase (AO). This internal process is in...

  16. Molecular Cloning and Expression of Bacterial Mercuric Reductase ...

    African Journals Online (AJOL)

    In order to characterize the bacterial mercuric reductase (merA) gene, mercury resistant (Hgr) Escherichia coli strains have been isolated from various mercury contaminated sites of India. Their minimum inhibitory concentration (MIC) for Hg and zone of inhibition for different antibiotics were measured, and finally mer operon ...

  17. The Polymorphisms in Methylenetetrahydrofolate Reductase, Methionine Synthase, Methionine Synthase Reductase, and the Risk of Colorectal Cancer

    Science.gov (United States)

    Zhou, Daijun; Mei, Qiang; Luo, Han; Tang, Bo; Yu, Peiwu

    2012-01-01

    Polymorphisms in genes involved in folate metabolism may modulate the risk of colorectal cancer (CRC), but data from published studies are conflicting. The current meta-analysis was performed to address a more accurate estimation. A total of 41 (17,552 cases and 26,238 controls), 24(8,263 cases and 12,033 controls), 12(3,758 cases and 5,646 controls), and 13 (5,511 cases and 7,265 controls) studies were finally included for the association between methylenetetrahydrofolate reductase (MTHFR) C677T and A1289C, methione synthase reductase (MTRR) A66G, methionine synthase (MTR) A2756G polymorphisms and the risk of CRC, respectively. The data showed that the MTHFR 677T allele was significantly associated with reduced risk of CRC (OR = 0.93, 95%CI 0.90-0.96), while the MTRR 66G allele was significantly associated with increased risk of CRC (OR = 1.11, 95%CI 1.01-1.18). Sub-group analysis by ethnicity revealed that MTHFR C677T polymorphism was significantly associated with reduced risk of CRC in Asians (OR = 0.80, 95%CI 0.72-0.89) and Caucasians (OR = 0.84, 95%CI 0.76-0.93) in recessive genetic model, while the MTRR 66GG genotype was found to significantly increase the risk of CRC in Caucasians (GG vs. AA: OR = 1.18, 95%CI 1.03-1.36). No significant association was found between MTHFR A1298C and MTR A2756G polymorphisms and the risk of CRC. Cumulative meta-analysis showed no particular time trend existed in the summary estimate. Probability of publication bias was low across all comparisons illustrated by the funnel plots and Egger's test. Collectively, this meta-analysis suggested that MTHFR 677T allele might provide protection against CRC in worldwide populations, while MTRR 66G allele might increase the risk of CRC in Caucasians. Since potential confounders could not be ruled out completely, further studies were needed to confirm these results. PMID:22719222

  18. Identification of 5α-reductase isoenzymes in canine skin.

    Science.gov (United States)

    Bernardi de Souza, Lucilene; Paradis, Manon; Zamberlam, Gustavo; Benoit-Biancamano, Marie-Odile; Price, Christopher

    2015-10-01

    Alopecia X in dogs is a noninflammatory alopecia that may be caused by a hormonal dysfunction. It may be similar to androgenic alopecia in men that is caused by the effect of dihydrotestosterone (DHT). The 5α-reductase isoenzymes, 5αR1 and 5αR2, and a recently described 5αR3, are responsible for the conversion of testosterone into DHT. However, which 5α-reductases are present in canine skin has not yet been described. The main objective of this study was to determine the pattern of expression of 5α-reductase genes in canine skin. Skin biopsies were obtained from healthy, intact young-mature beagles (three males, four females) at three anatomical sites normally affected by alopecia X (dorsal neck, back of thighs and base of tail) and two sites generally unaffected (dorsal head and ventral thorax). Prostate samples (n = 3) were collected as positive controls for 5α-reductase mRNA abundance measurement by real-time PCR. We detected mRNA encoding 5αR1 and 5αR3 but not 5αR2. There were no significant differences in 5αR1 and 5αR3 mRNA levels between the different anatomical sites, irrespective of gender (P > 0.05). Moreover, the mean mRNA abundance in each anatomical site did not differ between males and females (P > 0.05). To the best of the authors' knowledge, this is the first study demonstrating the expression of 5α-reductases in canine skin and the expression of 5αR3 in this tissue. These results may help to elucidate the pathogenesis of alopecia X and to determine more appropriate treatments for this disorder. © 2015 ESVD and ACVD.

  19. Crystal structures of pinoresinol-lariciresinol and phenylcoumaran benzylic ether reductases and their relationship to isoflavone reductases

    Science.gov (United States)

    Min, Tongpil; Kasahara, Hiroyuki; Bedgar, Diana L.; Youn, Buhyun; Lawrence, Paulraj K.; Gang, David R.; Halls, Steven C.; Park, HaJeung; Hilsenbeck, Jacqueline L.; Davin, Laurence B.; hide

    2003-01-01

    Despite the importance of plant lignans and isoflavonoids in human health protection (e.g. for both treatment and prevention of onset of various cancers) as well as in plant biology (e.g. in defense functions and in heartwood development), systematic studies on the enzymes involved in their biosynthesis have only recently begun. In this investigation, three NADPH-dependent aromatic alcohol reductases were comprehensively studied, namely pinoresinol-lariciresinol reductase (PLR), phenylcoumaran benzylic ether reductase (PCBER), and isoflavone reductase (IFR), which are involved in central steps to the various important bioactive lignans and isoflavonoids. Of particular interest was in determining how differing regio- and enantiospecificities are achieved with the different enzymes, despite each apparently going through similar enone intermediates. Initially, the three-dimensional x-ray crystal structures of both PLR_Tp1 and PCBER_Pt1 were solved and refined to 2.5 and 2.2 A resolutions, respectively. Not only do they share high gene sequence similarity, but their structures are similar, having a continuous alpha/beta NADPH-binding domain and a smaller substrate-binding domain. IFR (whose crystal structure is not yet obtained) was also compared (modeled) with PLR and PCBER and was deduced to have the same overall basic structure. The basis for the distinct enantio-specific and regio-specific reactions of PCBER, PLR, and IFR, as well as the reaction mechanism and participating residues involved (as identified by site-directed mutagenesis), are discussed.

  20. Triterpenes and meroterpenes from Ganoderma lucidum with inhibitory activity against HMGs reductase, aldose reductase and α-glucosidase.

    Science.gov (United States)

    Chen, Baosong; Tian, Jin; Zhang, Jinjin; Wang, Kai; Liu, Li; Yang, Bo; Bao, Li; Liu, Hongwei

    2017-07-01

    Seven new compounds including four lanostane triterpenoids, lucidenic acids Q-S (1-3) and methyl ganoderate P (4), and three triterpene-farnesyl hydroquinone conjugates, ganolucinins A-C (5-7), one new natural product ganomycin J (8), and 73 known compounds (9-81) were isolated from fruiting bodies of Ganoderma lucidum. The structures of the compounds 1-8 were determined by spectroscopic methods. Bioactivities of compounds isolated were assayed against HMG-CoA reductase, aldose reductase, α-glucosidase, and PTP1B. Ganolucidic acid η (39), ganoderenic acid K (44), ganomycin J (8), and ganomycin B (61) showed strong inhibitory activity against HMG-CoA reductase with IC 50 of 29.8, 16.5, 30.3 and 14.3μM, respectively. Lucidumol A (67) had relatively good effect against aldose reductase with IC 50 of 19.1μM. Farnesyl hydroquinones ganomycin J (8), ganomycin B (61), ganomycin I (62), and triterpene-farnesyl hydroquinone conjugates ganoleuconin M (76) and ganoleuconin O (79) possessed good inhibitory activity against α-glucosidase with IC 50 in the range of 7.8 to 21.5μM. This work provides chemical and biological evidence for the usage of extracts of G. lucidum as herbal medicine and food supplements for the control of hyperglycemic and hyperlipidemic symptoms. Copyright © 2017. Published by Elsevier B.V.

  1. Recombinant pinoresinol/lariciresinol reductase, recombinant dirigent protein, and methods of use

    Science.gov (United States)

    Lewis, Norman G.; Davin, Laurence B.; Dinkova-Kostova, Albena T.; Fujita, Masayuki; Gang, David R.; Sarkanen, Simo; Ford, Joshua D.

    2001-04-03

    Dirigent proteins and pinoresinol/lariciresinol reductases have been isolated, together with cDNAs encoding dirigent proteins and pinoresinol/lariciresinol reductases. Accordingly, isolated DNA sequences are provided which code for the expression of dirigent proteins and pinoresinol/lariciresinol reductases. In other aspects, replicable recombinant cloning vehicles are provided which code for dirigent proteins or pinoresinol/lariciresinol reductases or for a base sequence sufficiently complementary to at least a portion of dirigent protein or pinoresinol/lariciresinol reductase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding dirigent protein or pinoresinol/lariciresinol reductase. Thus, systems and methods are provided for the recombinant expression of dirigent proteins and/or pinoresinol/lariciresinol reductases.

  2. Radical SAM, A Novel Protein Superfamily Linking Unresolved Steps in Familiar Biosynthetic Pathways with Radical Mechanisms: Functional Characterization Using New Analysis and Information Visualization Methods

    Energy Technology Data Exchange (ETDEWEB)

    Sofia, Heidi J.; Chen, Guang; Hetzler, Elizabeth G.; Reyes Spindola, Jorge F.; Miller, Nancy E.

    2001-03-01

    A large protein superfamily with over 500 members has been discovered and analyzed using powerful new bioinformatics and information visualization methods. Evidence exists that these proteins generate a 5?-deoxyadenosyl radical by reductive cleavage of S-adenosylmethionine (SAM) through an unusual Fe-S center. Radical SAM superfamily proteins function in DNA precursor, vitamin, cofactor, antibiotic, and herbicide biosynthesis in a collection of basic and familiar pathways. One of the members is interferon-inducible and is considered a candidate drug target for osteoporosis. The identification of this superfamily suggests that radical-based catalysis is important in a number of previously well-studied but unresolved biochemical pathways.

  3. Evolution of the B3 DNA binding superfamily: new insights into REM family gene diversification.

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    Elisson A C Romanel

    Full Text Available BACKGROUND: The B3 DNA binding domain includes five families: auxin response factor (ARF, abscisic acid-insensitive3 (ABI3, high level expression of sugar inducible (HSI, related to ABI3/VP1 (RAV and reproductive meristem (REM. The release of the complete genomes of the angiosperm eudicots Arabidopsis thaliana and Populus trichocarpa, the monocot Orysa sativa, the bryophyte Physcomitrella patens,the green algae Chlamydomonas reinhardtii and Volvox carteri and the red algae Cyanidioschyzon melorae provided an exceptional opportunity to study the evolution of this superfamily. METHODOLOGY: In order to better understand the origin and the diversification of B3 domains in plants, we combined comparative phylogenetic analysis with exon/intron structure and duplication events. In addition, we investigated the conservation and divergence of the B3 domain during the origin and evolution of each family. CONCLUSIONS: Our data indicate that showed that the B3 containing genes have undergone extensive duplication events, and that the REM family B3 domain has a highly diverged DNA binding. Our results also indicate that the founding member of the B3 gene family is likely to be similar to the ABI3/HSI genes found in C. reinhardtii and V. carteri. Among the B3 families, ABI3, HSI, RAV and ARF are most structurally conserved, whereas the REM family has experienced a rapid divergence. These results are discussed in light of their functional and evolutionary roles in plant development.

  4. Prevertebrate Local Gene Duplication Facilitated Expansion of the Neuropeptide GPCR Superfamily.

    Science.gov (United States)

    Yun, Seongsik; Furlong, Michael; Sim, Mikang; Cho, Minah; Park, Sumi; Cho, Eun Bee; Reyes-Alcaraz, Arfaxad; Hwang, Jong-Ik; Kim, Jaebum; Seong, Jae Young

    2015-11-01

    In humans, numerous genes encode neuropeptides that comprise a superfamily of more than 70 genes in approximately 30 families and act mainly through rhodopsin-like G protein-coupled receptors (GPCRs). Two rounds of whole-genome duplication (2R WGD) during early vertebrate evolution greatly contributed to proliferation within gene families; however, the mechanisms underlying the initial emergence and diversification of these gene families before 2R WGD are largely unknown. In this study, we analyzed 25 vertebrate rhodopsin-like neuropeptide GPCR families and their cognate peptides using phylogeny, synteny, and localization of these genes on reconstructed vertebrate ancestral chromosomes (VACs). Based on phylogeny, these GPCR families can be divided into five distinct clades, and members of each clade tend to be located on the same VACs. Similarly, their neuropeptide gene families also tend to reside on distinct VACs. Comparison of these GPCR genes with those of invertebrates including Drosophila melanogaster, Caenorhabditis elegans, Branchiostoma floridae, and Ciona intestinalis indicates that these GPCR families emerged through tandem local duplication during metazoan evolution prior to 2R WGD. Our study describes a presumptive evolutionary mechanism and development pathway of the vertebrate rhodopsin-like GPCR and cognate neuropeptide families from the urbilaterian ancestor to modern vertebrates. © The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  5. The Role of Immunoglobulin Superfamily Cell Adhesion Molecules in Cancer Metastasis

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    Chee Wai Wong

    2012-01-01

    Full Text Available Metastasis is a major clinical problem and results in a poor prognosis for most cancers. The metastatic pathway describes the process by which cancer cells give rise to a metastatic lesion in a new tissue or organ. It consists of interconnecting steps all of which must be successfully completed to result in a metastasis. Cell-cell adhesion is a key aspect of many of these steps. Adhesion molecules belonging to the immunoglobulin superfamily (Ig-SF commonly play a central role in cell-cell adhesion, and a number of these molecules have been associated with cancer progression and a metastatic phenotype. Surprisingly, the contribution of Ig-SF members to metastasis has not received the attention afforded other cell adhesion molecules (CAMs such as the integrins. Here we examine the steps in the metastatic pathway focusing on how the Ig-SF members, melanoma cell adhesion molecule (MCAM, L1CAM, neural CAM (NCAM, leukocyte CAM (ALCAM, intercellular CAM-1 (ICAM-1 and platelet endothelial CAM-1 (PECAM-1 could play a role. Although much remains to be understood, this review aims to raise the profile of Ig-SF members in metastasis formation and prompt further research that could lead to useful clinical outcomes.

  6. Correlated Mutation in the Evolution of Catalysis in Uracil DNA Glycosylase Superfamily

    Science.gov (United States)

    Xia, Bo; Liu, Yinling; Guevara, Jose; Li, Jing; Jilich, Celeste; Yang, Ye; Wang, Liangjiang; Dominy, Brian N.; Cao, Weiguo

    2017-04-01

    Enzymes in Uracil DNA glycosylase (UDG) superfamily are essential for the removal of uracil. Family 4 UDGa is a robust uracil DNA glycosylase that only acts on double-stranded and single-stranded uracil-containing DNA. Based on mutational, kinetic and modeling analyses, a catalytic mechanism involving leaving group stabilization by H155 in motif 2 and water coordination by N89 in motif 3 is proposed. Mutual Information analysis identifies a complexed correlated mutation network including a strong correlation in the EG doublet in motif 1 of family 4 UDGa and in the QD doublet in motif 1 of family 1 UNG. Conversion of EG doublet in family 4 Thermus thermophilus UDGa to QD doublet increases the catalytic efficiency by over one hundred-fold and seventeen-fold over the E41Q and G42D single mutation, respectively, rectifying the strong correlation in the doublet. Molecular dynamics simulations suggest that the correlated mutations in the doublet in motif 1 position the catalytic H155 in motif 2 to stabilize the leaving uracilate anion. The integrated approach has important implications in studying enzyme evolution and protein structure and function.

  7. Relative Stabilities of Conserved and Non-Conserved Structures in the OB-Fold Superfamily

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    Andrei T. Alexandrescu

    2009-05-01

    Full Text Available The OB-fold is a diverse structure superfamily based on a β-barrel motif that is often supplemented with additional non-conserved secondary structures. Previous deletion mutagenesis and NMR hydrogen exchange studies of three OB-fold proteins showed that the structural stabilities of sites within the conserved β-barrels were larger than sites in non-conserved segments. In this work we examined a database of 80 representative domain structures currently classified as OB-folds, to establish the basis of this effect. Residue-specific values were obtained for the number of Cα-Cα distance contacts, sequence hydrophobicities, crystallographic B-factors, and theoretical B-factors calculated from a Gaussian Network Model. All four parameters point to a larger average flexibility for the non-conserved structures compared to the conserved β-barrels. The theoretical B-factors and contact densities show the highest sensitivity.Our results suggest a model of protein structure evolution in which novel structural features develop at the periphery of conserved motifs. Core residues are more resistant to structural changes during evolution since their substitution would disrupt a larger number of interactions. Similar factors are likely to account for the differences in stability to unfolding between conserved and non-conserved structures.

  8. A unique uracil-DNA binding protein of the uracil DNA glycosylase superfamily.

    Science.gov (United States)

    Sang, Pau Biak; Srinath, Thiruneelakantan; Patil, Aravind Goud; Woo, Eui-Jeon; Varshney, Umesh

    2015-09-30

    Uracil DNA glycosylases (UDGs) are an important group of DNA repair enzymes, which pioneer the base excision repair pathway by recognizing and excising uracil from DNA. Based on two short conserved sequences (motifs A and B), UDGs have been classified into six families. Here we report a novel UDG, UdgX, from Mycobacterium smegmatis and other organisms. UdgX specifically recognizes uracil in DNA, forms a tight complex stable to sodium dodecyl sulphate, 2-mercaptoethanol, urea and heat treatment, and shows no detectable uracil excision. UdgX shares highest homology to family 4 UDGs possessing Fe-S cluster. UdgX possesses a conserved sequence, KRRIH, which forms a flexible loop playing an important role in its activity. Mutations of H in the KRRIH sequence to S, G, A or Q lead to gain of uracil excision activity in MsmUdgX, establishing it as a novel member of the UDG superfamily. Our observations suggest that UdgX marks the uracil-DNA for its repair by a RecA dependent process. Finally, we observed that the tight binding activity of UdgX is useful in detecting uracils in the genomes. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  9. Diversity of the Superfamily of Phloem Lectins (Phloem Protein 2) in Angiosperms1

    Science.gov (United States)

    Dinant, Sylvie; Clark, Anna M.; Zhu, Yanmin; Vilaine, Françoise; Palauqui, Jean-Christophe; Kusiak, Chantal; Thompson, Gary A.

    2003-01-01

    Phloem protein 2 (PP2) is one of the most abundant and enigmatic proteins in the phloem sap. Although thought to be associated with structural P-protein, PP2 is translocated in the assimilate stream where its lectin activity or RNA-binding properties can exert effects over long distances. Analyzing the diversity of these proteins in vascular plants led to the identification of PP2-like genes in species from 17 angiosperm and gymnosperm genera. This wide distribution of PP2 genes in the plant kingdom indicates that they are ancient and common in vascular plants. Their presence in cereals and gymnosperms, both of which lack structural P-protein, also supports a wider role for these proteins. Within this superfamily, PP2 proteins have considerable size polymorphism. This is attributable to variability in the length of the amino terminus that extends from a highly conserved domain. The conserved PP2 domain was identified in the proteins encoded by six genes from several cucurbits, celery (Apium graveolens), and Arabidopsis that are specifically expressed in the sieve element-companion cell complex. The acquisition of additional modular domains in the amino-terminal extensions of other PP2-like proteins could reflect divergence from its phloem function. PMID:12529520

  10. Genome and transcriptome-wide analyses of cellulose synthase gene superfamily in soybean.

    Science.gov (United States)

    Nawaz, Muhammad Amjad; Rehman, Hafiz Mamoon; Baloch, Faheem Shehzad; Ijaz, Babar; Ali, Muhammad Amjad; Khan, Iqrar Ahmad; Lee, Jeong Dong; Chung, Gyuhwa; Yang, Seung Hwan

    2017-08-01

    The plant cellulose synthase gene superfamily belongs to the category of type-2 glycosyltransferases, and is involved in cellulose and hemicellulose biosynthesis. These enzymes are vital for maintaining cell-wall structural integrity throughout plant life. Here, we identified 78 putative cellulose synthases (CS) in the soybean genome. Phylogenetic analysis against 40 reference Arabidopsis CS genes clustered soybean CSs into seven major groups (CESA, CSL A, B, C, D, E and G), located on 19 chromosomes (except chromosome 18). Soybean CS expansion occurred in 66 duplication events. Additionally, we identified 95 simple sequence repeat makers related to 44 CSs. We next performed digital expression analysis using publically available datasets to understand potential CS functions in soybean. We found that CSs were highly expressed during soybean seed development, a pattern confirmed with an Affymatrix soybean IVT array and validated with RNA-seq profiles. Within CS groups, CESAs had higher relative expression than CSLs. Soybean CS models were designed based on maximum average RPKM values. Gene co-expression networks were developed to explore which CSs could work together in soybean. Finally, RT-PCR analysis confirmed the expression of 15 selected CSs during all four seed developmental stages. Copyright © 2017 Elsevier GmbH. All rights reserved.

  11. Microbial biodegradation of biuret: defining biuret hydrolases within the isochorismatase superfamily.

    Science.gov (United States)

    Robinson, Serina L; Badalamenti, Jonathan P; Dodge, Anthony G; Tassoulas, Lambros J; Wackett, Lawrence P

    2018-03-12

    Biuret is a minor component of urea fertilizer and an intermediate in s-triazine herbicide biodegradation. The microbial metabolism of biuret has never been comprehensively studied. Here, we enriched and isolated bacteria from a potato field that grew on biuret as a sole nitrogen source. We sequenced the genome of the fastest-growing isolate, Herbaspirillum sp. BH-1 and identified genes encoding putative biuret hydrolases (BHs). We purified and characterized a functional BH enzyme from Herbaspirillum sp. BH-1 and two other bacteria from divergent phyla. The BH enzymes reacted exclusively with biuret in the range of 2-11 µmol min -1 mg -1 protein. We then constructed a global protein superfamily network to map structure-function relationships in the BH subfamily and used this to mine > 7000 genomes. High-confidence BH sequences were detected in Actinobacteria, Alpha- and Beta-proteobacteria, and some fungi, archaea and green algae, but not animals or land plants. Unexpectedly, no cyanuric acid hydrolase homologs were detected in > 90% of genomes with BH homologs, suggesting BHs may have arisen independently of s-triazine ring metabolism. This work links genotype to phenotype by enabling accurate genome-mining to predict microbial utilization of biuret. Importantly, it advances understanding of the microbial capacity for biuret biodegradation in agricultural systems. © 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.

  12. Identification of the GTPase superfamily in Mycoplasma synoviae and Mycoplasma hyopneumoniae

    Directory of Open Access Journals (Sweden)

    Clayton Luiz Borges

    2007-01-01

    Full Text Available Mycoplasmas are the smallest known prokaryotes with self-replication ability. They are obligate parasites, taking up many molecules of their hosts and acting as pathogens in men, animals, birds and plants. Mycoplasma hyopneumoniae is the infective agent of swine mycoplasmosis and Mycoplasma synoviae is responsible for subclinical upper respiratory infections that may result in airsacculitis and synovitis in chickens and turkeys. These highly infectious organisms present a worldwide distribution and are responsible for major economic problems. Proteins of the GTPase superfamily occur in all domains of life, regulating functions such as protein synthesis, cell cycle and differentiation. Despite their functional diversity, all GTPases are believed to have evolved from a single common ancestor. In this work we have identified mycoplasma GTPases by searching the complete genome databases of Mycoplasma synoviae and Mycoplasma hyopneumoniae, J (non-pathogenic and 7448 (pathogenic strains. Fifteen ORFs encoding predicted GTPases were found in M. synoviae and in the two strains of M. hyopneumoniae. Searches for conserved G domains in GTPases were performed and the sequences were classified into families. The GTPase phylogenetic analysis showed that the subfamilies were well resolved into clades. The presence of GTPases in the three strains suggests the importance of GTPases in 'minimalist' genomes.

  13. Plum, an immunoglobulin superfamily protein, regulates axon pruning by facilitating TGF-β signaling.

    Science.gov (United States)

    Yu, Xiaomeng M; Gutman, Itai; Mosca, Timothy J; Iram, Tal; Ozkan, Engin; Garcia, K Christopher; Luo, Liqun; Schuldiner, Oren

    2013-05-08

    Axon pruning during development is essential for proper wiring of the mature nervous system, but its regulation remains poorly understood. We have identified an immunoglobulin superfamily (IgSF) transmembrane protein, Plum, that is cell autonomously required for axon pruning of mushroom body (MB) γ neurons and for ectopic synapse refinement at the developing neuromuscular junction in Drosophila. Plum promotes MB γ neuron axon pruning by regulating the expression of Ecdysone Receptor-B1, a key initiator of axon pruning. Genetic analyses indicate that Plum acts to facilitate signaling of Myoglianin, a glial-derived TGF-β, on MB γ neurons upstream of the type-I TGF-β receptor Baboon. Myoglianin, Baboon, and Ecdysone Receptor-B1 are also required for neuromuscular junction ectopic synapse refinement. Our study highlights both IgSF proteins and TGF-β facilitation as key promoters of developmental axon elimination and demonstrates a mechanistic conservation between MB axon pruning during metamorphosis and the refinement of ectopic larval neuromuscular connections. Copyright © 2013 Elsevier Inc. All rights reserved.

  14. BOC, an Ig superfamily member, associates with CDO to positively regulate myogenic differentiation

    Science.gov (United States)

    Kang, Jong-Sun; Mulieri, Philip J.; Hu, Yulan; Taliana, Lavinia; Krauss, Robert S.

    2002-01-01

    CDO is a cell surface receptor-like protein that positively regulates myogenic differentiation. Reported here is the identification of BOC, which, with CDO, defines a newly recognized subfamily within the immunoglobulin superfamily. cdo and boc are co-expressed in muscle precursors in the developing mouse embryo. Like CDO, BOC accelerates differentiation of cultured myoblast cell lines and participates in a positive feedback loop with the myogenic transcription factor, MyoD. CDO and BOC form complexes in a cis fashion via association of both their ectodomains and their intracellular domains. A soluble fusion protein that contains the entire BOC ectodomain functions similarly to full-length BOC to promote myogenic differentiation, indicating that the intracellular region is dispensable for its activity in this system. Furthermore, a dominant-negative form of CDO inhibits the pro-myogenic effects of soluble BOC, suggesting that BOC is dependent on CDO for its activity. CDO and BOC are proposed to be components of a receptor complex that mediates some of the cell–cell interactions between muscle precursors that are required for myogenesis. PMID:11782431

  15. Structure of TTHA1623, a novel metallo-β-lactamase superfamily protein from Thermus thermophilus HB8

    International Nuclear Information System (INIS)

    Yamamura, Akihiro; Okada, Akitoshi; Kameda, Yasuhiro; Ohtsuka, Jun; Nakagawa, Noriko; Ebihara, Akio; Nagata, Koji; Tanokura, Masaru

    2009-01-01

    The crystal structures of TTHA1623 from T. thermophilus HB8 in an iron-bound and a zinc-bound form have been determined to 2.8 and 2.2 Å resolution, respectively. TTHA1623 is a metallo-β-lactamase superfamily protein from the extremely thermophilic bacterium Thermus thermophilus HB8. Homologues of TTHA1623 exist in a wide range of bacteria and archaea and one eukaryote, Giardia lamblia, but their function remains unknown. To analyze the structural properties of TTHA1623, the crystal structures of its iron-bound and zinc-bound forms have been determined to 2.8 and 2.2 Å resolution, respectively. TTHA1623 possesses an αββα-fold similar to that of other metallo-β-lactamase superfamily proteins with glyoxalase II-type metal coordination. However, TTHA1623 exhibits a putative substrate-binding pocket with a unique shape

  16. Systematics of some Lower and Middle Devonian spiriferid brachiopods from Gaspe with a revision of the superfamily Delthyridoidea

    DEFF Research Database (Denmark)

    Bizzarro, Martin; Lespérance, P.J.

    1999-01-01

    The component subfamilies of the Delthyridoidea are critically reviewed and subjected to phylogenetic analysis. This shows the presence of two clades, assigned to the Delthyrididae and Acrospiriferidae, within the superfamily. The subfamilial categories are redefined mainly on the basis of the ch......The component subfamilies of the Delthyridoidea are critically reviewed and subjected to phylogenetic analysis. This shows the presence of two clades, assigned to the Delthyrididae and Acrospiriferidae, within the superfamily. The subfamilial categories are redefined mainly on the basis...... of the characters used in the phylogenetic analysis. The spiriferid, mainly delthyridide, Gaspe fauna is formally revised and redescribed. This new taxonomic treatment leads to more precise biostratigraphy and to the recognition of a new subfamily, the Gaspespiriferinae, based on the new genus Gaspespirifer. Five...... new species aye described: Howellella (Howellella) forillonensis, Brachyspirifer (Brachyspirifer) briseboisi, Paraspirifer desbiensi, Brevispirifer florentinus, and B. quebecensis, The occurrence of Brevispirifer species with Middle Devonian chonetaceans confirms the presence of marine Eifelian strata...

  17. Systematics of some Lower and Middle Devonian spiriferid brachiopods from Gaspe with a revision of the superfamily Delthyridoidea

    DEFF Research Database (Denmark)

    Bizzarro, Martin; Lespérance, P.J.

    1999-01-01

    The component subfamilies of the Delthyridoidea are critically reviewed and subjected to phylogenetic analysis. This shows the presence of two clades, assigned to the Delthyrididae and Acrospiriferidae, within the superfamily. The subfamilial categories are redefined mainly on the basis of the ch...... Brachyspirifer, Paraspirifer, and Vandercammenina are in Gaspe. This reinforces the hypothesis that Gaspe served as a stepping stone for Rhenish species invading North America in Pragian and Emsian times, as previously suggested by bivalve biogeography....

  18. Comparative genomic study of ALDH gene superfamily in Gossypium: A focus on Gossypium hirsutum under salt stress.

    Directory of Open Access Journals (Sweden)

    Yating Dong

    Full Text Available Aldehyde dehydrogenases (ALDHs are a superfamily of enzymes which play important role in the scavenging of active aldehydes molecules. In present work, a comprehensive whole-genomic study of ALDH gene superfamily was carried out for an allotetraploid cultivated cotton species, G. hirsutum, as well as in parallel relative to their diploid progenitors, G. arboreum and G. raimondii. Totally, 30 and 58 ALDH gene sequences belong to 10 families were identified from diploid and allotetraploid cotton species, respectively. The gene structures among the members from same families were highly conserved. Whole-genome duplication and segmental duplication might be the major driver for the expansion of ALDH gene superfamily in G. hirsutum. In addition, the expression patterns of GhALDH genes were diverse across tissues. Most GhALDH genes were induced or repressed by salt stress in upland cotton. Our observation shed lights on the molecular evolutionary properties of ALDH genes in diploid cottons and their alloallotetraploid derivatives. It may be useful to mine key genes for improvement of cotton response to salt stress.

  19. Evolutionary analysis of the ENTH/ANTH/VHS protein superfamily reveals a coevolution between membrane trafficking and metabolism

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    De Craene Johan-Owen

    2012-07-01

    Full Text Available Abstract Background Membrane trafficking involves the complex regulation of proteins and lipids intracellular localization and is required for metabolic uptake, cell growth and development. Different trafficking pathways passing through the endosomes are coordinated by the ENTH/ANTH/VHS adaptor protein superfamily. The endosomes are crucial for eukaryotes since the acquisition of the endomembrane system was a central process in eukaryogenesis. Results Our in silico analysis of this ENTH/ANTH/VHS superfamily, consisting of proteins gathered from 84 complete genomes representative of the different eukaryotic taxa, revealed that genomic distribution of this superfamily allows to discriminate Fungi and Metazoa from Plantae and Protists. Next, in a four way genome wide comparison, we showed that this discriminative feature is observed not only for other membrane trafficking effectors, but also for proteins involved in metabolism and in cytokinesis, suggesting that metabolism, cytokinesis and intracellular trafficking pathways co-evolved. Moreover, some of the proteins identified were implicated in multiple functions, in either trafficking and metabolism or trafficking and cytokinesis, suggesting that membrane trafficking is central to this co-evolution process. Conclusions Our study suggests that membrane trafficking and compartmentalization were not only key features for the emergence of eukaryotic cells but also drove the separation of the eukaryotes in the different taxa.

  20. The Association between Gene-Environment Interactions and Diseases Involving the Human GST Superfamily with SNP Variants

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    Antoinesha L. Hollman

    2016-03-01

    Full Text Available Exposure to environmental hazards has been associated with diseases in humans. The identification of single nucleotide polymorphisms (SNPs in human populations exposed to different environmental hazards, is vital for detecting the genetic risks of some important human diseases. Several studies in this field have been conducted on glutathione S-transferases (GSTs, a phase II detoxification superfamily, to investigate its role in the occurrence of diseases. Human GSTs consist of cytosolic and microsomal superfamilies that are further divided into subfamilies. Based on scientific search engines and a review of the literature, we have found a large amount of published articles on human GST super- and subfamilies that have greatly assisted in our efforts to examine their role in health and disease. Because of its polymorphic variations in relation to environmental hazards such as air pollutants, cigarette smoke, pesticides, heavy metals, carcinogens, pharmaceutical drugs, and xenobiotics, GST is considered as a significant biomarker. This review examines the studies on gene-environment interactions related to various diseases with respect to single nucleotide polymorphisms (SNPs found in the GST superfamily. Overall, it can be concluded that interactions between GST genes and environmental factors play an important role in human diseases.

  1. Galatheoidea are not monophyletic - molecular and morphological phylogeny of the squat lobsters (Decapoda: Anomura) with recognition of a new superfamily.

    Science.gov (United States)

    Schnabel, K E; Ahyong, S T; Maas, E W

    2011-02-01

    The monophyletic status of the squat lobster superfamily Galatheoidea has come under increasing doubt by studies using evidence as diverse as larval and adult somatic morphology, sperm ultrastructure, and molecular data. Here we synthesize phylogenetic data from these diverse strands, with the addition of new molecular and morphological data to examine the phylogeny of the squat lobsters and assess the status of the Galatheoidea. A total of 64 species from 16 of the 17 currently recognised anomuran families are included. Results support previous work pointing towards polyphyly in the superfamily Galatheoidea and Paguroidea, specifically, suggesting independent origins of the Galatheidae+Porcellanidae and the Chirostylidae+Kiwaidae. Morphological characters are selected that support clades resolved in the combined analysis and the taxonomic status of Galatheoidea sensu lato is revised. Results indicate that Chirostylidae are more closely related to an assemblage including Aegloidea, Lomisoidea and Paguroidea than to the remaining Galatheoidea and are referred to the superfamily Chirostyloidea to include the Chirostylidae and Kiwaidae. A considerable amount of research highlighting morphological differences supporting this split is discussed. The Galatheoidea sensu stricto is restricted to the families Galatheidae and Porcellanidae, and diagnoses for both Chirostyloidea and Galatheoidea are provided. Present results highlight the need for a detailed revision of a number of taxa, challenge some currently used morphological synapomorphies, and emphasise the need for integrated studies with wide taxon sampling and multiple data sources to resolve complex phylogenetic questions. Copyright © 2010 Elsevier Inc. All rights reserved.

  2. Data on the phosphorylation state of the catalytic serine of enzymes in the α-D-phosphohexomutase superfamily

    Directory of Open Access Journals (Sweden)

    Yingying Lee

    2017-02-01

    Full Text Available Most enzymes in the α-D-phosphohexomutase superfamily catalyze the reversible conversion of 1- to 6-phosphosugars. They play important roles in carbohydrate and sugar nucleotide metabolism, and participate in the biosynthesis of polysaccharides, glycolipids, and other exoproducts. Mutations in genes encoding these enzymes are associated with inherited metabolic diseases in humans, including glycogen storage disease and congenital disorders of glycosylation. Enzymes in the superfamily share a highly conserved active site serine that participates in the multi-step phosphoryl transfer reaction. Here we provide data on the effects of various phosphosugar ligands on the phosphorylation of this serine, as monitored by electrospray ionization mass spectrometry (ESI-MS data on the intact proteins. We also show data on the longevity of the phospho-enzyme under various solution conditions in one member of the superfamily from Pseudomonas aeruginosa, and present inhibition data for several ligands. These data should be useful for the production of homogeneous samples of phosphorylated or unphosphorylated proteins, which are essential for biophysical characterization of these enzymes.

  3. Recent structural insights into the function of copper nitrite reductases.

    Science.gov (United States)

    Horrell, Sam; Kekilli, Demet; Strange, Richard W; Hough, Michael A

    2017-11-15

    Copper nitrite reductases (CuNiR) carry out the first committed step of the denitrification pathway of the global nitrogen cycle, the reduction of nitrite (NO 2 - ) to nitric oxide (NO). As such, they are of major agronomic and environmental importance. CuNiRs occur primarily in denitrifying soil bacteria which carry out the overall reduction of nitrate to dinitrogen. In this article, we review the insights gained into copper nitrite reductase (CuNiR) function from three dimensional structures. We particularly focus on developments over the last decade, including insights from serial femtosecond crystallography using X-ray free electron lasers (XFELs) and from the recently discovered 3-domain CuNiRs.

  4. The evolution of the ribonucleotide reductases: much ado about oxygen.

    Science.gov (United States)

    Poole, Anthony M; Logan, Derek T; Sjöberg, Britt-Marie

    2002-08-01

    Ribonucleotide reduction is the only known biological means for de novo production of deoxyribonucleotides, the building blocks of DNA. These are produced from ribonucleotides, the building blocks of RNA, and the direction of this reaction has been taken to support the idea that, in evolution, RNA preceded DNA as genetic material. However, an understanding of the evolutionary relationships among the three modern-day classes of ribonucleotide reductase and how the first reductase arose early in evolution is still far off. We propose that the diversification of this class of enzymes is inherently tied to microbial colonization of aerobic and anaerobic niches. The work is of broader interest, as it also sheds light on the process of adaptation to oxygenic environments consequent to the evolution of atmospheric oxygen.

  5. Intraethnic variation in steroid-5-alpha-reductase polymorphisms in ...

    Indian Academy of Sciences (India)

    in prostate cancer patients: a potential factor implicated in. 5-alpha-reductase inhibitor treatment. Luis Alberto Henríquez-Hernández, Almudena Valenciano, Palmira Foro-Arnalot, María Jesús Álvarez-Cubero,. José Manuel Cozar, José Francisco Suárez-Novo, Manel Castells-Esteve, Pablo Fernández-Gonzalo,.

  6. Cloning and characterization of a nitrite reductase gene related to ...

    African Journals Online (AJOL)

    STORAGESEVER

    2010-03-01

    Mar 1, 2010 ... BL21 (DE3) strain with the recombinant expression vector pET-28A-GhNiR. NiR activity assay showed that the crude GhNiR protein had obvious activity to NaNO2 substrate. Key words: Cotton, nitrite reductase, prokaryotic expression, semi-quantitative RT-PCR, GenBank Accession. No: GQ389691.

  7. Xylose reductase from the thermophilic fungus Talaromyces emersonii

    Indian Academy of Sciences (India)

    Prakash

    action of xylose reductase (EC 1.1.1.21) and xylitol dehydrogenase (EC 1.1.1.9) is required to convert D-xylose to D-xylulose and all enzymes of the D-xylose pathway can be used in the L-arabinose pathway, where arabitol is oxidized by NAD+-dependent arabitol dehydrogenase (EC. 1.1.1.12) producing L-xylulose. This is ...

  8. Phylogenetic approach for inferring the origin and functional evolution of bacterial ADP-ribosylation superfamily.

    Science.gov (United States)

    Chellapandi, P; Sakthishree, S; Bharathi, M

    2013-09-01

    Bacterial ADP-ribosyltransferases (BADPRTs) are extensively contributed to determine the strain-specific virulence state and pathogenesis in human hosts. Understanding molecular evolution and functional diversity of the BADPRTs is an important standpoint to describe the fundamental behind in the vaccine designing for bacterial infections. In the present study, we have evaluated the origin and functional evolution of conserved domains within the BADPRTs by analyzing their sequence-function relationship. To represent the evolution history of BADPRTs, phylogenetic trees were constructed based on their protein sequence, structure and conserved domains using different evolutionary programs. Sequence divergence and genetic diversity were studied herein to deduce the functional evolution of conserved domains across the family and superfamily. The results of sequence similarity search have shown that three hypothetical proteins (above 90%) were identical to the members of BADPRTs and their functions were annotated by phylogenetic approach. Phylogenetic analysis of this study has revealed the family members of BADPRTs were phylogenetically related to one another, functionally diverged within the same family, and dispersed into closely related bacteria. The presence of core substitution pattern in the conserved domains would determine the family-specific function of BADPRTs. Functional diversity of the BADPRTs was exclusively distinguished by Darwinian positive selection (diphtheria toxin C and pertussis toxin S) and neutral selection (arginine ADP-ribosyltransferase, enterotoxin A and binary toxin A) acting on the existing domains. Many of the family members were sharing their sequence-specific features from members in the arginine ADP-ribosyltransferase family. Conservative functions of members in the BADPRTs have shown to be expanded only within closely related families, and retained as such in pathogenic bacteria by evolutionary process (domain duplication or

  9. Fauna Europaea: Coleoptera 2 (excl. series Elateriformia, Scarabaeiformia, Staphyliniformia and superfamily Curculionoidea)

    Science.gov (United States)

    Alonso Zarazaga, Miguel-Angel; Slipinski, Adam; Nilsson, Anders; Jelínek, Josef; Taglianti, Augusto Vigna; Turco, Federica; Otero, Carlos; Canepari, Claudio; Kral, David; Liberti, Gianfranco; Sama, Gianfranco; Nardi, Gianluca; Löbl, Ivan; Horak, Jan; Kolibac, Jiri; Háva, Jirí; Sapiejewski, Maciej; Jäch, Manfred; Bologna, Marco Alberto; Biondi, Maurizio; Nikitsky, Nikolai B.; Mazzoldi, Paolo; Zahradnik, Petr; Wegrzynowicz, Piotr; Constantin, Robert; Gerstmeier, Roland; Zhantiev, Rustem; Fattorini, Simone; Tomaszewska, Wioletta; Rücker, Wolfgang H.; Vazquez-Albalate, Xavier; Cassola, Fabio; Angelini, Fernando; Johnson, Colin; Schawaller, Wolfgang; Regalin, Renato; Baviera, Cosimo; Rocchi, Saverio; Cianferoni, Fabio; Beenen, Ron; Schmitt, Michael; Sassi, David; Kippenberg, Horst; Zampetti, Marcello Franco; Trizzino, Marco; Chiari, Stefano; Carpaneto, Giuseppe Maria; Sabatelli, Simone

    2015-01-01

    Abstract Fauna Europaea provides a public web-service with an index of scientific names (including synonyms) of all living European land and freshwater animals, their geographical distribution at country level (up to the Urals, excluding the Caucasus region), and some additional information. The Fauna Europaea project covers about 230,000 taxonomic names, including 130,000 accepted species and 14,000 accepted subspecies, which is much more than the originally projected number of 100,000 species. This represents a huge effort by more than 400 contributing specialists throughout Europe and is a unique (standard) reference suitable for many users in science, government, industry, nature conservation and education. Coleoptera represent a huge assemblage of holometabolous insects, including as a whole more than 200 recognized families and some 400,000 described species worldwide. Basic information is summarized on their biology, ecology, economic relevance, and estimated number of undescribed species worldwide. Little less than 30,000 species are listed from Europe. The Coleoptera 2 section of the Fauna Europaea database (Archostemata, Myxophaga, Adephaga and Polyphaga excl. the series Elateriformia, Scarabaeiformia, Staphyliniformia and the superfamily Curculionoidea) encompasses 80 families (according to the previously accepted family-level systematic framework) and approximately 13,000 species. Tabulations included a complete list of the families dealt with, the number of species in each, the names of all involved specialists, and, when possible, an estimate of the gaps in terms of total number of species at an European level. A list of some recent useful references is appended. Most families included in the Coleoptera 2 Section have been updated in the most recent release of the Fauna Europaea index, or are ready to be updated as soon as the FaEu data management environment completes its migration from Zoological Museum Amsterdam to Berlin Museum für Naturkunde

  10. Diverticulitis and Crohn's disease have distinct but overlapping tumor necrosis superfamily 15 haplotypes.

    Science.gov (United States)

    Connelly, Tara M; Choi, Christine S; Berg, Arthur S; Harris, Leonard; Coble, Joel; Koltun, Walter A

    2017-06-15

    Diverticulitis (DD) and Crohn's disease (CD) have overlapping features including bowel structuring, inflammation, and infection. Tumor necrosis superfamily 15 (TNFSF15) is an immunoregulatory, anti-angiogenic gene. CD has been previously associated with a haplotype of five TNFSF15 single-nucleotide polymorphism alleles: rs3810936 (G allele), rs6478108 (A), rs6478109 (G), rs7848647 (G), and rs7869487 (A). We aimed to determine the TNFSF15 risk haplotype for DD versus controls with a subgroup analysis of youthful DD patients (aged ≤55 y) versus older controls (aged ≥55 y). A total of 148 diverticulitis patients (90 aged ≤55 y) and 200 controls (87 aged ≥55 y) were genotyped using our custom-designed Illumina Veracode microarray chip. Genotypes from rs3810936, rs6478108, rs6478109, rs7848647, rs7869487 and two additional TNFSF15 single nucleotide polymorphisms, rs3810936 and rs11554257, were analyzed. PHASE version 2.1, R with HaploStats and the Broad Institute's Haploview program were used for statistics and imputed haplotype frequency. Permutation corrected for multiple comparisons. The CD GAGGA haplotype was significantly associated with diverticulitis (P = 0.03) in the all DD versus all controls comparison. A second haplotype, rs6478108 (A), rs6478109 (G), rs7869487 (A), and rs4263839 (G), was also associated with DD in this cohort (P = 0.025). A third haplotype rs6478108 (A), rs6478109 (G), rs7848647 (G) and rs7869487 (A), rs4263839 (G) was demonstrated in the DD 55 comparison (P = 0.045). Distinct but overlapping TNFSF15 haplotypes were demonstrated in diverticulitis patients versus healthy controls when compared with the known Crohn's risk haplotype suggesting similar but distinct genetic predispositions. This study strengthens the role for a genetic predisposition to diverticulitis that involves the TNFSF15 gene. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Phosphatidylcholine transfer protein interacts with thioesterase superfamily member 2 to attenuate insulin signaling.

    Science.gov (United States)

    Ersoy, Baran A; Tarun, Akansha; D'Aquino, Katharine; Hancer, Nancy J; Ukomadu, Chinweike; White, Morris F; Michel, Thomas; Manning, Brendan D; Cohen, David E

    2013-07-30

    Phosphatidylcholine transfer protein (PC-TP) is a phospholipid-binding protein that is enriched in liver and that interacts with thioesterase superfamily member 2 (THEM2). Mice lacking either protein exhibit improved hepatic glucose homeostasis and are resistant to diet-induced diabetes. Insulin receptor substrate 2 (IRS2) and mammalian target of rapamycin complex 1 (mTORC1) are key effectors of insulin signaling, which is attenuated in diabetes. We found that PC-TP inhibited IRS2, as evidenced by insulin-independent IRS2 activation after knockdown, genetic ablation, or chemical inhibition of PC-TP. In addition, IRS2 was activated after knockdown of THEM2, providing support for a role for the interaction of PC-TP with THEM2 in suppressing insulin signaling. Additionally, we showed that PC-TP bound to tuberous sclerosis complex 2 (TSC2) and stabilized the components of the TSC1-TSC2 complex, which functions to inhibit mTORC1. Preventing phosphatidylcholine from binding to PC-TP disrupted interactions of PC-TP with THEM2 and TSC2, and disruption of the PC-TP-THEM2 complex was associated with increased activation of both IRS2 and mTORC1. In livers of mice with genetic ablation of PC-TP or that had been treated with a PC-TP inhibitor, steady-state amounts of IRS2 were increased, whereas those of TSC2 were decreased. These findings reveal a phospholipid-dependent mechanism that suppresses insulin signaling downstream of its receptor.

  12. DIHYDROFOLATE REDUCTASE AS A VERSATILE DRUG TARGET IN HEALTHCARE

    Directory of Open Access Journals (Sweden)

    Naira Rashid

    2016-09-01

    Full Text Available Dihydrofolate reductase is one of the important enzymes for thymidylate and purine synthesis. It has been used as a drug target for treatment of various diseases. A large number of pharmaceutical drugs have been designed to inhibit the activity of dihydrofolate reductase. However, over the period of time some organisms have developed resistance against some of these drugs. There is also a chance of cross reactivity for these drugs, as they may target the dihydrofolate reductase enzyme of other organisms. Although using NMR spectroscopy, phylogenetic sequence analysis, comparative sequence analysis between dihydrofolate enzymes of various organisms and molecular modeling studies, a lot has been unraveled about the difference in the structure of this enzyme in various organisms, yet there is a need for deeper understanding of these differences so as to design drugs that are specific to their targets and reduce the chance for cross reactivity. The dihydrofolate enzyme can also be explored for treatment of various other diseases that are associated with the folate cycle.

  13. Nitrate reductase gene involvement in hexachlorobiphenyl dechlorination by Phanerochaete chrysosporium

    International Nuclear Information System (INIS)

    De, Supriyo; Perkins, Michael; Dutta, Sisir K.

    2006-01-01

    Polychlorobiphenyl (PCB) degradation usually occurs through reductive dechlorination under anaerobic conditions and phenolic ring cleavage under aerobic conditions. In this paper, we provide evidence of nitrate reductase (NaR) mediated dechlorination of hexachlorobiphenyl (PCB-153) in Phanerochaete chrysosporium under non-ligninolytic condition and the gene involved. The NaR enzyme and its cofactor, molybdenum (Mo), were found to mediate reductive dechlorination of PCBs even in aerobic condition. Tungsten (W), a competitive inhibitor of this enzyme, was found to suppress this dechlorination. Chlorine release assay provided further evidence of this nitrate reductase mediated dechlorination. Commercially available pure NaR enzyme from Aspergillus was used to confirm these results. Through homology search using TBLASTN program, NaR gene was identified, primers were designed and the RT-PCR product was sequenced. The NaR gene was then annotated in the P. chrysosporium genome (GenBank accession no. AY700576). This is the first report regarding the presence of nitrate reductase gene in this fungus with the explanation why this fungus can dechlorinate PCBs even in aerobic condition. These fungal inoculums are used commercially as pellets in sawdust for enhanced bioremediation of PCBs at the risk of depleting soil nitrates. Hence, the addition of nitrates to the pellets will reduce this risk as well as enhance its activity

  14. Multivariate sequence analysis reveals additional function impacting residues in the SDR superfamily.

    Science.gov (United States)

    Tiwari, Pratibha; Singh, Noopur; Dixit, Aparna; Choudhury, Devapriya

    2014-10-01

    The "extended" type of short chain dehydrogenases/reductases (SDR), share a remarkable similarity in their tertiary structures inspite of being highly divergent in their functions and sequences. We have carried out principal component analysis (PCA) on structurally equivalent residue positions of 10 SDR families using information theoretic measures like Jensen-Shannon divergence and average shannon entropy as variables. The results classify residue positions in the SDR fold into six groups, one of which is characterized by low Shannon entropies but high Jensen-Shannon divergence against the reference family SDR1E, suggesting that these positions are responsible for the specific functional identities of individual SDR families, distinguishing them from the reference family SDR1E. Site directed mutagenesis of three residues from this group in the enzyme UDP-Galactose 4-epimerase belonging to SDR1E shows that the mutants promote the formation of NADH containing abortive complexes. Finally, molecular dynamics simulations have been used to suggest a mechanism by which the mutants interfere with the re-oxidation of NADH leading to the formation of abortive complexes. © 2014 Wiley Periodicals, Inc.

  15. Sucrose mimics the light induction of Arabidopsis nitrate reductase gene transcription

    DEFF Research Database (Denmark)

    Cheng, Chi-Lien; Acedo, Gregoria N; Kristensen, Michael

    1992-01-01

    can replace light in eliciting an increase of nitrate reductase mRNA accumulation in dark-adapted green Arabidopsis plants. We show further that sucrose alone is sufficient for the full expression of nitrate reductase genes in etiolated Arabidopsis plants. Finally, using a reporter gene, we show......Nitrate reductase, the first enzyme in nitrate assimilation, is located at the crossroad of two energy-consuming pathways: nitrate assimilation and carbon fixation. Light, which regulates the expression of many higher-plant carbon fixation genes, also regulates nitrate reductase gene expression....... Located in the cytosol, nitrate reductase obtains its reductant not from photosynthesis but from carbohydrate catabolism. This relationship prompted us to investigate the indirect role that light might play, via photosynthesis, in the regulation of nitrate reductase gene expression. We show that sucrose...

  16. Identification and characterization of trans-3-hydroxy-l-proline dehydratase and Δ1-pyrroline-2-carboxylate reductase involved in trans-3-hydroxy-l-proline metabolism of bacteria

    Directory of Open Access Journals (Sweden)

    Seiya Watanabe

    2014-01-01

    Full Text Available trans-4-Hydroxy-l-proline (T4LHyp and trans-3-hydroxy-l-proline (T3LHyp occur mainly in collagen. A few bacteria can convert T4LHyp to α-ketoglutarate, and we previously revealed a hypothetical pathway consisting of four enzymes at the molecular level (J Biol Chem (2007 282, 6685–6695; J Biol Chem (2012 287, 32674–32688. Here, we first found that Azospirillum brasilense has the ability to grow not only on T4LHyp but also T3LHyp as a sole carbon source. In A. brasilense cells, T3LHyp dehydratase and NAD(PH-dependent Δ1-pyrroline-2-carboxylate (Pyr2C reductase activities were induced by T3LHyp (and d-proline and d-lysine but not T4LHyp, and no effect of T3LHyp was observed on the expression of T4LHyp metabolizing enzymes: a hypothetical pathway of T3LHyp → Pyr2C → l-proline was proposed. Bacterial T3LHyp dehydratase, encoded to LhpH gene, was homologous with the mammalian enzyme. On the other hand, Pyr2C reductase encoded to LhpI gene was a novel member of ornithine cyclodeaminase/μ-crystallin superfamily, differing from known bacterial protein. Furthermore, the LhpI enzymes of A. brasilense and another bacterium showed several different properties, including substrate and coenzyme specificities. T3LHyp was converted to proline by the purified LhpH and LhpI proteins. Furthermore, disruption of LhpI gene from A. brasilense led to loss of growth on T3LHyp, d-proline and d-lysine, indicating that this gene has dual metabolic functions as a reductase for Pyr2C and Δ1-piperidine-2-carboxylate in these pathways, and that the T3LHyp pathway is not linked to T4LHyp and l-proline metabolism.

  17. Gamma-irradiation activates biochemical systems: induction of nitrate reductase activity in plant callus.

    OpenAIRE

    Pandey, K N; Sabharwal, P S

    1982-01-01

    Gamma-irradiation induced high levels of nitrate reductase activity (NADH:nitrate oxidoreductase, EC 1.6.6.1) in callus of Haworthia mirabilis Haworth. Subcultures of gamma-irradiated tissues showed autonomous growth on minimal medium. We were able to mimic the effects of gamma-irradiation by inducing nitrate reductase activity in unirradiated callus with exogenous auxin and kinetin. These results revealed that induction of nitrate reductase activity by gamma-irradiation is mediated through i...

  18. Immunological comparison of the NADH:nitrate reductase from different cucumber tissues

    Directory of Open Access Journals (Sweden)

    Jolanta Marciniak

    2014-01-01

    Full Text Available Soluble nitrate reductase from cucumber roots (Cucumis sativus L. was isolated and purified with blue-Sepharose 4B. Specific antibodies against the NR protein were raised by immunization of a goat. Using polyclonal antibodies anti-NR properties of the nitrate reductase from various cucumber tissues were examined. Experiments showed difference in immuno-logical properties of nitrate reductase (NR from cotyledon roots and leaves.

  19. In search for function of two human orphan SDR enzymes: hydroxysteroid dehydrogenase like 2 (HSDL2) and short-chain dehydrogenase/reductase-orphan (SDR-O).

    Science.gov (United States)

    Kowalik, Dorota; Haller, Ferdinand; Adamski, Jerzy; Moeller, Gabriele

    2009-11-01

    The protein superfamily of short-chain dehydrogenases/reductases (SDRs) today comprises over 20,000 members found in pro- and eukaryotes. Despite low amino acid sequence identity (only 15-30%), they share several similar characteristics in conformational structures, the N-terminal cofactor (NAD(P)/NAD(P)H) binding region being the most conserved. The enzymes catalyze oxido-reductive reactions and have a broad spectrum of substrates. Not all recently identified SDRs have been analyzed in detail yet, and we therefore characterized two rudimentarily annotated human SDR candidates: an orphan SDR (SDR-O) and hydroxysteroid dehydrogenase like 2 (HSDL2). We analyzed the amino acid sequence for cofactor preference, performed subcellular localization studies, and a screening for substrates of the enzymes, including steroid hormones and retinoids. None of both tested proteins showed a significant conversion of steroid hormones. However, the peroxisomal localization of human HSDL2 may suggest an involvement in fatty acid metabolism. For SDR-O a weak conversion of retinal into retinol was detectable in the presence of the cofactor NADH.

  20. Structure of a short-chain dehydrogenase/reductase (SDR) within a genomic island from a clinical strain of Acinetobacter baumannii

    Energy Technology Data Exchange (ETDEWEB)

    Shah, Bhumika S., E-mail: bhumika.shah@mq.edu.au; Tetu, Sasha G. [Macquarie University, Research Park Drive, Sydney, NSW 2109 (Australia); Harrop, Stephen J. [University of New South Wales, Sydney, NSW 2052 (Australia); Paulsen, Ian T.; Mabbutt, Bridget C. [Macquarie University, Research Park Drive, Sydney, NSW 2109 (Australia)

    2014-09-25

    The structure of a short-chain dehydrogenase encoded within genomic islands of A. baumannii strains has been solved to 2.4 Å resolution. This classical SDR incorporates a flexible helical subdomain. The NADP-binding site and catalytic side chains are identified. Over 15% of the genome of an Australian clinical isolate of Acinetobacter baumannii occurs within genomic islands. An uncharacterized protein encoded within one island feature common to this and other International Clone II strains has been studied by X-ray crystallography. The 2.4 Å resolution structure of SDR-WM99c reveals it to be a new member of the classical short-chain dehydrogenase/reductase (SDR) superfamily. The enzyme contains a nucleotide-binding domain and, like many other SDRs, is tetrameric in form. The active site contains a catalytic tetrad (Asn117, Ser146, Tyr159 and Lys163) and water molecules occupying the presumed NADP cofactor-binding pocket. An adjacent cleft is capped by a relatively mobile helical subdomain, which is well positioned to control substrate access.

  1. Structure of a short-chain dehydrogenase/reductase (SDR) within a genomic island from a clinical strain of Acinetobacter baumannii

    International Nuclear Information System (INIS)

    Shah, Bhumika S.; Tetu, Sasha G.; Harrop, Stephen J.; Paulsen, Ian T.; Mabbutt, Bridget C.

    2014-01-01

    The structure of a short-chain dehydrogenase encoded within genomic islands of A. baumannii strains has been solved to 2.4 Å resolution. This classical SDR incorporates a flexible helical subdomain. The NADP-binding site and catalytic side chains are identified. Over 15% of the genome of an Australian clinical isolate of Acinetobacter baumannii occurs within genomic islands. An uncharacterized protein encoded within one island feature common to this and other International Clone II strains has been studied by X-ray crystallography. The 2.4 Å resolution structure of SDR-WM99c reveals it to be a new member of the classical short-chain dehydrogenase/reductase (SDR) superfamily. The enzyme contains a nucleotide-binding domain and, like many other SDRs, is tetrameric in form. The active site contains a catalytic tetrad (Asn117, Ser146, Tyr159 and Lys163) and water molecules occupying the presumed NADP cofactor-binding pocket. An adjacent cleft is capped by a relatively mobile helical subdomain, which is well positioned to control substrate access

  2. Histochemical localization of glutathione dependent NBT-reductase in mouse skin.

    Science.gov (United States)

    Shukla, Y

    2001-09-01

    Localization of the glutathione dependent Nitroblue tetrazolium (NBT) reductase in fresh frozen sections of mouse skin and possible dependence of NBT reductase on tissue thiol levels has been investigated. The fresh frozen tissue sections (8 m thickness) were prepared and incubated in medium containing NBT, reduced glutathione (GSH) and phosphate buffer. The staining for GSH was performed with mercury orange. The activity of the NBT-reductase in mouse skin has been found to be localized in the areas rich in glutathione and actively proliferating area of the skin. The activity of the NBT-reductase seems to be dependent on the glutathione contents.

  3. Functional Identification and Structure Determination of Two Novel Prolidases from cog1228 in the Amidohydrolase Superfamily

    Energy Technology Data Exchange (ETDEWEB)

    Xiang, Dao Feng; Patskovsky, Yury; Xu, Chengfu; Fedorov, Alexander A.; Fedorov, Elena V.; Sisco, Abby A.; Sauder, J. Michael; Burley, Stephen K.; Almo, Steven C.; Raushel, Frank M. (Einstein); (TAM); (Lilly)

    2010-12-07

    Two uncharacterized enzymes from the amidohydrolase superfamily belonging to cog1228 were cloned, expressed, and purified to homogeneity. The two proteins, Sgx9260c (gi|44242006) and Sgx9260b (gi|44479596), were derived from environmental DNA samples originating from the Sargasso Sea. The catalytic function and substrate profiles for Sgx9260c and Sgx9260b were determined using a comprehensive library of dipeptides and N-acyl derivative of L-amino acids. Sgx9260c catalyzes the hydrolysis of Gly-L-Pro, L-Ala-L-Pro, and N-acyl derivatives of L-Pro. The best substrate identified to date is N-acetyl-L-Pro with a value of k{sub cat}/K{sub m} of 3 x 10{sup 5} M{sup -1} s{sup -1}. Sgx9260b catalyzes the hydrolysis of L-hydrophobic L-Pro dipeptides and N-acyl derivatives of L-Pro. The best substrate identified to date is N-propionyl-L-Pro with a value of k{sub cat}/K{sub m} of 1 x 10{sup 5} M{sup -1} s{sup -1}. Three-dimensional structures of both proteins were determined by X-ray diffraction methods (PDB codes 3MKV and 3FEQ). These proteins fold as distorted ({beta}/{alpha})8-barrels with two divalent cations in the active site. The structure of Sgx9260c was also determined as a complex with the N-methylphosphonate derivative of L-Pro (PDB code 3N2C). In this structure the phosphonate moiety bridges the binuclear metal center, and one oxygen atom interacts with His-140. The {alpha}-carboxylate of the inhibitor interacts with Tyr-231. The proline side chain occupies a small substrate binding cavity formed by residues contributed from the loop that follows {beta}-strand 7 within the ({beta}/{alpha})8-barrel. A total of 38 other proteins from cog1228 are predicted to have the same substrate profile based on conservation of the substrate binding residues. The structure of an evolutionarily related protein, Cc2672 from Caulobacter crecentus, was determined as a complex with the N-methylphosphonate derivative of L-arginine (PDB code 3MTW).

  4. Biochemistry and Crystal Structure of Ectoine Synthase: A Metal-Containing Member of the Cupin Superfamily.

    Directory of Open Access Journals (Sweden)

    Nils Widderich

    Full Text Available Ectoine is a compatible solute and chemical chaperone widely used by members of the Bacteria and a few Archaea to fend-off the detrimental effects of high external osmolarity on cellular physiology and growth. Ectoine synthase (EctC catalyzes the last step in ectoine production and mediates the ring closure of the substrate N-gamma-acetyl-L-2,4-diaminobutyric acid through a water elimination reaction. However, the crystal structure of ectoine synthase is not known and a clear understanding of how its fold contributes to enzyme activity is thus lacking. Using the ectoine synthase from the cold-adapted marine bacterium Sphingopyxis alaskensis (Sa, we report here both a detailed biochemical characterization of the EctC enzyme and the high-resolution crystal structure of its apo-form. Structural analysis classified the (SaEctC protein as a member of the cupin superfamily. EctC forms a dimer with a head-to-tail arrangement, both in solution and in the crystal structure. The interface of the dimer assembly is shaped through backbone-contacts and weak hydrophobic interactions mediated by two beta-sheets within each monomer. We show for the first time that ectoine synthase harbors a catalytically important metal co-factor; metal depletion and reconstitution experiments suggest that EctC is probably an iron-dependent enzyme. We found that EctC not only effectively converts its natural substrate N-gamma-acetyl-L-2,4-diaminobutyric acid into ectoine through a cyclocondensation reaction, but that it can also use the isomer N-alpha-acetyl-L-2,4-diaminobutyric acid as its substrate, albeit with substantially reduced catalytic efficiency. Structure-guided site-directed mutagenesis experiments targeting amino acid residues that are evolutionarily highly conserved among the extended EctC protein family, including those forming the presumptive iron-binding site, were conducted to functionally analyze the properties of the resulting EctC variants. An assessment of

  5. A Survey of the ATP-Binding Cassette (ABC Gene Superfamily in the Salmon Louse (Lepeophtheirus salmonis.

    Directory of Open Access Journals (Sweden)

    Greta Carmona-Antoñanzas

    Full Text Available Salmon lice, Lepeophtheirus salmonis (Krøyer, 1837, are fish ectoparasites causing significant economic damage in the mariculture of Atlantic salmon, Salmo salar Linnaeus, 1758. The control of L. salmonis at fish farms relies to a large extent on treatment with anti-parasitic drugs. A problem related to chemical control is the potential for development of resistance, which in L. salmonis is documented for a number of drug classes including organophosphates, pyrethroids and avermectins. The ATP-binding cassette (ABC gene superfamily is found in all biota and includes a range of drug efflux transporters that can confer drug resistance to cancers and pathogens. Furthermore, some ABC transporters are recognised to be involved in conferral of insecticide resistance. While a number of studies have investigated ABC transporters in L. salmonis, no systematic analysis of the ABC gene family exists for this species. This study presents a genome-wide survey of ABC genes in L. salmonis for which, ABC superfamily members were identified through homology searching of the L. salmonis genome. In addition, ABC proteins were identified in a reference transcriptome of the parasite generated by high-throughput RNA sequencing (RNA-seq of a multi-stage RNA library. Searches of both genome and transcriptome allowed the identification of a total of 33 genes / transcripts coding for ABC proteins, of which 3 were represented only in the genome and 4 only in the transcriptome. Eighteen sequences were assigned to ABC subfamilies known to contain drug transporters, i.e. subfamilies B (4 sequences, C (11 and G (2. The results suggest that the ABC gene family of L. salmonis possesses fewer members than recorded for other arthropods. The present survey of the L. salmonis ABC gene superfamily will provide the basis for further research into potential roles of ABC transporters in the toxicity of salmon delousing agents and as potential mechanisms of drug resistance.

  6. A Survey of the ATP-Binding Cassette (ABC) Gene Superfamily in the Salmon Louse (Lepeophtheirus salmonis).

    Science.gov (United States)

    Carmona-Antoñanzas, Greta; Carmichael, Stephen N; Heumann, Jan; Taggart, John B; Gharbi, Karim; Bron, James E; Bekaert, Michaël; Sturm, Armin

    2015-01-01

    Salmon lice, Lepeophtheirus salmonis (Krøyer, 1837), are fish ectoparasites causing significant economic damage in the mariculture of Atlantic salmon, Salmo salar Linnaeus, 1758. The control of L. salmonis at fish farms relies to a large extent on treatment with anti-parasitic drugs. A problem related to chemical control is the potential for development of resistance, which in L. salmonis is documented for a number of drug classes including organophosphates, pyrethroids and avermectins. The ATP-binding cassette (ABC) gene superfamily is found in all biota and includes a range of drug efflux transporters that can confer drug resistance to cancers and pathogens. Furthermore, some ABC transporters are recognised to be involved in conferral of insecticide resistance. While a number of studies have investigated ABC transporters in L. salmonis, no systematic analysis of the ABC gene family exists for this species. This study presents a genome-wide survey of ABC genes in L. salmonis for which, ABC superfamily members were identified through homology searching of the L. salmonis genome. In addition, ABC proteins were identified in a reference transcriptome of the parasite generated by high-throughput RNA sequencing (RNA-seq) of a multi-stage RNA library. Searches of both genome and transcriptome allowed the identification of a total of 33 genes / transcripts coding for ABC proteins, of which 3 were represented only in the genome and 4 only in the transcriptome. Eighteen sequences were assigned to ABC subfamilies known to contain drug transporters, i.e. subfamilies B (4 sequences), C (11) and G (2). The results suggest that the ABC gene family of L. salmonis possesses fewer members than recorded for other arthropods. The present survey of the L. salmonis ABC gene superfamily will provide the basis for further research into potential roles of ABC transporters in the toxicity of salmon delousing agents and as potential mechanisms of drug resistance.

  7. Intracellular mediators of transforming growth factor β superfamily signaling localize to endosomes in chicken embryo and mouse lenses in vivo

    Directory of Open Access Journals (Sweden)

    Ishii Shunsuke

    2007-06-01

    Full Text Available Abstract Background Endocytosis is a key regulator of growth factor signaling pathways. Recent studies showed that the localization to endosomes of intracellular mediators of growth factor signaling may be required for their function. Although there is substantial evidence linking endocytosis and growth factor signaling in cultured cells, there has been little study of the endosomal localization of signaling components in intact tissues or organs. Results Proteins that are downstream of the transforming growth factor-β superfamily signaling pathway were found on endosomes in chicken embryo and postnatal mouse lenses, which depend on signaling by members of the TGFβ superfamily for their normal development. Phosphorylated Smad1 (pSmad1, pSmad2, Smad4, Smad7, the transcriptional repressors c-Ski and TGIF and the adapter molecules Smad anchor for receptor activation (SARA and C184M, localized to EEA-1- and Rab5-positive vesicles in chicken embryo and/or postnatal mouse lenses. pSmad1 and pSmad2 also localized to Rab7-positive late endosomes. Smad7 was found associated with endosomes, but not caveolae. Bmpr1a conditional knock-out lenses showed decreased nuclear and endosomal localization of pSmad1. Many of the effectors in this pathway were distributed differently in vivo from their reported distribution in cultured cells. Conclusion Based on the findings reported here and data from other signaling systems, we suggest that the localization of activated intracellular mediators of the transforming growth factor-β superfamily to endosomes is important for the regulation of growth factor signaling.

  8. Genome-wide analysis of the expansin gene superfamily reveals grapevine-specific structural and functional characteristics.

    Directory of Open Access Journals (Sweden)

    Silvia Dal Santo

    Full Text Available BACKGROUND: Expansins are proteins that loosen plant cell walls in a pH-dependent manner, probably by increasing the relative movement among polymers thus causing irreversible expansion. The expansin superfamily (EXP comprises four distinct families: expansin A (EXPA, expansin B (EXPB, expansin-like A (EXLA and expansin-like B (EXLB. There is experimental evidence that EXPA and EXPB proteins are required for cell expansion and developmental processes involving cell wall modification, whereas the exact functions of EXLA and EXLB remain unclear. The complete grapevine (Vitis vinifera genome sequence has allowed the characterization of many gene families, but an exhaustive genome-wide analysis of expansin gene expression has not been attempted thus far. METHODOLOGY/PRINCIPAL FINDINGS: We identified 29 EXP superfamily genes in the grapevine genome, representing all four EXP families. Members of the same EXP family shared the same exon-intron structure, and phylogenetic analysis confirmed a closer relationship between EXP genes from woody species, i.e. grapevine and poplar (Populus trichocarpa, compared to those from Arabidopsis thaliana and rice (Oryza sativa. We also identified grapevine-specific duplication events involving the EXLB family. Global gene expression analysis confirmed a strong correlation among EXP genes expressed in mature and green/vegetative samples, respectively, as reported for other gene families in the recently-published grapevine gene expression atlas. We also observed the specific co-expression of EXLB genes in woody organs, and the involvement of certain grapevine EXP genes in berry development and post-harvest withering. CONCLUSION: Our comprehensive analysis of the grapevine EXP superfamily confirmed and extended current knowledge about the structural and functional characteristics of this gene family, and also identified properties that are currently unique to grapevine expansin genes. Our data provide a model for the

  9. Methylenetetrahydrofolate reductase (MTHFR) deficiency presenting as a rash.

    LENUS (Irish Health Repository)

    Crushell, Ellen

    2012-09-01

    We report on the case of a 2-year-old girl recently diagnosed with Methylenetetrahydrofolate reductase (MTHFR) deficiency who originally presented in the neonatal period with a distinctive rash. At 11 weeks of age she developed seizures, she had acquired microcephaly and developmental delay. The rash deteriorated dramatically following commencement of phenobarbitone; both rash and seizures abated following empiric introduction of pyridoxine and folinic acid as treatment of possible vitamin responsive seizures. We postulate that phenobarbitone in combination with MTHFR deficiency may have caused her rash to deteriorate and subsequent folinic acid was helpful in treating the rash and preventing further acute neurological decline as commonly associated with this condition.

  10. Applications of Carboxylic Acid Reductases in Oleaginous Microbes

    Energy Technology Data Exchange (ETDEWEB)

    Resch, Michael G.; Linger, Jeffrey; McGeehan, John; Tyo, Keith; Beckham, Gregg

    2016-05-26

    Carboxylic acid reductases (CARs) are recently emerging reductive enzymes for the direct production of aldehydes from biologically-produced carboxylic acids. Recent work has demonstrated that these powerful enzymes are able to reduce a very broad range of volatile- to long-chain fatty acids as well as aromatic acids. Here, we express four CAR enzymes from different fungal origins to test their activity against fatty acids commonly produced in oleaginous microbes. These in vitro results will inform metabolic engineering strategies to conduct mild biological reduction of carboxylic acids in situ, which is conventionally done via hydrotreating catalysis at high temperatures and hydrogen pressures.

  11. Mutations in a Conserved Domain of E. coli MscS to the Most Conserved Superfamily Residue Leads to Kinetic Changes.

    Directory of Open Access Journals (Sweden)

    Hannah R Malcolm

    Full Text Available In Escherichia coli (E. coli the mechanosensitive channel of small conductance, MscS, gates in response to membrane tension created from acute external hypoosmotic shock, thus rescuing the bacterium from cell lysis. E. coli MscS is the most well studied member of the MscS superfamily of channels, whose members are found throughout the bacterial and plant kingdoms. Homology to the pore lining helix and upper vestibule domain of E. coli MscS is required for inclusion into the superfamily. Although highly conserved, in the second half of the pore lining helix (TM3B, E. coli MscS has five residues significantly different from other members of the superfamily. In superfamilies such as this, it remains unclear why variations within such a homologous region occur: is it tolerance of alternate residues, or does it define functional variance within the superfamily? Point mutations (S114I/T, L118F, A120S, L123F, F127E/K/T and patch clamp electrophysiology were used to study the effect of changing these residues in E. coli MscS on sensitivity and gating. The data indicate that variation at these locations do not consistently lead to wildtype channel phenotypes, nor do they define large changes in mechanosensation, but often appear to effect changes in the E. coli MscS channel gating kinetics.

  12. Generic revision of the large-winged mite superfamily Galumnoidea (Acari, Oribatida) of the world.

    Science.gov (United States)

    Ermilov, Sergey G; Klimov, Pavel B

    2017-11-27

    Genus-level taxa in the oribatid mite superfamily Galumnoidea (Acari, Oribatida) are revised based on morphology of adults and a previously published phylogenetic analysis. We give a concise overview of the general morphology of Galumnoidea, diagnoses and a key for families, genera, and subgenera, and a taxonomic list with two families, 39 genera, 9 non-nominal subgenera, and 590 species. The following nomenclatorial changes to genus-group taxa resulted from our revision: Allogalumna (Allogalumna) Grandjean, 1936 (=Xenogalumna Balogh, 1960(b) syn. nov.), Flagellozetes (Cosmogalumna) Aoki, 1988 comb. nov. (from Galumna (Cosmogalumna)), Flagellozetes (Variogalumna) Mahunka, 1995 stat. nov., Galumna (Galumna) Heyden, 1826 (=Rostrogalumna Engelbrecht, 1973 syn. nov.), Pilogalumna Grandjean, 1956(a) (=Disparagalumna Hammer, 1973 syn. nov.), Trichogalumna (Tanzanycha) Koçak & Kemal, 2008 stat. nov., Galumnella Berlese, 1916(a) (=Monogalumnella Mahunka, 1986 syn. nov. =Trichogalumnella Mahunka, 1992 syn. nov., =Bigalumnella Mahunka, 1994 syn. nov.). The following changes are made to species-group nomenclature: Angulogalumna areolata (Starý, 2005) comb. nov. (from Cuspidogalumna) et stat. ressur. (=Galumna (Angulogalumna) staryi Subías, 2010 syn. nov. replacement name for secondary homonym Galumna (Angulogalumna) areolata (Starý, 2005)), Allogalumna (Allogalumna) longula (Balogh, 1960(b)) comb. nov. (from Xenogalumna), Flagellozetes (Cosmogalumna) areticulata (Ermilov, Sandmann, Klarner, Widyastuti & Scheu, 2015(d)) comb. nov. (from Galumna (Cosmogalumna)), F. (C.) dongnaiensis (Ermilov & Anichkin, 2013) comb. nov. (from Galumna (Cosmogalumna)), F. (C.) ekaterinae (Ermilov & Friedrich, 2016(b)) comb. nov. (from Galumna (Cosmogalumna)), F. (C.) hiroyoshii (Nakamura & Fujikawa, 2004) comb. nov. (from Galumna (Cosmogalumna)), F. (C.) ornata (Aoki, 1988) comb. nov. (from Galumna (Cosmogalumna)), F. (C.) imperfectus (Aoki & Hu, 1993) comb. nov. (from Galumna (Cosmogalumna

  13. Functions of Flavin Reductase and Quinone Reductase in 2,4,6-Trichlorophenol Degradation by Cupriavidus necator JMP134▿

    OpenAIRE

    Belchik, Sara Mae; Xun, Luying

    2007-01-01

    The tcpRXABCYD operon of Cupriavidus necator JMP134 is involved in the degradation of 2,4,6-trichlorophenol (2,4,6-TCP), a toxic pollutant. TcpA is a reduced flavin adenine dinucleotide (FADH2)-dependent monooxygenase that converts 2,4,6-TCP to 6-chlorohydroxyquinone. It has been implied via genetic analysis that TcpX acts as an FAD reductase to supply TcpA with FADH2, whereas the function of TcpB in 2,4,6-TCP degradation is still unclear. In order to provide direct biochemical evidence for t...

  14. Analysis of nitrate reductase mRNA expression and nitrate reductase activity in response to nitrogen supply

    OpenAIRE

    Gholamreza Kavoosi; Sadegh Balotf; Homeira Eshghi; Hasan Hasani

    2014-01-01

    Nitrate is one of the major sources of nitrogen for the growth of plants. It is taken up by plant roots and transported to the leaves where it is reduced to nitrite in the. The main objective of this research was to investigate stimulatory effects of sodium nitrate, potassium nitrate, ammonia and urea on the production/generation of the nitrate reductase mRNA in Triticum aestivum plants. The plants were grown in standard nutrient solution for 21 days and then starved in a media without nitrat...

  15. Melanophore migration and survival during zebrafish adult pigment stripe development require the immunoglobulin superfamily adhesion molecule Igsf11.

    Directory of Open Access Journals (Sweden)

    Dae Seok Eom

    Full Text Available The zebrafish adult pigment pattern has emerged as a useful model for understanding the development and evolution of adult form as well as pattern-forming mechanisms more generally. In this species, a series of horizontal melanophore stripes arises during the larval-to-adult transformation, but the genetic and cellular bases for stripe formation remain largely unknown. Here, we show that the seurat mutant phenotype, consisting of an irregular spotted pattern, arises from lesions in the gene encoding Immunoglobulin superfamily member 11 (Igsf11. We find that Igsf11 is expressed by melanophores and their precursors, and we demonstrate by cell transplantation and genetic rescue that igsf11 functions autonomously to this lineage in promoting adult stripe development. Further analyses of cell behaviors in vitro, in vivo, and in explant cultures ex vivo demonstrate that Igsf11 mediates adhesive interactions and that mutants for igsf11 exhibit defects in both the migration and survival of melanophores and their precursors. These findings identify the first in vivo requirements for igsf11 as well as the first instance of an immunoglobulin superfamily member functioning in pigment cell development and patterning. Our results provide new insights into adult pigment pattern morphogenesis and how cellular interactions mediate pattern formation.

  16. Evolution of plant virus movement proteins from the 30K superfamily and of their homologs integrated in plant genomes

    Energy Technology Data Exchange (ETDEWEB)

    Mushegian, Arcady R., E-mail: mushegian2@gmail.com [Division of Molecular and Cellular Biosciences, National Science Foundation, 4201 Wilson Boulevard, Arlington, VA 22230 (United States); Elena, Santiago F., E-mail: sfelena@ibmcp.upv.es [Instituto de Biología Molecular y Celular de Plantas, CSIC-UPV, 46022 València (Spain); The Santa Fe Institute, Santa Fe, NM 87501 (United States)

    2015-02-15

    Homologs of Tobacco mosaic virus 30K cell-to-cell movement protein are encoded by diverse plant viruses. Mechanisms of action and evolutionary origins of these proteins remain obscure. We expand the picture of conservation and evolution of the 30K proteins, producing sequence alignment of the 30K superfamily with the broadest phylogenetic coverage thus far and illuminating structural features of the core all-beta fold of these proteins. Integrated copies of pararetrovirus 30K movement genes are prevalent in euphyllophytes, with at least one copy intact in nearly every examined species, and mRNAs detected for most of them. Sequence analysis suggests repeated integrations, pseudogenizations, and positive selection in those provirus genes. An unannotated 30K-superfamily gene in Arabidopsis thaliana genome is likely expressed as a fusion with the At1g37113 transcript. This molecular background of endopararetrovirus gene products in plants may change our view of virus infection and pathogenesis, and perhaps of cellular homeostasis in the hosts. - Highlights: • Sequence region shared by plant virus “30K” movement proteins has an all-beta fold. • Most euphyllophyte genomes contain integrated copies of pararetroviruses. • These integrated virus genomes often include intact movement protein genes. • Molecular evidence suggests that these “30K” genes may be selected for function.

  17. Evolutionary Expansion of the Amidohydrolase Superfamily in Bacteria in Response to the Synthetic Compounds Molinate and Diuron

    Science.gov (United States)

    Sugrue, Elena; Fraser, Nicholas J.; Hopkins, Davis H.; Carr, Paul D.; Khurana, Jeevan L.; Oakeshott, John G.; Scott, Colin

    2015-01-01

    The amidohydrolase superfamily has remarkable functional diversity, with considerable structural and functional annotation of known sequences. In microbes, the recent evolution of several members of this family to catalyze the breakdown of environmental xenobiotics is not well understood. An evolutionary transition from binuclear to mononuclear metal ion coordination at the active sites of these enzymes could produce large functional changes such as those observed in nature, but there are few clear examples available to support this hypothesis. To investigate the role of binuclear-mononuclear active-site transitions in the evolution of new function in this superfamily, we have characterized two recently evolved enzymes that catalyze the hydrolysis of the synthetic herbicides molinate (MolA) and phenylurea (PuhB). In this work, the crystal structures, mutagenesis, metal ion analysis, and enzyme kinetics of both MolA and PuhB establish that these enzymes utilize a mononuclear active site. However, bioinformatics and structural comparisons reveal that the closest putative ancestor of these enzymes had a binuclear active site, indicating that a binuclear-mononuclear transition has occurred. These proteins may represent examples of evolution modifying the characteristics of existing catalysts to satisfy new requirements, specifically, metal ion rearrangement leading to large leaps in activity that would not otherwise be possible. PMID:25636851

  18. Crystal structure and potential physiological role of zebra fish thioesterase superfamily member 2 (fTHEM2)

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Shanshan; Li, Han; Gao, Feng; Zhou, Ying, E-mail: zhouying@moon.ibp.ac.cn

    2015-08-07

    Thioesterase superfamily member 2 (THEM2) is an essential protein for mammalian cell proliferation. It belongs to the hotdog-fold thioesterase superfamily and catalyzes hydrolysis of thioester bonds of acyl-CoA in vitro, while its in vivo function remains unrevealed. In this study, Zebra fish was selected as a model organism to facilitate the investigations on THEM2. First, we solved the crystal structure of recombinant fTHEM2 at the resolution of 1.80 Å, which displayed a similar scaffolding as hTHEM2. Second, functional studies demonstrated that fTHEM2 is capable of hydrolyzing palmitoyl-CoA in vitro. In addition, injection of morpholino against fTHEM2 at one-cell stage resulted in distorted early embryo development, including delayed cell division, retarded development and increased death rate. The above findings validated our hypothesis that fTHEM2 could serve as an ideal surrogate for studying the physiological functions of THEM2. - Highlights: • The crystal structure of recombinant fTHEM2 is presented. • fTHEM2 is capable of hydrolyzing palmitoyl-CoA. • The influence of fTHEM2 on early embryo development is demonstrated.

  19. The Unique Role of the ECERIFERUM2-LIKE Clade of the BAHD Acyltransferase Superfamily in Cuticular Wax Metabolism.

    Science.gov (United States)

    Haslam, Tegan M; Gerelle, Wesley K; Graham, Sean W; Kunst, Ljerka

    2017-06-13

    The elongation of very-long-chain fatty acids is a conserved process used for the production of many metabolites, including plant cuticular waxes. The elongation of precursors of the most abundant cuticular wax components of some plants, however, is unique in requiring ECERIFERUM2-LIKE (CER2-LIKE) proteins. CER2-LIKEs are a clade within the BAHD superfamily of acyltransferases. They are known to be required for cuticular wax production in both Arabidopsis and maize based on mutant studies. Heterologous expression of Arabidopsis and rice CER2-LIKEs in Saccharomyces cerevisiae has demonstrated that they modify the chain-length specificity of elongation when paired with particular condensing enzymes. Despite sequence homology, CER2-LIKEs are distinct from the BAHD superfamily in that they do not appear to use acyl transfer activity to fulfill their biological function. Here, we review the discovery and characterization of CER2-LIKEs, propose several models to explain their function, and explore the importance of CER2-LIKE proteins for the evolution of plant cuticles.

  20. The Unique Role of the ECERIFERUM2-LIKE Clade of the BAHD Acyltransferase Superfamily in Cuticular Wax Metabolism

    Directory of Open Access Journals (Sweden)

    Tegan M. Haslam

    2017-06-01

    Full Text Available The elongation of very-long-chain fatty acids is a conserved process used for the production of many metabolites, including plant cuticular waxes. The elongation of precursors of the most abundant cuticular wax components of some plants, however, is unique in requiring ECERIFERUM2-LIKE (CER2-LIKE proteins. CER2-LIKEs are a clade within the BAHD superfamily of acyltransferases. They are known to be required for cuticular wax production in both Arabidopsis and maize based on mutant studies. Heterologous expression of Arabidopsis and rice CER2-LIKEs in Saccharomyces cerevisiae has demonstrated that they modify the chain-length specificity of elongation when paired with particular condensing enzymes. Despite sequence homology, CER2-LIKEs are distinct from the BAHD superfamily in that they do not appear to use acyl transfer activity to fulfill their biological function. Here, we review the discovery and characterization of CER2-LIKEs, propose several models to explain their function, and explore the importance of CER2-LIKE proteins for the evolution of plant cuticles.

  1. Combining protein sequence, structure, and dynamics: A novel approach for functional evolution analysis of PAS domain superfamily.

    Science.gov (United States)

    Dong, Zheng; Zhou, Hongyu; Tao, Peng

    2018-02-01

    PAS domains are widespread in archaea, bacteria, and eukaryota, and play important roles in various functions. In this study, we aim to explore functional evolutionary relationship among proteins in the PAS domain superfamily in view of the sequence-structure-dynamics-function relationship. We collected protein sequences and crystal structure data from RCSB Protein Data Bank of the PAS domain superfamily belonging to three biological functions (nucleotide binding, photoreceptor activity, and transferase activity). Protein sequences were aligned and then used to select sequence-conserved residues and build phylogenetic tree. Three-dimensional structure alignment was also applied to obtain structure-conserved residues. The protein dynamics were analyzed using elastic network model (ENM) and validated by molecular dynamics (MD) simulation. The result showed that the proteins with same function could be grouped by sequence similarity, and proteins in different functional groups displayed statistically significant difference in their vibrational patterns. Interestingly, in all three functional groups, conserved amino acid residues identified by sequence and structure conservation analysis generally have a lower fluctuation than other residues. In addition, the fluctuation of conserved residues in each biological function group was strongly correlated with the corresponding biological function. This research suggested a direct connection in which the protein sequences were related to various functions through structural dynamics. This is a new attempt to delineate functional evolution of proteins using the integrated information of sequence, structure, and dynamics. © 2017 The Protein Society.

  2. Tracing the Evolutionary History of the CAP Superfamily of Proteins Using Amino Acid Sequence Homology and Conservation of Splice Sites.

    Science.gov (United States)

    Abraham, Anup; Chandler, Douglas E

    2017-10-01

    Proteins of the CAP superfamily play numerous roles in reproduction, innate immune responses, cancer biology, and venom toxicology. Here we document the breadth of the CAP (Cysteine-RIch Secretory Protein (CRISP), Antigen 5, and Pathogenesis-Related) protein superfamily and trace the major events in its evolution using amino acid sequence homology and the positions of exon/intron borders within their genes. Seldom acknowledged in the literature, we find that many of the CAP subfamilies present in mammals, where they were originally characterized, have distinct homologues in the invertebrate phyla. Early eukaryotic CAP genes contained only one exon inherited from prokaryotic predecessors and as evolution progressed an increasing number of introns were inserted, reaching 2-5 in the invertebrate world and 5-15 in the vertebrate world. Focusing on the CRISP subfamily, we propose that these proteins evolved in three major steps: (1) origination of the CAP/PR/SCP domain in bacteria, (2) addition of a small Hinge domain to produce the two-domain SCP-like proteins found in roundworms and anthropoids, and (3) addition of an Ion Channel Regulatory domain, borrowed from invertebrate peptide toxins, to produce full length, three-domain CRISP proteins, first seen in insects and later to diversify into multiple subtypes in the vertebrate world.

  3. THE EFFECTS OF AN ALDOSE REDUCTASE INHIBITOR ON THE PROGRESSION OF DIABETIC-RETINOPATHY

    NARCIS (Netherlands)

    TROMP, A; HOOYMANS, JMM; BARENDSEN, BC; VONDOORMAAL, JJ

    1991-01-01

    The polyol pathway has long been associated with diabetic retinopathy. Glucose is converted to sorbitol with the aid of the enzyme aldose reductase. Aldose reductase inhibitors can prevent changes induced by diabetes. A total of 30 patients with minimal background retinopathy were randomly divided

  4. Characterization and regulation of Leishmania major 3-hydroxy-3-methylglutaryl-CoA reductase

    DEFF Research Database (Denmark)

    Montalvetti, A; Pena Diaz, Javier; Hurtado, R

    2000-01-01

    In eukaryotes the enzyme 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase catalyses the synthesis of mevalonic acid, a common precursor to all isoprenoid compounds. Here we report the isolation and overexpression of the gene coding for HMG-CoA reductase from Leishmania major. The protein from L...

  5. HMG-CoA-reductase inhibitors and neuropathy : reports to the Netherlands Pharmacovigilance Centre

    NARCIS (Netherlands)

    de Langen, J J; van Puijenbroek, E P

    2006-01-01

    The number of patients taking HMG-CoA-reductase inhibitors for hypercholesterolaemia is growing rapidly. Treatment with HMG-CoA-reductase inhibitors significantly reduces the risk of cardiovascular morbidity and mortality, but may rarely cause serious adverse drug reactions (ADRs). The most serious

  6. Bioactivation of lapachol responsible for DNA scission by NADPH-cytochrome P450 reductase.

    Science.gov (United States)

    Kumagai, Y; Tsurutani, Y; Shinyashiki, M; Homma-Takeda, S; Nakai, Y; Yoshikawa, T; Shimojo, N

    1997-09-01

    The reduction of the naphthoquinone derivative, lapachol, which is responsible for its bioactivation was examined using microsomal preparations and NADPH-cytochrome P450 reductase (P450 reductase). Phenobarbital (PB) pretreatment resulted in an induction of enzyme activities for cytochrome c reduction (1.54 times) and lapachol reduction (1.20 times) by hepatic microsomal preparation of rats. The specific activity of lapachol reduction by purified P450 reductase showed 56-fold higher than that by untreated liver microsomes. Addition of antibody against P450 reductase (2 mg of IgG/mg of protein) to the microsomal incubation mixture caused an immunoinhibition of cytochrome c (32%) and lapachol (19%) reduction activities, suggesting that P450 reductase catalyzes lapachol reduction. Generation of superoxide anion radical (1321 nmol/mg per min) in approximately equivalent amounts of with NADPH consumption (941 nmol/mg per min) was detected during metabolism of lapachol by P450 reductase. Electron spin resonance (ESR) experiments confirmed generation of superoxide anion radical and hydroxyl radical as these 5,5'-dimethyl-1-pyrroline N-oxide (DMPO) adducts. Incubation of lapachol with P450 reductase caused a cleavage of DNA which was reduced in the presence of Cu,Zn-superoxide dismutase (Cu,Zn-SOD), catalase(1), and hydroxyl radical scavengers such as dimethyl sulfoxide (DMSO) and thiourea. Taken together, these results indicate that lapachol is bioactivated by P450 reductase to reactive species, which promote DNA scission through the redox cycling based generation of superoxide anion radical.

  7. Bioinformatics approach of three partial polyprenol reductase genes in Kandelia obovata

    Science.gov (United States)

    Basyuni, M.; Wati, R.; Sagami, H.; Oku, H.; Baba, S.

    2018-03-01

    This present study describesthe bioinformatics approach to analyze three partial polyprenol reductase genes from mangrove plant, Kandeliaobovataas well aspredictedphysical and chemical properties, potential peptide, subcellular localization, and phylogenetic. The diversity was noted in the physical and chemical properties of three partial polyprenol reductase genes. The values of chloroplast were relatively high, showed that chloroplast transit peptide occurred in mangrove polyprenol reductase. The target peptide value of mitochondria varied from 0.088 to 0.198 indicated it was possible to be present. These results suggested the importance of understanding the diversity of physicochemical properties of the different amino acids in polyprenol reductase. The subcellular localization of two partial genes located in the plasma membrane. To confirm the homology among the polyprenol reductase in the database, a dendrogram was drawn. The phylogenetic tree depicts that there are three clusters, the partial genes of K. obovata joined the largest one: C23157 was close to Ricinus communis polyprenol reductase. Whereas, C23901 and C24171 were grouped with Ipomoea nil polyprenol reductase, suggested that these polyprenol reductase genes form distinct separation into tropical habitat plants.

  8. The role of thioredoxin reductases in brain development.

    Directory of Open Access Journals (Sweden)

    Jonna Soerensen

    Full Text Available The thioredoxin-dependent system is an essential regulator of cellular redox balance. Since oxidative stress has been linked with neurodegenerative disease, we studied the roles of thioredoxin reductases in brain using mice with nervous system (NS-specific deletion of cytosolic (Txnrd1 and mitochondrial (Txnrd2 thioredoxin reductase. While NS-specific Txnrd2 null mice develop normally, mice lacking Txnrd1 in the NS were significantly smaller and displayed ataxia and tremor. A striking patterned cerebellar hypoplasia was observed. Proliferation of the external granular layer (EGL was strongly reduced and fissure formation and laminar organisation of the cerebellar cortex was impaired in the rostral portion of the cerebellum. Purkinje cells were ectopically located and their dendrites stunted. The Bergmann glial network was disorganized and showed a pronounced reduction in fiber strength. Cerebellar hypoplasia did not result from increased apoptosis, but from decreased proliferation of granule cell precursors within the EGL. Of note, neuron-specific inactivation of Txnrd1 did not result in cerebellar hypoplasia, suggesting a vital role for Txnrd1 in Bergmann glia or neuronal precursor cells.

  9. Interspecific variation for thermal dependence of glutathione reductase in sainfoin.

    Science.gov (United States)

    Kidambi, S P; Mahan, J R; Matches, A G

    1990-05-01

    Understanding the biochemical and physiological consequences of species variation would expedite improvement in agronomically useful genotypes of sainfoin (Onobrychis spp.) Information on variation among sainfoin species is lacking on thermal dependence of glutathione reductase (B.C. 1.6.4.2.), which plays an important role in the protection of plants from both high and low temperature stresses by preventing harmful oxidation of enzymes and membranes. Our objective was to investigate the interspecific variation for thermal dependency of glutathione reductase in sainfoin. Large variation among species was found for: (i) the minimum apparent Km (0.4-2.5 μM NADPH), (ii) the temperature at which the minimum apparent Km was observed (15°-5°C), and (iii) the thermal kinetic windows (2°-30°C width) over a 15°-45°C temperature gradient. In general, tetraploid species had narrower (≤17°C) thermal kinetic windows than did diploid species (∼30°C), with one exception among the diploids. Within the tetraploid species, the cultivars of O. viciifolia had a broader thermal kinetic window (≥7°C) than the plant introduction (PI 212241, >2 °C) itself.

  10. Optimisation of nitrate reductase enzyme activity to synthesise silver nanoparticles.

    Science.gov (United States)

    Khodashenas, Bahareh; Ghorbani, Hamid Reza

    2016-06-01

    Today, the synthesis of silver nanoparticles (Ag NPs) is very common since it has many applications in different areas. The synthesis of these nanoparticles is done by means of physical, chemical, or biological methods. However, due to its inexpensive and environmentally friendly features, the biological method is more preferable. In the present study, using nitrate reductase enzyme available in the Escherichia coli (E. coli) bacterium, the biosynthesis of Ag NPs was investigated. In addition, the activity of the nitrate reductase enzyme was optimised by changing its cultural conditions, and the effects of silver nitrate (AgNO(3)) concentration and enzyme amount on nanoparticles synthesis were studied. Finally, the produced nanoparticles were studied using ultraviolet -visible (UV-Vis) spectrophotometer, dynamic light scattering technique, and transmission electron microscopy. UV-Visible spectrophotometric study showed the characteristic peak for Ag NPs at wavelength 405-420 nm for 1 mM metal precursor solution (AgNO(3)) with 1, 5, 10, and 20 cc supernatant and 435 nm for 0.01M AgNO(3) with 20 cc supernatant. In this study, it was found that there is a direct relationship between the AgNO(3) concentration and the size of produced Ag NPs.

  11. Crystal structure of human quinone reductase type 2, a metalloflavoprotein.

    Science.gov (United States)

    Foster, C E; Bianchet, M A; Talalay, P; Zhao, Q; Amzel, L M

    1999-08-03

    In mammals, two separate but homologous cytosolic quinone reductases have been identified: NAD(P)H:quinone oxidoreductase type 1 (QR1) (EC 1.6.99.2) and quinone reductase type 2 (QR2). Although QR1 and QR2 are nearly 50% identical in protein sequence, they display markedly different catalytic properties and substrate specificities. We report here two crystal structures of QR2: in its native form and bound to menadione (vitamin K(3)), a physiological substrate. Phases were obtained by molecular replacement, using our previously determined rat QR1 structure as the search model. QR2 shares the overall fold of the major catalytic domain of QR1, but lacks the smaller C-terminal domain. The FAD binding sites of QR1 and QR2 are very similar, but their hydride donor binding sites are considerably different. Unexpectedly, we found that QR2 contains a specific metal binding site, which is not present in QR1. Two histidine nitrogens, one cysteine thiol, and a main chain carbonyl group are involved in metal coordination. The metal binding site is solvent-accessible, and is separated from the FAD cofactor by a distance of about 13 A.

  12. The effect of ionic and non-ionic surfactants on the growth, nitrate reductase and nitrite reductase activities of Spirodela polyrrhiza (L. Schleiden

    Directory of Open Access Journals (Sweden)

    Józef Buczek

    2014-01-01

    Full Text Available Inclusion into the medium of 5 mg•dm-3 of non-ionic (ENF or ionic (DBST surfactant caused 50-60% inhibition of nitrite reductase MR activity in S. polyrrhiza. At the same time, increased accumulation of NO2- in the plant tissues and lowering of the total and soluble protein contents were found. DBST also lowered the nitrate reductase (NR activity and the dry mass of the plants.

  13. Characterization of the reductase domain of rat neuronal nitric oxide synthase generated in the methylotrophic yeast Pichia pastoris. Calmodulin response is complete within the reductase domain itself.

    Science.gov (United States)

    Gachhui, R; Presta, A; Bentley, D F; Abu-Soud, H M; McArthur, R; Brudvig, G; Ghosh, D K; Stuehr, D J

    1996-08-23

    Rat neuronal NO synthase (nNOS) is comprised of a flavin-containing reductase domain and a heme-containing oxygenase domain. Calmodulin binding to nNOS increases the rate of electron transfer from NADPH into its flavins, triggers electron transfer from flavins to the heme, activates NO synthesis, and increases reduction of artificial electron acceptors such as cytochrome c. To investigate what role the reductase domain plays in calmodulin's activation of these functions, we overexpressed a form of the nNOS reductase domain (amino acids 724-1429) in the yeast Pichia pastoris that for the first time exhibits a complete calmodulin response. The reductase domain was purified by 2',5'-ADP affinity chromatography yielding 25 mg of pure protein per liter of culture. It contained 1 FAD and 0.8 FMN per molecule. Most of the protein as isolated contained an air-stable flavin semiquinone radical that was sensitive to FeCN6 oxidation. Anaerobic titration of the FeCN6-oxidized reductase domain with NADPH indicated the flavin semiquinone re-formed after addition of 1-electron equivalent and the flavins could accept up to 3 electrons from NADPH. Calmodulin binding to the recombinant reductase protein increased its rate of NADPH-dependent flavin reduction and its rate of electron transfer to cytochrome c, FeCN6, or dichlorophenolindophenol to fully match the rate increases achieved when calmodulin bound to native full-length nNOS. Calmodulin's activation of the reductase protein was associated with an increase in domain tryptophan and flavin fluorescence. We conclude that many of calmodulin's actions on native nNOS can be fully accounted for through its interaction with the nNOS reductase domain itself.

  14. Sulforaphane promotes murine hair growth by accelerating the degradation of dihydrotestosterone.

    Science.gov (United States)

    Sasaki, Mari; Shinozaki, Shohei; Shimokado, Kentaro

    2016-03-25

    Dihydrotestosterone (DHT) causes the regression of human hair follicles in the parietal scalp, leading to androgenic alopecia (AGA). Sulforaphane (SFN) increases the expression of DHT degrading enzymes, such as 3α-hydroxysteroid dehydrogenases (3α-HSDs), and, therefore, SFN treatment may improve AGA. To determine the effects of SFN on hair growth, we administered SFN (10 mg/kg BW, IP) or vehicle (DMSO) to ob/ob mice for six weeks and examined hair regeneration and the plasma levels of testosterone and DHT. We also tested the effects of SFN on the expression of two forms of 3α-HSD, aldo-keto reductase 1c21 and dehydrogenase/reductase (SDR family) member 9, both in vitro and in vivo. SNF significantly enhanced hair regeneration in ob/ob mice. The mice treated with SFN showed lower plasma levels of testosterone and DHT than those treated with vehicle. SFN increased the mRNA and protein levels of the two forms of 3α-HSD in the liver of the mice and in cultured murine hepatocyte Hepa1c1c7 cells. These results suggest that SFN treatment increases the amount of 3α-HSDs in the liver, accelerates the degradation of blood DHT, and subsequently blocks the suppression of hair growth by DHT. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. The role of Cercospora zeae-maydis homologs of Rhodobacter sphaeroides 1O2-resistance genes in resistance to the photoactivated toxin cercosporin.

    Science.gov (United States)

    Beseli, Aydin; Goulart da Silva, Marilia; Daub, Margaret E

    2015-01-01

    The photosynthetic bacterium Rhodobacter sphaeroides and plant pathogenic fungus Cercospora nicotianae have been used as models for understanding resistance to singlet oxygen ((1)O(2)), a highly toxic reactive oxygen species. In Rhodobacter and Cercospora, (1)O(2) is derived, respectively, from photosynthesis and from the (1)O(2)-generating toxin cercosporin which the fungus produces to parasitize plants. We identified common genes recovered in transcriptome studies of putative (1)O(2)-resistance genes in these two systems, suggesting common (1)O(2)-resistance mechanisms. To determine if the Cercospora homologs of R. sphaeroides (1)O(2)-resistance genes are involved in resistance to cercosporin, we expressed the genes in the cercosporin-sensitive fungus Neurospora crassa and assayed for increases in cercosporin resistance. Neurospora crassa transformants expressing genes encoding aldo/keto reductase, succinyl-CoA ligase, O-acetylhomoserine (thiol) lyase, peptide methionine sulphoxide reductase and glutathione S-transferase did not have elevated levels of cercosporin resistance. Several transformants expressing aldehyde dehydrogenase were significantly more resistant to cercosporin. Expression of the transgene and enzyme activity did not correlate with resistance, however. We conclude that although the genes tested in this study are important in (1)O(2) resistance in R. sphaeroides, their Cercospora homologs are not involved in resistance to (1)O(2) generated from cercosporin. © FEMS 2014. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  16. Biotransformation and bioactivation reactions - 2015 literature highlights.

    Science.gov (United States)

    Baillie, Thomas A; Dalvie, Deepak; Rietjens, Ivonne M C M; Cyrus Khojasteh, S

    2016-05-01

    Since 1972, Drug Metabolism Reviews has been recognized as one of the principal resources for researchers in pharmacological, pharmaceutical and toxicological fields to keep abreast of advances in drug metabolism science in academia and the pharmaceutical industry. With a distinguished list of authors and editors, the journal covers topics ranging from relatively mature fields, such as cytochrome P450 enzymes, to a variety of emerging fields. We hope to continue this tradition with the current compendium of mini-reviews that highlight novel biotransformation processes that were published during the past year. Each review begins with a summary of the article followed by our comments on novel aspects of the research and their biological implications. This collection of highlights is not intended to be exhaustive, but rather to be illustrative of recent research that provides new insights or approaches that advance the field of drug metabolism. Abbreviations NAPQI N-acetyl-p-benzoquinoneimine ALDH aldehyde dehydrogenase AO aldehyde oxidase AKR aldo-keto reductase CES carboxylesterase CSB cystathionine β-synthase CSE cystathionine γ-lyase P450 cytochrome P450 DHPO 2,3-dihydropyridin-4-one ESI electrospray FMO flavin monooxygenase GSH glutathione GSSG glutathione disulfide ICPMS inductively coupled plasma mass spectrometry i.p. intraperitoneal MDR multidrug-resistant NNAL 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol NNK 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone oaTOF orthogonal acceleration time-of-flight PBK physiologically based kinetic PCP pentachlorophenol SDR short-chain dehydrogenase/reductase SULT sulfotransferase TB tuberculosis.

  17. Individualized supplementation of folic acid according to polymorphisms of methylenetetrahydrofolate reductase (MTHFR), methionine synthase reductase (MTRR) reduced pregnant complications.

    Science.gov (United States)

    Li, Xiujuan; Jiang, Jing; Xu, Min; Xu, Mei; Yang, Yan; Lu, Wei; Yu, Xuemei; Ma, Jianlin; Pan, Jiakui

    2015-01-01

    This study aimed to detect the genotype distributions and allele frequencies of methylenetetrahydrofolate reductase (MTHFR) C677T, A1298C and methionine synthase reductase (MTRR) A66G polymorphisms of pregnant women in Jiaodong region in China, and to investigate whether folic acid supplementation affect the pregnancy complications. A total of 7,812 pregnant women from the Jiaodong region in Shandong province in China. By using Taqman-MGB, 2,928 pregnant women (case group) were tested for the genotype distributions and allele frequencies of MTHFR C677T, A1298C and MTRR A66G polymorphisms. Folic acid metabolism ability was ranked at four levels and then pregnant women in different rank group were supplemented with different doses of folic acid. Their pregnancy complications were followed up and compared with 4,884 pregnant women without folic acid supplementation (control group) in the same hospital. The allele frequencies of MTHFR C677T were 49.1 and 50.9%; those of MTHFR A1298C were 80.2 and 19.8%, and those of MTRR A66G were 74.1 and 25.9%. After supplemented with folic acid, the complication rates in different age groups were significantly reduced, especially for gestational diabetes mellitus and hypertension. Periconceptional folic acid supplementation and healthcare following gene polymorphism testing may be a powerful measure to decrease congenital malformations. © 2015 S. Karger AG, Basel.

  18. 5,10-Methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MTRR), and methionine synthase reductase (MTR) gene polymorphisms and adult meningioma risk.

    Science.gov (United States)

    Zhang, Jun; Zhou, Yan-Wen; Shi, Hua-Ping; Wang, Yan-Zhong; Li, Gui-Ling; Yu, Hai-Tao; Xie, Xin-You

    2013-11-01

    The causes of meningiomas are not well understood. Folate metabolism gene polymorphisms have been shown to be associated with various human cancers. It is still controversial and ambiguous between the functional polymorphisms of folate metabolism genes 5,10-methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MTRR), and methionine synthase reductase (MTR) and risk of adult meningioma. A population-based case–control study involving 600 meningioma patients (World Health Organization [WHO] Grade I, 391 cases; WHO Grade II, 167 cases; WHO Grade III, 42 cases) and 600 controls was done for the MTHFR C677T and A1298C, MTRR A66G, and MTR A2756G variants in Chinese Han population. The folate metabolism gene polymorphisms were determined by using a polymerase chain reaction–restriction fragment length polymorphism assay. Meningioma cases had a significantly lower frequency of MTHFR 677 TT genotype [odds ratio (OR) = 0.49, 95 % confidence interval (CI) 0.33–0.74; P = 0.001] and T allele (OR = 0.80, 95 % CI 0.67–0.95; P = 0.01) than controls. A significant association between risk of meningioma and MTRR 66 GG (OR = 1.41, 95 % CI 1.02–1.96; P = 0.04) was also observed. When stratifying by the WHO grade of meningioma, no association was found. Our study suggested that MTHFR C677T and MTRR A66G variants may affect the risk of adult meningioma in Chinese Han population.

  19. A new cotton SDR family gene encodes a polypeptide possessing aldehyde reductase and 3-ketoacyl-CoA reductase activities.

    Science.gov (United States)

    Pang, Yu; Song, Wen-Qiang; Chen, Fang-Yuan; Qin, Yong-Mei

    2010-03-01

    To understand regulatory mechanisms of cotton fiber development, microarray analysis has been performed for upland cotton (Gossypium hirsutum). Based on this, a cDNA (GhKCR3) encoding a polypeptide belonging to short-chain alcohol dehydrogenase/reductase family was isolated and cloned. It contains an open reading frame of 987 bp encoding a polypeptide of 328 amino acid residues. Following its overexpression in bacterial cells, the purified recombinant protein specifically uses NADPH to reduce a variety of short-chain aldehydes. A fragment between Gly180 and Gly191 was found to be essential for its catalytic activity. Though the GhKCR3 gene shares low sequence similarities to the ortholog of Saccharomyces cerevisiae YBR159w that encodes 3-ketoacyl-CoA reductase (KCR) catalyzing the second step of fatty acid elongation, it was surprisingly able to complement the yeast ybr159wDelta mutant. Gas chromatography-mass spectrometry analysis showed that very long-chain fatty acids, especially C26:0, were produced in the ybr159wDelta mutant cells expressing GhKCR3. Applying palmitoyl-CoA and malonyl-CoA as substrates, GhKCR3 showed KCR activity in vitro. Quantitative real time-PCR analysis indicated GhKCR3 transcripts accumulated in rapidly elongating fibers, roots, and stems. Our results suggest that GhKCR3 is probably a novel KCR contributing to very long-chain fatty acid biosynthesis in plants.

  20. Computational Identification of the Paralogs and Orthologs of Human Cytochrome P450 Superfamily and the Implication in Drug Discovery

    Directory of Open Access Journals (Sweden)

    Shu-Ting Pan

    2016-06-01

    Full Text Available The human cytochrome P450 (CYP superfamily consisting of 57 functional genes is the most important group of Phase I drug metabolizing enzymes that oxidize a large number of xenobiotics and endogenous compounds, including therapeutic drugs and environmental toxicants. The CYP superfamily has been shown to expand itself through gene duplication, and some of them become pseudogenes due to gene mutations. Orthologs and paralogs are homologous genes resulting from speciation or duplication, respectively. To explore the evolutionary and functional relationships of human CYPs, we conducted this bioinformatic study to identify their corresponding paralogs, homologs, and orthologs. The functional implications and implications in drug discovery and evolutionary biology were then discussed. GeneCards and Ensembl were used to identify the paralogs of human CYPs. We have used a panel of online databases to identify the orthologs of human CYP genes: NCBI, Ensembl Compara, GeneCards, OMA (“Orthologous MAtrix” Browser, PATHER, TreeFam, EggNOG, and Roundup. The results show that each human CYP has various numbers of paralogs and orthologs using GeneCards and Ensembl. For example, the paralogs of CYP2A6 include CYP2A7, 2A13, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 2F1, 2J2, 2R1, 2S1, 2U1, and 2W1; CYP11A1 has 6 paralogs including CYP11B1, 11B2, 24A1, 27A1, 27B1, and 27C1; CYP51A1 has only three paralogs: CYP26A1, 26B1, and 26C1; while CYP20A1 has no paralog. The majority of human CYPs are well conserved from plants, amphibians, fishes, or mammals to humans due to their important functions in physiology and xenobiotic disposition. The data from different approaches are also cross-validated and validated when experimental data are available. These findings facilitate our understanding of the evolutionary relationships and functional implications of the human CYP superfamily in drug discovery.

  1. The TULIP superfamily of eukaryotic lipid-binding proteins as a mediator of lipid sensing and transport.

    Science.gov (United States)

    Alva, Vikram; Lupas, Andrei N

    2016-08-01

    The tubular lipid-binding (TULIP) superfamily has emerged in recent years as a major mediator of lipid sensing and transport in eukaryotes. It currently encompasses three protein families, SMP-like, BPI-like, and Takeout-like, which share a common fold. This fold consists of a long helix wrapped in a highly curved anti-parallel β-sheet, enclosing a central, lipophilic cavity. The SMP-like proteins, which include subunits of the ERMES complex and the extended synaptotagmins (E-Syts), appear to be mainly located at membrane contacts sites (MCSs) between organelles, mediating inter-organelle lipid exchange. The BPI-like proteins, which include the bactericidal/permeability-increasing protein (BPI), the LPS (lipopolysaccharide)-binding protein (LBP), the cholesteryl ester transfer protein (CETP), and the phospholipid transfer protein (PLTP), are either involved in innate immunity against bacteria through their ability to sense lipopolysaccharides, as is the case for BPI and LBP, or in lipid exchange between lipoprotein particles, as is the case for CETP and PLTP. The Takeout-like proteins, which are comprised of insect juvenile hormone-binding proteins and arthropod allergens, transport, where known, lipid hormones to target tissues during insect development. In all cases, the activity of these proteins is underpinned by their ability to bind large, hydrophobic ligands in their central cavity and segregate them away from the aqueous environment. Furthermore, where they are involved in lipid exchange, recent structural studies have highlighted their ability to establish lipophilic, tubular channels, either between organelles in the case of SMP domains or between lipoprotein particles in the case of CETP. Here, we review the current knowledge on the structure, versatile functions, and evolution of the TULIP superfamily. We propose a deep evolutionary split in this superfamily, predating the Last Eukaryotic Common Ancestor, between the SMP-like proteins, which act on

  2. Tumor Necrosis Factor (TNF) Receptor Superfamily Member 1b on CD8+ T Cells and TNF Receptor Superfamily Member 1a on Non-CD8+ T Cells Contribute Significantly to Upper Genital Tract Pathology Following Chlamydial Infection.

    Science.gov (United States)

    Manam, Srikanth; Thomas, Joshua D; Li, Weidang; Maladore, Allison; Schripsema, Justin H; Ramsey, Kyle H; Murthy, Ashlesh K

    2015-06-15

    We demonstrated previously that tumor necrosis factor α (TNF-α)-producing Chlamydia-specific CD8(+) T cells cause oviduct pathological sequelae. In the current study, we used wild-type C57BL/6J (WT) mice with a deficiency in genes encoding TNF receptor superfamily member 1a (TNFR1; TNFR1 knockout [KO] mice), TNF receptor superfamily member 1b (TNFR2; TNFR2 KO mice), and both TNFR1 and TNFR2 (TNFR1/2 double KO [DKO] mice) and mix-match adoptive transfers of CD8(+) T cells to study chlamydial pathogenesis. TNFR1 KO, TNFR2 KO, and TNFR1/2 DKO mice displayed comparable clearance of primary or secondary genital Chlamydia muridarum infection but significantly reduced oviduct pathology, compared with WT animals. The Chlamydia-specific total cellular cytokine response in splenic and draining lymph nodes and the antibody response in serum were comparable between the WT and KO animals. However, CD8(+) T cells from TNFR2 KO mice displayed significantly reduced activation (CD11a expression and cytokine production), compared with TNFR1 KO or WT animals. Repletion of TNFR2 KO mice with WT CD8(+) T cells but not with TNFR2 KO CD8(+) T cells and repletion of TNFR1 KO mice with either WT or TNFR1 KO CD8(+) T cells restored oviduct pathology to WT levels in both KO groups. Collectively, these results demonstrate that TNFR2-bearing CD8(+) T cells and TNFR1-bearing non-CD8(+) T cells contribute significantly to oviduct pathology following genital chlamydial infection. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  3. Thermolabile methylenetetrahydrofolate reductase in patients with coronary artery disease.

    Science.gov (United States)

    Kang, S S; Wong, P W; Zhou, J M; Sora, J; Lessick, M; Ruggie, N; Grcevich, G

    1988-07-01

    Thermostability of lymphocyte methylenetetrahydrofolate reductase (MTHFR) was determined in 21 patients aged less than 50 years with proven coronary artery disease, and in 21 age- and sex-matched controls without clinical evidence of vascular disease. The mean +/- SD of residual activity after heat inactivation at 46 degrees C for five minutes was 37.6% +/- 5.6% in the controls. In contrast, patients with coronary artery disease could be divided into two subgroups. Fifteen of them had 38.1 +/- 5.9% residual activity which was similar to that of the controls. In six of them the mean +/- SD residual activity after heat inactivation was 13.6% +/- 5.1% which was below 2 SD of the normal mean. These observations suggested that thermolabile MTHFR was associated with development of coronary artery disease.

  4. Diterpenoids with thioredoxin reductase inhibitory activities from Jatropha multifida.

    Science.gov (United States)

    Zhu, Jian-Yong; Zhang, Chun-Yang; Dai, Jing-Jing; Rahman, Khalid; Zhang, Hong

    2017-12-01

    Chemical investigation of the Jatropha multifida has led to the isolation of nine diterpenoids (1-9), including a new jatromulone A, four podocarpane diterpenoids (2-5), two lathyrane-type diterpenoids (6 and 7) and two dinorditerpenoids (8 and 9). Their structures were elucidated by spectroscopic analysis, and the absolute configurations of 1 were determined by CD analysis. All of the diterpenoids were screened for inhibitory activity against thioredoxin reductase (TrxR), which is a potential target for cancer chemotherapy with redox balance and antioxidant functions. Compounds 6 and 7 exhibited stronger activity (IC 50 : 23.4 and 10.6 μM, respectively) than the positive control, curcumin (IC 50  = 25.0 μM). Compounds 2-9 were isolated from J. multifida for the first time.

  5. Methylenetetrahydrofolate reductase polymorphisms in myeloid leukemia patients from Northeastern Brazil

    Directory of Open Access Journals (Sweden)

    Cynara Gomes Barbosa

    2008-01-01

    Full Text Available Methylenetetrahydrofolate reductase (MTHFR: EC 1.5.1.20 polymorphisms are associated to acute lymphoid leukemia in different populations. We used the polymerase chain reaction and the restriction fragment length polymorphism method (PCR-RFLP to investigate MTHFR C677T and A1298C polymorphism frequencies in 67 patients with chronic myeloid leukemia (CML, 27 with acute myeloid leukemia FAB subtype M3 (AML-M3 and 100 apparently healthy controls. The MTHFR mutant allele frequencies were as follows: CML = 17.2% for C677T, 21.6% for A1298C; AML-M3 = 22.2% for C677T, 24.1% for A1298C; and controls = 20.5% for C677T, 21% for A1298C. Taken together, our results provide evidence that MTHFR polymorphisms have no influence on the development of CML or AML-M3.

  6. Aldose reductase inhibitors from the fruiting bodies of Ganoderma applanatum.

    Science.gov (United States)

    Lee, Sanghyun; Shim, Sang Hee; Kim, Ju Sun; Shin, Kuk Hyun; Kang, Sam Sik

    2005-06-01

    The isolation and characterization of rat lens aldose reductase (RLAR) inhibitors from the fruiting bodies of Ganoderma applanatum were conducted. Among the extracts and fractions from G. applanatum tested, the MeOH extract and EtOAc fraction were found to exhibit potent RLAR inhibition in vitro, their IC50 being 1.7 and 0.8 microg/ml, respectively. From the active EtOAc fraction, seven compounds with diverse structural moieties were isolated and identified as D-mannitol (1), 2-methoxyfatty acids (2), cerebrosides (3), daucosterol (4), 2,5-dihydroxyacetophenone (5), 2,5-dihydroxybenzoic acid (6), and protocatechualdehyde (7). Among them, protocatechualdehyde (7) was found to be the most potent RLAR inhibitor (IC50=0.7 microg/ml), and may be useful for the prevention and/or treatment of diabetic complications.

  7. Two methylenetetrahydrofolate reductase gene (MTHFR) polymorphisms, schizophrenia and bipolar disorder

    DEFF Research Database (Denmark)

    Jönsson, Erik G; Larsson, Kristina; Vares, Maria

    2008-01-01

    disorder. In a replication attempt the MTHFR C677T and A1298C SNPs were analyzed in three Scandinavian schizophrenia case-control samples. In addition, Norwegian patients with bipolar disorder were investigated. There were no statistically significant allele or genotype case-control differences....... The present Scandinavian results do not verify previous associations between the putative functional MTHFR gene polymorphisms and schizophrenia or bipolar disorder. However, when combined with previous studies in meta-analyses there is still evidence for association between the MTHFR C677T polymorphism......Recent meta-analyses of the methylenetetrahydrofolate reductase gene (MTHFR) have suggested association between two of its functional single gene polymorphisms (SNPs; C677T and A1298C) and schizophrenia. Studies have also suggested association between MTHFR C677T and A1298C variation and bipolar...

  8. Crystal structure of isoflavone reductase from alfalfa (Medicago sativa L.).

    Science.gov (United States)

    Wang, Xiaoqiang; He, Xianzhi; Lin, Jianqiao; Shao, Hui; Chang, Zhenzhan; Dixon, Richard A

    2006-05-19

    Isoflavonoids play important roles in plant defense and exhibit a range of mammalian health-promoting activities. Isoflavone reductase (IFR) specifically recognizes isoflavones and catalyzes a stereospecific NADPH-dependent reduction to (3R)-isoflavanone. The crystal structure of Medicago sativa IFR with deletion of residues 39-47 has been determined at 1.6A resolution. Structural analysis, molecular modeling and docking, and comparison with the structures of other NADPH-dependent enzymes, defined the putative binding sites for co-factor and substrate and potential key residues for enzyme activity and substrate specificity. Further mutagenesis has confirmed the role of Lys144 as a catalytic residue. This study provides a structural basis for understanding the enzymatic mechanism and substrate specificity of IFRs as well as the functions of IFR-like proteins.

  9. Lactococcus lactis Thioredoxin Reductase Is Sensitive to Light Inactivation

    DEFF Research Database (Denmark)

    Björnberg, Olof; Viennet, Thibault; Skjoldager, Nicklas

    2015-01-01

    enzymes belong to the same class of low-molecular weight thioredoxin reductases and display similar kcat values (∼25 s-1) with their cognate thioredoxin. Remarkably, however, the L. lactis enzyme is inactivated by visible light and furthermore reduces molecular oxygen 10 times faster than E. coli Trx......R. The rate of light inactivation under standardized conditions (λmax = 460 nm and 4 °C) was reduced at lowered oxygen concentrations and in the presence of iodide. Inactivation was accompanied by a distinct spectral shift of the flavin adenine dinucleotide (FAD) that remained firmly bound. High......-resolution mass spectrometric analysis of heat-extracted FAD from light-damaged TrxR revealed a mass increment of 13.979 Da, relative to that of unmodified FAD, corresponding to the addition of one oxygen atom and the loss of two hydrogen atoms. Tandem mass spectrometry confined the increase in mass...

  10. Aspects of ribonucleotide reductase regulation and genome stability

    DEFF Research Database (Denmark)

    Nielsen, Helena Berner Nedergaard

    yeast, and Sml1, Hug1, and Dif1 in budding yeast. An elevated, as well as a reduced dNTP pool is shown to lead to an increase in spontaneous mutation rates, hence regulation of RNR is very important in order to maintain genomic stability. No human inhibitory proteins have yet been identified to regulate......In all living cells, synthesis of the DNA building blocks, deoxyribonucleoside triphosphates (dNTPs), is tightly regulated to ensure a precise DNA replication to maintain genomic stability. Ribonucleotide reductase (RNR) is the enzyme responsible for reducing ribonucleotides to their deoxy forms...... the human RNR enzyme. In this study regulation of human RNR was investigated using a fission yeast strain that depended solely on the human genes of R1 and R2 for dNTP synthesis. Even though this strain could grow like wild-type fission yeast it was hypersensitive to hydroxyurea (HU) and depended...

  11. Two methylenetetrahydrofolate reductase gene (MTHFR) polymorphisms, schizophrenia and bipolar disorder

    DEFF Research Database (Denmark)

    Jönsson, Erik G; Larsson, Kristina; Vares, Maria

    2008-01-01

    Recent meta-analyses of the methylenetetrahydrofolate reductase gene (MTHFR) have suggested association between two of its functional single gene polymorphisms (SNPs; C677T and A1298C) and schizophrenia. Studies have also suggested association between MTHFR C677T and A1298C variation and bipolar...... disorder. In a replication attempt the MTHFR C677T and A1298C SNPs were analyzed in three Scandinavian schizophrenia case-control samples. In addition, Norwegian patients with bipolar disorder were investigated. There were no statistically significant allele or genotype case-control differences....... The present Scandinavian results do not verify previous associations between the putative functional MTHFR gene polymorphisms and schizophrenia or bipolar disorder. However, when combined with previous studies in meta-analyses there is still evidence for association between the MTHFR C677T polymorphism...

  12. Fatty acyl-CoA reductases of birds

    Directory of Open Access Journals (Sweden)

    Hellenbrand Janine

    2011-12-01

    Full Text Available Abstract Background Birds clean and lubricate their feathers with waxes that are produced in the uropygial gland, a holocrine gland located on their back above the tail. The type and the composition of the secreted wax esters are dependent on the bird species, for instance the wax ester secretion of goose contains branched-chain fatty acids and unbranched fatty alcohols, whereas that of barn owl contains fatty acids and alcohols both of which are branched. Alcohol-forming fatty acyl-CoA reductases (FAR catalyze the reduction of activated acyl groups to fatty alcohols that can be esterified with acyl-CoA thioesters forming wax esters. Results cDNA sequences encoding fatty acyl-CoA reductases were cloned from the uropygial glands of barn owl (Tyto alba, domestic chicken (Gallus gallus domesticus and domestic goose (Anser anser domesticus. Heterologous expression in Saccharomyces cerevisiae showed that they encode membrane associated enzymes which catalyze a NADPH dependent reduction of acyl-CoA thioesters to fatty alcohols. By feeding studies of transgenic yeast cultures and in vitro enzyme assays with membrane fractions of transgenic yeast cells two groups of isozymes with different properties were identified, termed FAR1 and FAR2. The FAR1 group mainly synthesized 1-hexadecanol and accepted substrates in the range between 14 and 18 carbon atoms, whereas the FAR2 group preferred stearoyl-CoA and accepted substrates between 16 and 20 carbon atoms. Expression studies with tissues of domestic chicken indicated that FAR transcripts were not restricted to the uropygial gland. Conclusion The data of our study suggest that the identified and characterized avian FAR isozymes, FAR1 and FAR2, can be involved in wax ester biosynthesis and in other pathways like ether lipid synthesis.

  13. TGF-b superfamily cytokine MIC-1/GDF15 is a physiological appetite and body weight regulator.

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    Vicky Wang-Wei Tsai

    Full Text Available The TGF-b superfamily cytokine MIC-1/GDF15 circulates in all humans and when overproduced in cancer leads to anorexia/cachexia, by direct action on brain feeding centres. In these studies we have examined the role of physiologically relevant levels of MIC-1/GDF15 in the regulation of appetite, body weight and basal metabolic rate. MIC-1/GDF15 gene knockout mice (MIC-1(-/- weighed more and had increased adiposity, which was associated with increased spontaneous food intake. Female MIC-1(-/- mice exhibited some additional alterations in reduced basal energy expenditure and physical activity, possibly owing to the associated decrease in total lean mass. Further, infusion of human recombinant MIC-1/GDF15 sufficient to raise serum levels in MIC-1(-/- mice to within the normal human range reduced body weight and food intake. Taken together, our findings suggest that MIC-1/GDF15 is involved in the physiological regulation of appetite and energy storage.

  14. KinMutRF: a random forest classifier of sequence variants in the human protein kinase superfamily

    DEFF Research Database (Denmark)

    Pons, Tirso; Vazquez, Miguel; Matey-Hernandez, María Luisa

    2016-01-01

    remains challenging: cells tolerate most genomic alterations and only a minor fraction disrupt molecular function sufficiently and drive disease. Results: KinMutRF is a novel random-forest method to automatically identify pathogenic variants in human kinases. Twenty six decision trees implemented......Background: The association between aberrant signal processing by protein kinases and human diseases such as cancer was established long time ago. However, understanding the link between sequence variants in the protein kinase superfamily and the mechanistic complex traits at the molecular level...... as a random forest ponder a battery of features that characterize the variants: a) at the gene level, including membership to a Kinbase group and Gene Ontology terms; b) at the PFAM domain level; and c) at the residue level, the types of amino acids involved, changes in biochemical properties, functional...

  15. A new family of polymerases related to superfamily A DNA polymerases and T7-like DNA-dependent RNA polymerases

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    Aravind L

    2008-10-01

    Full Text Available Abstract Using sequence profile methods and structural comparisons we characterize a previously unknown family of nucleic acid polymerases in a group of mobile elements from genomes of diverse bacteria, an algal plastid and certain DNA viruses, including the recently reported Sputnik virus. Using contextual information from domain architectures and gene-neighborhoods we present evidence that they are likely to possess both primase and DNA polymerase activity, comparable to the previously reported prim-pol proteins. These newly identified polymerases help in defining the minimal functional core of superfamily A DNA polymerases and related RNA polymerases. Thus, they provide a framework to understand the emergence of both DNA and RNA polymerization activity in this class of enzymes. They also provide evidence that enigmatic DNA viruses, such as Sputnik, might have emerged from mobile elements coding these polymerases. Reviewers This article was reviewed by Eugene Koonin and Mark Ragan.

  16. RECEPTOR SUPERFAMILY OF TUMOR NECROSIS FACTOR Α, AND HSP90 HEAT SHOCK PROTEIN: A MOLECULAR BASIS FOR INTERACTIONS

    Directory of Open Access Journals (Sweden)

    N. V. Ryazantseva

    2011-01-01

    Full Text Available Abstract.  A  study  was  performed  aiming  to  investigate  interactions  between  TNFα  receptor  (TNF1 superfamily and heat shock protein Hsp90, using a Jurkat tumor cell line. The tumor cells cultured in presence of Hsp90 inhibitor (17-AAG showed increased numbers of cells, presenting surface TNFR1 and FasR, which facilitate  triggering  of  programmed  cell  death.  It  was  also  revealed  that  Hsp90  blockage  under  the  in  vitro conditions causes a decrease in FasL, while not affecting TNFα and sTNFR1 production by the tumor cells. (Med. Immunol., 2011, vol. 13, N 2-3, pp 247-252 

  17. Mm19, a Mycoplasma meleagridis Major Surface Nuclease that Is Related to the RE_AlwI Superfamily of Endonucleases.

    Directory of Open Access Journals (Sweden)

    Elhem Yacoub

    Full Text Available Mycoplasma meleagridis infection is widespread in turkeys, causing poor growth and feathering, airsacculitis, osteodystrophy, and reduction in hatchability. Like most mycoplasma species, M. meleagridis is characterized by its inability to synthesize purine and pyrimidine nucleotides de novo. Consistent with this intrinsic deficiency, we here report the cloning, expression, and characterization of a M. meleagridis gene sequence encoding a major surface nuclease, referred to as Mm19. Mm19 consists of a 1941-bp ORF encoding a 646-amino-acid polypeptide with a predicted molecular mass of 74,825 kDa. BLASTP analysis revealed a significant match with the catalytic/dimerization domain of type II restriction enzymes of the RE_AlwI superfamily. This finding is consistent with the genomic location of Mm19 sequence, which dispalys characteristics of a typical type II restriction-modification locus. Like intact M. meleagridis cells, the E. coli-expressed Mm19 fusion product was found to exhibit a nuclease activity against plasmid DNA, double-stranded DNA, single-stranded DNA, and RNA. The Mm19-associated nuclease activity was consistently enhanced with Mg2+ divalent cations, a hallmark of type II restriction enzymes. A rabbit hyperimmune antiserum raised against the bacterially expressed Mm19 strongly reacted with M. meleagridis intact cells and fully neutralized the surface-bound nuclease activity. Collectively, the results show that M. meleagridis expresses a strong surface-bound nuclease activity, which is the product of a single gene sequence that is related to the RE_AlwI superfamily of endonucleases.

  18. A novel inhibitor of α9α10 nicotinic acetylcholine receptors from Conus vexillum delineates a new conotoxin superfamily.

    Directory of Open Access Journals (Sweden)

    Sulan Luo

    Full Text Available Conotoxins (CTxs selectively target a range of ion channels and receptors, making them widely used tools for probing nervous system function. Conotoxins have been previously grouped into superfamilies according to signal sequence and into families based on their cysteine framework and biological target. Here we describe the cloning and characterization of a new conotoxin, from Conus vexillum, named αB-conotoxin VxXXIVA. The peptide does not belong to any previously described conotoxin superfamily and its arrangement of Cys residues is unique among conopeptides. Moreover, in contrast to previously characterized conopeptide toxins, which are expressed initially as prepropeptide precursors with a signal sequence, a ''pro'' region, and the toxin-encoding region, the precursor sequence of αB-VxXXIVA lacks a ''pro'' region. The predicted 40-residue mature peptide, which contains four Cys, was synthesized in each of the three possible disulfide arrangements. Investigation of the mechanism of action of αB-VxXXIVA revealed that the peptide is a nicotinic acetylcholine receptor (nAChR antagonist with greatest potency against the α9α10 subtype. (1H nuclear magnetic resonance (NMR spectra indicated that all three αB-VxXXIVA isomers were poorly structured in aqueous solution. This was consistent with circular dichroism (CD results which showed that the peptides were unstructured in buffer, but adopted partially helical conformations in aqueous trifluoroethanol (TFE solution. The α9α10 nAChR is an important target for the development of analgesics and cancer chemotherapeutics, and αB-VxXXIVA represents a novel ligand with which to probe the structure and function of this protein.

  19. Genetic polymorphisms in glutathione S-transferase (GST) superfamily and arsenic metabolism in residents of the Red River Delta, Vietnam.

    Science.gov (United States)

    Agusa, Tetsuro; Iwata, Hisato; Fujihara, Junko; Kunito, Takashi; Takeshita, Haruo; Minh, Tu Binh; Trang, Pham Thi Kim; Viet, Pham Hung; Tanabe, Shinsuke

    2010-02-01

    To elucidate the role of genetic factors in arsenic metabolism, we investigated associations of genetic polymorphisms in the members of glutathione S-transferase (GST) superfamily with the arsenic concentrations in hair and urine, and urinary arsenic profile in residents in the Red River Delta, Vietnam. Genotyping was conducted for GST omega1 (GSTO1) Ala140Asp, Glu155del, Glu208Lys, Thr217Asn, and Ala236Val, GST omega2 (GSTO2) Asn142Asp, GST pi1 (GSTP1) Ile105Val, GST mu1 (GSTM1) wild/null, and GST theta1 (GSTT1) wild/null. There were no mutation alleles for GSTO1 Glu208Lys, Thr217Asn, and Ala236Val in this population. GSTO1 Glu155del hetero type showed higher urinary concentration of As(V) than the wild homo type. Higher percentage of DMA(V) in urine of GSTM1 wild type was observed compared with that of the null type. Strong correlations between GSTP1 Ile105Val and arsenic exposure level and profile were observed in this study. Especially, heterozygote of GSTP1 Ile105Val had a higher metabolic capacity from inorganic arsenic to monomethyl arsenic, while the opposite trend was observed for ability of metabolism from As(V) to As(III). Furthermore, other factors including sex, age, body mass index, arsenic level in drinking water, and genotypes of As (+3 oxidation state) methyltransferase (AS3MT) were also significantly co-associated with arsenic level and profile in the Vietnamese. To our knowledge, this is the first study indicating the associations of genetic factors of GST superfamily with arsenic metabolism in a Vietnamese population. Copyright 2009 Elsevier Inc. All rights reserved.

  20. Analysis of the Active-Site Mechanism of Tyrosyl-DNA Phosphodiesterase I: A Member of the Phospholipase D Superfamily

    Energy Technology Data Exchange (ETDEWEB)

    Gajewski, Stefan; Comeaux, Evan Q.; Jafari, Nauzanene; Bharatham, Nagakumar; Bashford, Donald; White, Stephen W.; van Waardenburg, Robert C.A.M. (UAB); (SJCH)

    2012-03-15

    Tyrosyl-DNA phosphodiesterase I (Tdp1) is a member of the phospholipase D superfamily that hydrolyzes 3'-phospho-DNA adducts via two conserved catalytic histidines - one acting as the lead nucleophile and the second acting as a general acid/base. Substitution of the second histidine specifically to arginine contributes to the neurodegenerative disease spinocerebellar ataxia with axonal neuropathy (SCAN1). We investigated the catalytic role of this histidine in the yeast protein (His432) using a combination of X-ray crystallography, biochemistry, yeast genetics, and theoretical chemistry. The structures of wild-type Tdp1 and His432Arg both show a phosphorylated form of the nucleophilic histidine that is not observed in the structure of His432Asn. The phosphohistidine is stabilized in the His432Arg structure by the guanidinium group that also restricts the access of nucleophilic water molecule to the Tdp1-DNA intermediate. Biochemical analyses confirm that His432Arg forms an observable and unique Tdp1-DNA adduct during catalysis. Substitution of His432 by Lys does not affect catalytic activity or yeast phenotype, but substitutions with Asn, Gln, Leu, Ala, Ser, and Thr all result in severely compromised enzymes and DNA topoisomerase I-camptothecin dependent lethality. Surprisingly, His432Asn did not show a stable covalent Tdp1-DNA intermediate that suggests another catalytic defect. Theoretical calculations revealed that the defect resides in the nucleophilic histidine and that the pK{sub a} of this histidine is crucially dependent on the second histidine and on the incoming phosphate of the substrate. This represents a unique example of substrate-activated catalysis that applies to the entire phospholipase D superfamily.

  1. Genetic polymorphisms in glutathione S-transferase (GST) superfamily and arsenic metabolism in residents of the Red River Delta, Vietnam

    International Nuclear Information System (INIS)

    Agusa, Tetsuro; Iwata, Hisato; Fujihara, Junko; Kunito, Takashi; Takeshita, Haruo; Tu Binh Minh; Pham Thi Kim Trang; Pham Hung Viet; Tanabe, Shinsuke

    2010-01-01

    To elucidate the role of genetic factors in arsenic metabolism, we investigated associations of genetic polymorphisms in the members of glutathione S-transferase (GST) superfamily with the arsenic concentrations in hair and urine, and urinary arsenic profile in residents in the Red River Delta, Vietnam. Genotyping was conducted for GST ω1 (GSTO1) Ala140Asp, Glu155del, Glu208Lys, Thr217Asn, and Ala236Val, GST ω2 (GSTO2) Asn142Asp, GST π1 (GSTP1) Ile105Val, GST μ1 (GSTM1) wild/null, and GST θ1 (GSTT1) wild/null. There were no mutation alleles for GSTO1 Glu208Lys, Thr217Asn, and Ala236Val in this population. GSTO1 Glu155del hetero type showed higher urinary concentration of As V than the wild homo type. Higher percentage of DMA V in urine of GSTM1 wild type was observed compared with that of the null type. Strong correlations between GSTP1 Ile105Val and arsenic exposure level and profile were observed in this study. Especially, heterozygote of GSTP1 Ile105Val had a higher metabolic capacity from inorganic arsenic to monomethyl arsenic, while the opposite trend was observed for ability of metabolism from As V to As III . Furthermore, other factors including sex, age, body mass index, arsenic level in drinking water, and genotypes of As (+ 3 oxidation state) methyltransferase (AS3MT) were also significantly co-associated with arsenic level and profile in the Vietnamese. To our knowledge, this is the first study indicating the associations of genetic factors of GST superfamily with arsenic metabolism in a Vietnamese population.

  2. Characterization of human short chain dehydrogenase/reductase SDR16C family members related to retinol dehydrogenase 10.

    Science.gov (United States)

    Adams, Mark K; Lee, Seung-Ah; Belyaeva, Olga V; Wu, Lizhi; Kedishvili, Natalia Y

    2017-10-01

    All-trans-retinoic acid (RA) is a bioactive derivative of vitamin A that serves as an activating ligand for nuclear transcription factors, retinoic acid receptors. RA biosynthesis is initiated by the enzymes that oxidize retinol to retinaldehyde. It is well established that retinol dehydrogenase 10 (RDH10, SDR16C4), which belongs to the 16C family of the short chain dehydrogenase/reductase (SDR) superfamily of proteins, is the major enzyme responsible for the oxidation of retinol to retinaldehyde for RA biosynthesis during embryogenesis. However, several lines of evidence point towards the existence of additional retinol dehydrogenases that contribute to RA biosynthesis in vivo. In close proximity to RDH10 gene on human chromosome 8 are located two genes that are phylogenetically related to RDH10. The predicted protein products of these genes, retinol dehydrogenase epidermal 2 (RDHE2, SDR16C5) and retinol dehydrogenase epidermal 2-similar (RDHE2S, SDR16C6), share 59% and 56% sequence similarity with RDH10, respectively. Previously, we showed that the single ortholog of the human RDHE2 and RDHE2S in frogs, Xenopus laevis rdhe2, oxidizes retinol to retinaldehyde and is essential for frog embryonic development. In this study, we explored the potential of each of the two human proteins to contribute to RA biosynthesis. The results of this study demonstrate that human RDHE2 exhibits a relatively low but reproducible activity when expressed in either HepG2 or HEK293 cells. Expression of the native RDHE2 is downregulated in the presence of elevated levels of RA. On the other hand, the protein encoded by the human RDHE2S gene is unstable when expressed in HEK293 cells. RDHE2S protein produced in Sf9 cells is stable but has no detectable catalytic activity towards retinol. We conclude that the human RDHE2S does not contribute to RA biosynthesis, whereas the low-activity RA-sensitive human RDHE2 may have a role in adjusting the cellular levels of RA in accord with

  3. Photooxidative Destruction of Chloroplasts Leads to Reduced Expression of Peroxisomal NADH-Dependent Hydroxypyruvate Reductase in Developing Cucumber Cotyledons 1

    Science.gov (United States)

    Schwartz, Brain W.; Daniel, Steven G.; Becker, Wayne M.

    1992-01-01

    Photooxidative destruction of chloroplasts by exposure of norflurazon-treated cucumber (Cucumis sativus L.) seedlings to white light leads to reduced levels of the nuclear-encoded, peroxisomal enzyme hydroxypyruvate reductase. The partial reduction in hydroxypyruvate reductase activity under photooxidative conditions is accompanied by reductions in levels of hydroxypyruvate reductase protein and transcript. The low level of hydroxypyruvate reductase gene expression in the dark is not affected by norflurazon, and nonphotooxidizing far-red light is able to induce significant increases in hydroxypyruvate reductase expression even in the presence of norflurazon. We conclude that intact plastids are required for maximal expression of hydroxypyruvate reductase in the light and that the plastids affect hydroxypyruvate reductase gene expression at a pretranslational level. ImagesFigure 2Figure 3Figure 4 PMID:16668940

  4. Comparison of the Stereospecificity and Immunoreactivity of NADH-Ferricyanide Reductases in Plant Membranes.

    Science.gov (United States)

    Fredlund, K. M.; Struglics, A.; Widell, S.; Askerlund, P.; Kader, J. C.; Moller, I. M.

    1994-11-01

    The substrate stereospecificity of NADH-ferricyanide reductase activities in the inner mitochondrial membrane and peroxisomal membrane of potato (Solanum tuberosum L.) tubers, spinach (Spinacea oleracea L.) leaf plasma membrane, and red beetroot (Beta vulgaris L.) tonoplast were all specific for the [beta]-hydrogen of NADH, whereas the reductases in wheat root (Triticum aestivum L.) endoplasmic reticulum and potato tuber outer mitochondrial membrane were both [alpha]-hydrogen specific. In all isolated membrane fractions one or several polypeptides with an apparent size of 45 to 55 kD cross-reacted with antibodies raised against a microsomal NADH-ferricyanide reductase on western blots.

  5. X-ray structural studies of quinone reductase 2 nanomolar range inhibitors

    OpenAIRE

    Pegan, Scott D; Sturdy, Megan; Ferry, Gilles; Delagrange, Philippe; Boutin, Jean A; Mesecar, Andrew D

    2011-01-01

    Quinone reductase 2 (QR2) is one of two members comprising the mammalian quinone reductase family of enzymes responsible for performing FAD mediated reductions of quinone substrates. In contrast to quinone reductase 1 (QR1) which uses NAD(P)H as its co-substrate, QR2 utilizes a rare group of hydride donors, N-methyl or N-ribosyl nicotinamide. Several studies have linked QR2 to the generation of quinone free radicals, several neuronal degenerative diseases, and cancer. QR2 has been also identi...

  6. A novel mechanism of iron-core formation by Pyrococcus furiosus archaeoferritin, a member of an uncharacterized branch of the ferritin-like superfamily

    NARCIS (Netherlands)

    Honarmand Ebrahimi, K.; Hagedoorn, P.L.; Van der Weel, L.; Verhaert, P.D.E.M.; Hagen, W.R.

    2012-01-01

    Storage of iron in a nontoxic and bioavailable form is essential for many forms of life. Three subfamilies of the ferritin-like superfamily, namely, ferritin, bacterioferritin, and Dps (DNA-binding proteins from starved cells), are able to store iron. Although the function of these iron-storage

  7. Aspergillus niger protein estA defines a new class of fungal esterases within the alfa/beta hydrolase fold superfamily of proteins

    NARCIS (Netherlands)

    Bourne, Y.; Hasper, A.A.; Chahinian, H.; Juin, M.; Graaff, de L.H.

    2004-01-01

    From the fungus Aspergillus niger, we identified a new gene encoding protein EstA, a member of the alpha/beta-hydrolase fold superfamily but of unknown substrate specificity. EstA was overexpressed and its crystal structure was solved by molecular replacement using a lipaseacetylcholinesterase

  8. Structure of the human CD97 gene: exon shuffling has generated a new type of seven-span transmembrane molecule related to the secretin receptor superfamily

    NARCIS (Netherlands)

    Hamann, J. [=Jörg; Hartmann, E.; van Lier, R. A.

    1996-01-01

    Recent cDNA cloning of EMR1 and CD97 suggests the existence of a new group of seven-span transmembrane (7-TM) molecules, likely encoded by a gene cluster on the short arm of chromosome 19. The membrane-spanning region of both molecules is homologous to the secretin receptor (SecR) superfamily, a

  9. Molecular identification and functional delineation of a glutathione reductase homolog from disk abalone (Haliotis discus discus): Insights as a potent player in host antioxidant defense.

    Science.gov (United States)

    Herath, H M L P B; Wickramasinghe, P D S U; Bathige, S D N K; Jayasooriya, R G P T; Kim, Gi-Young; Park, Myoung Ae; Kim, Chul; Lee, Jehee

    2017-01-01

    Glutathione reductase (GSR) is an enzyme that catalyzes the biochemical conversion of oxidized glutathione (GSSG) into the reduced form (GSH). Since the ratio between the two forms of glutathione (GSH/GSSG) is important for the optimal function of GSH to act as an antioxidant against H 2 O 2 , the contribution of GSR as an enzymatic regulatory agent to maintain the proper ratio is essential. Abalones are marine mollusks that frequently encounter environmental factors that can trigger the overproduction of reactive oxygen species (ROS) such as H 2 O 2 . Therefore, we conducted the current study to reveal the molecular and functional properties of a GSR homolog in the disk abalone, Haliotis discus discus. The identified cDNA sequence (2325 bp) has a 1356 bp long open reading frame (ORF), coding for a 909 bp long amino acid sequence, which harbors a pyridine nucleotide-disulfide oxidoreductase domain (171-246 aa), a pyridine nucleotide-disulfide oxidoreductase dimerization domain, and a NAD(P)(+)-binding Rossmann fold superfamily signature domain. Four functional residues: the FAD binding site, glutathione binding site, NADPH binding motif, and assembly domain were identified to be conserved among the other species. The recombinant abalone GSR (rAbGSR) exhibited detectable activity in a standard glutathione reductase activity assay. The optimum pH and optimal temperature for the reaction were found to be 7.0 and 50 °C, respectively, while the ionic strength of the medium had no effect. The enzymatic reaction was vastly inhibited by Cu +2 and Cd +2 ions. A considerable effect of cellular protection was detected with a disk diffusion assay conducted with rAbGSR. Moreover, an MTT assay and flow cytometry confirmed the significance of the protective role of rAbGSR in cell function. Furthermore, AbGSR was found to be ubiquitously distributed in different types of abalone tissues. AbGSR mRNA expression was significantly upregulated in response to three immune challenges

  10. Structures of Proline Utilization A (PutA) Reveal the Fold and Functions of the Aldehyde Dehydrogenase Superfamily Domain of Unknown Function*

    Science.gov (United States)

    Luo, Min; Gamage, Thameesha T.; Arentson, Benjamin W.; Schlasner, Katherine N.; Becker, Donald F.; Tanner, John J.

    2016-01-01

    Aldehyde dehydrogenases (ALDHs) catalyze the NAD(P)+-dependent oxidation of aldehydes to carboxylic acids and are important for metabolism and detoxification. Although the ALDH superfamily fold is well established, some ALDHs contain an uncharacterized domain of unknown function (DUF) near the C terminus of the polypeptide chain. Herein, we report the first structure of a protein containing the ALDH superfamily DUF. Proline utilization A from Sinorhizobium meliloti (SmPutA) is a 1233-residue bifunctional enzyme that contains the DUF in addition to proline dehydrogenase and l-glutamate-γ-semialdehyde dehydrogenase catalytic modules. Structures of SmPutA with a proline analog bound to the proline dehydrogenase site and NAD+ bound to the ALDH site were determined in two space groups at 1.7–1.9 Å resolution. The DUF consists of a Rossmann dinucleotide-binding fold fused to a three-stranded β-flap. The Rossmann domain resembles the classic ALDH superfamily NAD+-binding domain, whereas the flap is strikingly similar to the ALDH superfamily dimerization domain. Paradoxically, neither structural element performs its implied function. Electron density maps show that NAD+ does not bind to the DUF Rossmann fold, and small-angle X-ray scattering reveals a novel dimer that has never been seen in the ALDH superfamily. The structure suggests that the DUF is an adapter domain that stabilizes the aldehyde substrate binding loop and seals the substrate-channeling tunnel via tertiary structural interactions that mimic the quaternary structural interactions found in non-DUF PutAs. Kinetic data for SmPutA indicate a substrate-channeling mechanism, in agreement with previous studies of other PutAs. PMID:27679491

  11. Structures of Proline Utilization A (PutA) Reveal the Fold and Functions of the Aldehyde Dehydrogenase Superfamily Domain of Unknown Function.

    Science.gov (United States)

    Luo, Min; Gamage, Thameesha T; Arentson, Benjamin W; Schlasner, Katherine N; Becker, Donald F; Tanner, John J

    2016-11-11

    Aldehyde dehydrogenases (ALDHs) catalyze the NAD(P) + -dependent oxidation of aldehydes to carboxylic acids and are important for metabolism and detoxification. Although the ALDH superfamily fold is well established, some ALDHs contain an uncharacterized domain of unknown function (DUF) near the C terminus of the polypeptide chain. Herein, we report the first structure of a protein containing the ALDH superfamily DUF. Proline utilization A from Sinorhizobium meliloti (SmPutA) is a 1233-residue bifunctional enzyme that contains the DUF in addition to proline dehydrogenase and l-glutamate-γ-semialdehyde dehydrogenase catalytic modules. Structures of SmPutA with a proline analog bound to the proline dehydrogenase site and NAD + bound to the ALDH site were determined in two space groups at 1.7-1.9 Å resolution. The DUF consists of a Rossmann dinucleotide-binding fold fused to a three-stranded β-flap. The Rossmann domain resembles the classic ALDH superfamily NAD + -binding domain, whereas the flap is strikingly similar to the ALDH superfamily dimerization domain. Paradoxically, neither structural element performs its implied function. Electron density maps show that NAD + does not bind to the DUF Rossmann fold, and small-angle X-ray scattering reveals a novel dimer that has never been seen in the ALDH superfamily. The structure suggests that the DUF is an adapter domain that stabilizes the aldehyde substrate binding loop and seals the substrate-channeling tunnel via tertiary structural interactions that mimic the quaternary structural interactions found in non-DUF PutAs. Kinetic data for SmPutA indicate a substrate-channeling mechanism, in agreement with previous studies of other PutAs. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Mo and W bis-MGD enzymes: nitrate reductases and formate dehydrogenases.

    Science.gov (United States)

    Moura, José J G; Brondino, Carlos D; Trincão, José; Romão, Maria João

    2004-10-01

    Molybdenum and tungsten are second- and third-row transition elements, respectively, which are found in a mononuclear form in the active site of a diverse group of enzymes that generally catalyze oxygen atom transfer reactions. Mononuclear Mo-containing enzymes have been classified into three families: xanthine oxidase, DMSO reductase, and sulfite oxidase. The proteins of the DMSO reductase family present the widest diversity of properties among its members and our knowledge about this family was greatly broadened by the study of the enzymes nitrate reductase and formate dehydrogenase, obtained from different sources. We discuss in this review the information of the better characterized examples of these two types of Mo enzymes and W enzymes closely related to the members of the DMSO reductase family. We briefly summarize, also, the few cases reported so far for enzymes that can function either with Mo or W at their active site.

  13. Survival and Psychomotor Development With Early Betaine Treatment in Patients With Severe Methylenetetrahydrofolate Reductase Deficiency

    NARCIS (Netherlands)

    Diekman, Eugene F.; de Koning, Tom J.; Verhoeven-Duif, Nanda M.; Rovers, Maroeska M.; van Hasselt, Peter M.

    IMPORTANCE The impact of betaine treatment on outcome in patients with severe methylenetetrahydrofolate reductase (MTHFR) deficiency is presently unclear. OBJECTIVE To investigate the effect of betaine treatment on development and survival in patients with severe MTHFR deficiency. DATA SOURCES

  14. Survival and psychomotor development with early betaine treatment in patients with severe methylenetetrahydrofolate reductase deficiency

    NARCIS (Netherlands)

    Diekman, E.F.; Koning, T.J. de; Verhoeven-Duif, N.M.; Rovers, M.M.; Hasselt, P.M. van

    2014-01-01

    IMPORTANCE The impact of betaine treatment on outcome in patients with severe methylenetetrahydrofolate reductase (MTHFR) deficiency is presently unclear. OBJECTIVE To investigate the effect of betaine treatment on development and survival in patients with severe MTHFR deficiency. DATA SOURCES

  15. Bioinformatics analysis of the predicted polyprenol reductase genes in higher plants

    Science.gov (United States)

    Basyuni, M.; Wati, R.

    2018-03-01

    The present study evaluates the bioinformatics methods to analyze twenty-four predicted polyprenol reductase genes from higher plants on GenBank as well as predicted the structure, composition, similarity, subcellular localization, and phylogenetic. The physicochemical properties of plant polyprenol showed diversity among the observed genes. The percentage of the secondary structure of plant polyprenol genes followed the ratio order of α helix > random coil > extended chain structure. The values of chloroplast but not signal peptide were too low, indicated that few chloroplast transit peptide in plant polyprenol reductase genes. The possibility of the potential transit peptide showed variation among the plant polyprenol reductase, suggested the importance of understanding the variety of peptide components of plant polyprenol genes. To clarify this finding, a phylogenetic tree was drawn. The phylogenetic tree shows several branches in the tree, suggested that plant polyprenol reductase genes grouped into divergent clusters in the tree.

  16. A soluble 3-hydroxy-3-methylglutaryl-CoA reductase in the protozoan Trypanosoma cruzi

    DEFF Research Database (Denmark)

    Pena Diaz, Javier; Montalvetti, A; Camacho, A

    1997-01-01

    cellular distribution of enzymic activity was investigated after differential centrifugation of Trypanosoma cell extracts. Reductase activity was primarily associated with the cellular soluble fraction because 95% of the total cellular activity was recovered in the supernatant and was particularly...

  17. The 1-hydroxy-2-methyl-butenyl 4-diphosphate reductase gene from ...

    African Journals Online (AJOL)

    The 1-hydroxy-2-methyl-butenyl 4-diphosphate reductase gene from Taxus media: Cloning, characterization and functional identification. Y Sun, M Chen, J Tang, W Liu, C Yang, Y Yang, X Lan, M Hsieh, Z Liao ...

  18. The Importance of Homozygous Polymorphisms of Methylenetetrahydrofolate Reductase Gene in Romanian Patients with Idiopathic Venous Thromboembolism

    OpenAIRE

    Hotoleanu, Cristina; Trifa, Adrian; Popp, Radu; Fodor, Daniela

    2013-01-01

    Background: Methylenetetrahydrofolate reductase (MTHFR) polymorphisms have recently raised the interest as a possible thrombophilic factors. Aims: We aimed to assess the frequency of the methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C polymorphisms in idiopathic venous thromboembolism (VTE) in a Romanian population and the associated risk of VTE. Study Design: We performed a case-control transversal study including 90 patients diagnosed with VTE and 75 sex- an...

  19. Aldose Reductase Inhibitory Activity of Compounds from??Zea mays L.

    OpenAIRE

    Kim, Tae Hyeon; Kim, Jin Kyu; Kang, Young-Hee; Lee, Jae-Yong; Kang, Il Jun; Lim, Soon Sung

    2013-01-01

    Aldose reductase (AR) inhibitors have a considerable therapeutic potential against diabetes complications and do not increase the risk of hypoglycemia. Through bioassay-guided fractionation of an EtOH extract of the kernel from purple corn (Zea mays L.), 7 nonanthocyanin phenolic compounds (compound 1?7) and 5 anthocyanins (compound 8?12) were isolated. These compounds were investigated by rat lens aldose reductase (RLAR) inhibitory assays. Kinetic analyses of recombinant human aldose reducta...

  20. Genome sequence analysis of predicted polyprenol reductase gene from mangrove plant kandelia obovata

    Science.gov (United States)

    Basyuni, M.; Sagami, H.; Baba, S.; Oku, H.

    2018-03-01

    It has been previously reported that dolichols but not polyprenols were predominated in mangrove leaves and roots. Therefore, the occurrence of larger amounts of dolichol in leaves of mangrove plants implies that polyprenol reductase is responsible for the conversion of polyprenol to dolichol may be active in mangrove leaves. Here we report the early assessment of probably polyprenol reductase gene from genome sequence of mangrove plant Kandelia obovata. The functional assignment of the gene was based on a homology search of the sequences against the non-redundant (nr) peptide database of NCBI using Blastx. The degree of sequence identity between DNA sequence and known polyprenol reductase was confirmed using the Blastx probability E-value, total score, and identity. The genome sequence data resulted in three partial sequences, termed c23157 (700 bp), c23901 (960 bp), and c24171 (531 bp). The c23157 gene showed the highest similarity (61%) to predicted polyprenol reductase 2- like from Gossypium raimondii with E-value 2e-100. The second gene was c23901 to exhibit high similarity (78%) to the steroid 5-alpha-reductase Det2 from J. curcas with E-value 2e-140. Furthermore, the c24171 gene depicted highest similarity (79%) to the polyprenol reductase 2 isoform X1 from Jatropha curcas with E- value 7e-21.The present study suggested that the c23157, c23901, and c24171, genes may encode predicted polyprenol reductase. The c23157, c23901, c24171 are therefore the new type of predicted polyprenol reductase from K. obovata.

  1. The mechanism for oxygen reduction in cytochrome c dependent nitric oxide reductase (cNOR) as obtained from a combination of theoretical and experimental results.

    Science.gov (United States)

    Blomberg, Margareta R A; Ädelroth, Pia

    2017-11-01

    Bacterial NO-reductases (NOR) belong to the heme-copper oxidase (HCuO) superfamily, in which most members are O 2 -reducing, proton-pumping enzymes. This study is one in a series aiming to elucidate the reaction mechanisms of the HCuOs, including the mechanisms for cellular energy conservation. One approach towards this goal is to compare the mechanisms for the different types of HCuOs, cytochrome c oxidase (CcO) and NOR, reducing the two substrates O 2 and NO. Specifically in this study, we describe the mechanism for oxygen reduction in cytochrome c dependent NOR (cNOR). Hybrid density functional calculations were performed on large cluster models of the cNOR binuclear active site. Our results are used, together with published experimental information, to construct a free energy profile for the entire catalytic cycle. Although the overall reaction is quite exergonic, we show that during the reduction of molecular oxygen in cNOR, two of the reduction steps are endergonic with high barriers for proton uptake, which is in contrast to oxygen reduction in CcO, where all reduction steps are exergonic. This difference between the two enzymes is suggested to be important for their differing capabilities for energy conservation. An additional result from this study is that at least three of the four reduction steps are initiated by proton transfer to the active site, which is in contrast to CcO, where electrons always arrive before the protons to the active site. The roles of the non-heme metal ion and the redox-active tyrosine in the active site are also discussed. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Purification and characterization of (+)dihydroflavonol (3-hydroxyflavanone) 4-reductase from flowers of Dahlia variabilis.

    Science.gov (United States)

    Fischer, D; Stich, K; Britsch, L; Grisebach, H

    1988-07-01

    Individual flowers from inflorescences of Dahlia variabilis (cv Scarlet Star) in young developmental stages contained relatively high activity of (+)-dihydroflavonol (DHF) 4-reductase. The DHF reductase was purified from such flowers to apparent homogeneity by a five-step procedure. This included affinity adsorption on Blue Sepharose and elution of the enzyme with NADP+. By gel filtration and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis it was shown that DHF reductase contains only one polypeptide chain with a Mr of about 41,000. The reductase required NADPH as cofactor and catalyzed transfer of the pro-S hydrogen of NADPH to the substrate. Flavanones and dihydroflavonols (3-hydroxyflavanones) were substrates for DHF reductase with pH optima of about 6.0 for flavanones and of about 6.8 for dihydroflavonols. Flavanones were reduced to the corresponding flavan-4-ols and (+)-dihydroflavonols to flavan-3,4-cis-diols. Apparent Michaelis constants determined for (2S)-naringenin, (2S)-eriodicytol, (+)-dihydrokaempferol, (+)-dihydroquercetin, and NADPH were, respectively, 2.3, 2, 10, 15, and 42 microM. V/Km values were higher for dihydroflavonols than for flavanones. Conversion of dihydromyricetin to leucodelphinidin was also catalyzed by the enzyme at a low rate, whereas flavones and flavonols were not accepted as substrates. DHF reductase was not inhibited by metal chelators.

  3. Genome-wide characterization of the aldehyde dehydrogenase gene superfamily in soybean and its potential role in drought stress response.

    Science.gov (United States)

    Wang, Wei; Jiang, Wei; Liu, Juge; Li, Yang; Gai, Junyi; Li, Yan

    2017-07-07

    Aldehyde dehydrogenases (ALDHs) represent a group of enzymes that detoxify aldehydes by facilitating their oxidation to carboxylic acids, and have been shown to play roles in plant response to abiotic stresses. However, the comprehensive analysis of ALDH superfamily in soybean (Glycine max) has been limited. In present study, a total of 53 GmALDHs were identified in soybean, and grouped into 10 ALDH families according to the ALDH Gene Nomenclature Committee and phylogenetic analysis. These groupings were supported by their gene structures and conserved motifs. Soybean ALDH superfamily expanded mainly by whole genome duplication/segmental duplications. Gene network analysis identified 1146 putative co-functional genes of 51 GmALDHs. Gene Ontology (GO) enrichment analysis suggested the co-functional genes of these 51 GmALDHs were enriched (FDR soybean tissues. The expression levels of 13 GmALDHs were significantly up-regulated and 14 down-regulated in response to water deficit. The occurrence frequencies of three drought-responsive cis-elements (ABRE, CRT/DRE, and GTGCnTGC/G) were compared in GmALDH genes that were up-, down-, or non-regulated by water deficit. Higher frequency of these three cis-elements was observed for the group of up-regulated GmALDH genes as compared to the group of down- or non- regulated GmALDHs by drought stress, implying their potential roles in the regulation of soybean response to drought stress. A total of 53 ALDH genes were identified in soybean genome and their phylogenetic relationships and duplication patterns were analyzed. The potential functions of GmALDHs were predicted by analyses of their co-functional gene networks, gene expression profiles, and cis-regulatory elements. Three GmALDH genes, including GmALDH3H2, GmALDH12A2 and GmALDH18B3, were highly induced by drought stress in soybean leaves. Our study provides a foundation for future investigations of GmALDH gene function in soybean.

  4. Association study between methylenetetrahydrofolate reductase gene polymorphisms and Graves' disease.

    Science.gov (United States)

    Mao, Renfang; Fan, Yihui; Zuo, Lulu; Geng, Dongfeng; Meng, Fantao; Zhu, Jing; Li, Qiang; Qiao, Hong; Jin, Yan; Bai, Jing; Fu, Songbin

    2010-10-01

    5,10-Methylenetetrahydrofolate reductase (MTHFR) catalyzes the metabolism of folate and nucleotides, which are essential for DNA synthesis and methylation. It is highly polymorphic, and its variant genotypes result in lower enzymatic activity and higher plasma homocysteine. Previous studies have provided evidence that a high prevalence of MTHFR gene polymorphisms is frequently detected in patients with autoimmune disease, suggesting a novel genetic association with autoimmune disorders. However, the genetic association between MTHFR and Graves' disease (GD), one of the most common autoimmune diseases, has not been studied. Here, we designed a clinic-based case-control study including 199 GD cases and 235 healthy controls to examine the associations between three common MTHFR polymorphisms (i.e., C677T, A1298C, and G1793A) and GD. Surprisingly, logistic regression analysis shows MTHFR 677CT + TT genotypes are associated with an approximately 42% reduction in the risk of GD in women (adjusted OR = 0.58, 95% CI = 0.3-0.9), compared to the CC genotype, indicating a significant protective effect of 677CT + TT genotypes. Our result provides epidemiological evidence that MTHFR mutation (C677T) protects women from GD. The protective effect, possibly obtained by influencing DNA methylation, should be confirmed in a large number of cohorts. Copyright © 2010 John Wiley & Sons, Ltd.

  5. A second target of benzamide riboside: dihydrofolate reductase.

    Science.gov (United States)

    Roussel, Breton; Johnson-Farley, Nadine; Kerrigan, John E; Scotto, Kathleen W; Banerjee, Debabrata; Felczak, Krzysztof; Pankiewicz, Krzysztof W; Gounder, Murugesan; Lin, HongXia; Abali, Emine Ercikan; Bertino, Joseph R

    2012-11-01

    Dihydrofolate reductase (DHFR) is an essential enzyme involved in de novo purine and thymidine biosynthesis. For several decades, selective inhibition of DHFR has proven to be a potent therapeutic approach in the treatment of various cancers including acute lymphoblastic leukemia, non-Hodgkin's lymphoma, osteogenic sarcoma, carcinoma of the breast, and head and neck cancer. Therapeutic success with DHFR inhibitor methotrexate (MTX) has been compromised in the clinic, which limits the success of MTX treatment by both acquired and intrinsic resistance mechanisms. We report that benzamide riboside (BR), via anabolism to benzamide adenine dinucleotide (BAD) known to potently inhibit inosine monophosphate dehydrogenase (IMPDH), also inhibits cell growth through a mechanism involving downregulation of DHFR protein. Evidence to support this second site of action of BR includes the finding that CCRF-CEM/R human T-cell lymphoblasic leukemia cells, resistant to MTX as a consequence of gene amplification and overexpression of DHFR, are more resistant to BR than are parental cells. Studies of the mechanism by which BR lowers DHFR showed that BR, through its metabolite BAD, reduced NADP and NADPH cellular levels by inhibiting nicotinamide adenine dinucleotide kinase (NADK). As consequence of the lack of NADPH, DHFR was shown to be destabilized. We suggest that, inhibition of NADK is a new approach to downregulate DHFR and to inhibit cell growth.

  6. Old and new inhibitors of quinone reductase 2.

    Science.gov (United States)

    Ferry, Gilles; Hecht, Sabrina; Berger, Sylvie; Moulharat, Natacha; Coge, Francis; Guillaumet, Gérald; Leclerc, Véronique; Yous, Saïd; Delagrange, Philippe; Boutin, Jean A

    2010-07-30

    Quinone reductase 2 is a cytosolic enzyme which catalyses the reduction of quinones, such as menadione and coenzymes Q. Despite a relatively close sequence-based resemblance to NAD(P)H:quinone oxidoreductase 1 (QR1), it has many different features. QR2 is the third melatonin binding site (MT3). It is inhibited in the micromolar range by melatonin, and does not accept conventional phosphorylated nicotinamides as hydride donors. QR2 has a powerful capacity to activate quinones leading to unexpected toxicity situations. In the present paper, we report the characterization of three QR2 modulators: melatonin, resveratrol and S29434. The latter compound inhibits QR2 activity with an IC(50) in the low nanomolar range. The potency of the modulators ranged as follows, from the least to the most potent: melatonin

  7. Antiproliferative and quinone reductase-inducing activities of withanolides derivatives.

    Science.gov (United States)

    García, Manuela E; Nicotra, Viviana E; Oberti, Juan C; Ríos-Luci, Carla; León, Leticia G; Marler, Laura; Li, Guannan; Pezzuto, John M; van Breemen, Richard B; Padrón, José M; Hueso-Falcón, Idaira; Estévez-Braun, Ana

    2014-07-23

    Two new and five known withanolides (jaborosalactones 2, 3, 4, 5, and 24) were isolated from the leaves of Jaborosa runcinata Lam. We also obtained some derivatives from jaborosalactone 5, which resulted to be the major isolated metabolite. The natural compounds as well as derivatives were evaluated for their antiproliferative activity and the induction of quinone reductase 1 (QR1; NQ01) activity. Structure-activity relationships revealed valuable information on the pharmacophore of withanolide-type compounds. Three compounds of this series showed significantly higher antiproliferative activity than jaborosalactone 5. The effect of these compounds on the cell cycle was determined. Furthermore, the ability of major compounds to induce QR1 was evaluated. It was found that all the active test compounds are monofunctional inducers that interact with Keap1. The most promising derivatives prepared from jaborosalactone 5 include (23R)-4β,12β,21-trihydroxy-1,22-dioxo-12,23-cycloergostan-2,5,17,24-tetraen-26,23-olide (18) and (23R)-21-acetoxy-12β-hydroxy-1,22-dioxo-12,23-cycloergostan-2,5,17,24-tetraen-26,23-lactame (20). Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  8. Structure, function, and mechanism of cytosolic quinone reductases.

    Science.gov (United States)

    Bianchet, Mario A; Erdemli, Sabri Bora; Amzel, L Mario

    2008-01-01

    Quinone reductases type 1 (QR1) are FAD-containing enzymes that catalyze the reduction of many quinones, including menadione (Vit K3), to hydroquinones using reducing equivalents provided by NAD(P)H. The reaction proceeds with a ping-pong mechanism in which the NAD(P)H and the substrate occupy alternatively overlapping regions of the same binding site and participate in a double hydride transfer: one from NAD(P)H to the FAD of the enzyme, and one from the FADH(2) of the enzyme to the quinone substrate. The main function of QR1 is probably the detoxification of dietary quinones but it may also contribute to the reduction of vitamin K for its involvement in blood coagulation. In addition, the same reaction that QR1 uses in the detoxification of quinones, activates some compounds making them cytotoxic. Since QR1 is elevated in many tumors, this property has encouraged the development of chemotherapeutic compounds that become cytotoxic after reduction by QR1. The structures of QR1 alone, and in complexes with substrates, inhibitors, and chemotherapeutic prodrugs, combined with biochemical and mechanistic studies have provided invaluable insight into the mechanism of the enzyme as well as suggestions for the improvements of the chemotherapeutic prodrugs. Similar information is beginning to accumulate about another related enzyme, QR2.

  9. 5 alpha-reductase inhibitors and prostatic disease.

    Science.gov (United States)

    Schröder, F H

    1994-08-01

    5 alpha-Reductase inhibitors are a new class of substances with very specific effects on type I and type II 5 alpha R which may be of use in the treatment of skin disease, such as male pattern baldness, male acne and hirsutism, as well as prostatic hyperplasia and prostate cancer. At least two types of 5 alpha R inhibitors with a different pH optimum have been described. cDNA encoding for both the type I and the type II enzyme has been cloned. Most of the orally effective 5 alpha R inhibitors belong to the class of 4-azasteroids. The radical substituted in the 17 position of the steroid ring seems to be related to species specific variations and to the types of 5 alpha R enzymes in different species and organ systems. 5 alpha R inhibitors lead to a decrease of plasma DHT by about 65% while there is a slight rise in plasma testosterone. The decrease of tissue DHT in the ventral prostate of the intact rat, the dog and in humans is more pronounced and amounts to about 85%. There is a reciprocal rise of tissue T in these systems. The application of an inhibitor of 5 alpha R type II leads to a shrinkage of BPH in men by about 30%. In the rat a similar shrinkage accompanied by a significant decrease of total organ DNA occurs. This decrease, however, is not as pronounced as can be achieved with castration.(ABSTRACT TRUNCATED AT 250 WORDS)

  10. Increased 5α-reductase activity in idiopathic hirsutism

    International Nuclear Information System (INIS)

    Serafini, P.; Lobo, R.A.

    1985-01-01

    In vitro, genital skin 5α-reductase activity (5α-RA) was measured in ten hirsute women with normal androgen levels (idiopathic hirsutism (IH)) and in ten hirsute women with elevated androgen levels (polycystic ovary syndrome (PCO)) in order to determine the influence of secreted androgens on 5α-RA. In vitro 5α-RA was assessed by incubations of skin with 14 C-testosterone (T) for 2 hours, after which steroids were separated and the radioactivity of dihydrotestosterone (DHT) and 5α-androstane 3α-17β-estradiol (3α-diol) in specific eluates were determined. All androgens were normal in IH with the exception of higher levels of 3α-diol glucuronide which were similar to the levels of PCO. The conversion ratio (CR) of T to DHT in IH and PCO were similar, yet significantly greater than the CR of control subjects. The CR of T to 3α-diol in IH and PCO were similar, yet higher than in control subjects. Serum androgens showed no correlation with 5α-RA, while the CR of T to DHT showed a significant positive correlation with the Ferriman and Gallwey score. The increased 5α-RA in IH appears to be independent of serum androgen levels and is, therefore, an inherent abnormality. The term idiopathic is a misnomer, because hirsutism in these patients may be explained on the basis of increased skin 5α-RA

  11. Cheminformatics Models for Inhibitors of Schistosoma mansoni Thioredoxin Glutathione Reductase

    Directory of Open Access Journals (Sweden)

    Sonam Gaba

    2014-01-01

    Full Text Available Schistosomiasis is a neglected tropical disease caused by a parasite Schistosoma mansoni and affects over 200 million annually. There is an urgent need to discover novel therapeutic options to control the disease with the recent emergence of drug resistance. The multifunctional protein, thioredoxin glutathione reductase (TGR, an essential enzyme for the survival of the pathogen in the redox environment has been actively explored as a potential drug target. The recent availability of small-molecule screening datasets against this target provides a unique opportunity to learn molecular properties and apply computational models for discovery of activities in large molecular libraries. Such a prioritisation approach could have the potential to reduce the cost of failures in lead discovery. A supervised learning approach was employed to develop a cost sensitive classification model to evaluate the biological activity of the molecules. Random forest was identified to be the best classifier among all the classifiers with an accuracy of around 80 percent. Independent analysis using a maximally occurring substructure analysis revealed 10 highly enriched scaffolds in the actives dataset and their docking against was also performed. We show that a combined approach of machine learning and other cheminformatics approaches such as substructure comparison and molecular docking is efficient to prioritise molecules from large molecular datasets.

  12. Determination of Nitrate Reductase Assay Depending on the Microbial Growth

    International Nuclear Information System (INIS)

    El-Kabbany, H.M.

    2012-01-01

    A rapid micro-dilution assay for determination of the antimicrobial susceptibility of different bacterial isolates was developed. This assay is based on the ability of the most of viable organisms to reduce nitrates. The MIC or MBC could be determined by nitrate reductase (NR) only after 30 to 90 min of incubation depending on the behaviour of microbial growth. Bacterial viability is detected by a positive nitrite reduction rather than visible turbidity. The nitrate reduction assay was compared with standard micro-assay using 250 isolates of different taxa against 10 antibiotics belonging to different classes. An excellent agreement of 82.5 % was found between the two methods and only 17.5 % of 1794 trials showed difference in the determined MIC by tow-dilution interval above or below the MIC determined by the turbidimetric method under the same test conditions. However, the nitrate reduction assay was more rapid and sensitive in detecting viable bacteria and so, established an accurate estimate of the minimal inhibitory concentration (MIC) or the minimal bacterial concentration (MBC). The nitrate reduction assay offers the additional advantage that it could be used to determine the MBC without having to subculture the broth. 232 cases of resistance were detected by NR and 4 different media were tested for susceptibility test. The bacterial isolates were exposed to ultra violet (UV) light for different period

  13. Sinorhizobium meliloti Phage ΦM9 Defines a New Group of T4 Superfamily Phages with Unusual Genomic Features but a Common T=16 Capsid.

    Science.gov (United States)

    Johnson, Matthew C; Tatum, Kelsey B; Lynn, Jason S; Brewer, Tess E; Lu, Stephen; Washburn, Brian K; Stroupe, M Elizabeth; Jones, Kathryn M

    2015-11-01

    Relatively little is known about the phages that infect agriculturally important nitrogen-fixing rhizobial bacteria. Here we report the genome and cryo-electron microscopy structure of the Sinorhizobium meliloti-infecting T4 superfamily phage ΦM9. This phage and its close relative Rhizobium phage vB_RleM_P10VF define a new group of T4 superfamily phages. These phages are distinctly different from the recently characterized cyanophage-like S. meliloti phages of the ΦM12 group. Structurally, ΦM9 has a T=16 capsid formed from repeating units of an extended gp23-like subunit that assemble through interactions between one subunit and the adjacent E-loop insertion domain. Though genetically very distant from the cyanophages, the ΦM9 capsid closely resembles that of the T4 superfamily cyanophage Syn9. ΦM9 also has the same T=16 capsid architecture as the very distant phage SPO1 and the herpesviruses. Despite their overall lack of similarity at the genomic and structural levels, ΦM9 and S. meliloti phage ΦM12 have a small number of open reading frames in common that appear to encode structural proteins involved in interaction with the host and which may have been acquired by horizontal transfer. These proteins are predicted to encode tail baseplate proteins, tail fibers, tail fiber assembly proteins, and glycanases that cleave host exopolysaccharide. Despite recent advances in the phylogenetic and structural characterization of bacteriophages, only a small number of phages of plant-symbiotic nitrogen-fixing soil bacteria have been studied at the molecular level. The effects of phage predation upon beneficial bacteria that promote plant growth remain poorly characterized. First steps in understanding these soil bacterium-phage dynamics are genetic, molecular, and structural characterizations of these groups of phages. The T4 superfamily phages are among the most complex phages; they have large genomes packaged within an icosahedral head and a long, contractile tail

  14. De novo Sequencing and Analysis of Lemongrass Transcriptome Provides First Insights into the Essential Oil Biosynthesis of Aromatic Grasses

    Directory of Open Access Journals (Sweden)

    Seema Meena

    2016-07-01

    Full Text Available Aromatic grasses of the genus Cymbopogon (Poaceae family represent unique group of plants that produce diverse composition of monoterpene rich essential oils, which have great value in flavour, fragrance, cosmetic and aromatherapy industries. Despite the commercial importance of these natural aromatic oils, their biosynthesis at the molecular level remains unexplored. As the first step towards understanding the essential oil biosynthesis, we performed de novo transcriptome assembly and analysis of C. flexuosus (lemongrass by employing Illumina sequencing. Mining of transcriptome data and subsequent phylogenetic analysis led to identification of terpene synthases (TPS, pyrophosphatases (PPase, alcohol dehydrogenases (ADH, aldo-keto reductases (AKR, carotenoid cleavage dioxygenases (CCD, alcohol acetyltransferases (AAT and aldehyde dehydrogenases (ALDH, which are potentially involved in essential oil biosynthesis. Comparative essential oil profiling and mRNA expression analysis in three Cymbopogon species (C. flexuosus, aldehyde type; C. martinii, alcohol type; and C. winterianus, intermediate type with varying essential oil composition indicated the involvement of identified candidate genes in the formation of alcohols, aldehydes and acetates. Molecular modeling and docking further supported the role of identified enzymes in aroma formation in Cymbopogon. Also, simple sequence repeats (SSRs were found in the transcriptome with many linked to terpene pathway genes including the genes potentially involved in aroma biosynthesis. This work provides the first insights into the essential oil biosynthesis of aromatic grasses, and the identified candidate genes and markers can be a great resource for biotechnological and molecular breeding approaches to modulate the essential oil composition.

  15. Oxidative Stress and Carbonyl Lesions in Ulcerative Colitis and Associated Colorectal Cancer

    Directory of Open Access Journals (Sweden)

    Zhiqi Wang

    2016-01-01

    Full Text Available Oxidative stress has long been known as a pathogenic factor of ulcerative colitis (UC and colitis-associated colorectal cancer (CAC, but the effects of secondary carbonyl lesions receive less emphasis. In inflammatory conditions, reactive oxygen species (ROS, such as superoxide anion free radical (O2∙-, hydrogen peroxide (H2O2, and hydroxyl radical (HO∙, are produced at high levels and accumulated to cause oxidative stress (OS. In oxidative status, accumulated ROS can cause protein dysfunction and DNA damage, leading to gene mutations and cell death. Accumulated ROS could also act as chemical messengers to activate signaling pathways, such as NF-κB and p38 MAPK, to affect cell proliferation, differentiation, and apoptosis. More importantly, electrophilic carbonyl compounds produced by lipid peroxidation may function as secondary pathogenic factors, causing further protein and membrane lesions. This may in turn exaggerate oxidative stress, forming a vicious cycle. Electrophilic carbonyls could also cause DNA mutations and breaks, driving malignant progression of UC. The secondary lesions caused by carbonyl compounds may be exceptionally important in the case of host carbonyl defensive system deficit, such as aldo-keto reductase 1B10 deficiency. This review article updates the current understanding of oxidative stress and carbonyl lesions in the development and progression of UC and CAC.

  16. Methyl jasmonate: putative mechanisms of action on cancer cells cycle, metabolism, and apoptosis.

    Science.gov (United States)

    Cesari, Italo Mario; Carvalho, Erika; Figueiredo Rodrigues, Mariana; Mendonça, Bruna Dos Santos; Amôedo, Nivea Dias; Rumjanek, Franklin David

    2014-01-01

    Methyl jasmonate (MJ), an oxylipid that induces defense-related mechanisms in plants, has been shown to be active against cancer cells both in vitro and in vivo, without affecting normal cells. Here we review most of the described MJ activities in an attempt to get an integrated view and better understanding of its multifaceted modes of action. MJ (1) arrests cell cycle, inhibiting cell growth and proliferation, (2) causes cell death through the intrinsic/extrinsic proapoptotic, p53-independent apoptotic, and nonapoptotic (necrosis) pathways, (3) detaches hexokinase from the voltage-dependent anion channel, dissociating glycolytic and mitochondrial functions, decreasing the mitochondrial membrane potential, favoring cytochrome c release and ATP depletion, activating pro-apoptotic, and inactivating antiapoptotic proteins, (4) induces reactive oxygen species mediated responses, (5) stimulates MAPK-stress signaling and redifferentiation in leukemia cells, (6) inhibits overexpressed proinflammatory enzymes in cancer cells such as aldo-keto reductase 1 and 5-lipoxygenase, and (7) inhibits cell migration and shows antiangiogenic and antimetastatic activities. Finally, MJ may act as a chemosensitizer to some chemotherapics helping to overcome drug resistant. The complete lack of toxicity to normal cells and the rapidity by which MJ causes damage to cancer cells turn MJ into a promising anticancer agent that can be used alone or in combination with other agents.

  17. De Novo Sequencing and Analysis of Lemongrass Transcriptome Provide First Insights into the Essential Oil Biosynthesis of Aromatic Grasses.

    Science.gov (United States)

    Meena, Seema; Kumar, Sarma R; Venkata Rao, D K; Dwivedi, Varun; Shilpashree, H B; Rastogi, Shubhra; Shasany, Ajit K; Nagegowda, Dinesh A

    2016-01-01

    Aromatic grasses of the genus Cymbopogon (Poaceae family) represent unique group of plants that produce diverse composition of monoterpene rich essential oils, which have great value in flavor, fragrance, cosmetic, and aromatherapy industries. Despite the commercial importance of these natural aromatic oils, their biosynthesis at the molecular level remains unexplored. As the first step toward understanding the essential oil biosynthesis, we performed de novo transcriptome assembly and analysis of C. flexuosus (lemongrass) by employing Illumina sequencing. Mining of transcriptome data and subsequent phylogenetic analysis led to identification of terpene synthases, pyrophosphatases, alcohol dehydrogenases, aldo-keto reductases, carotenoid cleavage dioxygenases, alcohol acetyltransferases, and aldehyde dehydrogenases, which are potentially involved in essential oil biosynthesis. Comparative essential oil profiling and mRNA expression analysis in three Cymbopogon species (C. flexuosus, aldehyde type; C. martinii, alcohol type; and C. winterianus, intermediate type) with varying essential oil composition indicated the involvement of identified candidate genes in the formation of alcohols, aldehydes, and acetates. Molecular modeling and docking further supported the role of identified protein sequences in aroma formation in Cymbopogon. Also, simple sequence repeats were found in the transcriptome with many linked to terpene pathway genes including the genes potentially involved in aroma biosynthesis. This work provides the first insights into the essential oil biosynthesis of aromatic grasses, and the identified candidate genes and markers can be a great resource for biotechnological and molecular breeding approaches to modulate the essential oil composition.

  18. Facile fabrication of CdSe/CdS quantum dots and their application on the screening of colorectal cancer

    Energy Technology Data Exchange (ETDEWEB)

    Cao, Hongfeng; Dong, Quanjin, E-mail: qjdong1508@163.com [Zhejiang Provincial People’s Hospital, Department of Colorectal Surgery (China); Hu, Li [Nanjing University of Science and Technology, School of Environmental and Biological Engineering (China); Tu, Shiliang; Chai, Rui; Dai, Qiaoqiong [Zhejiang Provincial People’s Hospital, Department of Colorectal Surgery (China)

    2015-11-15

    In this paper, a facile aqueous route to water-soluble CdSe/CdS quantum dots (QDs) under mild conditions has been developed. The samples were characterized by means of transmission electron microscopy, energy-dispersive X-ray spectroscopy, and photoluminescence (PL) spectroscopy. The PL property of the QDs can be controlled by adjusting the reaction time. The CdSe/CdS QDs after 48-h reaction with size of 5 nm have the strongest PL intensity located at 553 nm, and the highest quantum yield of 19.9 %. The obtained QDs were applied for the colorectal cancer screening. The QDs could be conjugated with antibody of aldo-keto reductase family 1, member B10 (AKR1B10) for the detection of AKR1B10. The AKR1B10 in PBS/5 % serum solution with concentration of 1 ng/mL could be well calibrated, and the limit of detection could be lower than 0.05 ng/mL.

  19. Stereochemical inversion of (S)-reticuline by a cytochrome P450 fusion in opium poppy.

    Science.gov (United States)

    Farrow, Scott C; Hagel, Jillian M; Beaudoin, Guillaume A W; Burns, Darcy C; Facchini, Peter J

    2015-09-01

    The gateway to morphine biosynthesis in opium poppy (Papaver somniferum) is the stereochemical inversion of (S)-reticuline since the enzyme yielding the first committed intermediate salutaridine is specific for (R)-reticuline. A fusion between a cytochrome P450 (CYP) and an aldo-keto reductase (AKR) catalyzes the S-to-R epimerization of reticuline via 1,2-dehydroreticuline. The reticuline epimerase (REPI) fusion was detected in opium poppy and in Papaver bracteatum, which accumulates thebaine. In contrast, orthologs encoding independent CYP and AKR enzymes catalyzing the respective synthesis and reduction of 1,2-dehydroreticuline were isolated from Papaver rhoeas, which does not accumulate morphinan alkaloids. An ancestral relationship between these enzymes is supported by a conservation of introns in the gene fusions and independent orthologs. Suppression of REPI transcripts using virus-induced gene silencing in opium poppy reduced levels of (R)-reticuline and morphinan alkaloids and increased the overall abundance of (S)-reticuline and its O-methylated derivatives. Discovery of REPI completes the isolation of genes responsible for known steps of morphine biosynthesis.

  20. Facile fabrication of CdSe/CdS quantum dots and their application on the screening of colorectal cancer

    Science.gov (United States)

    Cao, Hongfeng; Dong, Quanjin; Hu, Li; Tu, Shiliang; Chai, Rui; Dai, Qiaoqiong

    2015-11-01

    In this paper, a facile aqueous route to water-soluble CdSe/CdS quantum dots (QDs) under mild conditions has been developed. The samples were characterized by means of transmission electron microscopy, energy-dispersive X-ray spectroscopy, and photoluminescence (PL) spectroscopy. The PL property of the QDs can be controlled by adjusting the reaction time. The CdSe/CdS QDs after 48-h reaction with size of 5 nm have the strongest PL intensity located at 553 nm, and the highest quantum yield of 19.9 %. The obtained QDs were applied for the colorectal cancer screening. The QDs could be conjugated with antibody of aldo-keto reductase family 1, member B10 (AKR1B10) for the detection of AKR1B10. The AKR1B10 in PBS/5 % serum solution with concentration of 1 ng/mL could be well calibrated, and the limit of detection could be lower than 0.05 ng/mL.

  1. Involvement of an octose ketoreductase and two acyltransferases in the biosynthesis of paulomycins

    Science.gov (United States)

    Li, Jine; Wang, Min; Ding, Yong; Tang, Yue; Zhang, Zhiguo; Chen, Yihua

    2016-02-01

    C-4 hydroxyethyl branched octoses have been observed in polysaccharides of several genera of gram negative bacteria and in various antibiotics produced by gram positive bacteria. The C-4 hydroxyethyl branch was proposed to be converted from C-4 acetyl branch by an uncharacterized ketoreduction step. Paulomycins (PAUs) are glycosylated antibiotics with potent inhibitory activity against gram positive bacteria and are structurally defined by its unique C-4‧ hydroxyethyl branched paulomycose moiety. A novel aldo-keto-reductase, Pau7 was characterized as the enzyme catalyzing the stereospecific ketoreduction of 7‧-keto of PAU E (1) to give the C-4‧ hydroxyethyl branched paulomycose moiety of PAU F (2). An acyltransferase Pau6 further decorates the C-4‧ hydroxyethyl branch of paulomycose moiety of 2 by attaching various fatty acyl chains to 7‧-OH to generate diverse PAUs. In addition, another acyltransferase Pau24 was proposed to be responsible for the 13-O-acetylation of PAUs.

  2. Lignin-associated metagene expression in a lignocellulose-digesting termite.

    Science.gov (United States)

    Sethi, Amit; Slack, Jeffrey M; Kovaleva, Elena S; Buchman, George W; Scharf, Michael E

    2013-01-01

    Lignin is a component of plant biomass that presents a significant obstacle to biofuel production. It is composed of a highly stable phenylpropanoid matrix that upon degradation, releases toxic metabolites. Termites have specialized digestive systems that overcome the lignin barrier in wood lignocellulose to efficiently release fermentable simple sugars; however, how termites specifically degrade lignin and tolerate its toxic byproducts remains unknown. Here, using the termite Reticulitermes flavipes and its symbiotic (protozoan) gut fauna as a model system, we used high throughput Roche 454-titanium pyrosequencing and proteomics approaches to (i) experimentally compare the effects of diets containing varying degrees of lignin complexity on host-symbiont digestome composition, (ii) deeply sample host and symbiont lignocellulase diversity, and (iii) identify promising lignocellulase candidates for functional characterization. In addition to revealing over 9500 differentially expressed transcripts related to a wide range of physiological processes, our findings reveal two detoxification enzyme families not generally considered in connection with lignocellulose digestion: aldo-keto reductases and catalases. Recombinant versions of two host enzymes from these enzyme families, which apparently play no roles in cellulose or hemicellulose digestion, significantly enhance lignocellulose saccharification by cocktails of host and symbiont cellulases. These hypothesis-driven results provide important new insights into (i) dietary lignin as a xenobiotic challenge, (ii) the complex mechanisms used by termites to cope with their lignin-rich diets, and (iii) novel lignin-targeted enzymatic approaches to enhance biofuel and biomaterial production. Copyright © 2012 Elsevier Ltd. All rights reserved.

  3. Methyl Jasmonate: Putative Mechanisms of Action on Cancer Cells Cycle, Metabolism, and Apoptosis

    Directory of Open Access Journals (Sweden)

    Italo Mario Cesari

    2014-01-01

    Full Text Available Methyl jasmonate (MJ, an oxylipid that induces defense-related mechanisms in plants, has been shown to be active against cancer cells both in vitro and in vivo, without affecting normal cells. Here we review most of the described MJ activities in an attempt to get an integrated view and better understanding of its multifaceted modes of action. MJ (1 arrests cell cycle, inhibiting cell growth and proliferation, (2 causes cell death through the intrinsic/extrinsic proapoptotic, p53-independent apoptotic, and nonapoptotic (necrosis pathways, (3 detaches hexokinase from the voltage-dependent anion channel, dissociating glycolytic and mitochondrial functions, decreasing the mitochondrial membrane potential, favoring cytochrome c release and ATP depletion, activating pro-apoptotic, and inactivating antiapoptotic proteins, (4 induces reactive oxygen species mediated responses, (5 stimulates MAPK-stress signaling and redifferentiation in leukemia cells, (6 inhibits overexpressed proinflammatory enzymes in cancer cells such as aldo-keto reductase 1 and 5-lipoxygenase, and (7 inhibits cell migration and shows antiangiogenic and antimetastatic activities. Finally, MJ may act as a chemosensitizer to some chemotherapics helping to overcome drug resistant. The complete lack of toxicity to normal cells and the rapidity by which MJ causes damage to cancer cells turn MJ into a promising anticancer agent that can be used alone or in combination with other agents.

  4. Nitrate reductase from Spinacea oleracea. FAD and the reactivation of the enzyme treated with p-Hydroxymercuribenzoate.

    Science.gov (United States)

    Castillo, F; de la Rosa, F F; Palacián, E

    1977-12-01

    Spinach nitrate reductase complex previously inactivated by treatment with mercurials p-hydroxymercuribenzoate or p-hydroxymercuriphenyl sulphonate can be reactivated by incubation with dithioerythritol. The reactivation of NADH-diaphorase seems to be FAD-dependent, whereas that of FNH2-nitrate reductase is not. The requirement of FAD for NADH-inactivation of nitrate reductase treated with p-hydroxymercuribenzoate disappears after treatment with dithioerythritol.

  5. Induction of aldose reductase and sorbitol in renal inner medullary cells by elevated extracellular NaCl.

    OpenAIRE

    Bagnasco, S M; Uchida, S; Balaban, R S; Kador, P F; Burg, M B

    1987-01-01

    Aldose reductase [aldehyde reductase 2; alditol:NAD(P)+ 1-oxidoreductase, EC 1.1.1.21] catalyzes conversion of glucose to sorbitol. Although its activity is implicated in the progression of ocular and neurological complications of diabetes, the normal function of the enzyme in most cells is unknown. Both aldose reductase activity and substantial levels of sorbitol were previously reported in renal inner medullary cells. In this tissue, the extracellular NaCl concentration normally is high and...

  6. Mutations in SERPINB7, Encoding a Member of the Serine Protease Inhibitor Superfamily, Cause Nagashima-type Palmoplantar Keratosis

    Science.gov (United States)

    Kubo, Akiharu; Shiohama, Aiko; Sasaki, Takashi; Nakabayashi, Kazuhiko; Kawasaki, Hiroshi; Atsugi, Toru; Sato, Showbu; Shimizu, Atsushi; Mikami, Shuji; Tanizaki, Hideaki; Uchiyama, Masaki; Maeda, Tatsuo; Ito, Taisuke; Sakabe, Jun-ichi; Heike, Toshio; Okuyama, Torayuki; Kosaki, Rika; Kosaki, Kenjiro; Kudoh, Jun; Hata, Kenichiro; Umezawa, Akihiro; Tokura, Yoshiki; Ishiko, Akira; Niizeki, Hironori; Kabashima, Kenji; Mitsuhashi, Yoshihiko; Amagai, Masayuki

    2013-01-01

    “Nagashima-type” palmoplantar keratosis (NPPK) is an autosomal recessive nonsyndromic diffuse palmoplantar keratosis characterized by well-demarcated diffuse hyperkeratosis with redness, expanding on to the dorsal surfaces of the palms and feet and the Achilles tendon area. Hyperkeratosis in NPPK is mild and nonprogressive, differentiating NPPK clinically from Mal de Meleda. We performed whole-exome and/or Sanger sequencing analyses of 13 unrelated NPPK individuals and identified biallelic putative loss-of-function mutations in SERPINB7, which encodes a cytoplasmic member of the serine protease inhibitor superfamily. We identified a major causative mutation of c.796C>T (p.Arg266∗) as a founder mutation in Japanese and Chinese populations. SERPINB7 was specifically present in the cytoplasm of the stratum granulosum and the stratum corneum (SC) of the epidermis. All of the identified mutants are predicted to cause premature termination upstream of the reactive site, which inhibits the proteases, suggesting a complete loss of the protease inhibitory activity of SERPINB7 in NPPK skin. On exposure of NPPK lesional skin to water, we observed a whitish spongy change in the SC, suggesting enhanced water permeation into the SC due to overactivation of proteases and a resultant loss of integrity of the SC structure. These findings provide an important framework for developing pathogenesis-based therapies for NPPK. PMID:24207119

  7. Identification of a WNT5A-Responsive Degradation Domain in the Kinesin Superfamily Protein KIF26B

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    Edith P. Karuna

    2018-04-01

    Full Text Available Noncanonical WNT pathways function independently of the β-catenin transcriptional co-activator to regulate diverse morphogenetic and pathogenic processes. Recent studies showed that noncanonical WNTs, such as WNT5A, can signal the degradation of several downstream effectors, thereby modulating these effectors’ cellular activities. The protein domain(s that mediates the WNT5A-dependent degradation response, however, has not been identified. By coupling protein mutagenesis experiments with a flow cytometry-based degradation reporter assay, we have defined a protein domain in the kinesin superfamily protein KIF26B that is essential for WNT5A-dependent degradation. We found that a human disease-causing KIF26B mutation located at a conserved amino acid within this domain compromises the ability of WNT5A to induce KIF26B degradation. Using pharmacological perturbation, we further uncovered a role of glycogen synthase kinase 3 (GSK3 in WNT5A regulation of KIF26B degradation. Lastly, based on the identification of the WNT5A-responsive domain, we developed a new reporter system that allows for efficient profiling of WNT5A-KIF26B signaling activity in both somatic and stem cells. In conclusion, our study identifies a new protein domain that mediates WNT5A-dependent degradation of KIF26B and provides a new tool for functional characterization of noncanonical WNT5A signaling in cells.

  8. GH97 is a new family of glycoside hydrolases, which is related to the α-galactosidase superfamily

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    Naumoff Daniil G

    2005-08-01

    Full Text Available Abstract Background As a rule, about 1% of genes in a given genome encode glycoside hydrolases and their homologues. On the basis of sequence similarity they have been grouped into more than ninety GH families during the last 15 years. The GH97 family has been established very recently and initially included only 18 bacterial proteins. However, the evolutionary relationship of the genes encoding proteins of this family remains unclear, as well as their distribution among main groups of the living organisms. Results The extensive search of the current databases allowed us to double the number of GH97 family proteins. Five subfamilies were distinguished on the basis of pairwise sequence comparison and phylogenetic analysis. Iterative sequence analysis revealed the relationship of the GH97 family with the GH27, GH31, and GH36 families of glycosidases, which belong to the α-galactosidase superfamily, as well as a more distant relationship with some other glycosidase families (GH13 and GH20. Conclusion The results of this study show an unexpected sequence similarity of GH97 family proteins with glycoside hydrolases from several other families, that have (β/α8-barrel fold of the catalytic domain and a retaining mechanism of the glycoside bond hydrolysis. These data suggest a common evolutionary origin of glycosidases representing different families and clans.

  9. Hind limb scaling of kangaroos and wallabies (superfamily Macropodoidea): implications for hopping performance, safety factor and elastic savings.

    Science.gov (United States)

    McGowan, C P; Skinner, J; Biewener, A A

    2008-02-01

    The aim of this study was to examine hind limb scaling of the musculoskeletal system in the Macropodoidea, the superfamily containing wallabies and kangaroos, to re-examine the effect of size on the locomotor mechanics and physiology of marsupial hopping. Morphometric musculoskeletal analyses were conducted of 15 species and skeletal specimens of 21 species spanning a size range from 0.8 to 80 kg that included representatives of 12 of the 16 extant genera of macropodoids. We found that unlike other groups, macropodoids are able to match force demands associated with increasing body size primarily through a combination of positive allometry in muscle area and muscle moment arms. Isometric scaling of primary hind limb bones suggests, however, that larger species experience relatively greater bone stresses. Muscle to tendon area ratios of the ankle extensors scale with strong positive allometry, indicating that peak tendon stresses also increase with increasing body size but to a lesser degree than previously reported. Consistent with previous morphological and experimental studies, large macropodoids are therefore better suited for elastic strain energy recovery but operate at lower safety factors, which likely poses an upper limit to body size. Scaling patterns for extant macropodoids suggest that extinct giant kangaroos (approximately 250 kg) were likely limited in locomotor capacity.

  10. Extracellular Ribonuclease from Bacillus licheniformis (Balifase, a New Member of the N1/T1 RNase Superfamily

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    Yulia Sokurenko

    2016-01-01

    Full Text Available The N1/T1 RNase superfamily comprises enzymes with well-established antitumor effects, such as ribotoxins secreted by fungi, primarily by Aspergillus and Penicillium species, and bacterial RNase secreted by B. pumilus (binase and B. amyloliquefaciens (barnase. RNase is regarded as an alternative to classical chemotherapeutic agents due to its selective cytotoxicity towards tumor cells. New RNase with a high degree of structural similarity with binase (73% and barnase (74% was isolated and purified from Bacillus licheniformis (balifase, calculated molecular weight 12421.9 Da, pI 8.91. The protein sample with enzymatic activity of 1.5 × 106 units/A280 was obtained. The physicochemical properties of balifase are similar to those of barnase. However, in terms of its gene organization and promoter activity, balifase is closer to binase. The unique feature of balifase gene organization consists in the fact that genes of RNase and its inhibitor are located in one operon. Similarly to biosynthesis of binase, balifase synthesis is induced under phosphate starvation; however, in contrast to binase, balifase does not form dimers under natural conditions. We propose that the highest stability of balifase among analyzed RNase types allows the protein to retain its structure without oligomerization.

  11. A Streptomyces-specific member of the metallophosphatase superfamily contributes to spore dormancy and interaction with Aspergillus proliferans.

    Science.gov (United States)

    Lamp, Jessica; Weber, Maren; Cingöz, Gökhan; Ortiz de Orué Lucana, Darío; Schrempf, Hildgund

    2013-05-01

    We have identified, cloned and characterized a formerly unknown protein from Streptomyces lividans spores. The deduced protein belongs to a novel member of the metallophosphatase superfamily and contains a phosphatase domain and predicted binding sites for divalent ions. Very close relatives are encoded in the genomic DNA of many different Streptomyces species. As the deduced related homologues diverge from other known phosphatase types, we named the protein MptS (metallophosphatase type from Streptomyces). Comparative physiological and biochemical investigations and analyses by fluorescence microscopy of the progenitor strain, designed mutants carrying either a disruption of the mptS gene or the reintroduced gene as fusion with histidine codons or the egfp gene led to the following results: (i) the mptS gene is transcribed in the course of aerial mycelia formation. (ii) The MptS protein is produced during the late stages of growth, (iii) accumulates within spores, (iv) functions as an active enzyme that releases inorganic phosphate from an artificial model substrate, (v) is required for spore dormancy and (vi) MptS supports the interaction amongst Streptomyces lividans spores with conidia of the fungus Aspergillus proliferans. We discuss the possible role(s) of MptS-dependent enzymatic activity and the implications for spore biology. © 2013 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  12. Myelin-oligodendrocyte glycoprotein is a member of a subset of the immunoglobulin superfamily encoded within the major histocompatibility complex

    Energy Technology Data Exchange (ETDEWEB)

    Pham-Dinh, D.; Dautigny, A. (Institut des Neurosciences, Paris (France)); Mattei, M.G.; Roeckel, N. (Institut National de la Sante et de la Recherche Medicale Unite, Marseille (France)); Nussbaum, J.H.; Roussel, G. (Centre National de la Recherche Scientifique Unite, Strasbourg (France)); Pontarotti, P. (Centre Natinal de la Recherche Scientifique Unite, Toulouse (France)); Mather, I.H. (Univ. of Maryland, College Park, MD (United States)); Artzt, K. (Univ. of Texas, Austin, TX (United States)); Lindahl, K.F. (Univ. of Texas Southwestern Medical Center, Dallas, TX (United States))

    1993-09-01

    Myelin/oligodendrocyte glycoprotein (MOG) is found on the surface of myelinating oligodendrocytes and external lamellae of myelin sheaths in the central nervous system, and it is target antigen in experimental autoimmune encephalomyelitis and multiple sclerosis. The authors have isolated bovine, mouse, and rat MOG cDNA clones and shown that the developmental pattern of MOG expression in the rat central nervous system coincides with the late stages of myelination. The amino-terminal, extracellular domain of MOG has characteristics of an immunoglobulin variable domain and is 46% and 41% identical with the amino terminus of bovine butyrophilin (expressed in the lactating mammary gland) and B-G antigens of the chicken major histocompatibility complex (MHC), respectively; these proteins thus form a subset of the immunoglobulin superfamily. The homology between MOG and B-G extends beyond their structure and genetic mapping to their ability to induce strong antibody responses and has implications for the role of MOG in pathological, autoimmune conditions. The authors colocalized the MOG and BT genes to the human MHC on chromosome 6p21.3-p22. The mouse MOG gene was mapped to the homologous band C of chromosome 17, within the M region of the mouse MHC. 38 refs., 6 figs.

  13. Exploring the role of cellular homologous of the 30K-superfamily of plant virus movement proteins.

    Science.gov (United States)

    Carrasco, José L; Sánchez-Navarro, Jesús A; Elena, Santiago F

    2018-02-21

    Genes orthologous to the 30K-superfamily of movement proteins (MP) from plant viruses have been recently discovered by bioinformatics analyses as integrated elements in the genome of most vascular plants. However, their functional relevance for plants is still unclear. Here, we undertake some preliminary steps into the functional characterization of one of these putative MP genes found in Arabidopsis thaliana. We found that the AtMP gene is expressed at different stages of the plant development, with accumulation being highest in flowers but lowest in mature siliques. We also found down-regulation of the gene may result in a small delay in plant development and in an exacerbation of the negative effect of salinity in germination efficiency. We have also explored whether changes in expression of the endogenous AtMP have any effect on susceptibility to infection with several viruses, and found that the infectivity of tobacco rattle tobravirus was strongly dependent on the expression of the endogenous AtMP. Finally, we have cloned the endogenous MP from four different plant species into an expression vector that allows for specifically assessing their activity as cell-to-cell movement proteins and have shown that though some may still retain the ancestral activity, they do so in a quite inefficient manner, thus suggesting they have acquired a novel function during adaptation to the host genome. Copyright © 2018 Elsevier B.V. All rights reserved.

  14. New insights into potential functions for the protein 4.1superfamily of proteins in kidney epithelium

    Energy Technology Data Exchange (ETDEWEB)

    Calinisan, Venice; Gravem, Dana; Chen, Ray Ping-Hsu; Brittin,Sachi; Mohandas, Narla; Lecomte, Marie-Christine; Gascard, Philippe

    2005-06-17

    Members of the protein 4.1 family of adapter proteins are expressed in a broad panel of tissues including various epithelia where they likely play an important role in maintenance of cell architecture and polarity and in control of cell proliferation. We have recently characterized the structure and distribution of three members of the protein 4.1 family, 4.1B, 4.1R and 4.1N, in mouse kidney. We describe here binding partners for renal 4.1 proteins, identified through the screening of a rat kidney yeast two-hybrid system cDNA library. The identification of putative protein 4.1-based complexes enables us to envision potential functions for 4.1 proteins in kidney: organization of signaling complexes, response to osmotic stress, protein trafficking, and control of cell proliferation. We discuss the relevance of these protein 4.1-based interactions in kidney physio-pathology in the context of their previously identified functions in other cells and tissues. Specifically, we will focus on renal 4.1 protein interactions with beta amyloid precursor protein (beta-APP), 14-3-3 proteins, and the cell swelling-activated chloride channel pICln. We also discuss the functional relevance of another member of the protein 4.1 superfamily, ezrin, in kidney physiopathology.

  15. A super-family of transcriptional activators regulates bacteriophage packaging and lysis in Gram-positive bacteria

    Science.gov (United States)

    Quiles-Puchalt, Nuria; Tormo-Más, María Ángeles; Campoy, Susana; Toledo-Arana, Alejandro; Monedero, Vicente; Lasa, Íñigo; Novick, Richard P.; Christie, Gail E.; Penadés, José R.

    2013-01-01

    The propagation of bacteriophages and other mobile genetic elements requires exploitation of the phage mechanisms involved in virion assembly and DNA packaging. Here, we identified and characterized four different families of phage-encoded proteins that function as activators required for transcription of the late operons (morphogenetic and lysis genes) in a large group of phages infecting Gram-positive bacteria. These regulators constitute a super-family of proteins, here named late transcriptional regulators (Ltr), which share common structural, biochemical and functional characteristics and are unique to this group of phages. They are all small basic proteins, encoded by genes present at the end of the early gene cluster in their respective phage genomes and expressed under cI repressor control. To control expression of the late operon, the Ltr proteins bind to a DNA repeat region situated upstream of the terS gene, activating its transcription. This involves the C-terminal part of the Ltr proteins, which control specificity for the DNA repeat region. Finally, we show that the Ltr proteins are the only phage-encoded proteins required for the activation of the packaging and lysis modules. In summary, we provide evidence that phage packaging and lysis is a conserved mechanism in Siphoviridae infecting a wide variety of Gram-positive bacteria. PMID:23771138

  16. Role of a new member of IGFBP superfamily, IGFBP-rP10, in proliferation and differentiation of osteoblastic cells

    International Nuclear Information System (INIS)

    Shibata, Yasuaki; Tsukazaki, Tomoo; Hirata, Kazunari; Xin Cheng; Yamaguchi, Akira

    2004-01-01

    Bone regeneration is critically regulated by various molecules. To identify the new genes involved in bone regeneration, we performed microarray-based gene expression analysis using a mouse bone regeneration model. We identified a new member of the IGFBP superfamily, designated IGFBP-rP10, whose expression is up-regulated at the early phase of bone regeneration. IGFBP-rP10 consists of an IGFBP homologous domain followed by a Kazal-type protein inhibitor domain and an immunoglobulin G-like domain. A real-time-based RT-PCR analysis demonstrated that various tissues including bone expressed IGFBP-rP10 mRNA in various degrees, and confirmed an up-regulation at the early phase of bone regeneration. In situ hybridization revealed that osteoblastic cells expressed IGFPB-rP10 mRNA during bone regeneration. Bone morphogenetic protein-2 increased the expression level of IGFBP-rP10 mRNA in various cells including C3H10T1/2, MC3T3-E1, C2C12, and primary murine osteoblastic cells. The addition of recombinant mouse IGFBP-rP10 promoted the proliferation of these cells but failed to stimulate alkaline phosphatase activity. These results suggest that IGFBP-rP10 is involved in the proliferation of osteoblasts during bone formation and bone regeneration

  17. Molecular and biochemical characterization of the Fe(III) chelate reductase gene family in Arabidopsis thaliana.

    Science.gov (United States)

    Wu, Huilan; Li, Lihua; Du, Juan; Yuan, Youxi; Cheng, Xudong; Ling, Hong-Qing

    2005-09-01

    Iron chelate reductase is required for iron acquisition from soil and for metabolism in plants. In the genome of Arabidopsis thaliana there are eight genes classified into the iron chelate reductase gene family (AtFROs) based on sequence homology with AtFRO2 (a ferric chelate reductase in Arabidopsis). They are localized on chromosome 1 (three AtFROs) and chromosome 5 (five AtFROs) of Arabidopsis and show a high level of amino acid sequence similarity to each other. An assay for ferric chelate reductase activity revealed that AtFRO2, AtFRO3, AtFRO4, AtFRO5, AtFRO7 and AtFRO8 conferred significantly increased iron reduction activity compared with the control when expressed in yeast cells, indicating that the six AtFROs encode iron chelate reductases functioning in iron homeostasis in Arabidopsis. AtFRO2 displayed the highest iron reduction activity among the AtFROs investigated, further demonstrating that AtFRO2 is a major iron reductase gene in Arabidopsis. AtFRO2 and AtFRO3 were mainly expressed in roots of Arabidopsis, AtFRO5 and AtFRO6 in shoots and flowers, and AtFRO7 in cotyledons and trichomes, whereas the transcription of AtFRO8 was specific for leaf veins. Considering the tissue-specific expression profiles of AtFRO genes, we suggest that AtFRO2 and AtFRO3 are two Fe(III) chelate reductases mainly functioning in iron acquisition and metabolism in Arabidopsis roots, while AtFRO5, AtFRO6, AtFRO7 and AtFRO8 are required for iron homeostasis in different tissues of shoots.

  18. Production and Characterization of Monoclonal Antibodies against NADPH-Cytochrome P-450 Reductases from Helianthus tuberosus1

    Science.gov (United States)

    Lesot, Agnès; Benveniste, Irène; Hasenfratz, Marie-Paule; Durst, Francis

    1992-01-01

    Monoclonal antibodies (mAbs) against a plant NADPH-cytochrome P-450 (Cyt P-450) reductase from Jerusalem artichoke (Helianthus tuberosus) tuber were prepared. These antibodies were produced by hybridoma resulting from the fusion of spleen cells from a rat immunized with a purified preparation of the reductase and mouse myeloma cells. The mAbs thus obtained were screened for their interaction with the reductases, first in western dots and then in blots, and for their ability to inhibit the NADPH-cytochrome c (Cyt c) reductase activity from Jerusalem artichoke microsomes. Among the 11 clones giving a positive response on western blots, only 6 were also able to inhibit microsomal NADPH-Cyt c reductase activity, and the microsomal Cyt P-450 monooxygenase activities dependent upon electrons transferred by the reductase. Thus, two families of mAbs were characterized: a family of mAbs that interact with epitopes of the reductase implicated in the reduction of Cyt P-450 by NADPH (binding sites for NADPH, flavin mononucleotide, flavin adenine dinucleotide, and Cyt P-450), and a structural family, whose members recognize epitopes outside the active site of the reductases. These mAbs specifically recognize the reductase, and all of them interact with all of the isoforms, indicating that important primary or secondary structural analogies exist between the isoforms, not only at the active site, but also at the level of epitopes not directly associated with catalytic activity. Images Figure 1 Figure 2 Figure 3 PMID:16653138

  19. Anatomo-pathological aspects of parasitism by nematodes of the superfamily Metastrongyloidea in wild crab-eating fox (Cerdocyon thous in Midwestern Brazil

    Directory of Open Access Journals (Sweden)

    Jair Alves Ferreira Júnior

    Full Text Available ABSTRACT: Nematodes of the superfamily Metastrongyloidea affect the respiratory, cardiovascular, and nervous systems of domestic carnivores and are uncommonly detected in wild animals. This report describes the lesions associated with pulmonary parasitism by nematodes of the superfamily Metastrongyloidea in a wild crab-eating fox ( Cerdocyon thous in the Federal District, Brazil. Grossly, there was pulmonary hyperemia, edema, and emphysema. Microscopically, there was granulomatous arteritis associated with intravascular metastrongylid. The anatomical location, characteristic lesion, and histological features of the parasite suggested that the nematode involved in this case is Angiostrongylus vasorum . This worm is frequently reported parasitizing pulmonary arteries of domestic canids but is uncommonly described in wild canids in Midwestern Brazil.

  20. Methylenetetrahydrofolate reductase genotype association with the risk of follicular lymphoma.

    Science.gov (United States)

    Ismail, Said I; Ababneh, Nida A; Khader, Yousef; Abu-Khader, Ahmad A; Awidi, Abdullah

    2009-12-01

    The metabolism of folate is essential in DNA synthesis, and polymorphisms of genes involved in such metabolism have been implicated in many types of cancer. Among these, the methylene tetrahydrofolate reductase gene (MTHFR) encodes an enzyme that converts folate to a methyl donor used for DNA methylation. We studied the association between the different genotypes of the two most common MTHFR polymorphisms, C677T and A1298C, and the risk of follicular lymphoma (FL). For this purpose, 55 previously diagnosed FL patients and 170 normal control subjects were examined using polymerase chain reaction followed by restriction fragment length polymorphism. The frequency of the A1298C CC homozygous mutant genotype was significantly higher in patients with FL than in control subjects (OR = 3.51, 95% CI = 1.39-8.86, P = 0.008). No such association was found for the heterozygous A1298C AC genotype (OR = 1.08, 95% CI = 0.55-2.12, P = 0.83). On the other hand, no significant association was found for either the C677T CT heterozygous genotype (OR = 0.79, 95% CI = 0.42-1.51, P = 0.49) or the C677T TT homozygous mutant genotype (OR = 0.55, 95% CI = 0.12-2.65, P = 0.46). The present findings add to the very few reports suggesting a link between the A1298C CC homozygous MTHFR genotype and a higher risk of developing FL, and the first such in a Jordanian population.

  1. Sepiapterin Reductase Mediates Chemical Redox Cycling in Lung Epithelial Cells*

    Science.gov (United States)

    Yang, Shaojun; Jan, Yi-Hua; Gray, Joshua P.; Mishin, Vladimir; Heck, Diane E.; Laskin, Debra L.; Laskin, Jeffrey D.

    2013-01-01

    In the lung, chemical redox cycling generates highly toxic reactive oxygen species that can cause alveolar inflammation and damage to the epithelium, as well as fibrosis. In this study, we identified a cytosolic NADPH-dependent redox cycling activity in mouse lung epithelial cells as sepiapterin reductase (SPR), an enzyme important for the biosynthesis of tetrahydrobiopterin. Human SPR was cloned and characterized. In addition to reducing sepiapterin, SPR mediated chemical redox cycling of bipyridinium herbicides and various quinones; this activity was greatest for 1,2-naphthoquinone followed by 9,10-phenanthrenequinone, 1,4-naphthoquinone, menadione, and 2,3-dimethyl-1,4-naphthoquinone. Whereas redox cycling chemicals inhibited sepiapterin reduction, sepiapterin had no effect on redox cycling. Additionally, inhibitors such as dicoumarol, N-acetylserotonin, and indomethacin blocked sepiapterin reduction, with no effect on redox cycling. Non-redox cycling quinones, including benzoquinone and phenylquinone, were competitive inhibitors of sepiapterin reduction but noncompetitive redox cycling inhibitors. Site-directed mutagenesis of the SPR C-terminal substrate-binding site (D257H) completely inhibited sepiapterin reduction but had minimal effects on redox cycling. These data indicate that SPR-mediated reduction of sepiapterin and redox cycling occur by distinct mechanisms. The identification of SPR as a key enzyme mediating chemical redox cycling suggests that it may be important in generating cytotoxic reactive oxygen species in the lung. This activity, together with inhibition of sepiapterin reduction by redox-active chemicals and consequent deficiencies in tetrahydrobiopterin, may contribute to tissue injury. PMID:23640889

  2. Rapid Identification of Aldose Reductase Inhibitory Compounds from Perilla frutescens

    Directory of Open Access Journals (Sweden)

    Ji Hun Paek

    2013-01-01

    Full Text Available The ethyl acetate (EtOAc soluble fraction of methanol extracts of Perilla frutescens (P. frutescens inhibits aldose reductase (AR, the key enzyme in the polyol pathway. Our investigation of inhibitory compounds from the EtOAc soluble fraction of P. frutescens was followed by identification of the inhibitory compounds by a combination of HPLC microfractionation and a 96-well enzyme assay. This allowed the biological activities to be efficiently matched with selected HPLC peaks. Structural analyses of the active compounds were performed by LC-MSn. The main AR inhibiting compounds were tentatively identified as chlorogenic acid and rosmarinic acid by LC-MSn. A two-step high speed counter current chromatography (HSCCC isolation method was developed with a solvent system of n-hexane-ethyl acetate-methanol-water at 1.5 : 5 : 1 : 5, v/v and 3 : 7 : 5 : 5, v/v. The chemical structures of the isolated compounds were determined by 1H- and 13C-nuclear magnetic resonance spectrometry (NMR. The main compounds inhibiting AR in the EtOAc fraction of methanol extracts of P. frutescens were identified as chlorogenic acid (2 (IC50 = 3.16 μM, rosmarinic acid (4 (IC50 = 2.77 μM, luteolin (5 (IC50 = 6.34 μM, and methyl rosmarinic acid (6 (IC50 = 4.03 μM.

  3. Methylenetetrahydrofolate Reductase gene polymorphism in children with allergic rhinitis.

    Science.gov (United States)

    Dogru, M; Aydin, H; Aktas, A; Cırık, A A

    2015-01-01

    Methylenetetrahydrofolate Reductase (MTHFR) polymorphisms by impairing folate metabolism may influence the development of allergic diseases. The results of studies evaluating the relationship between MTHFR polymorphisms and atopic disease are controversial. The aim of this study was to investigate the association between the polymorphisms of C677T and A1298C for MTHFR gene and allergic rhinitis (AR) in children. Ninety patients followed up with diagnosis of allergic rhinitis in our clinic and 30 children with no allergic diseases were included in the study. All participants were genotyped for the MTHFR (C677T) and (A1298C) polymorphisms. Vitamin b12, folate and homocysteine levels were measured. The mean age of patients was 9.2±2.9 years; 66.7% of the patients were male. There was no significant difference between patient and control groups regarding gender, age and atopy history of the family (p>0.05). The frequency of homozygotes for MTHFR C677T polymorphism in the patient and control groups was 3.3% and 10%, respectively. The frequency of homozygotes for MTHFR A1298C polymorphism among groups was 26.7% and 16.7%, respectively. The association between allergic rhinitis and polymorphisms of C677T and A1298C for MTHFR gene was not statistically significant in patients compared with controls (p>0.05). There were no statistically significant differences between the patients and the control group in terms of serum vitamin b12, folate and homocysteine levels (p>0.05). We found no evidence for an association between allergic rhinitis and polymorphisms of C677T and A1298C for MTHFR gene in children. Further studies investigating the relationship between MTHFR polymorphism and AR are required. Copyright © 2014 SEICAP. Published by Elsevier Espana. All rights reserved.

  4. Methylenetetrahydrofolate reductase gene polymorphisms in Egyptian Turner Syndrome patients.

    Science.gov (United States)

    Ismail, Manal F; Zarouk, Waheba A; Ruby, Mona O; Mahmoud, Wael M; Gad, Randa S

    2015-01-01

    Folate metabolism dysfunctions can result in DNA hypomethylation and abnormal chromosome segregation. Two common polymorphisms of the methylenetetrahydrofolate reductase (MTHFR) encoding gene (C677T and A1298C) reduce MTHFR activity, but when associated with aneuploidy, the results are conflicting. Turner Syndrome (TS) is an interesting model for investigating the association between MTHFR gene polymorphisms and nondisjunction because of the high frequency of chromosomal mosaicism in this syndrome. To investigate the association of MTHFR gene C677T and A1298C polymorphisms in TS patients and their mothers and to correlate these polymorphisms with maternal risk of TS offspring. MTHFR C677T and A1298C polymorphisms were genotyped in 33 TS patients, their mothers and 15 healthy females with their mothers as controls using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and sequencing technique. Genotype and allele frequencies of both C677T and A1298C were not significantly different between TS cases and controls. There were no significant differences in C677T genotype distribution between the TS mothers and controls (p=1). The MTHFR 1298AA and 1298AC genotypes were significantly increased in TS mothers Vs. control mothers (p=0.002). The C allele frequency of the A1298C polymorphism was significantly different between the TS mothers and controls (p=0.02). The association of A1298C gene polymorphism in TS patients was found to increase with increasing age of both mothers (p=0.026) and fathers (p=0.044) of TS cases. Our findings suggest a strong association between maternal MTHFR A1298C and risk of TS in Egypt.

  5. The Reaction Mechanism of Methyl-Coenzyme M Reductase

    Science.gov (United States)

    Wongnate, Thanyaporn; Ragsdale, Stephen W.

    2015-01-01

    Methyl-coenzyme M reductase (MCR) is a nickel tetrahydrocorphinoid (coenzyme F430) containing enzyme involved in the biological synthesis and anaerobic oxidation of methane. MCR catalyzes the conversion of methyl-2-mercaptoethanesulfonate (methyl-SCoM) and N-7-mercaptoheptanoylthreonine phosphate (CoB7SH) to CH4 and the mixed disulfide CoBS-SCoM. In this study, the reaction of MCR from Methanothermobacter marburgensis, with its native substrates was investigated using static binding, chemical quench, and stopped-flow techniques. Rate constants were measured for each step in this strictly ordered ternary complex catalytic mechanism. Surprisingly, in the absence of the other substrate, MCR can bind either substrate; however, only one binary complex (MCR·methyl-SCoM) is productive whereas the other (MCR·CoB7SH) is inhibitory. Moreover, the kinetic data demonstrate that binding of methyl-SCoM to the inhibitory MCR·CoB7SH complex is highly disfavored (Kd = 56 mm). However, binding of CoB7SH to the productive MCR·methyl-SCoM complex to form the active ternary complex (CoB7SH·MCR(NiI)·CH3SCoM) is highly favored (Kd = 79 μm). Only then can the chemical reaction occur (kobs = 20 s−1 at 25 °C), leading to rapid formation and dissociation of CH4 leaving the binary product complex (MCR(NiII)·CoB7S−·SCoM), which undergoes electron transfer to regenerate Ni(I) and the final product CoBS-SCoM. This first rapid kinetics study of MCR with its natural substrates describes how an enzyme can enforce a strictly ordered ternary complex mechanism and serves as a template for identification of the reaction intermediates. PMID:25691570

  6. Methylenetetrahy-drofolate Reductase Gene Polymorphism in Patients Receiving Hemodialysis

    Directory of Open Access Journals (Sweden)

    Ermina Kiseljaković

    2010-04-01

    Full Text Available Methylenetetrahydrofolate Reductase (MTHFR is key enzyme in metabolism of homocysteine. Homozygotes for mutation (TT genotype have hyperhomocysteinemia, risk factor for atherosclerosis development. The aim of the study was to find out distribution of genotype frequencies of C677T MTHFR among patients on maintenance hemodialysis. Possible association of alleles and genotypes of C677T polymorphism of the MTHFR gene with age of onset, duration of dialysis and cause of kidney failure was studied also. Cross-sectional study includes 80 patients from Clinic of Hemodialysis KUCS in Sarajevo. In order to perform genotyping, isolated DNA was analyzed by RFLP-PCR and gel-electrophoresis. From total of 80 patients, 42.5% (n=24 were female, 57.5% (n=46 were male, mean age 54.59±1.78 years and duration of dialysis 79.92±6.32 months. Genotype distribution was: CC 51.2% (n=41, CT 37.5% (n=30 and TT 11.2% (n=9. Patients with wild-type genotype have longer duration of dialysis in month (87.1 ± 63.93 comparing to TT genotype patients (67.06 ± 39.3, with no statistical significance. T allele frequency was significantly higher in group of vascular and congenital cause of kidney failure (Pearson X2 =6.049, P<0.05 comparing to inflammation etiology group. Genotype distribution results are within the results other studies in Europe. Obtained results indicate that C677T polymorphism is not associated with onset, duration and cause of kidney failure in our hemodialysis population. There is an association of T allele of the MTHFR gene and vascular and congenital cause kidney failure.

  7. Hyperhomocysteinaemia, methylenetetrahydrofolate reductase polymorphism and risk of coronary artery disease.

    Science.gov (United States)

    Kerkeni, Mohsen; Addad, Faouzi; Chauffert, Maryline; Myara, Anne; Gerhardt, Marie; Chevenne, Didier; Trivin, François; Farhat, Mohamed Ben; Miled, Abdelhedi; Maaroufi, Khira

    2006-05-01

    Hyperhomocysteinaemia is an independent, graded risk factor for coronary artery disease (CAD). The methylenetetrahydrofolate reductase (MTHFR) polymorphism is associated with hyperhomcysteinaemia and may therefore influence individual susceptibility to CAD. We have investigated this risk factor in a Tunisian Arab population. Polymerase chain reaction-restriction fragment length polymorphism analysis was used to detect the C677T and A1298C variants of the MTHFR gene in 100 patients with CAD and 120 healthy controls. The severity of CAD was expressed as the number of affected vessels. Plasma total homocysteine (tHcy) concentration was determined using a direct chemiluminescence assay. MTHFR CC, CT and TT genotype frequencies in the CAD group were significantly different from those observed in the control group (49%, 35% and 16% versus 48.3%, 45.8% and 5.8%, respectively; P = 0.031). However, MTHFR AA, AC and CC genotypes frequencies in the CAD group were not significantly different from the control group ( P = 0.568). Patients with CAD showed higher plasma tHcy concentrations than patients without CAD (15.86 +/- 8.63 micromol/L versus 11.90 +/- 3.25 micromol/L, P MTHFR polymorphisms and the number of stenosed vessels. Patients with the MTHFR TT genotype had higher plasma tHcy, serum creatinine, cholesterol and triglyceride concentrations than patients with the MTHFR CC genotype. The C677T polymorphism of the MTHFR gene is associated with hyperhomocysteinaemia, lipid dysregulation and the presence of CAD in this Tunisian Arab population.

  8. Increased 5. cap alpha. -reductase activity in idiopathic hirsutism

    Energy Technology Data Exchange (ETDEWEB)

    Serafini, P.; Lobo, R.A.

    1985-01-01

    In vitro, genital skin 5..cap alpha..-reductase activity (5..cap alpha..-RA) was measured in ten hirsute women with normal androgen levels (idiopathic hirsutism (IH)) and in ten hirsute women with elevated androgen levels (polycystic ovary syndrome (PCO)) in order to determine the influence of secreted androgens on 5..cap alpha..-RA. In vitro 5..cap alpha..-RA was assessed by incubations of skin with /sup 14/C-testosterone (T) for 2 hours, after which steroids were separated and the radioactivity of dihydrotestosterone (DHT) and 5..cap alpha..-androstane 3..cap alpha..-17..beta..-estradiol (3..cap alpha..-diol) in specific eluates were determined. All androgens were normal in IH with the exception of higher levels of 3..cap alpha..-diol glucuronide which were similar to the levels of PCO. The conversion ratio (CR) of T to DHT in IH and PCO were similar, yet significantly greater than the CR of control subjects. The CR of T to 3..cap alpha..-diol in IH and PCO were similar, yet higher than in control subjects. Serum androgens showed no correlation with 5..cap alpha..-RA, while the CR of T to DHT showed a significant positive correlation with the Ferriman and Gallwey score. The increased 5..cap alpha..-RA in IH appears to be independent of serum androgen levels and is, therefore, an inherent abnormality. The term idiopathic is a misnomer, because hirsutism in these patients may be explained on the basis of increased skin 5..cap alpha..-RA.

  9. Rapid Identification of Aldose Reductase Inhibitory Compounds from Perilla frutescens

    Science.gov (United States)

    Paek, Ji Hun; Shin, Kuk Hyun; Kang, Young-Hee; Lee, Jae-Yong; Lim, Soon Sung

    2013-01-01

    The ethyl acetate (EtOAc) soluble fraction of methanol extracts of Perilla frutescens (P. frutescens) inhibits aldose reductase (AR), the key enzyme in the polyol pathway. Our investigation of inhibitory compounds from the EtOAc soluble fraction of P. frutescens was followed by identification of the inhibitory compounds by a combination of HPLC microfractionation and a 96-well enzyme assay. This allowed the biological activities to be efficiently matched with selected HPLC peaks. Structural analyses of the active compounds were performed by LC-MSn. The main AR inhibiting compounds were tentatively identified as chlorogenic acid and rosmarinic acid by LC-MSn. A two-step high speed counter current chromatography (HSCCC) isolation method was developed with a solvent system of n-hexane-ethyl acetate-methanol-water at 1.5 : 5 : 1 : 5, v/v and 3 : 7 : 5 : 5, v/v. The chemical structures of the isolated compounds were determined by 1H- and 13C-nuclear magnetic resonance spectrometry (NMR). The main compounds inhibiting AR in the EtOAc fraction of methanol extracts of P. frutescens were identified as chlorogenic acid (2) (IC50 = 3.16 μM), rosmarinic acid (4) (IC50 = 2.77 μM), luteolin (5) (IC50 = 6.34 μM), and methyl rosmarinic acid (6) (IC50 = 4.03 μM). PMID:24308003

  10. Mycobacterium smegmatis SftH exemplifies a distinctive clade of superfamily II DNA-dependent ATPases with 3′ to 5′ translocase and helicase activities

    OpenAIRE

    Yakovleva, Lyudmila; Shuman, Stewart

    2012-01-01

    Bacterial DNA helicases are nucleic acid-dependent NTPases that play important roles in DNA replication, recombination and repair. We are interested in the DNA helicases of Mycobacteria, a genus of the phylum Actinobacteria, which includes the human pathogen Mycobacterium tuberculosis and its avirulent relative Mycobacterium smegmatis. Here, we identify and characterize M. smegmatis SftH, a superfamily II helicase with a distinctive domain structure, comprising an N-terminal NTPase domain and...

  11. Interactive surface in the PapD chaperone cleft is conserved in pilus chaperone superfamily and essential in subunit recognition and assembly.

    OpenAIRE

    Slonim, L N; Pinkner, J S; Brändén, C I; Hultgren, S J

    1992-01-01

    The assembly of adhesive pili in Gram-negative bacteria is modulated by specialized periplasmic chaperone systems. PapD is the prototype member of the superfamily of periplasmic pilus chaperones. Previously, the alignment of chaperone sequences superimposed on the three dimensional structure of PapD revealed the presence of invariant, conserved and variable amino acids. Representative residues that protruded into the PapD cleft were targeted for site directed mutagenesis to investigate the pi...

  12. A histone-like protein of mycobacteria possesses ferritin superfamily protein-like activity and protects against DNA damage by Fenton reaction.

    Directory of Open Access Journals (Sweden)

    Masaki Takatsuka

    Full Text Available Iron is an essential metal for living organisms but its level must be strictly controlled in cells, because ferrous ion induces toxicity by generating highly active reactive oxygen, hydroxyl radicals, through the Fenton reaction. In addition, ferric ion shows low solubility under physiological conditions. To overcome these obstacles living organisms possess Ferritin superfamily proteins that are distributed in all three domains of life: bacteria, archaea, and eukaryotes. These proteins minimize hydroxyl radical formation by ferroxidase activity that converts Fe(2+ into Fe(3+ and sequesters iron by storing it as a mineral inside a protein cage. In this study, we discovered that mycobacterial DNA-binding protein 1 (MDP1, a histone-like protein, has similar activity to ferritin superfamily proteins. MDP1 prevented the Fenton reaction and protects DNA by the ferroxidase activity. The K(m values of the ferroxidase activity by MDP1 of Mycobacterium bovis bacillus Calmette-Guérin (BCG-3007c, Mycobacterium tuberculosis (Rv2986c, and Mycobacterium leprae (ML1683; ML-LBP were 0.292, 0.252, and 0.129 mM, respectively. Furthermore, one MDP1 molecule directly captured 81.4±19.1 iron atoms, suggesting the role of this protein in iron storage. This study describes for the first time a ferroxidase-iron storage protein outside of the ferritin superfamily proteins and the protective role of this bacterial protein from DNA damage.

  13. Structural Analysis of Papain-Like NlpC/P60 Superfamily Enzymes with a Circularly Permuted Topology Reveals Potential Lipid Binding Sites

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Qingping; Rawlings, Neil D.; Chiu, Hsiu-Ju; Jaroszewski, Lukasz; Klock, Heath E.; Knuth, Mark W.; Miller, Mitchell D.; Elsliger, Marc-Andre; Deacon, Ashley M.; Godzik, Adam; Lesley, Scott A.; Wilson, Ian A. (SG); (Wellcome)

    2012-07-11

    NlpC/P60 superfamily papain-like enzymes play important roles in all kingdoms of life. Two members of this superfamily, LRAT-like and YaeF/YiiX-like families, were predicted to contain a catalytic domain that is circularly permuted such that the catalytic cysteine is located near the C-terminus, instead of at the N-terminus. These permuted enzymes are widespread in virus, pathogenic bacteria, and eukaryotes. We determined the crystal structure of a member of the YaeF/YiiX-like family from Bacillus cereus in complex with lysine. The structure, which adopts a ligand-induced, 'closed' conformation, confirms the circular permutation of catalytic residues. A comparative analysis of other related protein structures within the NlpC/P60 superfamily is presented. Permutated NlpC/P60 enzymes contain a similar conserved core and arrangement of catalytic residues, including a Cys/His-containing triad and an additional conserved tyrosine. More surprisingly, permuted enzymes have a hydrophobic S1 binding pocket that is distinct from previously characterized enzymes in the family, indicative of novel substrate specificity. Further analysis of a structural homolog, YiiX (PDB 2if6) identified a fatty acid in the conserved hydrophobic pocket, thus providing additional insights into possible function of these novel enzymes.

  14. Bifunctional phosphoglucose/phosphomannose isomerases from the Archaea Aeropyrum pernix and Thermoplasma acidophilum constitute a novel enzyme family within the phosphoglucose isomerase superfamily.

    Science.gov (United States)

    Hansen, Thomas; Wendorff, Daniel; Schönheit, Peter

    2004-01-16

    The hyperthermophilic crenarchaeon Aeropyrum pernix contains phosphoglucose isomerase (PGI) activity. However, obvious homologs with significant identity to known PGIs could not be identified in the sequenced genome of this organism. The PGI activity from A. pernix was purified and characterized. Kinetic analysis revealed that, unlike all known PGIs, the enzyme catalyzed reversible isomerization not only of glucose 6-phosphate but also of epimeric mannose 6-phosphate at similar catalytic efficiency, thus defining the protein as bifunctional phosphoglucose/phosphomannose isomerase (PGI/PMI). The gene pgi/pmi encoding PGI/PMI (open reading frame APE0768) was identified by matrix-assisted laser desorption ionization time-of-flight analyses; the gene was overexpressed in Escherichia coli as functional PGI/PMI. Putative PGI/PMI homologs were identified in several (hyper)thermophilic archaea and two bacteria. The homolog from Thermoplasma acidophilum (Ta1419) was overexpressed in E. coli, and the recombinant enzyme was characterized as bifunctional PGI/PMI. PGI/PMIs showed low sequence identity to the PGI superfamily and formed a distinct phylogenetic cluster. However, secondary structure predictions and the presence of several conserved amino acids potentially involved in catalysis indicate some structural and functional similarity to the PGI superfamily. Thus, we propose that bifunctional PGI/PMI constitutes a novel protein family within the PGI superfamily.

  15. Association of Suicidality and Depression With 5α-Reductase Inhibitors.

    Science.gov (United States)

    Welk, Blayne; McArthur, Eric; Ordon, Michael; Anderson, Kelly K; Hayward, Jade; Dixon, Stephanie

    2017-05-01

    There have been concerns raised by patients and regulatory agencies regarding serious psychiatric adverse effects associated with 5α-reductase inhibitors. To determine if there is an increased risk of suicide, self-harm, or depression among older men starting a 5α-reductase inhibitor for prostatic enlargement. A population-based, retrospective, matched cohort study using linked administrative data for 93 197 men ages 66 years or older (median [IQR] age, 75 [70-80] years) in Ontario, Canada, who initiated a new prescription for a 5α-reductase inhibitor during the study period (2003 through 2013). Participants were matched (using a propensity score that included 44 of our 96 covariates that included medical comorbidities, medication usage, and health care system utilization) to an equal number of men not prescribed a 5α-reductase inhibitor. Duration of finasteride or dutasteride usage. Suicide. Secondary outcomes were self-harm and depression. Men who used 5α-reductase inhibitors were not at a significantly increased risk of suicide (HR, 0.88; 95% CI, 0.53-1.45). Risk of self-harm was significantly increased during the initial 18 months after 5α-reductase inhibitor initiation (HR, 1.88; 95% CI, 1.34-2.64), but not thereafter. Incident depression risk was elevated during the initial 18 months after 5α-reductase inhibitor initiation (HR, 1.94; 95% CI, 1.73-2.16), and continued to be elevated, but to a lesser degree, for the remainder of the follow-up period (HR, 1.22; 95% CI, 1.08-1.37). The absolute increases in the event rates for these 2 outcomes were 17 per 100 000 patient-years and 237 per 100 000 patient-years, respectively. The type of 5α-reductase inhibitor (finasteride or dutasteride) did not significantly modify the observed associations with suicide, self-harm, and depression. In a large cohort of men ages 66 years or older, we did not demonstrate an increased risk of suicide associated with 5α-reductase inhibitor use. However, the risk of

  16. Recombinant pinoresinol-lariciresinol reductases from western red cedar (Thuja plicata) catalyze opposite enantiospecific conversions.

    Science.gov (United States)

    Fujita, M; Gang, D R; Davin, L B; Lewis, N G

    1999-01-08

    Although the heartwood of woody plants represents the main source of fiber and solid wood products, essentially nothing is known about how the biological processes leading to its formation are initiated and regulated. Accordingly, a reverse transcription-polymerase chain reaction-guided cloning strategy was employed to obtain genes encoding pinoresinol-lariciresinol reductases from western red cedar (Thuja plicata) as a means to initiate the study of its heartwood formation. (+)-Pinoresinol-(+)-lariciresinol reductase from Forsythia intermedia was used as a template for primer construction for reverse transcription-polymerase chain reaction amplifications, which, when followed by homologous hybridization cloning, resulted in the isolation of two distinct classes of putative pinoresinol-lariciresinol reductase cDNA clones from western red cedar. A representative of each class was expressed as a fusion protein with beta-galactosidase and assayed for enzymatic activity. Using both deuterated and radiolabeled (+/-)-pinoresinols as substrates, it was established that each class of cDNA encoded a pinoresinol-lariciresinol reductase of different (opposite) enantiospecificity. Significantly, the protein from one class converted (+)-pinoresinol into (-)-secoisolariciresinol, whereas the other utilized the opposite (-)-enantiomer to give the corresponding (+)-form. This differential substrate specificity raises important questions about the role of each of these individual reductases in heartwood formation, such as whether they are expressed in different cells/tissues or at different stages during heartwood development.

  17. Structural and biochemical properties of cloned and expressed human and rat steroid 5α-reductases

    International Nuclear Information System (INIS)

    Andersson, S.; Russell, D.W.

    1990-01-01

    The microsomal enzyme steroid 5α-reductase is responsible for the conversion of testosterone into the more potent androgen dihydrotestosterone. In man, this steroid acts on a variety of androgen-responsive target tissues to mediate such diverse endocrine processes as male sexual differentiation in the fetus and prostatic growth in men. Here we describe the isolation, structure, and expression of a cDNA encoding the human steroid 5α-reductase. A rat cDNA was used as a hybridization probe to screen a human prostate cDNA library. A 2.1-kilobase cDNA was identified and DNA sequence analysis indicated that the human steroid 5α-reductase was a hydrophobic protein of 259 amino acids with a predicted molecular weight of 29,462. A comparison of the human and rat protein sequences revealed a 60% identity. Transfection of expression vectors containing the human and rat cDNAs into simian COS cells resulted in the synthesis of high levels of steroid 5α-reductase enzyme activity. Both enzymes expressed in COS cells showed similar substrate specificities for naturally occurring steroid hormones. However, synthetic 4-azasteroids demonstrated marked differences in their abilities to inhibit the human and rat steroid 5α-reductases

  18. INHIBITORY ACTIVITY OF FLAVONOIDS ON THE LENS ALDOSE REDUCTASE OF HEALTHY AND DIABETIC RATS

    Directory of Open Access Journals (Sweden)

    M. T. Goodarzi

    2006-05-01

    Full Text Available Aldose reductase is a critical enzyme in the polyol pathway that plays an important role in diabetes mellitus. Inhibition of the activity of this enzyme can prevent cataract in diabetic patients’lenses. In this study the inhibitory effect of two flavonoids, quercetin and naringin, in the activity of aldose reductase in streptozotocin-induced diabetic and healthy rats were investigated. Thirty male rats were divided in six groups. The first, second and third group were healthy rats that received water,quercetin and naringin, respectively. The fourth, fifth and sixth groups were streptozocin-induced diabetic rats that received water, quercetin and naringin, respectively. These rats were fed orally in a definite dose from each substance for 12 days. After this period rats were scarified and their lenses were separated and homogenized. The activity of aldose reductase was measured in each homogenized sample separately. The effect of feeding of these substances in blood sugar was also determined. Aldose reductase activity was reduced 73 and 69 percent in diabetic rats fed by quercetin and naringin, respectively, and the difference compared to control group was significant. In healthy rats this reduction was 63 and 59 percent, respectively, and the difference was significant compared to those who did not receive flavonoids. It was concluded that these substances were effective in reduction of aldose reductase activity in vivo and consequently could delay the progress of cataract.

  19. A Single Mutation Increases the Activity and Stability of Pectobacterium carotovorum Nitrile Reductase.

    Science.gov (United States)

    Zhou, Zheng; Li, Min; Xu, Jian-He; Zhang, Zhi-Jun

    2018-03-02

    Nitrile reductases are considered to be promising and environmentally benign nitrile-reducing biocatalysts to replace traditional metal catalysts. Unfortunately, the catalytic efficiencies of the nitrile reductases reported so far are very low. To date, all attempts to increase the catalytic activity of nitrile reductases by protein engineering have failed. In this work, we successfully increased the specific activity of a nitrile reductase from Pectobacterium carotovorum from 354 to 526 U g prot -1 by engineering the substrate binding pocket; moreover, the thermostability was also improved (≈2-fold), showing half-lives of 140 and 32 h at 30 and 40 °C, respectively. In the bioreduction of 2-amino-5-cyanopyrrolo[2,3-d]pyrimidin-4-one (preQ 0 ) to 2-amino-5-aminomethylpyrrolo[2,3-d]pyrimidin-4-one (preQ 1 ), the variant was advantageous over the wild-type enzyme with a higher reaction rate and complete conversion of the substrate within a shorter period. Homology modeling and docking analysis revealed some possible origins of the increased activity and stability. These results establish a solid basis for future engineering of nitrile reductases to increase the catalytic efficiency further, which is a prerequisite for applying these novel biocatalysts in synthetic chemistry. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Comparative modelling and molecular docking of nitrate reductase from Bacillus weihenstephanensis (DS45

    Directory of Open Access Journals (Sweden)

    R. Seenivasagan

    2016-07-01

    Full Text Available Nitrate reductase catalyses the oxidation of NAD(PH and the reduction of nitrate to nitrite. NR serves as a central point for the integration of metabolic pathways by governing the flux of reduced nitrogen through several regulatory mechanisms in plants, algae and fungi. Bacteria express nitrate reductases that convert nitrate to nitrite, but mammals lack these specific enzymes. The microbial nitrate reductase reduces toxic compounds to nontoxic compounds with the help of NAD(PH. In the present study, our results revealed that Bacillus weihenstephanensis expresses a nitrate reductase enzyme, which was made to generate the 3D structure of the enzyme. Six different modelling servers, namely Phyre2, RaptorX, M4T Server, HHpred, SWISS MODEL and Mod Web, were used for comparative modelling of the structure. The model was validated with standard parameters (PROCHECK and Verify 3D. This study will be useful in the functional characterization of the nitrate reductase enzyme and its docking with nitrate molecules, as well as for use with autodocking.

  1. Overexpression of Nitrate Reductase in Tobacco Delays Drought-Induced Decreases in Nitrate Reductase Activity and mRNA1

    Science.gov (United States)

    Ferrario-Méry, Sylvie; Valadier, Marie-Hélène; Foyer, Christine H.

    1998-01-01

    Transformed (cauliflower mosaic virus 35S promoter [35S]) tobacco (Nicotiana plumbaginifolia L.) plants constitutively expressing nitrate reductase (NR) and untransformed controls were subjected to drought for 5 d. Drought-induced changes in biomass accumulation and photosynthesis were comparable in both lines of plants. After 4 d of water deprivation, a large increase in the ratio of shoot dry weight to fresh weight was observed, together with a decrease in the rate of photosynthetic CO2 assimilation. Foliar sucrose increased in both lines during water stress, but hexoses increased only in leaves from untransformed controls. Foliar NO3− decreased rapidly in both lines and was halved within 2 d of the onset of water deprivation. Total foliar amino acids decreased in leaves of both lines following water deprivation. After 4 d of water deprivation no NR activity could be detected in leaves of untransformed plants, whereas about 50% of the original activity remained in the leaves of the 35S-NR transformants. NR mRNA was much more stable than NR activity. NR mRNA abundance increased in the leaves of the 35S-NR plants and remained constant in controls for the first 3 d of drought. On the 4th d, however, NR mRNA suddenly decreased in both lines. Rehydration at d 3 caused rapid recovery (within 24 h) of 35S-NR transcripts, but no recovery was observed in the controls. The phosphorylation state of the protein was unchanged by long-term drought. There was a strong correlation between maximal extractable NR activity and ambient photosynthesis in both lines. We conclude that drought first causes increased NR protein turnover and then accelerates NR mRNA turnover. Constitutive NR expression temporarily delayed drought-induced losses in NR activity. 35S-NR expression may therefore allow more rapid recovery of N assimilation following short-term water deficit. PMID:9576799

  2. D-Ribulose 5-Phosphate 3-Epimerase: Functional and Structural Relationships to Members of the Ribulose-Phosphate Binding (beta/alpha)8-Barrel Superfamily

    International Nuclear Information System (INIS)

    Akana, J.; Federov, A.; Federov, E.; Novak, W.; Babbitt, P.; Almo, S.; Gerlt, J.

    2006-01-01

    The 'ribulose phosphate binding' superfamily defined by the Structural Classification of Proteins (SCOP) database is considered the result of divergent evolution from a common (β/α) 8 -barrel ancestor. The superfamily includes D-ribulose 5-phosphate 3-epimerase (RPE), orotidine 5'-monophosphate decarboxylase (OMPDC), and 3-keto-L-gulonate 6-phosphate decarboxylase (KGPDC), members of the OMPDC suprafamily, as well as enzymes involved in histidine and tryptophan biosynthesis that utilize phosphorylated metabolites as substrates. We now report studies of the functional and structural relationships of RPE to the members of the superfamily. As suggested by the results of crystallographic studies of the RPEs from rice and Plasmodium falciparum, the RPE from Streptococcus pyogenes is activated by Zn 2+ which binds with a stoichiometry of one ion per polypeptide. Although wild type RPE has a high affinity for Zn 2+ and inactive apoenzyme cannot be prepared, the affinity for Zn 2+ is decreased by alanine substitutions for the two histidine residues that coordinate the Zn 2+ ion (H34A and H67A); these mutant proteins can be prepared in an inactive, metal-free form and activated by exogenous Zn 2+ . The crystal structure of the RPE was solved at 1.8 Angstroms resolution in the presence of D-xylitol 5-phosphate, an inert analogue of the D-xylulose 5-phosphate substrate. This structure suggests that the 2,3-enediolate intermediate in the 1,1-proton transfer reaction is stabilized by bidentate coordination to the Zn 2+ that also is liganded to His 34, Asp 36, His 67, and Asp 176; the carboxylate groups of the Asp residues are positioned also to function as the acid/base catalysts. Although the conformation of the bound analogue resembles those of ligands bound in the active sites of OMPDC and KGPDC, the identities of the active site residues that coordinate the essential Zn 2+ and participate as acid/base catalysts are not conserved. We conclude that only the phosphate

  3. Genome-wide characterization and expression analysis of the aldehyde dehydrogenase (ALDH) gene superfamily under abiotic stresses in cotton.

    Science.gov (United States)

    Guo, Xinlei; Wang, Yuanyuan; Lu, Hejun; Cai, Xiaoyan; Wang, Xingxing; Zhou, Zhongli; Wang, Chunying; Wang, Yuhong; Zhang, Zhenmei; Wang, Kunbo; Liu, Fang

    2017-09-10

    In plants, aldehyde dehydrogenases (ALDHs) function as 'aldehyde scavengers' by removing reactive aldehydes and thus play important roles in stress responses. To date, 30 ALDHs have been identified in Gossypium raimondii, whereas ALDHs have not been studied in Gossypium arboreum or in tetraploid cotton. In this study, we identified 30, 59 and 59 aldehyde dehydrogenase (ALDH) genes from G. arboreum, G. hirsutum and G. barbadense, respectively. Gene structure analysis revealed that members of the same family exhibit similar exon-intron structures and structural domains, and all members of the ALDH18 family possess a distinct AA-kinase domain. Synteny analysis showed that segmental and tandem duplications have played an important role in the expansion and evolution of ALDHs in cotton. Phylogenetic and synteny analysis between G. arboreum and G. raimondii demonstrated that all GaALDHs and GrALDHs are orthologous and that most GaALDHs are located in syntenic blocks corresponding to those of G. raimondii, implying that these genes appeared before the divergence of G. arboreum and G. raimondii and that no expansion of the ALDH superfamily has occurred in these two cotton species. Quantitative real-time PCR analysis revealed that the majority of GaALDHs and GhALDHs are up-regulated under conditions of high salinity and drought, indicating that these genes may be stress responsive. The findings of this study, based on genome-wide identification of ALDHs in Gossypium and analysis of their evolution and expression, provide a foundation for further analysis of ALDHs and suggest potential target genes for improving stress resistance in cotton. Copyright © 2017. Published by Elsevier B.V.

  4. Genome-wide identification and analysis of the aldehyde dehydrogenase (ALDH) gene superfamily in apple (Malus × domestica Borkh.).

    Science.gov (United States)

    Li, Xiaoqin; Guo, Rongrong; Li, Jun; Singer, Stacy D; Zhang, Yucheng; Yin, Xiangjing; Zheng, Yi; Fan, Chonghui; Wang, Xiping

    2013-10-01

    Aldehyde dehydrogenases (ALDHs) represent a protein superfamily encoding NAD(P)(+)-dependent enzymes that oxidize a wide range of endogenous and exogenous aliphatic and aromatic aldehydes. In plants, they are involved in many biological processes and play a role in the response to environmental stress. In this study, a total of 39 ALDH genes from ten families were identified in the apple (Malus × domestica Borkh.) genome. Synteny analysis of the apple ALDH (MdALDH) genes indicated that segmental and tandem duplications, as well as whole genome duplications, have likely contributed to the expansion and evolution of these gene families in apple. Moreover, synteny analysis between apple and Arabidopsis demonstrated that several MdALDH genes were found in the corresponding syntenic blocks of Arabidopsis, suggesting that these genes appeared before the divergence of lineages that led to apple and Arabidopsis. In addition, phylogenetic analysis, as well as comparisons of exon-intron and protein structures, provided further insight into both their evolutionary relationships and their putative functions. Tissue-specific expression analysis of the MdALDH genes demonstrated diverse spatiotemporal expression patterns, while their expression profiles under abiotic stress and various hormone treatments indicated that many MdALDH genes were responsive to high salinity and drought, as well as different plant hormones. This genome-wide identification, as well as characterization of evolutionary relationships and expression profiles, of the apple MdALDH genes will not only be useful for the further analysis of ALDH genes and their roles in stress response, but may also aid in the future improvement of apple stress tolerance. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  5. Structure and mechanism of benzaldehyde dehydrogenase from Pseudomonas putida ATCC 12633, a member of the Class 3 aldehyde dehydrogenase superfamily.

    Science.gov (United States)

    Zahniser, Megan P D; Prasad, Shreenath; Kneen, Malea M; Kreinbring, Cheryl A; Petsko, Gregory A; Ringe, Dagmar; McLeish, Michael J

    2017-03-01

    Benzaldehyde dehydrogenase from Pseudomonas putida (PpBADH) belongs to the Class 3 aldehyde dehydrogenase (ALDH) family. The Class 3 ALDHs are unusual in that they are generally dimeric (rather than tetrameric), relatively non-specific and utilize both NAD+ and NADP+. To date, X-ray structures of three Class 3 ALDHs have been determined, of which only two have cofactor bound, both in the NAD+ form. Here we report the crystal structure of PpBADH in complex with NADP+ and a thioacyl intermediate adduct. The overall architecture of PpBADH resembles that of most other members of the ALDH superfamily, and the cofactor binding residues are well conserved. Conversely, the pattern of cofactor binding for the rat Class 3 ALDH differs from that of PpBADH and other ALDHs. This has been interpreted in terms of a different mechanism for the rat enzyme. Comparison with the PpBADH structure, as well as multiple sequence alignments, suggest that one of two conserved glutamates, at positions 215 (209 in rat) and 337 (333 in rat), would act as the general base necessary to hydrolyze the thioacyl intermediate. While the latter is the general base in the rat Class 3 ALDH, site-specific mutagenesis indicates that Glu215 is the likely candidate for PpBADH, a result more typical of the Class 1 and 2 ALDH families. Finally, this study shows that hydride transfer is not rate limiting, lending further credence to the suggestion that PpBADH is more similar to the Class 1 and 2 ALDHs than it is to other Class 3 ALDHs. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  6. Novel evolutionary lineages of the invertebrate oxytocin/vasopressin superfamily peptides and their receptors in the common octopus (Octopus vulgaris)

    Science.gov (United States)

    Kanda, Atsuhiro; Satake, Honoo; Kawada, Tsuyoshi; Minakata, Hiroyuki

    2004-01-01

    The common octopus, Octopus vulgaris, is the first invertebrate species that was shown to possess two oxytocin/vasopressin (OT/VP) superfamily peptides, octopressin (OP) and cephalotocin (CT). Previously, we cloned a GPCR (G-protein-coupled receptor) specific to CT [CTR1 (CT receptor 1)]. In the present study, we have identified an additional CTR, CTR2, and a novel OP receptor, OPR. Both CTR2 and OPR include domains and motifs typical of GPCRs, and the intron– exon structures are in accord with those of OT/VP receptor genes. CTR2 and OPR expressed in Xenopus oocytes induced calcium-mediated inward chloride current in a CT- and OP-specific manner respectively. Several regions and residues, which are requisite for binding of the vertebrate OT/VP receptor family with their ligands, are highly conserved in CTRs, but not in OPR. These different sequences between CTRs and OPR, as well as the amino acid residues of OP and CT at positions 2–5, were presumed to play crucial roles in the binding selectivity to their receptors, whereas the difference in the polarity of OT/VP family peptide residues at position 8 confers OT and VP with the binding specificity in vertebrates. CTR2 mRNA was present in various peripheral tissues, and OPR mRNA was detected in both the nervous system and peripheral tissues. Our findings suggest that the CT and OP genes, similar to the OT/VP family, evolved through duplication, but the ligand–receptor selectivity were established through different evolutionary lineages from those of their vertebrate counterparts. PMID:15504101

  7. The short mRNA isoform of the immunoglobulin superfamily, member 1 gene encodes an intracellular glycoprotein.

    Directory of Open Access Journals (Sweden)

    Ying Wang

    Full Text Available Mutations in the immunoglobulin superfamily, member 1 gene (IGSF1/Igsf1 cause an X-linked form of central hypothyroidism. The canonical form of IGSF1 is a transmembrane glycoprotein with 12 immunoglobulin (Ig loops. The protein is co-translationally cleaved into two sub-domains. The carboxyl-terminal domain (CTD, which contains the last 7 Ig loops, is trafficked to the plasma membrane. Most pathogenic mutations in IGSF1 map to the portion of the gene encoding the CTD. IGSF1/Igsf1 encodes a variety of transcripts. A little studied, but abundant splice variant encodes a truncated form of the protein, predicted to contain the first 2 Ig loops of the full-length IGSF1. The protein (hereafter referred to as IGSF1 isoform 2 or IGSF1-2 is likely retained in most individuals with IGSF1 mutations. Here, we characterized basic biochemical properties of the protein as a foray into understanding its potential function. IGSF1-2, like the IGSF1-CTD, is a glycoprotein. In both mouse and rat, the protein is N-glycosylated at a single asparagine residue in the first Ig loop. Contrary to earlier predictions, neither the murine nor rat IGSF1-2 is secreted from heterologous or homologous cells. In addition, neither protein associates with the plasma membrane. Rather, IGSF1-2 appears to be retained in the endoplasmic reticulum. Whether the protein plays intracellular functions or is trafficked through the secretory pathway under certain physiologic or pathophysiologic conditions has yet to be determined.

  8. Mouse RC/BTB2, a Member of the RCC1 Superfamily, Localizes to Spermatid Acrosomal Vesicles

    Science.gov (United States)

    Shen, Xuening; Nagarkatti-Gude, David R.; Hess, Rex A.; Henderson, Scott C.; Strauss, Jerome F.; Zhang, Zhibing

    2012-01-01

    Mouse RC/BTB2 is an unstudied protein of the RCC1 (Regulator of Chromosome Condensation) superfamily. Because of the significant remodeling of chromatin that occurs during spermiogenesis, we characterized the expression and localization of mouse RC/BTB2 in the testis and male germ cells. The Rc/btb2 gene yields two major transcripts: 2.3 kb Rc/btb2-s, present in most somatic tissues examined; and 2.5 kb Rc/btb2-t, which contains a unique non-translated exon in its 5′-UTR that is only detected in the testis. During the first wave of spermatogenesis, Rc/btb2-t mRNA is expressed from day 8 after birth, reaching highest levels of expression at day 30 after birth. The full-length protein contains three RCC1 domains in the N-terminus, and a BTB domain in the C-terminus. In the testis, the protein is detectable from day 12, but is progressively up-regulated to day 30 and day 42 after birth. In spermatids, some of the protein co-localizes with acrosomal markers sp56 and peanut lectin, indicating that it is an acrosomal protein. A GFP-tagged RCC1 domain is present throughout the cytoplasm of transfected CHO cells. However, both GFP-tagged, full-length RC/BTB2 and a GFP-tagged BTB domain localize to vesicles in close proximity to the nuclear membrane, suggesting that the BTB domain might play a role in mediating full-length RC/BTB2 localization. Since RCC1 domains associate with Ran, a small GTPase that regulates molecular trafficking, it is possible that RC/BTB2 plays a role in transporting proteins during acrosome formation. PMID:22768142

  9. Sexual side effects of 5-α-reductase inhibitors finasteride and dutasteride: A comprehensive review.

    Science.gov (United States)

    Fertig, Raymond M; Gamret, A Caresse; Darwin, Evan; Gaudi, Sudeep

    2017-11-11

    The 5-α-reductase inhibitors finasteride and dutasteride are frequently used in the treatment of androgenetic alopecia and benign prostatichyperplasia. These drugs are effective at reducing levels of dihydrotestosterone, the primary androgen responsible for the pathogenesis of both these conditions. However, finasteride and dutasteride have also been shown to produce an increase in the incidence of sexual dysfunction, namely, impotence, decreased libido, and ejaculation disorder. The purpose of this study is to review the existing medical literature with regard to the sexual side effects of 5-α-reductase inhibitor therapy. This review is an extensive look at the sexual effects of 5-α-reductase inhibitors and compares outcomes for finasteride versus dutasteride in addition to comparing sexualside effects for each of the different dosages prescribed of finasteride and dutasteride.

  10. Atorvastatin calcium: an addition to HMG-CoA reductase inhibitors.

    Science.gov (United States)

    Chong, P H; Seeger, J D

    1997-01-01

    Atorvastatin calcium is an HMG-coenzyme A (CoA) reductase inhibitor that was approved by the Food and Drug Administration on December 17, 1996. Like other such agents, it inhibits the action of HMG-CoA reductase and thereby decreases endogenous cholesterol synthesis, leading to a decrease in circulating low-density lipoprotein cholesterol. In addition to its effect on lipoprotein profile, atorvastatin reduces triglycerides to a greater extent than other HMG-CoA reductase inhibitors. These actions occur in a dose-dependent fashion. The adverse effect profile is similar to that of other agents in this class. Indications for atorvastatin include primary hypercholesterolemia as well as other lipid disorders.

  11. Molecular and phenotypic characterization of transgenic soybean expressing the Arabidopsis ferric chelate reductase gene, FRO2.

    Science.gov (United States)

    Vasconcelos, Marta; Eckert, Helene; Arahana, Venancio; Graef, George; Grusak, Michael A; Clemente, Tom

    2006-10-01

    Soybean (Glycine max Merr.) production is reduced under iron-limiting calcareous soils throughout the upper Midwest regions of the US. Like other dicotyledonous plants, soybean responds to iron-limiting environments by induction of an active proton pump, a ferric iron reductase and an iron transporter. Here we demonstrate that heterologous expression of the Arabidopsis thaliana ferric chelate reductase gene, FRO2, in transgenic soybean significantly enhances Fe(+3) reduction in roots and leaves. Root ferric reductase activity was up to tenfold higher in transgenic plants and was not subjected to post-transcriptional regulation. In leaves, reductase activity was threefold higher in the transgenic plants when compared to control. The enhanced ferric reductase activity led to reduced chlorosis, increased chlorophyll concentration and a lessening in biomass loss in the transgenic events between Fe treatments as compared to control plants grown under hydroponics that mimicked Fe-sufficient and Fe-deficient soil environments. However, the data indicate that constitutive FRO2 expression under non-iron stress conditions may lead to a decrease in plant productivity as reflected by reduced biomass accumulation in the transgenic events under non-iron stress conditions. When grown at Fe(III)-EDDHA levels greater than 10 microM, iron concentration in the shoots of transgenic plants was significantly higher than control. The same observation was found in the roots in plants grown at iron levels higher than 32 microM Fe(III)-EDDHA. These results suggest that heterologous expression of an iron chelate reductase in soybean can provide a route to alleviate iron deficiency chlorosis.

  12. Characterisation of a desmosterol reductase involved in phytosterol dealkylation in the silkworm, Bombyx mori.

    Directory of Open Access Journals (Sweden)

    Leonora F Ciufo

    Full Text Available Most species of invertebrate animals cannot synthesise sterols de novo and many that feed on plants dealkylate phytosterols (mostly C(29 and C(28 yielding cholesterol (C(27. The final step of this dealkylation pathway involves desmosterol reductase (DHCR24-catalysed reduction of desmosterol to cholesterol. We now report the molecular characterisation in the silkworm, Bombyx mori, of such a desmosterol reductase involved in production of cholesterol from phytosterol, rather than in de novo synthesis of cholesterol. Phylogenomic analysis of putative desmosterol reductases revealed the occurrence of various clades that allowed for the identification of a strong reductase candidate gene in Bombyx mori (BGIBMGA 005735. Following PCR-based cloning of the cDNA (1.6 kb and its heterologous expression in Saccharomyces cerevisae, the recombinant protein catalysed reduction of desmosterol to cholesterol in an NADH- and FAD-dependent reaction.Conceptual translation of the cDNA, that encodes a 58.9 kDa protein, and database searching, revealed that the enzyme belongs to an FAD-dependent oxidoreductase family. Western blotting revealed reductase protein expression exclusively in the microsomal subcellular fraction and primarily in the gut. The protein is peripherally associated with microsomal membranes. 2D-native gel and PAGE analysis revealed that the reductase is part of a large complex with molecular weight approximately 250 kDa. The protein occurs in midgut microsomes at a fairly constant level throughout development in the last two instars, but is drastically reduced during the wandering stage in preparation for metamorphosis. Putative Broad Complex transcription factor-binding sites detectable upstream of the DHCR24 gene may play a role in this down-regulation.

  13. Inhibition of human anthracycline reductases by emodin — A possible remedy for anthracycline resistance

    International Nuclear Information System (INIS)

    Hintzpeter, Jan; Seliger, Jan Moritz; Hofman, Jakub; Martin, Hans-Joerg; Wsol, Vladimir; Maser, Edmund

    2016-01-01

    The clinical application of anthracyclines, like daunorubicin and doxorubicin, is limited by two factors: dose-related cardiotoxicity and drug resistance. Both have been linked to reductive metabolism of the parent drug to their metabolites daunorubicinol and doxorubicinol, respectively. These metabolites show significantly less anti-neoplastic properties as their parent drugs and accumulate in cardiac tissue leading to chronic cardiotoxicity. Therefore, we aimed to identify novel and potent natural inhibitors for anthracycline reductases, which enhance the anticancer effect of anthracyclines by preventing the development of anthracycline resistance. Human enzymes responsible for the reductive metabolism of daunorubicin were tested for their sensitivity towards anthrachinones, in particular emodin and anthraflavic acid. Intense inhibition kinetic data for the most effective daunorubicin reductases, including IC 50 - and K i -values, the mode of inhibition, as well as molecular docking, were compiled. Subsequently, a cytotoxicity profile and the ability of emodin to reverse daunorubicin resistance were determined using multiresistant A549 lung cancer and HepG2 liver cancer cells. Emodin potently inhibited the four main human daunorubicin reductases in vitro. Further, we could demonstrate that emodin is able to synergistically sensitize human cancer cells towards daunorubicin at clinically relevant concentrations. Therefore, emodin may yield the potential to enhance the therapeutic effectiveness of anthracyclines by preventing anthracycline resistance via inhibition of the anthracycline reductases. In symphony with its known pharmacological properties, emodin might be a compound of particular interest in the management of anthracycline chemotherapy efficacy and their adverse effects. - Highlights: • Natural and synthetic compounds were identified as inhibitors for human daunorubicin reductases. • Emodin is a potent inhibitor for human daunorubicin reductases.

  14. The electron transfer reactions of NADPH: cytochrome P450 reductase with nonphysiological oxidants.

    Science.gov (United States)

    Cénas, N; Anusevicius, Z; Bironaité, D; Bachmanova, G I; Archakov, A I; Ollinger, K

    1994-12-01

    The steady-state kinetics of oxidation of rat liver NADPH: cytochrome P450 reductase (EC 1.6.2.4) by quinones, aromatic nitrocompounds, ferricyanide, Fe(EDTA)-, and cytochrome c has been studied. The logarithms of bimolecular rate constants of reduction (kcat/Km) of quinones and nitrocompounds increase with the increase in their single-electronreduction potential (E1(7)), reaching a maximum value at E1(7) > -0.15 V. The reactivities of nitroaromatics are about by an order of magnitude lower than the reactivities of quinones. For a series of nitroaromatics including the compounds with previously undetermined E1(7) values, an orthogonality was found between their reactivities toward cytochrome P450 reductase, flavocytochrome b2 (EC 1.1.2.3), and the NADPH: adrenodoxin reductase (EC 1.18.1.2)-adrenodoxin system. This indicates the absence of significant specific interactions during these reactions. The effects of ionic strength on reaction kinetics and the character of inhibition by a product of reaction, NADP+, are in accordance with the reduction of oxidants at the negatively charged site in the surroundings of FMN of P450 reductase. Quinones inactivate oxidized reductase modifying the NADP(H) binding site. The redox cycling of quinones markedly slows the inactivation. The kinetic data presented are consistent with an outer-sphere electron transfer mechanism. The analysis of kinetics of reduction of cytochrome c, ferricyanide, and Fe(EDTA)- using the model of Mauk et al. (A. G. Mauk, R. A. Scott, and H. B. Gray (1980) J. Am. Chem. Soc. 102, 4360-4363) gives calculated distances of FMN from the surface of protein globule, 0.33-0.63 nm. The data from nitroreductase reactions of cytochrome P450 reductase, flavocytochrome b2, and adrenodoxin were used for approximate evaluation of previously unknown E1(7) of nitrocompounds.

  15. Structure of diaminohydroxyphosphoribosylaminopyrimidine deaminase/5-amino-6-(5-phosphoribosylamino)uracil reductase from Acinetobacter baumannii.

    Science.gov (United States)

    Dawson, Alice; Trumper, Paul; Chrysostomou, Georgios; Hunter, William N

    2013-06-01

    The bifunctional diaminohydroxyphosphoribosylaminopyrimidine deaminase/5-amino-6-(5-phosphoribosylamino)uracil reductase (RibD) represents a potential antibacterial drug target. The structure of recombinant Acinetobacter baumannii RibD is reported in orthorhombic and tetragonal crystal forms at 2.2 and 2.0 Å resolution, respectively. Comparisons with orthologous structures in the Protein Data Bank indicated close similarities. The tetragonal crystal form was obtained in the presence of guanosine monophosphate, which surprisingly was observed to occupy the adenine-binding site of the reductase domain.

  16. Chloroplast Fe(III) chelate reductase activity is essential for seedling viability under iron limiting conditions

    OpenAIRE

    Jeong, Jeeyon; Cohu, Christopher; Kerkeb, Loubna; Pilon, Marinus; Connolly, Erin L.; Guerinot, Mary Lou

    2008-01-01

    Photosynthesis, heme biosynthesis, and Fe-S cluster assembly all take place in the chloroplast, and all require iron. Reduction of iron via a membrane-bound Fe(III) chelate reductase is required before iron transport across membranes in a variety of systems, but to date there has been no definitive genetic proof that chloroplasts have such a reduction system. Here we report that one of the eight members of the Arabidopsis ferric reductase oxidase (FRO) family, FRO7, localizes to the chloropla...

  17. Why Is Mammalian Thioredoxin Reductase 1 So Dependent upon the Use of Selenium?

    OpenAIRE

    Lothrop, Adam P.; Snider, Gregg W.; Ruggles, Erik L.; Hondal, Robert J.

    2014-01-01

    Cytosolic thioredoxin reductase 1 (TR1) is the best characterized of the class of high-molecular weight (M r) thioredoxin reductases (TRs). TR1 is highly dependent upon the rare amino acid selenocysteine (Sec) for the reduction of thioredoxin (Trx) and a host of small molecule substrates, as mutation of Sec to cysteine (Cys) results in a large decrease in catalytic activity for all substrate types. Previous work in our lab and others has shown that the mitochondrial TR (TR3) is much less depe...

  18. Crystallization and preliminary X-ray diffraction studies of ferredoxin reductase from Leptospira interrogans

    International Nuclear Information System (INIS)

    Nascimento, Alessandro S.; Ferrarezi, Thiago; Catalano-Dupuy, Daniela L.; Ceccarelli, Eduardo A.; Polikarpov, Igor

    2006-01-01

    Crystals adequate for X-ray diffraction analysis have been prepared from L. interrogans ferredoxin-NADP + reductase. Ferredoxin-NADP + reductase (FNR) is an FAD-containing enzyme that catalyzes electron transfer between NADP(H) and ferredoxin. Here, results are reported of the recombinant expression, purification and crystallization of FNR from Leptospira interrogans, a parasitic bacterium of animals and humans. The L. interrogans FNR crystals belong to a primitive monoclinic space group and diffract to 2.4 Å resolution at a synchrotron source

  19. Crystallization and preliminary X-ray diffraction studies of ferredoxin reductase from Leptospira interrogans

    Energy Technology Data Exchange (ETDEWEB)

    Nascimento, Alessandro S.; Ferrarezi, Thiago [Instituto de Física de São Carlos, Universidade de São Paulo, Av. Trabalhador Saocarlense 400, São Carlos, SP, 13560-970 (Brazil); Catalano-Dupuy, Daniela L.; Ceccarelli, Eduardo A. [Facultad de Ciencias Bioquímicas y Farmacéuticas, Molecular Biology Division, Instituto de Biología Molecular y Celular de Rosario (IBR), CONICET, Universidad Nacional de Rosario, Suipacha 531, S2002LRK Rosario (Argentina); Polikarpov, Igor, E-mail: ipolikarpov@if.sc.usp.br [Instituto de Física de São Carlos, Universidade de São Paulo, Av. Trabalhador Saocarlense 400, São Carlos, SP, 13560-970 (Brazil)

    2006-07-01

    Crystals adequate for X-ray diffraction analysis have been prepared from L. interrogans ferredoxin-NADP{sup +} reductase. Ferredoxin-NADP{sup +} reductase (FNR) is an FAD-containing enzyme that catalyzes electron transfer between NADP(H) and ferredoxin. Here, results are reported of the recombinant expression, purification and crystallization of FNR from Leptospira interrogans, a parasitic bacterium of animals and humans. The L. interrogans FNR crystals belong to a primitive monoclinic space group and diffract to 2.4 Å resolution at a synchrotron source.

  20. Is decreased libido associated with the use of HMG-CoA-reductase inhibitors?

    Science.gov (United States)

    de Graaf, L; Brouwers, A H P M; Diemont, W L

    2004-09-01

    To describe patients with decreased libido during use of a HMG-CoA-reductase-inhibitor, and to discuss causality and pharmacological hypotheses for this association by analysis of the adverse drug reactions (ADR) database of the Netherlands Pharmacovigilance Centre Lareb. Eight patients were identified as having decreased libido during use of statins. In two of these cases testosterone levels were determined and appeared to be decreased. Decreased libido is a probable adverse drug reaction of HMG-CoA-reductase-inhibitors and is reversible. The ADR may be caused by low serum testosterone levels, mainly due to intracellular cholesterol depletion.

  1. Alpha 1-blockers vs 5 alpha-reductase inhibitors in benign prostatic hyperplasia. A comparative review

    DEFF Research Database (Denmark)

    Andersen, J T

    1995-01-01

    During recent years, pharmacological treatment of symptomatic benign prostatic hyperplasia (BPH) has become the primary treatment choice for an increasing number of patients. The 2 principal drug classes employed are alpha 1-blockers and 5 alpha-reductase inhibitors. Current information from...... of patients who will respond well to alpha 1-blockers have yet to be identified, and data concerning the long term effects of these drugs are not yet available. 5 alpha-Reductase inhibitors have a slow onset of effect, but treatment leads to improvement in symptoms, reduction of the size of the prostate gland...... or unwilling to undergo surgical resection of the prostate will benefit from such therapy....

  2. Biochemical and structural analysis of Gox2181, a new member of the SDR superfamily from Gluconobacter oxydans.

    Science.gov (United States)

    Liu, Xu; Yuan, Zuanning; Adam Yuan, Y; Lin, Jinping; Wei, Dongzhi

    2011-11-18

    Gluconobacter oxydans enable to oxidize sugars and polyols incompletely to corresponding materials with potential industrial applications, containing around 75 putative dehydrogenases. One of these putative dehydrogenases, Gox2181, was cloned and expressed in Escherichia coli BL21 (DE3), and its X-ray crystal structure was determined to a resolution of 1.8 Å. Gox2181 formed a homo-tetramer in the crystal that was coincident with the apparent molecular mass determined in the solution. Gox2181 displayed α/β-folding patterns, the conserved catalytic tetrad of Asn119-Ser147-Tyr162-Lys166, and the NAD-binding pocket, which aligned well with the 'classical' type of short-chain dehydrogenase/reductase (SDR) enzymes. Gox2181 was denoted SDR51C based on the SDR nomenclature system. The purified recombinant Gox2181 was demonstrated to be NAD(H)-dependent and active towards a wide range of substrates, including sugar alcohols, secondary alcohols, ketones, and ketoses. Among the substrates tested, Gox2181 displayed preference for secondary hydroxyl or carbonyl groups, showing low K(m) values with d-arabitol and butanedione. Copyright © 2011 Elsevier Inc. All rights reserved.

  3. Escherichia coli ferredoxin-NADP+ reductase and oxygen-insensitive nitroreductase are capable of functioning as ferric reductase and of driving the Fenton reaction.

    Science.gov (United States)

    Takeda, Kouji; Sato, Junichi; Goto, Kazuyuki; Fujita, Takanori; Watanabe, Toshihiro; Abo, Mitsuru; Yoshimura, Etsuro; Nakagawa, Junichi; Abe, Akira; Kawasaki, Shinji; Niimura, Youichi

    2010-08-01

    Two free flavin-independent enzymes were purified by detecting the NAD(P)H oxidation in the presence of Fe(III)-EDTA and t-butyl hydroperoxide from E. coli. The enzyme that requires NADH or NADPH as an electron donor was a 28 kDa protein, and N-terminal sequencing revealed it to be oxygen-insensitive nitroreductase (NfnB). The second enzyme that requires NADPH as an electron donor was a 30 kDa protein, and N-terminal sequencing revealed it to be ferredoxin-NADP(+) reductase (Fpr). The chemical stoichiometry of the Fenton activities of both NfnB and Fpr in the presence of Fe(III)-EDTA, NAD(P)H and hydrogen peroxide was investigated. Both enzymes showed a one-electron reduction in the reaction forming hydroxyl radical from hydrogen peroxide. Also, the observed Fenton activities of both enzymes in the presence of synthetic chelate iron compounds were higher than their activities in the presence of natural chelate iron compounds. When the Fenton reaction occurs, the ferric iron must be reduced to ferrous iron. The ferric reductase activities of both NfnB and Fpr occurred with synthetic chelate iron compounds. Unlike NfnB, Fpr also showed the ferric reductase activity on an iron storage protein, ferritin, and various natural iron chelate compounds including siderophore. The Fenton and ferric reductase reactions of both NfnB and Fpr occurred in the absence of free flavin. Although the k(cat)/K(m) value of NfnB for Fe(III)-EDTA was not affected by free flavin, the k(cat)/K(m) value of Fpr for Fe(III)-EDTA was 12-times greater in the presence of free FAD than in the absence of free FAD.

  4. Hypothesis onSerenoa repens(Bartram) small extract inhibition of prostatic 5α-reductase through anin silicoapproach on 5β-reductase x-ray structure.

    Science.gov (United States)

    Governa, Paolo; Giachetti, Daniela; Biagi, Marco; Manetti, Fabrizio; De Vico, Luca

    2016-01-01

    Benign prostatic hyperplasia is a common disease in men aged over 50 years old, with an incidence increasing to more than 80% over the age of 70, that is increasingly going to attract pharmaceutical interest. Within conventional therapies, such as α -adrenoreceptor antagonists and 5 α -reductase inhibitor, there is a large requirement for treatments with less adverse events on, e.g., blood pressure and sexual function: phytotherapy may be the right way to fill this need. Serenoa repens standardized extract has been widely studied and its ability to reduce lower urinary tract symptoms related to benign prostatic hyperplasia is comprehensively described in literature. An innovative investigation on the mechanism of inhibition of 5 α -reductase by Serenoa repens extract active principles is proposed in this work through computational methods, performing molecular docking simulations on the crystal structure of human liver 5 β -reductase. The results confirm that both sterols and fatty acids can play a role in the inhibition of the enzyme, thus, suggesting a competitive mechanism of inhibition. This work proposes a further confirmation for the rational use of herbal products in the management of benign prostatic hyperplasia, and suggests computational methods as an innovative, low cost, and non-invasive process for the study of phytocomplex activity toward proteic targets.

  5. [Origin and evolution of parasitism in mites of the infraorder Eleutherengona (Acari: Prostigmata). Report II. Superfamily Cheyletoidea].

    Science.gov (United States)

    Bochkov, A V

    2009-01-01

    these eggs were laid by feather mites (Astigmata: Psoroptidia). These rounded shape eggs, however, are more similar with those of Cheyletoidea, than with the boomerang-shape eggs of feather mites. The position of the subfamily Ophioptinae associated with snakes of the superfamily Colubroidea in the core of the family Harpirhynchidae (bird parasites) is explained by the switching of its ancestor from passerine birds. Certain snakes feed on nestlings and adult birds, and most of these preys are small passerine birds.

  6. Functional Characterization of TaFUSCA3, a B3-Superfamily Transcription Factor Gene in the Wheat

    Directory of Open Access Journals (Sweden)

    Fusheng Sun

    2017-06-01

    Full Text Available The end-use quality of wheat, including its unique rheology and viscoelastic properties, is predominantly determined by the composition and concentration of gluten proteins. While, the mechanism regulating expression of the seed storage protein (SSP genes and other related genes in wheat remains unclear. In this study, we report on the cloning and functional identification of TaFUSCA3, a B3-superfamily transcription factor (TF gene in wheat. Sequence alignment indicated that wheat and barley FUSCA3 genes are highly conserved. Quantitative reverse-transcription (qRT-PCR analysis showed that the transcript of TaFUSCA3 was accumulated mostly in the stamens and the endosperms of immature wheat seeds. Yeast-one-hybrid results proved that the full-length TaFUSCA3 and its C-terminal region had transcriptional activities. Yeast-two-hybrid and bimolecular fluorescence complementation assays indicated that TaFUSCA3 could activate the expression of the high molecular weight glutenin subunit gene Glu-1Bx7 and interact with the seed-specific bZIP protein TaSPA. DNA-protein-interaction enzyme-linked immunosorbent assay demonstrated that TaFUSCA3 specifically recognizes the RY-box of the Glu-1Bx7 promoter region. Transient expression results showed that TaFUSCA3 could trans-activate the Glu-1Bx7 promoter, which contains eight RY-box sequences. TaFUSCA3 was unable to activate the downstream transcription when the RY-box was fully mutated. TaFUSCA3 could activate the transcription of the At2S3 gene promoter in a complementation of loss-of-function experiment using the Arabidopsis thaliana line fus3-3, which is a FUSCA3 mutant, demonstrating the evolutionary conservation of the TaFUSCA3 gene. In conclusion, the wheat B3-type TF, TaFUSCA3, is functional conserved between monocot and dicot, and could regulate SSP gene expression by interacting specifically with TaSPA.

  7. Phylogenomic and functional analyses of salmon lice aquaporins uncover the molecular diversity of the superfamily in Arthropoda.

    Science.gov (United States)

    Stavang, Jon Anders; Chauvigné, Francois; Kongshaug, Heidi; Cerdà, Joan; Nilsen, Frank; Finn, Roderick Nigel

    2015-08-19

    An emerging field in biomedical research is focusing on the roles of aquaporin water channels in parasites that cause debilitating or lethal diseases to their vertebrate hosts. The primary vectorial agents are hematophagous arthropods, including mosquitoes, flies, ticks and lice, however very little is known concerning the functional diversity of aquaporins in non-insect members of the Arthropoda. Here we conducted phylogenomic and functional analyses of aquaporins in the salmon louse, a marine ectoparasitic copepod that feeds on the skin and body fluids of salmonids, and used the primary structures of the isolated channels to uncover the genomic repertoires in Arthropoda. Genomic screening identified 7 aquaporin paralogs in the louse in contrast to 42 in its host the Atlantic salmon. Phylogenetic inference of the louse nucleotides and proteins in relation to orthologs identified in Chelicerata, Myriapoda, Crustacea and Hexapoda revealed that the arthropod aquaporin superfamily can be classified into three major grades (1) classical aquaporins including Big brain (Bib) and Prip-like (PripL) channels (2) aquaglyceroporins (Glp) and (3) unorthodox aquaporins (Aqp12-like). In Hexapoda, two additional subfamilies exist as Drip and a recently classified entomoglyceroporin (Eglp) group. Cloning and remapping the louse cDNAs to the genomic DNA revealed that they are encoded by 1-7 exons, with two of the Glps being expressed as N-terminal splice variants (Glp1_v1, -1_v2, -3_v1, -3_v2). Heterologous expression of the cRNAs in amphibian oocytes demonstrated that PripL transports water and urea, while Bib does not. Glp1_v1, -2, -3_v1 and -3_v2 each transport water, glycerol and urea, while Glp1_v2 and the Aqp12-like channels were retained intracellularly. Transcript abundance analyses revealed expression of each louse paralog at all developmental stages, except for glp1_v1, which is specific to preadult and adult males. Our data suggest that the aquaporin repertoires of

  8. Three spinach leaf nitrate reductase-3-hydroxy-3-methylglutaryl-CoA reductase kinases that are regulated by reversible phosphorylation and/or Ca2+ ions.

    Science.gov (United States)

    Douglas, P; Pigaglio, E; Ferrer, A; Halfords, N G; MacKintosh, C

    1997-07-01

    In spinach (Spinacea oleracea L.) leaf extracts, three protein kinases (PKI, PKII and PKIII) were identified each of which phosphorylated spinach nitrate reductase on serine-543, and inactivated the enzyme in the presence of nitrate reductase inhibitor, 14-3-3. PKIII was also very active in phosphorylating and inactivating Arabidopsis (Landsberg erecta) 3-hydroxy-3-methylglutaryl-coenzyme A reductase 1 (HMGR1). PKI and PKII phosphorylated HMGR1 more slowly than PKIII, compared with their relative rates of phosphorylation of nitrate reductase. HMGR1 identical with those that are seen after phosphorylation of serine-577 by the sucrose non-fermenting (SNF1)-like PK, 3-hydroxy-3-methylglutaryl-Co A reductase kinase A (HRK-A), from cauliflower [Dale, Arró, Becerra, Morrice, Boronat, Hardie and Ferrer (1995) Eur. J. Biochem. 233, 506-513]. PKI was Ca2+-dependent when prepared in the absence of protein phosphatase (PP) inhibitors, and largely Ca2+-dependent when prepared in the presence of PP inhibitors (NaF and EGTA). The Ca2+-independent portion of PKI was inactivated by either PP2A or PP2C, while the Ca2+-dependent portion of PKI became increasingly activated during storage, which we presume was mimicking the effect of an unidentified PP. These findings indicate that PK1 is regulated by two functionally distinct phosphorylations. PKI had a molecular mass of 45 kDa on gel filtration and was active towards substrate peptides that terminated at the +2 residue from the phosphorylation site, whereas PKIII was inactive towards these peptides. PKII was Ca2+-stimulated under all conditions tested. PKIII was Ca2+-indepdented, inactivated by PP2A or PP2C, had a requirement for a hydrophobic residue in the +4 position of peptide substrates, had a molecular mass by gel filtration of approximately 140 kDa, and an antibody against the rye SNF1-related PK (RKIN1) recognized a 58 kDa subunit in fractions containing PKIII. These properties of PKIII are identical with those reported

  9. Three spinach leaf nitrate reductase-3-hydroxy-3-methylglutaryl-CoA reductase kinases that are required by reversible phosphorylation and/or Ca2+ ions.

    Science.gov (United States)

    Douglas, P; Pigaglio, E; Ferrer, A; Halfords, N G; MacKintosh, C

    1997-01-01

    In spinach (Spinacea oleracea L.) leaf extracts, three protein kinases (PKI, PKII and PKIII) were identified each of which phosphorylated spinach nitrate reductase on serine-543, and inactivated the enzyme in the presence of nitrate reductase inhibitor, 14-3-3. PKIII was also very active in phosphorylating and inactivating Arabidopsis (Landsberg erecta) 3-hydroxy-3-methylglutaryl-coenzyme A reductase 1 (HMGR1). PKI and PKII phosphorylated HMGR1 more slowly than PKIII, compared with their relative rates of phosphorylation of nitrate reductase. HMGR1 identical with those that are seen after phosphorylation of serine-577 by the sucrose non-fermenting (SNF1)-like PK, 3-hydroxy-3-methylglutaryl-Co A reductase kinase A (HRK-A), from cauliflower [Dale, Arró, Becerra, Morrice, Boronat, Hardie and Ferrer (1995) Eur. J. Biochem. 233, 506-513]. PKI was Ca2+-dependent when prepared in the absence of protein phosphatase (PP) inhibitors, and largely Ca2+-dependent when prepared in the presence of PP inhibitors (NaF and EGTA). The Ca2+-independent portion of PKI was inactivated by either PP2A or PP2C, while the Ca2+-dependent portion of PKI became increasingly activated during storage, which we presume was mimicking the effect of an unidentified PP. These findings indicate that PK1 is regulated by two functionally distinct phosphorylations. PKI had a molecular mass of 45 kDa on gel filtration and was active towards substrate peptides that terminated at the +2 residue from the phosphorylation site, whereas PKIII was inactive towards these peptides. PKII was Ca2+-stimulated under all conditions tested. PKIII was Ca2+-indepdented, inactivated by PP2A or PP2C, had a requirement for a hydrophobic residue in the +4 position of peptide substrates, had a molecular mass by gel filtration of approximately 140 kDa, and an antibody against the rye SNF1-related PK (RKIN1) recognized a 58 kDa subunit in fractions containing PKIII. These properties of PKIII are identical with those reported

  10. Pre-clinical activity of PR-104 as monotherapy and in combination with sorafenib in hepatocellular carcinoma.

    Science.gov (United States)

    Abbattista, Maria R; Jamieson, Stephen M F; Gu, Yongchuan; Nickel, Jennifer E; Pullen, Susan M; Patterson, Adam V; Wilson, William R; Guise, Christopher P

    2015-01-01

    PR-104 is a clinical stage bioreductive prodrug that is converted in vivo to its cognate alcohol, PR-104A. This dinitrobenzamide mustard is reduced to activated DNA cross-linking metabolites (hydroxylamine PR-104H and amine PR-104M) under hypoxia by one-electron reductases and independently of hypoxia by the 2-electron reductase aldo-keto reductase 1C3 (AKR1C3). High expression of AKR1C3, along with extensive hypoxia, suggested the potential of PR-104 for treatment of hepatocellular carcinoma (HCC). However, a phase IB trial with sorafenib demonstrated significant toxicity that was ascribed in part to reduced PR-104A clearance, likely reflecting compromised glucuronidation in patients with advanced HCC. Here, we evaluate the activity of PR-104 in HCC xenografts (HepG2, PLC/PRF/5, SNU-398, Hep3B) in mice, which do not significantly glucuronidate PR-104A. Cell line differences in sensitivity to PR-104A in vitro under aerobic conditions could be accounted for by differences in both expression of AKR1C3 (high in HepG2 and PLC/PRF/5) and sensitivity to the major active metabolite PR-104H, to which PLC/PRF/5 was relatively resistant, while hypoxic selectivity of PR-104A cytotoxicity and reductive metabolism was greatest in the low-AKR1C3 SNU-398 and Hep3B lines. Expression of AKR1C3 in HepG2 and PLC/PRF/5 xenografts was in the range seen in 21 human HCC specimens. PR-104 monotherapy elicited significant reductions in growth of Hep3B and HepG2 xenografts, and the combination with sorafenib was significantly active in all 4 xenograft models. The results suggest that better-tolerated analogs of PR-104, without a glucuronidation liability, may have the potential to exploit AKR1C3 and/or hypoxia in HCC in humans.

  11. Differential stress-induced regulation of two quinone reductases in the brown rot Basidiomycete Gloeophyllum trabeum

    Science.gov (United States)

    Roni Cohen; Melissa R. Suzuki; Kenneth E. Hammel

    2004-01-01

    Quinone reductases (QRDs) have two important functions in the basidiomycete Gloeophyllum trabeum, which causes brown rot of wood. First, a QRD is required to generate biodegradative hydroxyl radicals via redox cycling between two G. trabeum extracellular metabolites, 2,5-dimethoxyhydroquinone (2,5-DMHQ) and 2,5-dimethoxy-1,4-benzoquinone (2,5- DMBQ). Second, because 2,...

  12. A Rational Approach to Identify Inhibitors of Mycobacterium tuberculosis Enoyl Acyl Carrier Protein Reductase

    Czech Academy of Sciences Publication Activity Database

    Chhabria, M. T.; Parmar, K. B.; Brahmkshatriya, Pathik

    2013-01-01

    Roč. 19, č. 21 (2013), s. 3878-3883 ISSN 1381-6128 Institutional support: RVO:61388963 Keywords : mycobacterium tuberculosis * enoyl acyl carrier protein reductase * pharmacophore modeling * molecular docking * binding interactions Subject RIV: FR - Pharmacology ; Medidal Chemistry Impact factor: 3.288, year: 2013

  13. Studies on the kinetic mechanism of nitrate reductase from spinach (Spinacea oleracea).

    Science.gov (United States)

    de la Rosa, F F; Palacián, E; Castillo, F

    1980-09-01

    Based on Lineweaver-Burk plots of the initial velocities, at different concentrations of NADH and nitrate, and product inhibition patterns, an Iso Ping Pong Bi Bi steady state kinetic mechanism is proposed for the spinach nitrate reductase. This mechanism incorporates the concept that the oxidized enzyme is present in two isomeric forms.

  14. Differential antioxidant and quinone reductase inducing activity of American, Asian, and Siberian ginseng

    Science.gov (United States)

    The antioxidant and quinone reductase (QR) inducing activities of American, Asian, and Siberian ginseng have been reported using various plant materials, solvents, and assays. To directly establish their comparative bioactivity, the effects of extracts obtained from acidified methanol (MeOH), a gas...

  15. Purification and properties of a NADPH-dependent erythrose reductase from the newly isolated Torula corallina.

    Science.gov (United States)

    Lee, Jung-Kul; Hong, Kwang-Won; Kim, Sang-Yong

    2003-01-01

    Torula corallina (KCCM-10171) is a yeast strain that is currently used for the industrial production of erythritol and has the highest erythritol yield ever reported for an erythritol-producing microorganism. Production of erythritol in T. corallina is catalyzed by erythrose reductase, an enzyme that converts erythrose to erythritol using NADPH as a cofactor. In this study, NADPH-dependent erythrose reductase was purified to homogeneity from the newly isolated T. corallina. The relative molecular weight of the erythrose reductase as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size exclusion chromatography was 35.4 and 71.0 kDa, respectively, indicating that the enzyme is dimeric. This enzyme catalyzed both erythrose reduction and erythritol oxidation; both enzyme activities required NADP(H). The pH and temperature optima for erythrose reduction and erythritol oxidation were 6.0, 40 degrees C and 8.0, 45 degrees C, respectively. The sequence of the first 10 amino acids of this enzyme was N-V-K-N-F-Y-Q-P-N-D. The affinity (K(m)( )()= 7.12 mM) of the enzyme for erythrose was comparable to that of other known erythrose reductases, and the specificity for erythrose was very high, resulting in no production of other polyols, which may explain the high erythritol yield observed in this strain.

  16. Fumarate-mediated inhibition of erythrose reductase, a key enzyme for erythritol production by Torula corallina.

    Science.gov (United States)

    Lee, Jung-Kul; Koo, Bong-Seong; Kim, Sang-Yong

    2002-09-01

    Torula corallina, a strain presently being used for the industrial production of erythritol, has the highest erythritol yield ever reported for an erythritol-producing microorganism. The increased production of erythritol by Torula corallina with trace elements such as Cu(2+) has been thoroughly reported, but the mechanism by which Cu(2+) increases the production of erythritol has not been studied. This study demonstrated that supplemental Cu(2+) enhanced the production of erythritol, while it significantly decreased the production of a major by-product that accumulates during erythritol fermentation, which was identified as fumarate by instrumental analyses. Erythrose reductase, a key enzyme that converts erythrose to erythritol in T. corallina, was purified to homogeneity by chromatographic methods, including ion-exchange and affinity chromatography. In vitro, purified erythrose reductase was significantly inhibited noncompetitively by increasing the fumarate concentration. In contrast, the enzyme activity remained almost constant regardless of Cu(2+) concentration. This suggests that supplemental Cu(2+) reduced the production of fumarate, a strong inhibitor of erythrose reductase, which led to less inhibition of erythrose reductase and a high yield of erythritol. This is the first report that suggests catabolite repression by a tricarboxylic acid cycle intermediate in T. corallina.

  17. Low activity of superoxide dismutase and high activity of glutathione reductase in erythrocytes from centenarians

    DEFF Research Database (Denmark)

    Andersen, Helle Raun; Jeune, B; Nybo, H

    1998-01-01

    aged between 60 and 79 years. MEASUREMENTS: enzyme activities of superoxide dismutase (CuZn-SOD), glutathione peroxidase, catalase and glutathione reductase (GR) in erythrocytes. Functional capacity among the centenarians was evaluated by Katz' index of activities of daily living, the Physical...

  18. The Activities of NADH-MethB Reductase and Glucose-6 ...

    African Journals Online (AJOL)

    The activities of NADH-MetHB reductase and G-6-PD were investigated in malaria patients in Calabar, Nigeria. Seventy malaria patients were selected for this study. Sixty-two age, sex – matched apparently healthy children were used as controls. Ages of subjects ranged from 6 months to 12 years (Mean = 5±1.3 years).

  19. Identification and characterization of an inborn error of metabolism caused by dihydrofolate reductase deficiency

    NARCIS (Netherlands)

    Banka, S.; Blom, H.J.; Walter, J.; Aziz, M.; Urquhart, J.; Clouthier, C.M.; Rice, G.I.; Brouwer, A.P.M. de; Hilton, E.; Vassallo, G.; Will, A.; Smith, D.E.; Smulders, Y.M.; Wevers, R.A.; Steinfeld, R.; Heales, S.; Crow, Y.J.; Pelletier, J.N.; Jones, S.; Newman, W.G.

    2011-01-01

    Dihydrofolate reductase (DHFR) is a critical enzyme in folate metabolism and an important target of antineoplastic, antimicrobial, and antiinflammatory drugs. We describe three individuals from two families with a recessive inborn error of metabolism, characterized by megaloblastic anemia and/or

  20. Evidence for a novel mechanism of time-resolved flavin fluorescence depolarization in glutathione reductase.

    NARCIS (Netherlands)

    Berg, van den P.A.W.; Hoek, van A.; Visser, A.J.W.G.

    2004-01-01

    Time-resolved flavin fluorescence anisotropy studies on glutathione reductase (GR) have revealed a remarkable new phenomenon: wild-type GR displays a rapid process of fluorescence depolarization, that is absent in mutant enzymes lacking a nearby tyrosine residue that blocks the NADPH-binding cleft.