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Sample records for hmg-coa reductase hmgr

  1. Transgenic Cotton Plants Expressing Double-stranded RNAs Target HMG-CoA Reductase (HMGR) Gene Inhibits the Growth, Development and Survival of Cotton Bollworms.

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    Tian, Geng; Cheng, Linlin; Qi, Xuewei; Ge, Zonghe; Niu, Changying; Zhang, Xianlong; Jin, Shuangxia

    2015-01-01

    RNA interference (RNAi) has been developed as a powerful technique in the research of functional genomics as well as plant pest control. In this report, double-stranded RNAs (dsRNA) targeting 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) gene, which catalyze a rate-limiting enzymatic reaction in the mevalonate pathway of juvenile hormone (JH) synthesis in cotton bollworm, was expressed in cotton plants via Agrobacterium tumefaciens-mediated transformation. PCR and Sothern analysis revealed the integration of HMGR gene into cotton genome. RT-PCR and qRT-PCR confirmed the high transcription level of dsHMGR in transgenic cotton lines. The HMGR expression both in transcription and translation level was significantly downregulated in cotton bollworms (helicoverpa armigera) larvae after feeding on the leaves of HMGR transgenic plants. The transcription level of HMGR gene in larvae reared on transgenic cotton leaves was as much as 80.68% lower than that of wild type. In addition, the relative expression level of vitellogenin (Vg, crucial source of nourishment for offspring embryo development) gene was also reduced by 76.86% when the insect larvae were fed with transgenic leaves. The result of insect bioassays showed that the transgenic plant harboring dsHMGR not only inhibited net weight gain but also delayed the growth of cotton bollworm larvae. Taken together, transgenic cotton plant expressing dsRNAs successfully downregulated HMGR gene and impaired the development and survival of target insect, which provided more option for plant pest control.

  2. Species-specific expansion and molecular evolution of the 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR gene family in plants.

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    Wei Li

    Full Text Available The terpene compounds represent the largest and most diverse class of plant secondary metabolites which are important in plant growth and development. The 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR; EC 1.1.1.34 is one of the key enzymes contributed to terpene biosynthesis. To better understand the basic characteristics and evolutionary history of the HMGR gene family in plants, a genome-wide analysis of HMGR genes from 20 representative species was carried out. A total of 56 HMGR genes in the 14 land plant genomes were identified, but no genes were found in all 6 algal genomes. The gene structure and protein architecture of all plant HMGR genes were highly conserved. The phylogenetic analysis revealed that the plant HMGRs were derived from one ancestor gene and finally developed into four distinct groups, two in the monocot plants and two in dicot plants. Species-specific gene duplications, caused mainly by segmental duplication, led to the limited expansion of HMGR genes in Zea mays, Gossypium raimondii, Populus trichocarpa and Glycine max after the species diverged. The analysis of Ka/Ks ratios and expression profiles indicated that functional divergence after the gene duplications was restricted. The results suggested that the function and evolution of HMGR gene family were dramatically conserved throughout the plant kingdom.

  3. Genome-Wide Identification and Comparative Analysis of the 3-Hydroxy-3-methylglutaryl Coenzyme A Reductase (HMGR Gene Family in Gossypium

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    Wei Liu

    2018-01-01

    Full Text Available Terpenes are the largest and most diverse class of secondary metabolites in plants and play a very important role in plant adaptation to environment. 3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR is a rate-limiting enzyme in the process of terpene biosynthesis in the cytosol. Previous study found the HMGR genes underwent gene expansion in Gossypium raimondii, but the characteristics and evolution of the HMGR gene family in Gossypium genus are unclear. In this study, genome-wide identification and comparative study of HMGR gene family were carried out in three Gossypium species with genome sequences, i.e., G. raimondii, Gossypium arboreum, and Gossypium hirsutum. In total, nine, nine and 18 HMGR genes were identified in G. raimondii, G. arboreum, and G. hirsutum, respectively. The results indicated that the HMGR genes underwent gene expansion and a unique gene cluster containing four HMGR genes was found in all the three Gossypium species. The phylogenetic analysis suggested that the expansion of HMGR genes had occurred in their common ancestor. There was a pseudogene that had a 10-bp deletion resulting in a frameshift mutation and could not be translated into functional proteins in G. arboreum and the A-subgenome of G. hirsutum. The expression profiles of the two pseudogenes showed that they had tissue-specific expression. Additionally, the expression pattern of the pseudogene in the A-subgenome of G. hirsutum was similar to its paralogous gene in the D-subgenome of G. hirsutum. Our results provide useful information for understanding cytosolic terpene biosynthesis in Gossypium species.

  4. Overexpressing 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR in the lactococcal mevalonate pathway for heterologous plant sesquiterpene production.

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    Adelene Ai-Lian Song

    Full Text Available Isoprenoids are a large and diverse group of metabolites with interesting properties such as flavour, fragrance and therapeutic properties. They are produced via two pathways, the mevalonate pathway or the 2-C-methyl-D-erythritol-4-phosphate (MEP pathway. While plants are the richest source of isoprenoids, they are not the most efficient producers. Escherichia coli and yeasts have been extensively studied as heterologous hosts for plant isoprenoids production. In the current study, we describe the usage of the food grade Lactococcus lactis as a potential heterologous host for the production of sesquiterpenes from a local herbaceous Malaysian plant, Persicaria minor (synonym Polygonum minus. A sesquiterpene synthase gene from P. minor was successfully cloned and expressed in L. lactis. The expressed protein was identified to be a β-sesquiphellandrene synthase as it was demonstrated to be functional in producing β-sesquiphellandrene at 85.4% of the total sesquiterpenes produced based on in vitro enzymatic assays. The recombinant L. lactis strain developed in this study was also capable of producing β-sesquiphellandrene in vivo without exogenous substrates supplementation. In addition, overexpression of the strain's endogenous 3-hydroxy-3-methylglutaryl coenzyme-A reductase (HMGR, an established rate-limiting enzyme in the eukaryotic mevalonate pathway, increased the production level of β-sesquiphellandrene by 1.25-1.60 fold. The highest amount achieved was 33 nM at 2 h post-induction.

  5. Effects of HMG-COA Reductase Inhibitor Therapy on LDL Cholesterol Blood Levels in Hyperlipidemia: A Longitudinal Retrospective Anlaysis Using a Department of Defense Integrated Database.

    Science.gov (United States)

    1998-05-21

    Kinlay, and Peter Ganz, Atherogenesis and Ischemic Heart Disease. American Journal of Cardiology 1997; 80(8B):3H-7H. 33. A. L. Lehninger , D. L. Nelson...of its Pharmacology and Therapeutic Potential in the Management of Hyperlipidaemias. Drugs 1997; 53(5):828-847. A. L. Lehninger , D. L. Nelson, and M

  6. Insight into the mechanism of polyphenols on the activity of HMGR by molecular docking.

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    Islam, Barira; Sharma, Charu; Adem, Abdu; Aburawi, Elhadi; Ojha, Shreesh

    2015-01-01

    Statins are hypolipidemic drugs that are effective in the treatment of hypercholesterolemia by attenuating cholesterol synthesis in the liver via competitive inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. Recently, dietary changes associated with drug therapy have garnered attention as novel drugs to mitigate or ameliorate hypercholesterolemia. The present study was undertaken to observe different dietary polyphenols that can bind to the active site of HMGR and inhibit it. Results from the 12 dietary polyphenols tested reveal that polyphenols can bind to HMGR and block the binding of nicotinamide adenine dinucleotide phosphate (NADP(+)). We observed that the rigidity of phenolic rings prevents the polyphenols from docking to the enzyme activity site. The presence of an ester linkage between the phenolic rings in (-)-epigallocatechin-3-gallate (EGCG) and the alkyl chain in curcumin allows them to orient in the active site of the HMGR and bind to the catalytic residues. EGCG and curcumin showed binding to the active site residues with a low GRID score, which may be a potential inhibitor of HMGR. Kaempferol showed binding to HMG-CoA, but with low binding affinity. These observations provide a rationale for the consistent hypolipidemic effect of EGCG and curcumin, which has been previously reported in several epidemiological and animal studies. Therefore, this study substantiates the mechanism of polyphenols on the activity of HMGR by molecular docking and provides the impetus for drug design involving further structure-function relationship studies.

  7. Insight into the mechanism of polyphenols on the activity of HMGR by molecular docking

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    Islam B

    2015-08-01

    Full Text Available Barira Islam,1,* Charu Sharma,2,* Abdu Adem,3 Elhadi Aburawi,1 Shreesh Ojha3 1Department of Paediatrics, 2Department of Internal Medicine, 3Department of Pharmacology and Therapeutics, College of Medicine and Health Sciences, United Arab Emirates University, Al-Ain, Abu Dhabi, United Arab Emirates *These authors contributed equally to this work Abstract: Statins are hypolipidemic drugs that are effective in the treatment of hypercholesterolemia by attenuating cholesterol synthesis in the liver via competitive inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA reductase. Recently, dietary changes associated with drug therapy have garnered attention as novel drugs to mitigate or ameliorate hypercholesterolemia. The present study was undertaken to observe different dietary polyphenols that can bind to the active site of HMGR and inhibit it. Results from the 12 dietary polyphenols tested reveal that polyphenols can bind to HMGR and block the binding of nicotinamide adenine dinucleotide phosphate (NADP+. We observed that the rigidity of phenolic rings prevents the polyphenols from docking to the enzyme activity site. The presence of an ester linkage between the phenolic rings in (–-epigallocatechin-3-gallate (EGCG and the alkyl chain in curcumin allows them to orient in the active site of the HMGR and bind to the catalytic residues. EGCG and curcumin showed binding to the active site residues with a low GRID score, which may be a potential inhibitor of HMGR. Kaempferol showed binding to HMG-CoA, but with low binding affinity. These observations provide a rationale for the consistent hypolipidemic effect of EGCG and curcumin, which has been previously reported in several epidemiological and animal studies. Therefore, this study substantiates the mechanism of polyphenols on the activity of HMGR by molecular docking and provides the impetus for drug design involving further structure–function relationship studies. Keywords: polyphenols

  8. The GATA and SORLIP motifs in the 3-hydroxy-3-methylglutaryl-CoA reductase promoter of Picrorhiza kurrooa for the control of light-mediated expression.

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    Kawoosa, Tabasum; Gahlan, Parul; Devi, Aribam Surbala; Kumar, Sanjay

    2014-03-01

    Light upregulates the expression of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) in Picrorhiza kurrooa, an endangered medicinal herb. Upstream sequences of HMGR of P. kurrooa (PropkHMGR) were analyzed in relation to its role in light-mediated regulation of gene expression. GATA motif in PropkHMGR exhibited stronger DNA-protein interaction with the nuclear extract of dark-exposed plants in contrast to SORLIP that exhibited stronger binding with the nuclear extract of light-exposed plants. Analysis of PropkHMGR (PropkHMGR-D1, -1,059/-1) and its deletion fragments PropkHMGR-D2 (-825/-1), PropkHMGR-D3 (-651/-1), PropkHMGR-D4 (-452/-1), and PropkHMGR-D5 (-101/-1) in Arabidopsis thaliana showed PropkHMGR to regulate gene expression [β-glucuronidase (GUS) was used as a reporter gene] at all the developmental stages but only in actively dividing tissues, excluding anthers. Whereas, PropkHMGR-D2 regulated GUS expression in relatively older seedlings but the expression was observed only in shoot apical meristem, root tips, and anthers. PropkHMGR-mediated gene expression was higher in dark as compared to that in the light in Arabidopsis across four temperatures studied. As opposed to the results in P. kurrooa, GATA motifs exhibited DNA-protein interaction with nuclear extract of light-exposed plants of Arabidopsis. SORLIP motifs in Arabidopsis also exhibited DNA-protein interaction with nuclear extract of light-exposed plants as in P. kurrooa. Data showed that (1) PropkHMGR regulated light-mediated gene expression and (2) GATA motif exhibited an inverse relationship between strength of DNA-protein interaction and the gene expression whereas the relationship was species specific for SORLIP.

  9. Hypercholesterolemia and 3-Hydroxy 3-Methylglutaryl Coenzyme A Reductase Regulation during Ageing

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    Laura Trapani

    2009-01-01

    Full Text Available We present here a brief description of the path that cholesterol covers from its intestinal absorption to its effect exerted on some enzyme regulation. Some mechanisms underlying hypercholesterolemia onset and, in particular, the role and the regulation of 3-hydroxy 3-methylglutaryl Coenzyme A reductase (HMGR during adult life and during aging, have been described. In addition some pharmacological interventions to control proper HMGR regulation and, in turn, cholesterol homeostasis maintenance will be introduced.

  10. Molecular cloning, characterization and expression analysis of 3-hydroxy-3-methylglutaryl coenzyme A reductase gene from Centella asiatica L.

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    Kalita, Ratna; Patar, Lochana; Shasany, Ajit Kumar; Modi, Mahendra K; Sen, Priyabrata

    2015-09-01

    3-Hydroxy-3-methylglutaryl-CoA reductases (HMGR) plays an important role in catalyzing the first committed step of isoprenoid biosynthesis in the mevelonic (MVA) pathway (catalyzes the conversion of HMG-CoA to MVA) in plants. The present manuscript reports the full length cDNA cloning of HMGR (CaHMGR, GenBank accession number: KJ939450.2) and its characterization from Centella asiatica. Sequence analysis indicated that the cDNA was of 1965 bp, which had an open reading frame of 1617 bp and encoded a protein containing 539 amino-acids with a mol wt of 57.9 kDa. A BLASTp search against non-redundant (nr) protein sequence showed that C. asiatica HMGR (CaHMGR) has 65-81% identity with HMGRs from different plant species and multi-alignment comparison analysis showed the presence of two motif each corresponding to HMG-CoA-binding and NADP(H)-binding. The Conserved Domain Database analysis predicted that CaHMGR belongs to Class I hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase. Three-dimensional modeling confirmed the novelty of CaHMGR with a spatial structure similar to Homo sapiens (PDB id: 1IDQ8_A). Tissue Expression analysis indicates that CaHMGR is ubiquitous albeit differentially expressed among different tissues analysed, Strong expression was recorded in the nodes and leaves and low in the roots. The present investigation confirmed that nodes are vital to terpenoid synthesis in C. asiatica. Thus, the cloning of full length CDS, characterization and structure-function analysis of HMGR gene in Centella facilitate to understand the HMGR's functions and regulatory mechanisms involved in mevalonate pathway in C. asiatica at genetic level.

  11. Involvement of tristetraprolin in transcriptional activation of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase by insulin

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    Ness, Gene C., E-mail: gness@hsc.usf.edu [Department of Molecular Medicine, College of Medicine, University of South Florida, Tampa, FL 33612 (United States); Edelman, Jeffrey L.; Brooks, Patricia A. [Department of Molecular Medicine, College of Medicine, University of South Florida, Tampa, FL 33612 (United States)

    2012-03-30

    Highlights: Black-Right-Pointing-Pointer siRNAs to tristetraprolin blocks transcription of HMGR in vivo in rat liver. Black-Right-Pointing-Pointer siRNAs to tristetraprolin inhibits insulin activation of HMGR transcription. Black-Right-Pointing-Pointer Insulin acts to rapidly increase tristetraprolin in liver nuclear extracts. -- Abstract: Several AU-rich RNA binding element (ARE) proteins were investigated for their possible effects on transcription of hepatic 3-hydroxy-3-methyglutaryl coenzyme A reductase (HMGR) in normal rats. Using in vivo electroporation, four different siRNAs to each ARE protein were introduced together with HMGR promoter (-325 to +20) luciferase construct and compared to saline controls. All four siRNAs to tristetraprolin (TTP) completely eliminated transcription from the HMGR promoter construct. Since insulin acts to rapidly increase hepatic HMGR transcription, the effect of TTP siRNA on induction by insulin was tested. The 3-fold stimulation by insulin was eliminated by this treatment. In comparison, siRNA to AU RNA binding protein/enoyl coenzyme A hydratase (AUH) had no effect. These findings indicate a role for TTP in the insulin-mediated activation of hepatic HMGR transcription.

  12. Mitochondrial localization of the mevalonate pathway enzyme 3-Hydroxy-3-methyl-glutaryl-CoA reductase in the Trypanosomatidae

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    Pena Diaz, Javier; Montalvetti, Andrea; Flores, Carmen-Lisset

    2004-01-01

    3-Hydroxy-3-methyl-glutaryl-CoA reductase (HMGR) is a key enzyme in the sterol biosynthesis pathway, but its subcellular distribution in the Trypanosomatidae family is somewhat controversial. Trypanosoma cruzi and Leishmania HMGRs are closely related in their catalytic domains to bacterial...... obtained with wild-type cells and transfectants overexpressing the enzyme established that HMGR in both T. cruzi and Leishmania major is localized primarily in the mitochondrion and that elimination of the mitochondrial targeting sequence in Leishmania leads to protein accumulation in the cytosolic...... compartment. Furthermore, T. cruzi HMGR is efficiently targeted to the mitochondrion in yeast cells. Thus, when the gene encoding T. cruzi HMGR was expressed in a hmg1 hmg2 mutant of Saccharomyces cerevisiae, the mevalonate auxotrophy of mutant cells was relieved, and immunoelectron analysis showed...

  13. Kinetic properties and inhibition of Trypanosoma cruzi 3-hydroxy-3-methylglutaryl CoA reductase

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    Hurtado-Guerrrero, Ramón; Pena Diaz, Javier; Montalvetti, Andrea

    2002-01-01

    A detailed kinetic analysis of the recombinant soluble enzyme 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) from Trypanosoma cruzi has been performed. The enzyme catalyzes the normal anabolic reaction and the reductant is NADPH. It also catalyzes the oxidation of mevalonate but at a lower...

  14. Proliferation and Morphogenesis of the Endoplasmic Reticulum Driven by the Membrane Domain of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase in Plant Cells.

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    Ferrero, Sergi; Grados-Torrez, Ricardo Enrique; Leivar, Pablo; Antolín-Llovera, Meritxell; López-Iglesias, Carmen; Cortadellas, Nuria; Ferrer, Joan Carles; Campos, Narciso

    2015-07-01

    The enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) has a key regulatory role in the mevalonate pathway for isoprenoid biosynthesis and is composed of an endoplasmic reticulum (ER)-anchoring membrane domain with low sequence similarity among eukaryotic kingdoms and a conserved cytosolic catalytic domain. Organized smooth endoplasmic reticulum (OSER) structures are common formations of hypertrophied tightly packed ER membranes devoted to specific biosynthetic and secretory functions, the biogenesis of which remains largely unexplored. We show that the membrane domain of plant HMGR suffices to trigger ER proliferation and OSER biogenesis. The proliferating membranes become highly enriched in HMGR protein, but they do not accumulate sterols, indicating a morphogenetic rather than a metabolic role for HMGR. The N-terminal MDVRRRPP motif present in most plant HMGR isoforms is not required for retention in the ER, which was previously proposed, but functions as an ER morphogenic signal. Plant OSER structures are morphologically similar to those of animal cells, emerge from tripartite ER junctions, and mainly build up beside the nuclear envelope, indicating conserved OSER biogenesis in high eukaryotes. Factors other than the OSER-inducing HMGR construct mediate the tight apposition of the proliferating membranes, implying separate ER proliferation and membrane association steps. Overexpression of the membrane domain of Arabidopsis (Arabidopsis thaliana) HMGR leads to ER hypertrophy in every tested cell type and plant species, whereas the knockout of the HMG1 gene from Arabidopsis, encoding its major HMGR isoform, causes ER aggregation at the nuclear envelope. Our results show that the membrane domain of HMGR contributes to ER morphogenesis in plant cells. © 2015 American Society of Plant Biologists. All Rights Reserved.

  15. Methyl farnesoate synthesis in the lobster mandibular organ: The roles of HMG-CoA reductase and farnesoic acid-O-methyltransferase

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    Li, Sheng; Friesen, Jon A.; Holford, Kenneth C.; Borst, David W.

    2009-01-01

    Eyestalk ablation (ESA) increases crustacean production of methyl farnesoate (MF), a juvenile hormone-like compound, but the biochemical steps involved are not completely understood. We measured the activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) and farnesoic acid-O-methyl transferase (FAOMeT), an early step and the last step in MF synthesis. ESA elevated hemolymph levels of MF in male lobsters. Enzyme activity suggested that increased MF production on day one was due largely to elevated HMGR activity while changes in FAOMeT activity closely paralleled changes in MF levels on day 14. Transcript levels for HMGR and FAOMeT changed little on day one, but both increased substantially on day 14. We treated ESA males with a partially purified mandibular organ inhibiting hormone (MOIH) and observed a significant decline in MF levels, FAOMeT activity, and FAOMeT-mRNA levels after 5 hours. However, no effect was observed on HMGR activity or its mRNA indicating that they must be regulated by a separate sinus gland peptide. We confirmed that lobster HMGR was not a phosphoprotein and was not regulated by reversible phosphorylation, an important mechanism for regulating other HMGRs. Nevertheless, molecular modeling indicated that the catalytic mechanisms of lobster and mammalian HMGR were similar. PMID:19778626

  16. Enhancing production of ergosterol in Pichia pastoris GS115 by over-expression of 3-hydroxy-3-methylglutaryl CoA reductase from Glycyrrhiza uralensis.

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    Liu, Ying; Zhu, Xiaoqing; Li, Wendong; Wen, Hao; Gao, Ya; Liu, Yong; Liu, Chunsheng

    2014-04-01

    The rate-limiting enzyme in the mevalonic acid (MVA) pathway which can lead to triterpenoid saponin glycyrrhizic acid (GA) is 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR). In order to reveal the effect of copy number variation in the HMGR gene on the MVA pathway, the HMGR gene from Glycyrrhiza uralensis Fisch. (GuHMGR) was cloned and over-expressed in Pichia pastoris GS115. Six recombinant P. pastoris strains containing different copy numbers of the GuHMGR gene were obtained and the content of ergosterol was analyzed by HPLC. The results showed that all the recombinant P. pastoris strains contained more ergosterol than the negative control and the strains with 8 and 44 copies contained significantly more ergosterol than the other strains. However, as the copy number increased, the content of ergosterol showed an increasing-decreasing-increasing pattern. This study provides a rationale for increasing the content of GA through over-expressing the GuHMGR gene in cultivars of G. uralensis.

  17. Thermodynamic and Structure Guided Design of Statin Based Inhibitors of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase

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    Sarver, Ronald W.; Bills, Elizabeth; Bolton, Gary; Bratton, Larry D.; Caspers, Nicole L.; Dunbar, James B.; Harris, Melissa S.; Hutchings, Richard H.; Kennedy, Robert M.; Larsen, Scott D.; Pavlovsky, Alexander; Pfefferkorn, Jeffrey A.; Bainbridge, Graeme (Pfizer)

    2008-10-02

    Clinical studies have demonstrated that statins, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) inhibitors, are effective at lowering mortality levels associated with cardiovascular disease; however, 2--7% of patients may experience statin-induced myalgia that limits compliance with a treatment regimen. High resolution crystal structures, thermodynamic binding parameters, and biochemical data were used to design statin inhibitors with improved HMGR affinity and therapeutic index relative to statin-induced myalgia. These studies facilitated the identification of imidazole 1 as a potent (IC{sub 50} = 7.9 nM) inhibitor with excellent hepatoselectivity (>1000-fold) and good in vivo efficacy. The binding of 1 to HMGR was found to be enthalpically driven with a {Delta}H of -17.7 kcal/M. Additionally, a second novel series of bicyclic pyrrole-based inhibitors was identified that induced order in a protein flap of HMGR. Similar ordering was detected in a substrate complex, but has not been reported in previous statin inhibitor complexes with HMGR.

  18. Exploration of natural product ingredients as inhibitors of human HMG-CoA reductase through structure-based virtual screening.

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    Lin, Shih-Hung; Huang, Kao-Jean; Weng, Ching-Feng; Shiuan, David

    2015-01-01

    Cholesterol plays an important role in living cells. However, a very high level of cholesterol may lead to atherosclerosis. HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase is the key enzyme in the cholesterol biosynthesis pathway, and the statin-like drugs are inhibitors of human HMG-CoA reductase (hHMGR). The present study aimed to virtually screen for potential hHMGR inhibitors from natural product to discover hypolipidemic drug candidates with fewer side effects and lesser toxicities. We used the 3D structure 1HWK from the PDB (Protein Data Bank) database of hHMGR as the target to screen for the strongly bound compounds from the traditional Chinese medicine database. Many interesting molecules including polyphenolic compounds, polisubstituted heterocyclics, and linear lipophilic alcohols were identified and their ADMET (absorption, disrtibution, metabolism, excretion, toxicity) properties were predicted. Finally, four compounds were obtained for the in vitro validation experiments. The results indicated that curcumin and salvianolic acid C can effectively inhibit hHMGR, with IC50 (half maximal inhibitory concentration) values of 4.3 µM and 8 µM, respectively. The present study also demonstrated the feasibility of discovering new drug candidates through structure-based virtual screening.

  19. The 3-hydroxy-3-methylglutaryl coenzyme-A reductases from fungi: a proposal as a therapeutic target and as a study model.

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    Andrade-Pavón, Dulce; Sánchez-Sandoval, Eugenia; Rosales-Acosta, Blanca; Ibarra, José Antonio; Tamariz, Joaquín; Hernández-Rodríguez, César; Villa-Tanaca, Lourdes

    2014-01-01

    The enzyme 3-hydroxy-3-methylglutaryl coenzyme-A reductase (HMGR) catalyzes the conversion of HMG-Co-A into mevalonate. This step is the limiting point for the synthesis of cholesterol in mammals and ergosterol in fungi. We describe in this article the genome organization of HMGR coding genes and those deduced from different fungi, recount the evidence showing statins as HMGR inhibitors for ergosterol synthesis and its effect in yeast viability, and propose fungal HMGR (HMGRf) as a model to study the use of pharmaceutical compounds to inhibit cholesterol and ergosterol synthesis. Bibliographical search and bioinformatic analyses were performed and discussed. HMGRfs belong to the class I with a high homology in the catalytic region. The sterol biosynthetic pathway in humans and fungi share many enzymes in the initial steps (such as the HMGR enzyme), but in the last steps enzymes are different rendering the two final products: cholesterol in mammals and ergosterol in fungi. With regards to inhibitors such as statins and other compounds, these affect also fungal viability. Since HMGR from Schizosaccharomyces pombe and Ustilago maydis are very similar to the human HMGR in the catalytic regions, we propose that fungal enzymes can be used to test inhibitors for a potential use in humans. We consider that HMGRf is a good therapeutic target to design and test new antifungal compounds. This manuscript is part of the series of works presented at the "V International Workshop: Molecular genetic approaches to the study of human pathogenic fungi" (Oaxaca, Mexico, 2012). Copyright © 2013 Revista Iberoamericana de Micología. Published by Elsevier Espana. All rights reserved.

  20. In vivo identification of promoter elements and transcription factors mediating activation of hepatic HMG-CoA reductase by T{sub 3}

    Energy Technology Data Exchange (ETDEWEB)

    Boone, Lindsey R.; Niesen, Melissa I. [Department of Molecular Medicine, College of Medicine, University of South Florida, Tampa, FL (United States); Jaroszeski, Mark [Department of Chemical and Biomedical Engineering, College of Engineering, University of South Florida, Tampa, FL (United States); Ness, Gene C., E-mail: gness@hsc.usf.edu [Department of Molecular Medicine, College of Medicine, University of South Florida, Tampa, FL (United States)

    2009-07-31

    The promoter elements and transcription factors necessary for triiodothyronine (T{sub 3}) induction of hepatic HMG-CoA reductase (HMGR) were investigated by transfecting rat livers with wild type and mutant HMGR promoter-luciferase constructs using in vivo electroporation. Mutations in the sterol response element (SRE), nuclear factor-y (NF-Y) site, and the newly identified upstream transcription factor-2 (USF-2) site essentially abolished the T{sub 3} response. Chromatin immunoprecipitation (ChIP) analysis demonstrated that T{sub 3} treatment caused a 4-fold increase in in vivo binding of USF-2 to the HMGR promoter. Co-transfection of the wild type HMGR promoter with siRNAs to USF-2, SREBP-2, or NF-Y nearly abolished the T{sub 3} induction, as measured by promoter activity. These data provide in vivo evidence for functional roles for USF-2, SREBP-2, and NF-Y in mediating the T{sub 3}-induction of hepatic HMGR transcription.

  1. Discovery and quantitative structure-activity relationship study of lepidopteran HMG-CoA reductase inhibitors as selective insecticides.

    Science.gov (United States)

    Zang, Yang-Yang; Li, Yuan-Mei; Yin, Yue; Chen, Shan-Shan; Kai, Zhen-Peng

    2017-09-01

    In a previous study we have demonstrated that insect 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) can be a potential selective insecticide target. Three series of inhibitors were designed on the basis of the difference in HMGR structures from Homo sapiens and Manduca sexta, with the aim of discovering potent selective insecticide candidates. An in vitro bioassay showed that gem-difluoromethylenated statin analogues have potent effects on JH biosynthesis of M. sexta and high selectivity between H. sapiens and M. sexta. All series II compounds {1,3,5-trisubstituted [4-tert-butyl 2-(5,5-difluoro-2,2-dimethyl-6-vinyl-4-yl) acetate] pyrazoles} have some effect on JH biosynthesis, whereas most of them are inactive on human HMGR. In particular, the IC50 value of compound II-12 (37.8 nm) is lower than that of lovastatin (99.5 nm) and similar to that of rosuvastatin (24.2 nm). An in vivo bioassay showed that I-1, I-2, I-3 and II-12 are potential selective insecticides, especially for lepidopteran pest control. A predictable and statistically meaningful CoMFA model of 23 inhibitors (20 as training sets and three as test sets) was obtained with a value of q2 and r2 of 0.66 and 0.996 respectively. The final model suggested that a potent insect HMGR inhibitor should contain suitable small and non-electronegative groups in the ring part, and electronegative groups in the side chain. Four analogues were discovered as potent selective lepidopteran HMGR inhibitors, which can specifically be used for lepidopteran pest control. The CoMFA model will be useful for the design of new selective insect HMGR inhibitors that are structurally related to the training set compounds. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  2. Differential induction and suppression of potato 3-hydroxy-3-methylglutaryl coenzyme A reductase genes in response to Phytophthora infestans and to its elicitor arachidonic acid.

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    Choi, D; Ward, B L; Bostock, R M

    1992-10-01

    Induction of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is essential for the biosynthesis of sesquiterpenoid phytoalexins and steroid derivatives in Solanaceous plants following stresses imposed by wounding and pathogen infection. To better understand this complex step in stress-responsive isoprenoid synthesis, we isolated three classes of cDNAS encoding HMGR (hmg1, hmg2, and hmg3) from a potato tuber library using a probe derived from an Arabidopsis HMGR cDNA. The potato cDNAs had extensive homology in portions of the protein coding regions but had low homology in the 3' untranslated regions. RNA gel blot analyses using gene-specific probes showed that hmg1 was strongly induced in tuber tissue by wounding, but the wound induction was strongly suppressed by treatment of the tissue with the fungal elicitor arachidonic acid or by inoculation with an incompatible or compatible race of the fungal pathogen Phytophtora infestans. The hmg2 and hmg3 mRNAs also accumulated in response to wounding, but in contrast to hmg1, these mRNAs were strongly enhanced by arachidonic acid or inoculation. Inoculation with a compatible race of P. infestans resulted in similar patterns in HMGR gene expression of hmg2 and hmg3 except that the magnitude and rate of the changes in mRNA levels were reduced relative to the incompatible interaction. The differential regulation of members of the HMGR gene family may explain in part the previously reported changes in HMGR enzyme activities following wounding and elicitor treatment. The suppression of hmg1 and the enhancement of hmg2 and hmg3 transcript levels following elicitor treatment or inoculation with the incompatible race parallel the suppression in steroid and stimulation of sesquiterpenoid accumulations observed in earlier investigations. The results are discussed in relation to the hypothesis that there are discrete organizational channels for sterol and sesquiterpene biosynthesis in potato and other Solanaceous species.

  3. Exploration of natural product ingredients as inhibitors of human HMG-CoA reductase through structure-based virtual screening

    Directory of Open Access Journals (Sweden)

    Lin SH

    2015-06-01

    Full Text Available Shih-Hung Lin,1 Kao-Jean Huang,1,2 Ching-Feng Weng,1 David Shiuan1 1Department of Life Science and Institute of Biotechnology, National Dong Hwa University, Hualien, Taiwan, Republic of China; 2Development Center of Biotechnology, Taipei, Taiwan, Republic of China Abstract: Cholesterol plays an important role in living cells. However, a very high level of cholesterol may lead to atherosclerosis. HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A reductase is the key enzyme in the cholesterol biosynthesis pathway, and the statin-like drugs are inhibitors of human HMG-CoA reductase (hHMGR. The present study aimed to virtually screen for potential hHMGR inhibitors from natural product to discover hypolipidemic drug candidates with fewer side effects and lesser toxicities. We used the 3D structure 1HWK from the PDB (Protein Data Bank database of hHMGR as the target to screen for the strongly bound compounds from the traditional Chinese medicine database. Many interesting molecules including polyphenolic compounds, polisubstituted heterocyclics, and linear lipophilic alcohols were identified and their ADMET (absorption, disrtibution, metabolism, excretion, toxicity properties were predicted. Finally, four compounds were obtained for the in vitro validation experiments. The results indicated that curcumin and salvianolic acid C can effectively inhibit hHMGR, with IC50 (half maximal inhibitory concentration values of 4.3 µM and 8 µM, respectively. The present study also demonstrated the feasibility of discovering new drug candidates through structure-based virtual screening. Keywords: HMG-CoA reductase, virtual screening, curcumin, salvianolic acid C

  4. 3-Hydroxy-3-methylglutaryl-coenzyme A reductase kinase and sucrose-phosphate synthase kinase activities in cauliflower florets: Ca2+ dependence and substrate specificities.

    Science.gov (United States)

    Toroser, D; Huber, S C

    1998-07-15

    Plant 3-hydroxy-3-methylglutaryl-CoA reductase(HMGR; EC 1.1.1.34) and sucrose-phosphate synthase (SPS; EC 2.4.1.14) and synthetic peptides designed from the known phosphorylation sites of plant HMGR (SAMS*: KSHMKYNRSTKDVK), rat acetyl-CoA carboxylase (SAMS: HMRSAMSGLHLVKRR), spinach SPS (SP2: GRRJRRISSVEJJDKK), and spinach NADH:nitrate reductase (NR6: GPTLKRTASTPFJNTTSK) were used to characterize kinase activities from cauliflower (Brassica oleracea L. ) inflorescences. The three major peaks of protein kinase activity resolved by anion-exchange FPLC are homologs of those observed previously in spinach leaves and thus are designated PKI, PKIV, and PKIII, listed in order of elution. PKIV was the most active in terms of phosphorylation and inactivation of recombinant Nicotiana HMGR and was also strictly Ca2+ dependent. The novel aspects are that PKIII has not been detected in previous cauliflower studies, that SAMS* is a more specific peptide substrate to identify potential HMGR kinases, and that the major HMGR kinase in cauliflower is Ca2+ dependent. Of the three major kinases that phosphorylated the SP2 peptide only PKI (partially Ca2+ sensitive) and PKIII (Ca2+ insensitive) inactivated native spinach leaf SPS. Cauliflower extracts contained endogenous SPS that was inactivated by endogenous kinase(s) in an ATP-dependent manner and this may be one of the substrate target proteins for PKI and/or PKIII. The substrate specificity of the three kinase peaks was studied using synthetic peptide variants of the SP2 sequence. All three kinases had a strong preference for peptides with a basic residue at P-6 (as in SP2 and SAMS*; SAMS has a free amino terminus at this position) or a Pro at P-7 (as in NR6). This requirement for certain residues at P-6 or P-7 was not recognized in earlier studies but appears to be a general requirement. In plant HMGR, a conserved His residue at P-6 is involved directly in catalysis and this may explain why substrates reduced HMGR phosphorylation

  5. methylglutaryl coenzyme A reductase gene from Jatr

    African Journals Online (AJOL)

    STORAGESEVER

    2009-08-04

    Aug 4, 2009 ... house at 27±1°C with 16 h light and 8 h dark photoperiod. Leaves were collected .... swiss-Model). The phylogenetic analysis of JcHMGR and HMGR from other species was aligned with CLUSTAL W (1.82) using de- fault parameters. ..... NADP(H)-binding motifs (GTVGGGT) whose architecture resembled a ...

  6. Bioactivity guided fractionation and hypolipidemic property of a novel HMG-CoA reductase inhibitor from Ficus virens Ait.

    Science.gov (United States)

    Iqbal, Danish; Khan, M Salman; Khan, Mohd Sajid; Ahmad, Saheem; Hussain, Md Sarfaraj; Ali, Mohd

    2015-03-04

    The current perspective for the search of 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitor has been shifted towards a natural agent also having antioxidant property. Thus, this study was intended to isolate and identify the bioactive compounds from methanolic extract of Ficus virens bark (FVBM) and to evaluate their antioxidant, HMG-CoA reductase inhibitory and hypolipidemic activity. Bioactivity guided fractionation and isolation of bioactive compound from FVBM extract has been done to isolate and characterize the potent HMG-CoA reductase (HMGR) inhibitor with antioxidant activity by using repeated extensive column chromatography followed by spectroscopic methods, including Infrared (IR), 1H & 13C nuclear magnetic resonance (NMR) and Mass spectrum analysis. The in vitro HMGR inhibition and enzyme kinetic assay was determined using HMG-CoA as substrate. In addition, antioxidant activity of the new isolated compound, was measured using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay and FRAP value. In-silico molecular informatics of HMGR enzyme type inhibition and pharmacokinetics data of the new compound was further evaluated through molecular docking and ADME-T studies. Further, in-vivo hypolipidemic property of FVBM extract and newly isolated compound was also analyzed in triton-WR 1339 induced rats. Thereby, we report the discovery of n-Octadecanyl-O-α-D-glucopyranosyl(6'→1″)-O-α-D-glucopyranoside (F18) as a novel HMG-CoA reductase inhibitor with strong antioxidant property. This inhibitor exhibited not only higher free radical scavenging activity but also marked HMG-CoA reductase inhibitory activity with an IC50 value of 84±2.8 ng/ml. This inhibitory activity concurred with kinetic study that showed inhibition constant (K i) of 84 ng/ml via an uncompetitive mode of inhibition. The inhibition was also corroborated by molecular docking analysis and in silico pharmacokinetics data. The in vivo study revealed that

  7. Arachidonic acid alters tomato HMG expression and fruit growth and induces 3-hydroxy-3-methylglutaryl coenzyme A reductase-independent lycopene accumulation

    Energy Technology Data Exchange (ETDEWEB)

    Rodriguez-Concepcion, M.; Gruissem, W. [Univ. of California, Berkeley, CA (United States). Dept. of Plant and Microbial Biology

    1999-01-01

    Regulation of isoprenoid end-product synthesis required for normal growth and development in plants is not well understood. To investigate the extent to which specific genes for the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) are involved in end-product regulation, the authors manipulated expression of the HMG1 and HMG2 genes in tomato (Lycopersicon esculentum) fruit using arachidonic acid (AA). In developing young fruit AA blocked fruit growth, inhibited HMG1, and activated HMG2 expression. These results are consistent with other reports indicating that HMG1 expression is closely correlated with growth processes requiring phytosterol production. In mature-green fruit AA strongly induced the expression of HMG2, PSY1 (the gene for phytoene synthase), and lycopene accumulation before the normal onset of carotenoid synthesis and ripening. The induction of lycopene synthesis was not blocked by inhibition of HMGR activity using mevinolin, suggesting that cytoplasmic HMGR is not required for carotenoid synthesis. Their results are consistent with the function of an alternative plastid isoprenoid pathway (the Rohmer pathway) that appears to direct the production of carotenoids during tomato fruit ripening.

  8. Identification of HMG-CoA Reductase Inhibitor Active Compound in Medicinal Forest Plants

    Directory of Open Access Journals (Sweden)

    Shelly Rahmania

    2017-08-01

    Full Text Available Cardiovascular disease is a leading cause of death worldwide, hypercholesterolemia is one of the causes. Three medicinal forest plants are potential natural resources to be developed as cholesterol-reducing herbal product, but scientific informations on their mechanism is still limited. The objective of this research is to explore the potency of the leaf of Jati Belanda (Guazuma ulmifolia, Jabon (Antocephalus macrophyllus, and Mindi (Melia azedarach as inhibitor of HMG-CoA reductase (HMGR, a key enzyme in the regulation of cholesterol biosynthesis. Samples were macerated in ethanol 96% and the filtrate was partitioned using n-hexane and chloroform to obtain the ethanolic flavonoid extract. The effect of each extracts on the HMG-CoA reductase activity were analyzed using HMGR assay kit. At concentration of 10 ppm the G.ulmifolia ethanolic extract showed the highest inhibitory activity as well as pravastatin control inhibitor.  The phenolic content of the ethanolic extracts of G.ulmifolia, A.macrophyllus, and M.azedarach were: 11.00, 34.83, and 13.67 mg gallic acid AE/g dried leaves, respectively. The flavonoid content of the ethanolic extracts of G.ulmifolia, A.macrophyllus, and M.azedarach were: 0.22, 0.64, and 0.78 mg QE/g dried leaves, respectively. Interestingly, G.ulmifolia extract the lowest concentration of phenolic and flavonoid content. HPLC analysis showed that all samples contain quercetin at similiar small concentrations (6.7%, 6.6%, and 7.0% for G.ulmifolia, A.macrophyllus, and M.azedarach, respectively. This indicating other active compounds may play some roles in this inhibitory action on HMG-CoA reductase activity. Further identification using LC-MS/MS showed that G.ulmifolia flavonoid extract contained an unidetified coumpound with molecural weight of 380.0723 Da.  

  9. Quinone Reductase 2 Is a Catechol Quinone Reductase

    Energy Technology Data Exchange (ETDEWEB)

    Fu, Yue; Buryanovskyy, Leonid; Zhang, Zhongtao (NYMEDCO)

    2008-09-05

    The functions of quinone reductase 2 have eluded researchers for decades even though a genetic polymorphism is associated with various neurological disorders. Employing enzymatic studies using adrenochrome as a substrate, we show that quinone reductase 2 is specific for the reduction of adrenochrome, whereas quinone reductase 1 shows no activity. We also solved the crystal structure of quinone reductase 2 in complexes with dopamine and adrenochrome, two compounds that are structurally related to catecholamine quinones. Detailed structural analyses delineate the mechanism of quinone reductase 2 specificity toward catechol quinones in comparison with quinone reductase 1; a side-chain rotational difference between quinone reductase 1 and quinone reductase 2 of a single residue, phenylalanine 106, determines the specificity of enzymatic activities. These results infer functional differences between two homologous enzymes and indicate that quinone reductase 2 could play important roles in the regulation of catecholamine oxidation processes that may be involved in the etiology of Parkinson disease.

  10. Variability in statin-induced changes in gene expression profiles of pancreatic cancer

    Czech Academy of Sciences Publication Activity Database

    Gbelcová, H.; Rimpelová, S.; Ruml, T.; Fenclova, M.; Kosek, V.; Hajslova, J.; Strnad, Hynek; Kolář, Michal; Vítek, L.

    2017-01-01

    Roč. 7 (2017), č. článku 44219. ISSN 2045-2322 R&D Projects: GA MŠk(CZ) LO1304 Institutional support: RVO:68378050 Keywords : hmg-coa reductase * lipid droplets * rho-gtpases * cell-death * in-vitro * risk * protein * association * simvastatin * apoptosis Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.259, year: 2016

  11. Evidence that biliverdin-IX beta reductase and flavin reductase are identical.

    OpenAIRE

    Shalloe, F; Elliott, G; Ennis, O; Mantle, T J

    1996-01-01

    A search of the database shows that human biliverdin-IX beta reductase and flavin reductase are identical. We have isolated flavin reductase from bovine erythrocytes and show that the activity co-elutes with biliverdin-IX beta reductase. Preparations of the enzyme that are electrophoretically homogeneous exhibit both flavin reductase and biliverdin-IX beta reductase activities; however, they are not capable of catalysing the reduction of biliverdin-IX alpha. Although there is little obvious s...

  12. Pseudoazurin-nitrite reductase interactions.

    Science.gov (United States)

    Impagliazzo, Antonietta; Krippahl, Ludwig; Ubbink, Marcellus

    2005-09-01

    The nitrite reductase-binding site on pseudoazurin has been determined by using NMR chemical-shift perturbations. It comprises residues in the hydrophobic patch surrounding the exposed copper ligand His81 as well as several positively charged residues. The binding site is similar for both redox states of pseudoazurin, despite differences in the binding mode. The results suggest that pseudoazurin binds in a well-defined orientation. Docking simulations provide a putative structure of the complex with a binding site on nitrite reductase that has several hydrophobic and polar residues as well as a ridge of negatively charged side chains and a copper-to-copper distance of 14 A.

  13. 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 ...

  14. The binding sites on human heme oxygenase-1 for cytochrome p450 reductase and biliverdin reductase.

    Science.gov (United States)

    Wang, Jinling; de Montellano, Paul R Ortiz

    2003-05-30

    Human heme oxygenase-1 (hHO-1) catalyzes the NADPH-cytochrome P450 reductase-dependent oxidation of heme to biliverdin, CO, and free iron. The biliverdin is subsequently reduced to bilirubin by biliverdin reductase. Earlier kinetic studies suggested that biliverdin reductase facilitates the release of biliverdin from hHO-1 (Liu, Y., and Ortiz de Montellano, P. R. (2000) J. Biol. Chem. 275, 5297-5307). We have investigated the binding of P450 reductase and biliverdin reductase to truncated, soluble hHO-1 by fluorescence resonance energy transfer and site-specific mutagenesis. P450 reductase and biliverdin reductase bind to truncated hHO-1 with Kd = 0.4 +/- 0.1 and 0.2 +/- 0.1 microm, respectively. FRET experiments indicate that biliverdin reductase and P450 reductase compete for binding to truncated hHO-1. Mutation of surface ionic residues shows that hHO-1 residues Lys18, Lys22, Lys179, Arg183, Arg198, Glu19, Glu127, and Glu190 contribute to the binding of cytochrome P450 reductase. The mutagenesis results and a computational analysis of the protein surfaces partially define the binding site for P450 reductase. An overlapping binding site including Lys18, Lys22, Lys179, Arg183, and Arg185 is similarly defined for biliverdin reductase. These results confirm the binding of biliverdin reductase to hHO-1 and define binding sites of the two reductases.

  15. Evidence that biliverdin-IX beta reductase and flavin reductase are identical.

    Science.gov (United States)

    Shalloe, F; Elliott, G; Ennis, O; Mantle, T J

    1996-01-01

    A search of the database shows that human biliverdin-IX beta reductase and flavin reductase are identical. We have isolated flavin reductase from bovine erythrocytes and show that the activity co-elutes with biliverdin-IX beta reductase. Preparations of the enzyme that are electrophoretically homogeneous exhibit both flavin reductase and biliverdin-IX beta reductase activities; however, they are not capable of catalysing the reduction of biliverdin-IX alpha. Although there is little obvious sequence identity between biliverdin-IX alpha reductase (BVR-A) and biliverdin-IX beta reductase (BVR-B), they do show weak immunological cross-reactivity. Both enzymes bind to 2',5'-ADP-Sepharose. PMID:8687377

  16. [Aldehyde reductase activity and blood aldo-keto reductase spectrum in adolescents with neuroendocrine obesity].

    Science.gov (United States)

    Kuleshova, D K; Davydov, V V; Shvets, V N

    2012-01-01

    Investigation of aldehyde-reductase activity and blood aldo-keto reductase spectrum has been performed in 13-15 and 16-18-years old adolescents with obesity to clear up the mechanisms of neuroendocrine obesity at the age of puberty. It has been established that basal aldehyde reductase activity and blood aldo-keto reductase spectrum of healthy adolescents in early puberty do not differ from those of healthy adolescents in late puberty. A decreased aldehyde reductase activity and some alterations in blood aldo-keto reductase spectrum have been observed in late puberty in adolescents with neuroendocrine obesity. In adolescents with obesity there have been registered some changes in blood aldo-keto reductase spectrum which are not accompanied by any alterations in its aldehyde reductase activity. The results obtained suggest that certain prerequisites are formed in late puberty to complicate the course of neuroendocrine obesity.

  17. 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.

  18. 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

  19. Genetics Home Reference: 5-alpha reductase deficiency

    Science.gov (United States)

    ... G. New mutations, hotspots, and founder effects in Brazilian patients with steroid 5alpha-reductase deficiency type 2. ... should consult with a qualified healthcare professional . About Selection Criteria for Links Data Files & API Site Map ...

  20. Methylenetetrahydrofolate Reductase A1298C Polymorphism and ...

    African Journals Online (AJOL)

    Epigenetic alterations in cancer-related genes are recognized to play an important role in BC carcinogenesis. Epidemiological studies have consistently supported that ... Methylenetetrahydrofolate reductase (MTHFR) enzyme is essential for DNA synthesis ...... disease: A common mutation in methylenetetrahydrofolate.

  1. Characterization of the quinine reductase activity of the ferrice reductase B protein from Paracoccus denitrificans.

    NARCIS (Netherlands)

    Sedlacek, V.; van Spanning, R.J.M.; Kucera, I.

    2009-01-01

    The ferric reductase B (FerB) protein of Paracoccus denitrificans exhibits activity of an NAD(P)H: Fe(III) chelate, chromate and quinone oxidoreductase. Sequence analysis places FerB in a family of soluble flavin-containing quinone reductases. The enzyme reduces a range of quinone substrates,

  2. 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.

  3. Characterization of the chlorate reductase from Pseudomonas chloritidismutans

    NARCIS (Netherlands)

    Wolterink, A.F.W.M.; Schiltz, E.; Hagedoorn, P.L.; Hagen, W.R.; Kengen, S.W.M.; Stams, A.J.M.

    2003-01-01

    A chlorate reductase has been purified from the chlorate-reducing strain Pseudomonas chloritidismutans. Comparison with the periplasmic (per)chlorate reductase of strain GR-1 showed that the cytoplasmic chlorate reductase of P. chloritidismutans reduced only chlorate and bromate. Differences were

  4. 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...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7375 Glutathione reductase assay. (a) Identification. A glutathione reductase assay is a device used to determine the...

  5. Methylenetetrahydrofolate reductase gene polymorphism in type 1 ...

    African Journals Online (AJOL)

    In patients with type-I diabetes mellitus folate deficiency is associated with endothelial dysfunction. So, polymorphism in genes involved in folate metabolism may have a role in vascular disease. This study was designed to evaluate the relationship between methylenetetrahydrofolate reductase (MTHFR) gene polymorphism ...

  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. Promiscuity and diversity in 3-ketosteroid reductases

    Science.gov (United States)

    Penning, Trevor M.; Chen, Mo; Jin, Yi

    2014-01-01

    Many steroid hormones contain a Δ4-3-ketosteroid functionality that undergoes sequential reduction by 5α- or 5β- steroid reductases to produce 5α- or 5β-dihydrosteroids; and a subsequent 3-keto-reduction to produce a series of isomeric tetrahydrosteroids. Apart from steroid 5α-reductase all the remaining enzymes involved in the two step reduction process in humans belong to the aldo-keto reductase (AKR) superfamily. The enzymes involved in 3-ketosteroid reduction are AKR1C1–AKR1C4. These enzymes are promiscuous and also catalyze 20-keto- and 17-keto-steroid reduction. Interest in these reactions exist since they regulate steroid hormone metabolism in the liver, and in steroid target tissues, they may regulate steroid hormone receptor occupancy. In addition many of the dihydrosteroids are not biologically inert. The same enzymes are also involved in the metabolism of synthetic steroids e.g., hormone replacement therapeutics, contraceptive agents and inhaled glucocorticoids, and may regulate drug efficacy at their cognate receptors. This article reviews these reactions and the structural basis for substrate diversity in AKR1C1–AKR1C4, ketosteroid reductases. This article is part of a Special Issue entitled ‘Steroid/Sterol signaling’. PMID:25500069

  8. Differential nitrate accumulation, nitrate reduction, nitrate reductase ...

    African Journals Online (AJOL)

    Differential nitrate accumulation, nitrate reduction, nitrate reductase activity, protein production and carbohydrate biosynthesis in response to potassium and sodium ... due to the positive effects of potassium on the enzyme activity, sugars transport, water and nutrient transport, protein synthesis and carbohydrate metabolism.

  9. Two Greek siblings with sepiapterin reductase deficiency.

    NARCIS (Netherlands)

    Verbeek, M.M.; Willemsen, M.A.A.P.; Wevers, R.A.; Lagerwerf, A.J.; Abeling, N.G.; Blau, N.; Thony, B.; Vargiami, E.; Zafeiriou, D.I.

    2008-01-01

    BACKGROUND: Sepiapterin reductase (SR) deficiency is a rare inherited disorder of neurotransmitter metabolism; less than 25 cases have been described in the literature so far. METHODS: We describe the clinical history and extensive cerebrospinal fluid (CSF) and urine examination of two Greek

  10. 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 ...

  11. Clipboard: Lymphohematopoietic licence: Sterol C-14 reductase ...

    Indian Academy of Sciences (India)

    Clipboard: Lymphohematopoietic licence: Sterol C-14 reductase activity of lamin B receptor (Lbr) is essential for neutrophil differentiation. Durgadas P Kasbekar. Volume 37 ... Keywords. Greenberg/HEM dysplasia; lymphohematopoietic progenitor cells; nuclear envelope; Pelger-Huët anomaly; promyelocyte differentiation ...

  12. Methylenetetrahydrofolate reductase gene polymorphism in type 1 ...

    African Journals Online (AJOL)

    Mohammed A AboElAsrar

    2012-05-05

    May 5, 2012 ... Elevated homocysteine is a known risk factor for vascular disease. So the polymorphism in methylenetetrahydrofolate reductase may have detrimental consequences [5]. In patients with type-I diabetes mellitus folate deficiency is associated with endothelial dysfunction and folate supplementa- tion improves ...

  13. Steroid 5alpha-reductase inhibitors.

    Science.gov (United States)

    Flores, Eugenio; Bratoeff, Eugene; Cabeza, Marisa; Ramirez, Elena; Quiroz, Alexandra; Heuze, Ivonne

    2003-05-01

    The objective of this study is to synthesize new steroidal compounds based on the progesterone skeleton with a high inhibitory activity for the enzyme 5alpha-reductase. Presently similar compounds are being used for the treatment of androgen dependent diseases such as: hirsutism, androgenic alopecia, bening prostatic hyperplasia and prostate cancer. Dihydrotestosterone 2 (Fig. (1)), a 5alpha-reduced metabolite of testosterone 1 has been implicated as a causative factor in the progression of these diseases, largely through the clinical evaluation of males who are genetically deficient of steroid 5alpha-reductase enzyme. As a result of this study, the inhibition of this enzyme has become a pharmacological strategy for the design and synthesis of new antiandrogenic drugs. The advent of finasteride 8 (Fig. (4)) a 5alpha-reductase inhibitor has grately alleviated the symptoms associated with benign prostatic hyperplasia. In our laboratory we recently synthesized several new 16beta-methyl-pregnadiene-3,20-diones derivatives 27 (Fig.(6)), 38-42 (Fig. (11)), 16beta-phenyl-pregnadiene-3,17a-dione derivatives 32-33 (Fig. (7)), 16beta-phenyl-pregnatriene-3,17a-diones, 30, 31 (Fig. (7)) and 16beta-methyl-pregnatriene-3,20-diones 43-46 (Fig. (11)). These compounds were evaluated as 5alpha-reductase inhibitors in the following biological models: Penicillium crustosum broths, the flank organs of gonadectomized male hamsters, the incorporation of radiolabeled sodium acetate into lipids, the effect of the new steroids on the reduction of the weight of the seminal vesicles and on the in vitro metabolism of [(3)H]T to [(3)H]DHT in seminal vesicles homogenates of gonadectomized male hamsters. All trienones 30, 31, and 43-46 in all biological models showed consistently a higher 5alpha-reductase inhibitory activity than the corresponding dienones 27, 32, 33 and 38-42. We believe that with these compounds the 5alpha-reductase enzyme is inactivated by an irreversible Michael type addition

  14. Biliverdin reductase: A major physiologic cytoprotectant

    Science.gov (United States)

    Barañano, David E.; Rao, Mahil; Ferris, Christopher D.; Snyder, Solomon H.

    2002-01-01

    Bilirubin, an abundant pigment that causes jaundice, has long lacked any clear physiologic role. It arises from enzymatic reduction by biliverdin reductase of biliverdin, a product of heme oxygenase activity. Bilirubin is a potent antioxidant that we show can protect cells from a 10,000-fold excess of H2O2. We report that bilirubin is a major physiologic antioxidant cytoprotectant. Thus, cellular depletion of bilirubin by RNA interference markedly augments tissue levels of reactive oxygen species and causes apoptotic cell death. Depletion of glutathione, generally regarded as a physiologic antioxidant cytoprotectant, elicits lesser increases in reactive oxygen species and cell death. The potent physiologic antioxidant actions of bilirubin reflect an amplification cycle whereby bilirubin, acting as an antioxidant, is itself oxidized to biliverdin and then recycled by biliverdin reductase back to bilirubin. This redox cycle may constitute the principal physiologic function of bilirubin. PMID:12456881

  15. Structural Elucidation of Chalcone Reductase and Implications for Deoxychalcone Biosynthesis

    Science.gov (United States)

    Bomati, Erin K.; Austin, Michael B.; Bowman, Marianne E.; Dixon, Richard A.; Noel, Joseph P.

    2010-01-01

    4,2′,4′,6′-tetrahydroxychalcone (chalcone) and 4,2′,4′-trihydroxychalcone (deoxychalcone) serve as precursors of ecologically important flavonoids and isoflavonoids. Deoxychalcone formation depends on chalcone synthase and chalcone reductase; however, the identity of the chalcone reductase substrate out of the possible substrates formed during the multistep reaction catalyzed by chalcone synthase remains experimentally elusive. We report here the three-dimensional structure of alfalfa chalcone reductase bound to the NADP+ cofactor and propose the identity and binding mode of its substrate, namely the non-aromatized coumaryl-trione intermediate of the chalcone synthase-catalyzed cyclization of the fully extended coumaryl-tetraketide thioester intermediate. In the absence of a ternary complex, the quality of the refined NADP+-bound chalcone reductase structure serves as a template for computer-assisted docking to evaluate the likelihood of possible substrates. Interestingly, chalcone reductase adopts the three-dimensional structure of the aldo/keto reductase superfamily. The aldo/keto reductase fold is structurally distinct from all known ketoreductases of fatty acid biosynthesis, which instead belong to the short-chain dehydrogenase/reductase superfamily. The results presented here provide structural support for convergent functional evolution of these two ketoreductases that share similar roles in the biosynthesis of fatty acids/polyketides. In addition, the chalcone reductase structure represents the first protein structure of a member of the aldo/ketoreductase 4 family. Therefore, the chalcone reductase structure serves as a template for the homology modeling of other aldo/ketoreductase 4 family members, including the reductase involved in morphine biosynthesis, namely codeinone reductase. PMID:15970585

  16. Cloning and nitrate induction of nitrate reductase mRNA

    OpenAIRE

    Cheng, Chi-Lien; Dewdney, Julia; Kleinhofs, Andris; Goodman, Howard M.

    1986-01-01

    Nitrate is the major source of nitrogen taken from the soil by higher plants but requires reduction to ammonia prior to incorporation into amino acids. The first enzyme in the reducing pathway is a nitrate-inducible enzyme, nitrate reductase (EC 1.6.6.1). A specific polyclonal antiserum raised against purified barley nitrate reductase has been used to immunoprecipitate in vivo labeled protein and in vitro translation products, demonstrating that nitrate induction increases nitrate reductase p...

  17. Structural and biochemical characterization of cinnamoyl-coa reductases

    Science.gov (United States)

    Cinnamoyl-coenzyme A reductase (CCR) catalyzes the reduction of hydroxycinnamoyl-coenzyme A (CoA) esters using NADPH to produce hydroxycinnamyl aldehyde precursors in lignin synthesis. The catalytic mechanism and substrate specificity of cinnamoyl-CoA reductases from sorghum (Sorghum bicolor), a str...

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

    African Journals Online (AJOL)

    GREGO

    nitrate transformation into nitrite (µg of NO2. -/min/g F.W) is observed when incubation period of enzyme is short (1 to 5 min). Key words: Extraction, dosage, nitrate reductase activity, callus, cotton. INTRODUCTION. Nitrate reductase (EC. 1.7.99.4) is an oxidoreductase enzyme involved in nitrogen assimilation in plant. It.

  19. Biliverdin Reductase: a Target for Cancer Therapy?

    Directory of Open Access Journals (Sweden)

    Peter eGibbs

    2015-06-01

    Full Text Available Biliverdin reductase (BVR is a multifunctional protein that is the primary source of the potent antioxidant, bilirubin. BVR regulates activities/functions in the insulin/IGF-1/IRK/PI3K/MAPK pathways. Activation of certain kinases in these pathways is/are hallmark(s of cancerous cells. The protein is a scaffold/bridge and intracellular transporter of kinases that regulate growth and proliferation of cells, including PKCs, ERK and Akt, and their targets including NF-κB, Elk1, HO-1 and iNOS. The scaffold and transport functions enable activated BVR to relocate from the cytosol to the nucleus or to the plasma membrane, depending on the activating stimulus. This enables the reductase to function in diverse signaling pathways. And, its expression at the transcript and protein levels are increased in human tumors and the infiltrating T-cells, monocytes and circulating lymphocytes, as well as the circulating and infiltrating macrophages. These functions suggest that the cytoprotective role of BVR may be permissive for cancer/tumor growth. In this review, we summarize the recent developments that define the pro-growth activities of BVR, particularly with respect to its input into the MAPK signaling pathway and present evidence that BVR-based peptides inhibit activation of protein kinases, including MEK, PKCδ and ERK as well as downstream targets including Elk1 and iNOS, and thus offers a credible novel approach to reduce cancer cell proliferation.

  20. [High throughput screening of active and stereoselective carbonyl reductases].

    Science.gov (United States)

    Zhang, Hang; Chen, Xi; Feng, Jinhui; Bao, Jinku; Wu, Qiaqing; Zhu, Dunming

    2015-02-01

    In this study, a fast carbonyl reductases colorimetric screening method for discovering stereoselective carbonyl reductases was established by combining the reverse alcohol oxidation with the azoreductase-catalyzed reduction of azo dye. When azo dye (Orange I , 4-(4-hydroxy-1-naphthylazo) benzenesulfonic acid) and azoreductase (AzoB) were added into the reaction system of alcohol oxidation catalyzed by carbonyl reductase, the produced NAD(P)H served as electron donor for the azoreductase to reduce the azo dye, resulting the color fade. Hence, the carbonyl reductases can be screened by the obvious color change. When chiral alcohol was used as the substrate, the activity and stereoselectivity of carbonyl reductases can be screened at the same time.

  1. Expression of 3-hydroxy-3-methylglutaryl-CoA reductase, p-hydroxybenzoate-m-geranyltransferase and genes of phenylpropanoid pathway exhibits positive correlation with shikonins content in arnebia [Arnebia euchroma (Royle Johnston

    Directory of Open Access Journals (Sweden)

    Sharma Madhu

    2010-11-01

    Full Text Available Abstract Background Geranyl pyrophosphate (GPP and p-hydroxybenzoate (PHB are the basic precursors involved in shikonins biosynthesis. GPP is derived from mevalonate (MVA and/or 2-C-methyl-D-erythritol 4-phosphate (MEP pathway(s, depending upon the metabolite and the plant system under consideration. PHB, however, is synthesized by only phenylpropanoid (PP pathway. GPP and PHB are central moieties to yield shikonins through the synthesis of m-geranyl-p-hydroxybenzoate (GHB. Enzyme p-hydroxybenzoate-m-geranyltransferase (PGT catalyses the coupling of GPP and PHB to yield GHB. The present research was carried out in shikonins yielding plant arnebia [Arnebia euchroma (Royle Johnston], wherein no molecular work has been reported so far. The objective of the work was to identify the preferred GPP synthesizing pathway for shikonins biosynthesis, and to determine the regulatory genes involved in the biosynthesis of GPP, PHB and GHB. Results A cell suspension culture-based, low and high shikonins production systems were developed to facilitate pathway identification and finding the regulatory gene. Studies with mevinolin and fosmidomycin, inhibitors of MVA and MEP pathway, respectively suggested MVA as a preferred route of GPP supply for shikonins biosynthesis in arnebia. Accordingly, genes of MVA pathway (eight genes, PP pathway (three genes, and GHB biosynthesis were cloned. Expression studies showed down-regulation of all the genes in response to mevinolin treatment, whereas gene expression was not influenced by fosmidomycin. Expression of all the twelve genes vis-à-vis shikonins content in low and high shikonins production system, over a period of twelve days at frequent intervals, identified critical genes of shikonins biosynthesis in arnebia. Conclusion A positive correlation between shikonins content and expression of 3-hydroxy-3-methylglutaryl-CoA reductase (AeHMGR and AePGT suggested critical role played by these genes in shikonins

  2. Monodehydroascorbate reductase mediates TNT toxicity in plants.

    Science.gov (United States)

    Johnston, Emily J; Rylott, Elizabeth L; Beynon, Emily; Lorenz, Astrid; Chechik, Victor; Bruce, Neil C

    2015-09-04

    The explosive 2,4,6-trinitrotoluene (TNT) is a highly toxic and persistent environmental pollutant. Due to the scale of affected areas, one of the most cost-effective and environmentally friendly means of removing explosives pollution could be the use of plants. However, mechanisms of TNT phytotoxicity have been elusive. Here, we reveal that phytotoxicity is caused by reduction of TNT in the mitochondria, forming a nitro radical that reacts with atmospheric oxygen, generating reactive superoxide. The reaction is catalyzed by monodehydroascorbate reductase 6 (MDHAR6), with Arabidopsis deficient in MDHAR6 displaying enhanced TNT tolerance. This discovery will contribute toward the remediation of contaminated sites. Moreover, in an environment of increasing herbicide resistance, with a shortage in new herbicide classes, our findings reveal MDHAR6 as a valuable plant-specific target. Copyright © 2015, American Association for the Advancement of Science.

  3. Structures of mammalian cytosolic quinone reductases.

    Science.gov (United States)

    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.

  4. Enhancement of nitrate reductase activity by benzyladenine in Agrostemma githago

    Energy Technology Data Exchange (ETDEWEB)

    Kende, H.; Hahn, H.; Kays, S.E.

    1971-01-01

    Nitrate reductase activity in excised embryos of Agrostemma githago increases in response to both NO/sub 3//sup -/ and cytokinins. Discussed was whether cytokinins affected nitrate reductase activity directly or through NO/sub 3//sup -/, either by amplifying the effect of low endogenous NO/sub 3//sup -/ levels, or by making NO/sub 3//sup -/ available for induction from a metabolically inactive compartment. Nitrate reductase activity was enhanced on the average by 50% after 1 hour of benzyladenine treatment. In some experiments, the cytokinin response was detectable as early as 30 minutes after addition of benzyladenine. Nitrate reductase activity increased linearly for 4 hours and began to decay 13 hours after start of the hormone treatment. When embryos were incubated in solutions containing mixtures of NO/sub 3//sup -/ and benzyladenine, additive responses were obtained. The effects of NO/sub 3//sup -/ and benzyladenine were counteracted by abscisic acid. The increase in nitrate reductase activity was inhibited at lower abscisic acid concentrations in embryos which were induced with NO/sub 3//sup -/, as compared to embryos treated with benzyladenine. Casein hydrolysate inhibited the development of nitrate reductase activity. The response to NO/sub 3//sup -/ was more susceptible to inhibition by casein hydrolysate than the response to the hormone. When NO/sub 3//sup -/ and benzyladenine were withdrawn from the medium after maximal enhancement of nitrate reductase activity, the level of the enzyme decreased rapidly. Nitrate reductase activity increased again as a result of a second treatment with benzyladenine but not with NO/sub 3//sup -/. At the time of the second exposure to benzyladenine, no NO/sub 3//sup -/ was detectable in extracts of Agrostemma embryos. This is taken as evidence that cytokinins enhance nitrate reductase activity directly and not through induction by NO/sub 3//sup -/. 11 references, 5 figures, 3 tables.

  5. [Fumarate reductase in the mitochondria of the trematode Calicophoron ijimai].

    Science.gov (United States)

    Iarygina, G V; Vykhrestiuk, N P; Burenina, E A

    1983-01-01

    The presence of active fumarate reductase system in mitochondria of the trematode Calicophoron ijimai was shown. Fumarate reductase activities in different collections of C. ijimai vary considerably. Maximum activity accounts for 47.7 +/- 1.0 nM/min/mg protein whereas minimum--for 15.1 +/- 0.1. Some properties of the enzyme were studied. The effect of thiabendazole, bitionol, oxinid and preparations of G-1026 and G-937 on the fumarate reductase activity was investigated. G-1026, G-937 preparations and bitionol have the strongest inhibitory effect on the enzyme. Thiabendazole inhibited but little the fumarate reductase reaction in C. ijimai. The enzyme activity was not affected by oxinid.

  6. 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...

  7. 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

  8. The nitric oxide reductase activity of cytochrome c nitrite reductase from Escherichia coli.

    Science.gov (United States)

    van Wonderen, Jessica H; Burlat, Bénédicte; Richardson, David J; Cheesman, Myles R; Butt, Julea N

    2008-04-11

    Cytochrome c nitrite reductase (NrfA) from Escherichia coli has a well established role in the respiratory reduction of nitrite to ammonium. More recently the observation that anaerobically grown E. coli nrf mutants were more sensitive to NO. than the parent strain led to the proposal that NrfA might also participate in NO. detoxification. Here we describe protein film voltammetry that presents a quantitative description of NrfA NO. reductase activity. NO. reduction is initiated at similar potentials to NrfA-catalyzed reduction of nitrite and hydroxylamine. All three activities are strongly inhibited by cyanide. Together these results suggest a common site for reduction of all three substrates as axial ligands to the lysine-coordinated NrfA heme rather than nonspecific NO. reduction at one of the four His-His coordinated hemes also present in each NrfA subunit. NO. reduction by NrfA is described by a K(m) of the order of 300 microm. The predicted turnover number of approximately 840 NO. s(-1) is much higher than that of the dedicated respiratory NO. reductases of denitrification and the flavorubredoxin and flavohemoglobin of E. coli that are also proposed to play roles in NO. detoxification. In considering the manner by which anaerobically growing E. coli might detoxify exogenously generated NO. encountered during invasion of a human host it appears that the periplasmically located NrfA should be effective in maintaining low NO. levels such that any NO. reaching the cytoplasm is efficiently removed by flavorubredoxin (K(m) approximately 0.4 microm).

  9. 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.

  10. 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.

  11. 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.

  12. The role of light in the inducation of nitrate reductase and nitrite reductase in cucumber seedlings

    Directory of Open Access Journals (Sweden)

    J. Buczek

    2015-01-01

    Full Text Available The activity of nitrate reductase (NR and nitrite reductase (NiR was investigated in vivo and in vitro in the roots and NR activity in 3-day-old cotyledons of cucumber seedlings. NR activity in the roots appears almost immediately after addition of nitrate ions to the induction medium, whereas, in the cotyledones NR induction is delayed. In general light enhances NR activity in the cotyledons and depresses it in the roots in experiments of short duration. Etiolation of the cotyledons reduces NR activity in the roots and leads to disappearance of the activity of this enzyme in the cotyledons, whereas the NR activity of roots kept in darkness, after transfer of the etiolated plants to light, increases threefold. In roots growing in darkness a delay in NiR induction is observed, while in those growing in ligth it occurs at the same time as NR induction. Chlormaphenicol (CAP, cycloheximide (CHI and actinomycin D (ACM applied at the beginning of the period of seedling induction with initrates inhibit NR activity in the cotyledons, whereas in the roots only CHI and ACM exert such an effect. To sum up, NR is synthesized in cucumber roots and cotyledons de novo on the cytoplasmic polyribosomes, and light per se is not indispensable for this synthesis, but it has an indirect influence on the activity level of NR and NiR both in the roots and the cotyledons.

  13. 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

  14. 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.

  15. 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.

  16. Substrate channeling between the human dihydrofolate reductase and thymidylate synthase.

    Science.gov (United States)

    Wang, Nuo; McCammon, J Andrew

    2016-01-01

    In vivo, as an advanced catalytic strategy, transient non-covalently bound multi-enzyme complexes can be formed to facilitate the relay of substrates, i. e. substrate channeling, between sequential enzymatic reactions and to enhance the throughput of multi-step enzymatic pathways. The human thymidylate synthase and dihydrofolate reductase catalyze two consecutive reactions in the folate metabolism pathway, and experiments have shown that they are very likely to bind in the same multi-enzyme complex in vivo. While reports on the protozoa thymidylate synthase-dihydrofolate reductase bifunctional enzyme give substantial evidences of substrate channeling along a surface "electrostatic highway," attention has not been paid to whether the human thymidylate synthase and dihydrofolate reductase, if they are in contact with each other in the multi-enzyme complex, are capable of substrate channeling employing surface electrostatics. This work utilizes protein-protein docking, electrostatics calculations, and Brownian dynamics to explore the existence and mechanism of the substrate channeling between the human thymidylate synthase and dihydrofolate reductase. The results show that the bound human thymidylate synthase and dihydrofolate reductase are capable of substrate channeling and the formation of the surface "electrostatic highway." The substrate channeling efficiency between the two can be reasonably high and comparable to that of the protozoa. © 2015 The Protein Society.

  17. Sulfite Reductase Activity in Extracts of Various Photosynthetic Bacteria

    Science.gov (United States)

    Peck, H. D.; Tedro, S.; Kamen, M. D.

    1974-01-01

    Extracts of representative bacterial strains from the various families of photosynthetic prokaryotes are demonstrated to possess significant levels of sulfite reductase [EC 1.8.99.1; hydrogen-sulfide: (acceptor)oxidoreductase] activity with reduced methyl viologen as electron donor, but not NADPH2. The enzyme is localized primarily in the soluble fraction of the extracts, in contrast to adenylysulfate reductase [EC 1.8.99.2; AMP, sulfite: (acceptor) oxidoreductase], which is bound normally in the membrane fractions of those bacteria in which it is found. Assignment of the sulfite reductase activities to the biosynthetic (“assimilatory”) pathway is suggested by levels of specific activity noted and ready solubility. PMID:4526215

  18. 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...... 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...... 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...

  19. Methemoglobin reductase activity in intact fish red blood cells

    DEFF Research Database (Denmark)

    Jensen, Frank B; Nielsen, Karsten

    2018-01-01

    Hb reductase activity in fish offsets their higher Hb autoxidation and higher likelihood of encountering elevated nitrite. Deoxygenation significantly raised the rates of RBC metHb reduction, and more so in rainbow trout than in carp. The temperature sensitivity of metHb reduction in rainbow trout RBCs......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...... of counteracting oxidation. This assay was used to compare metHb reduction in rainbow trout and carp RBCs under both oxygenated and deoxygenated conditions. Washing resulted in effective wash-out of nitrite to low and safe values (~2μM). The subsequent decline in [metHb] with time followed first-order kinetics...

  20. The Drosophila carbonyl reductase sniffer is an efficient 4-oxonon-2-enal (4ONE) reductase.

    Science.gov (United States)

    Martin, Hans-Jörg; Ziemba, Marta; Kisiela, Michael; Botella, José A; Schneuwly, Stephan; Maser, Edmund

    2011-05-30

    Studies with the fruit-fly Drosophila melanogaster demonstrated that the enzyme sniffer prevented oxidative stress-induced neurodegeneration. Mutant flies overexpressing sniffer had significantly extended life spans in a 99.5% oxygen atmosphere compared to wild-type flies. However, the molecular mechanism of this protection remained unclear. Sequence analysis and database searches identified sniffer as a member of the short-chain dehydrogenase/reductase superfamily with a 27.4% identity to the human enzyme carbonyl reductase type I (CBR1). As CBR1 catalyzes the reduction of the lipid peroxidation products 4HNE and 4ONE, we tested whether sniffer is able to metabolize these lipid derived aldehydes by carbonyl reduction. To produce recombinant enzyme, the coding sequence of sniffer was amplified from a cDNA-library, cloned into a bacterial expression vector and the His-tagged protein was purified by Ni-chelate chromatography. We found that sniffer catalyzed the NADPH-dependent carbonyl reduction of 4ONE (K(m)=24±2 μM, k(cat)=500±10 min(-1), k(cat)/K(m)=350 s(-1) mM(-1)) but not that of 4HNE. The reaction product of 4ONE reduction by sniffer was mainly 4HNE as shown by HPLC- and GC/MS analysis. Since 4HNE, though still a potent electrophile, is less neurotoxic and protein reactive than 4ONE, one mechanism by which sniffer exerts its neuroprotective effects in Drosophila after oxidative stress may be enzymatic reduction of 4ONE. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

  1. 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.

  2. Sulfur Isotope Effects of Dissimilatory Sulfite Reductase

    Directory of Open Access Journals (Sweden)

    William D. Leavitt

    2015-12-01

    Full Text Available The precise interpretation of environmental sulfur isotope records requires a quantitative understanding of the biochemical controls on sulfur isotope fractionation by the principle isotope-fractionating process within the S cycle, microbial sulfate reduction (MSR. Here we provide the only direct observation of the major (34S/32S and minor (33S/32S, 36S/32S sulfur isotope fractionations imparted by a central enzyme in the energy metabolism of sulfate reducers, dissimilatory sulfite reductase (DsrAB. Results from in vitro sulfite reduction experiments allow us to calculate the in vitro DsrAB isotope effect in 34S/32S (hereafter, 34εDsrAB to be 15.3±2‰, 2σ. The accompanying minor isotope effect in 33S, described as 33λDsrAB, is calculated to be 0.5150±0.0012, 2σ. These observations facilitate a rigorous evaluation of the isotopic fractionation associated with the dissimilatory MSR pathway, as well as of the environmental variables that govern the overall magnitude of fractionation by natural communities of sulfate reducers. The isotope effect induced by DsrAB upon sulfite reduction is a factor of 0.3 to 0.6 times prior indirect estimates, which have ranged from 25 to 53‰ in 34εDsrAB. The minor isotope fractionation observed from DsrAB is consistent with a kinetic or equilibrium effect. Our in vitro constraints on the magnitude of 34εDsrAB is similar to the median value of experimental observations compiled from all known published work, where 34εr-p = 16.1‰ (r – p indicates reactant versus product, n = 648. This value closely matches those of MSR operating at high sulfate reduction rates in both laboratory chemostat experiments (34εSO4-H2S = 17.3±1.5‰ and in modern marine sediments (34εSO4-H2S = 17.3±3.8‰. Targeting the direct isotopic consequences of a specific enzymatic processes is a fundamental step toward a biochemical foundation for reinterpreting the biogeochemical and geobiological sulfur isotope records in modern

  3. Mitochondrial Thioredoxin-Glutathione Reductase from Larval Taenia crassiceps (Cysticerci

    Directory of Open Access Journals (Sweden)

    Alberto Guevara-Flores

    2010-01-01

    Full Text Available Mitochondrial thioredoxin-glutathione reductase was purified from larval Taenia crassiceps (cysticerci. The preparation showed NADPH-dependent reductase activity with either thioredoxin or GSSG, and was able to perform thiol/disulfide exchange reactions. At 25∘C specific activities were 437  ±  27 mU mg-1 and 840  ±  49 mU mg-1 with thioredoxin and GSSG, respectively. Apparent Km values were 0.87  ±  0.04  μM, 41  ±  6  μM and 19  ±  10  μM for thioredoxin, GSSG and NADPH, respectively. Thioredoxin from eukaryotic sources was accepted as substrate. The enzyme reduced H2O2 in a NADPH-dependent manner, although with low catalytic efficiency. In the presence of thioredoxin, mitochondrial TGR showed a thioredoxin peroxidase-like activity. All disulfide reductase activities were inhibited by auranofin, suggesting mTGR is dependent on selenocysteine. The reductase activity with GSSG showed a higher dependence on temperature as compared with the DTNB reductase activity. The variation of the GSSG- and DTNB reductase activities on pH was dependent on the disulfide substrate. Like the cytosolic isoform, mTGR showed a hysteretic kinetic behavior at moderate or high GSSG concentrations, but it was less sensitive to calcium. The enzyme was able to protect glutamine synthetase from oxidative inactivation, suggesting that mTGR is competent to contend with oxidative stress.

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

    DEFF Research Database (Denmark)

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

    2003-01-01

    ) reductases reported previously. Downstream of the butA gene of L. pseudomesenteroides, but coding in the opposite orientation, a putative DNA recombinase was identified. A two-step PCR approach was used to construct FPR02, a butA mutant of the wild-type strain, CHCC2114. FPR02 had significantly reduced......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...

  5. NITRATE REDUCTASE ACTIVITY DURING HEAT SHOCK IN WINTER WHEAT

    Directory of Open Access Journals (Sweden)

    Klimenko S.B.

    2006-03-01

    Full Text Available Nitrates are the basic source of nitrogen for the majority of plants. Absorption and transformation of nitrates in plants are determined by external conditions and, first of all, temperature and light intensity. The influence of the temperature increasing till +40 0С on activity of nitrate reductase was studied. It is shown, that the rise of temperature was accompanied by sharp decrease of activity nitrate reductase in leaves of winter wheat, what, apparently, occurred for the account deactivations of enzyme and due to its dissociation.

  6. 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 ...

  7. 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.

  8. Sepiapterin reductase deficiency : A Treatable Mimic of Cerebral Palsy

    NARCIS (Netherlands)

    Friedman, Jennifer; Roze, Emmanuel; Abdenur, Jose E.; Chang, Richard; Gasperini, Serena; Saletti, Veronica; Wali, Gurusidheshwar M.; Eiroa, Hernan; Neville, Brian; Felice, Alex; Parascandalo, Ray; Zafeiriou, Dimitrios I.; Arrabal-Fernandez, Luisa; Dill, Patricia; Eichler, Florian S.; Echenne, Bernard; Gutierrez-Solana, Luis G.; Hoffmann, Georg F.; Hyland, Keith; Kusmierska, Katarzyna; Tijssen, Marina A. J.; Lutz, Thomas; Mazzuca, Michel; Penzien, Johann; Bwee Tien Poll-The, [No Value; Sykut-Cegielska, Jolanta; Szymanska, Krystyna; Thoeny, Beat; Blau, Nenad

    Objective: Sepiapterin reductase deficiency (SRD) is an under-recognized levodopa-responsive disorder. We describe clinical, biochemical, and molecular findings in a cohort of patients with this treatable condition. We aim to improve awareness of the phenotype and available diagnostic and

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

    African Journals Online (AJOL)

    STORAGESEVER

    2010-03-01

    Mar 1, 2010 ... somatic embryogenesis stages, and that the level of GhNiR mRNA was also higher in the cultivar with higher somatic ..... Planta, 183: 17-24. Alexander H, Treusch, Sven L, Arnulf K, Stephan CS, Hans-Peter K,. Christa S (2005). Novel genes for nitrite reductase and Amo-related proteins indicate a role of ...

  10. Xylose reductase from the thermophilic fungus Talaromyces emersonii

    Indian Academy of Sciences (India)

    Prakash

    National University of Ireland, Galway, University Road, Galway, Ireland. 2Shannon Applied Biotechnology Centre, Limerick Institute Technology, Moylish Park, Limerick, Ireland. *Corresponding authors (Fax, 0035361208208; Email, patrick.murray@lit.ie). Xylose reductase is involved in the first step of the fungal pentose ...

  11. 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

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

    African Journals Online (AJOL)

    olayemitoyin

    Bioinformatic analysis of dihydrofolate reductase predicted in the genome sequence of Lactobacillus pentosus KCA1. *Kingsley C. Anukam. 1 and Uche Oge. 2. 1TWAS Genomic Research Unit, Department of Medical Laboratory Science, 2Department of Physiology,. School of Basic Medical Sciences, University of Benin, ...

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

    African Journals Online (AJOL)

    The genome has open reading frames coding for the complete genes required for folate biosynthesis. Our previous study shows that rats fed with L. pentosus KCA1 led to enhancement of haematological parameters. Bioinformatic tool such as ClustalW algorithm was used to analyze dihydrofolate reductase (folA/dfrA) ...

  14. 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 ...

  15. 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, ...

  16. Molybdenum-containing nitrite reductases: Spectroscopic characterization and redox mechanism.

    Science.gov (United States)

    Wang, Jun; Keceli, Gizem; Cao, Rui; Su, Jiangtao; Mi, Zhiyuan

    2017-01-01

    This review summarizes the spectroscopic results, which will provide useful suggestions for future research. In addition, the fields that urgently need more information are also advised. Nitrite-NO-cGMP has been considered as an important signaling pathway of NO in human cells. To date, all the four known human molybdenum-containing enzymes, xanthine oxidase, aldehyde oxidase, sulfite oxidase, and mitochondrial amidoxime-reducing component, have been shown to function as nitrite reductases under hypoxia by biochemical, cellular, or animal studies. Various spectroscopic techniques have been applied to investigate the structure and catalytic mechanism of these enzymes for more than 20 years. We summarize the published data on the applications of UV-vis and EPR spectroscopies, and X-ray crystallography in studying nitrite reductase activity of the four human molybdenum-containing enzymes. UV-vis has provided useful information on the redox active centers of these enzymes. The utilization of EPR spectroscopy has been critical in determining the coordination and redox status of the Mo center during catalysis. Despite the lack of substrate-bound crystal structures of these nitrite reductases, valuable structural information has been obtained by X-ray crystallography. To fully understand the catalytic mechanisms of these physiologically/pathologically important nitrite reductases, structural studies on substrate-redox center interaction are needed.

  17. 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.

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

    African Journals Online (AJOL)

    Dihydroflavonol 4-reductase (DFR) gene is a key gene of anthocyanins biosynthesis pathway, which represent an importance pathway for orchid flower. In this study, cloning and expression analysis of DFR gene in Ascocenda spp. were carried out. Nucleotide analysis revealed that the Ascocenda DFR gene was 1,056 bp ...

  19. Aldose Reductase Inhibitory and Antiglycation Activities of Four ...

    African Journals Online (AJOL)

    Thonn., Punica granatum L., and Stevia rebaudiana Bertoni) standardized extracts and their major constituents (morusin, phyllanthin, punicalagin and stevioside) in the treatment of long-term diabetic complications by inhibition of aldose reductase (AR) enzyme and advanced glycation end products (AGEs) formation.

  20. 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

  1. Steroidal antiandrogens and 5alpha-reductase inhibitors.

    Science.gov (United States)

    Bratoeff, E; Ramírez, E; Murillo, E; Flores, G; Cabeza, M

    1999-12-01

    The purpose of this work is to synthesize a pregnane derivative with a high antiandrogenic effect or a high inhibitory activity for the enzyme 5 alpha-reductase type 2. Benign prostatic hyperplasia and prostate cancer are androgen dependent diseases which afflict a large percentage of the male population. Dihydrotestosterone 3, a 5 alpha-reductase metabolite of testosterone 2 has been implicated as a causative factor in the progression of these diseases, largely through the clinical evaluation of males who are genetically deficient of steroid 5 alpha-reductase enzyme. As a result of this study, the inhibition of this enzyme has become a pharmacological strategy for the design and synthesis of new drugs. The advent of finasteride 22 "figure 5" a 5 alpha-reductase inhibitor, has greatly alleviated the symptoms associated with benign prostatic hyperplasia. On the other hand, the discovery of cyproterone acetate 4 "figure 2" alone or in combination with the antiandrogens flutamide 14 "figure 3" or bicalutamide 21 has greatly reduced the misery of prostate cancer. Prostate cancer kills about 40,000 men in the USA and approximately 400,000 prostatectomies are performed each year. In our laboratory we have recently synthesized ten new progesterone derivatives 17 alpha-acyloyloxy-6-halo (chloro, bromo) 16 beta-methyl-4, 6-pregnadiene-3, 20-diones (54a-54e and 55a-55e), "figure 10". These steroids were evaluated as antiandrogens and exhibited a much higher activity than the commercially available cyproterone acetate 4. The same compounds were also evaluated as 5 alpha-reductase inhibitors and showed a slightly higher inhibitory activity than that of finasteride 22, the drug of choice today for the treatment of benign prostatic hyperplasia In another study we synthesized several new 4-halo (bromo and chloro) 17 alpha-benzoyloxy and also 4-halo-17 alpha-acetoxy progesterone derivatives (58-63) "figure 13". These compounds were prepared from the commercially available 17 alpha

  2. 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.

  3. Hydroxyurea-Mediated Cytotoxicity Without Inhibition of Ribonucleotide Reductase

    Directory of Open Access Journals (Sweden)

    Li Phing Liew

    2016-11-01

    Full Text Available In many organisms, hydroxyurea (HU inhibits class I ribonucleotide reductase, leading to lowered cellular pools of deoxyribonucleoside triphosphates. The reduced levels for DNA precursors is believed to cause replication fork stalling. Upon treatment of the hyperthermophilic archaeon Sulfolobus solfataricus with HU, we observe dose-dependent cell cycle arrest, accumulation of DNA double-strand breaks, stalled replication forks, and elevated levels of recombination structures. However, Sulfolobus has a HU-insensitive class II ribonucleotide reductase, and we reveal that HU treatment does not significantly impact cellular DNA precursor pools. Profiling of protein and transcript levels reveals modulation of a specific subset of replication initiation and cell division genes. Notably, the selective loss of the regulatory subunit of the primase correlates with cessation of replication initiation and stalling of replication forks. Furthermore, we find evidence for a detoxification response induced by HU treatment.

  4. Differential expression of disulfide reductase enzymes in a free-living platyhelminth (Dugesia dorotocephala.

    Directory of Open Access Journals (Sweden)

    Alberto Guevara-Flores

    Full Text Available A search of the disulfide reductase activities expressed in the adult stage of the free-living platyhelminth Dugesia dorotocephala was carried out. Using GSSG or DTNB as substrates, it was possible to obtain a purified fraction containing both GSSG and DTNB reductase activities. Through the purification procedure, both disulfide reductase activities were obtained in the same chromatographic peak. By mass spectrometry analysis of peptide fragments obtained after tryptic digestion of the purified fraction, the presence of glutathione reductase (GR, thioredoxin-glutathione reductase (TGR, and a putative thioredoxin reductase (TrxR was detected. Using the gold compound auranofin to selectively inhibit the GSSG reductase activity of TGR, it was found that barely 5% of the total GR activity in the D. dorotocephala extract can be assigned to GR. Such strategy did allow us to determine the kinetic parameters for both GR and TGR. Although It was not possible to discriminate DTNB reductase activity due to TrxR from that of TGR, a chromatofocusing experiment with a D. dorotocephala extract resulted in the obtention of a minor protein fraction enriched in TrxR, strongly suggesting its presence as a functional protein. Thus, unlike its parasitic counterparts, in the free-living platyhelminth lineage the three disulfide reductases are present as functional proteins, albeit TGR is still the major disulfide reductase involved in the reduction of both Trx and GSSG. This fact suggests the development of TGR in parasitic flatworms was not linked to a parasitic mode of life.

  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. Long term treatment with betaine in methylenetetrahydrofolate reductase deficiency.

    OpenAIRE

    Ronge, E; Kjellman, B

    1996-01-01

    A girl aged 7.5 years with deficiency of 5,10-methylenetetrahydrofolate reductase was treated from early infancy with betaine, 3-6 g daily. She has slight microcephaly, moderate developmental delay, and impaired vision but there have been no obvious signs of folate deficiency. From 4 years of age, she developed an unexplained extreme increase in appetite and weight. Recent magnetic resonance imaging of her brain was normal. The plasma methionine levels have been normal but in the lower range,...

  7. Carbon-deuterium bonds as probes of dihydrofolate reductase.

    Science.gov (United States)

    Thielges, Megan C; Case, David A; Romesberg, Floyd E

    2008-05-21

    Much effort has been directed toward understanding the contributions of electrostatics and dynamics to protein function and especially to enzyme catalysis. Unfortunately, these studies have been limited by the absence of direct experimental probes. We have been developing the use of carbon-deuterium bonds as probes of proteins and now report the application of the technique to the enzyme dihydrofolate reductase, which catalyzes a hydride transfer and has served as a paradigm for biological catalysis. We observe that the stretching absorption frequency of (methyl- d 3) methionine carbon-deuterium bonds shows an approximately linear dependence on solvent dielectric. Solvent and computational studies support the empirical interpretation of the stretching frequency in terms of local polarity. To begin to explore the use of this technique to study enzyme function and mechanism, we report a preliminary analysis of (methyl- d 3) methionine residues within dihydrofolate reductase. Specifically, we characterize the IR absorptions at Met16 and Met20, within the catalytically important Met20 loop, and Met42, which is located within the hydrophobic core of the enzyme. The results confirm the sensitivity of the carbon-deuterium bonds to their local protein environment, demonstrate that dihydrofolate reductase is electrostatically and dynamically heterogeneous, and lay the foundation for the direct characterization protein electrostatics and dynamics and, potentially, their contribution to catalysis.

  8. 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.

  9. Seven novel mutations in the methylenetetrahydrofolate reductase gene and genotype/phenotype correlations in severe methylenetetrahydrofolate reductase deficiency.

    OpenAIRE

    Goyette, P; Frosst, P.; Rosenblatt, D S; ROZEN, R.

    1995-01-01

    5-Methyltetrahydrofolate, the major form of folate in plasma, is a carbon donor for the remethylation of homocysteine to methionine. This form of folate is generated from 5,10-methylenetetrahydrofolate through the action of 5,10-methylenetetrahydrofolate reductase (MTHFR), a cytosolic flavoprotein. Patients with an autosomal recessive severe deficiency of MTHFR have homocystinuria and a wide range of neurological and vascular disturbances. We have recently described the isolation of a cDNA fo...

  10. Hepatocyte Hyperproliferation upon Liver-Specific Co-disruption of Thioredoxin-1, Thioredoxin Reductase-1, and Glutathione Reductase

    Directory of Open Access Journals (Sweden)

    Justin R. Prigge

    2017-06-01

    Full Text Available Energetic nutrients are oxidized to sustain high intracellular NADPH/NADP+ ratios. NADPH-dependent reduction of thioredoxin-1 (Trx1 disulfide and glutathione disulfide by thioredoxin reductase-1 (TrxR1 and glutathione reductase (Gsr, respectively, fuels antioxidant systems and deoxyribonucleotide synthesis. Mouse livers lacking both TrxR1 and Gsr sustain these essential activities using an NADPH-independent methionine-consuming pathway; however, it remains unclear how this reducing power is distributed. Here, we show that liver-specific co-disruption of the genes encoding Trx1, TrxR1, and Gsr (triple-null causes dramatic hepatocyte hyperproliferation. Thus, even in the absence of Trx1, methionine-fueled glutathione production supports hepatocyte S phase deoxyribonucleotide production. Also, Trx1 in the absence of TrxR1 provides a survival advantage to cells under hyperglycemic stress, suggesting that glutathione, likely via glutaredoxins, can reduce Trx1 disulfide in vivo. In triple-null livers like in many cancers, deoxyribonucleotide synthesis places a critical yet relatively low-volume demand on these reductase systems, thereby favoring high hepatocyte turnover over sustained hepatocyte integrity.

  11. Selenite reduction by Shewanella oneidensis MR-1 is mediated by fumarate reductase in periplasm

    Science.gov (United States)

    Li, Dao-Bo; Cheng, Yuan-Yuan; Wu, Chao; Li, Wen-Wei; Li, Na; Yang, Zong-Chuang; Tong, Zhong-Hua; Yu, Han-Qing

    2014-01-01

    In situ reduction of selenite to elemental selenium (Se(0)), by microorganisms in sediments and soils is an important process and greatly affects the environmental distribution and the biological effects of selenium. However, the mechanism behind such a biological process remains unrevealed yet. Here we use Shewanella oneidensis MR-1, a widely-distributed dissimilatory metal-reducing bacterium with a powerful and diverse respiration capability, to evaluate the involvement of anaerobic respiration system in the microbial selenite reduction. With mutants analysis, we identify fumarate reductase FccA as the terminal reductase of selenite in periplasm. Moreover, we find that such a reduction is dependent on central respiration c-type cytochrome CymA. In contrast, nitrate reductase, nitrite reductase, and the Mtr electron transfer pathway do not work as selenite reductases. These findings reveal a previously unrecognized role of anaerobic respiration reductases of S. oneidensis MR-1 in selenite reduction and geochemical cycles of selenium in sediments and soils.

  12. Induction of NADPH-Cytochroune P-450 (c) Reductase in Wounded Tissues from Helianthus tuberosus Tubers

    OpenAIRE

    Agnes, Lesot; Irene, Benveniste; Marie-Paule, Hasenfratz; Francis, Durst; C.N.R.S.,Institut de Biologie Moleculaire des Plantes. Departement d'Enzymologie Cellulaire et Moieculaire, Institut de Botanique

    1990-01-01

    Cytochrome P-450 is not self-sufficient for the catalysis of monooxygenase reaction but requires NADPH and NADPH-cytochrome P-450 (c) reductase. The activity of NADPH-cytochrome P-450 reductase was strongly enhanced by wounding and aging in Jerusalem artichoke (Helianthus tuberosus L.) tuber tissues. This stimulation was correlated with the synthesis of the enzyme protein based on i) quantitation of the reductase protein by Western blotting, ii) incor-poration of [^S]methionine into the immun...

  13. A substrate-bound structure of cyanobacterial biliverdin reductase identifies stacked substrates as critical for activity

    OpenAIRE

    Takao, Haruna; Hirabayashi, Kei; Nishigaya, Yuki; Kouriki, Haruna; Nakaniwa, Tetsuko; Hagiwara, Yoshinori; Harada, Jiro; Sato, Hideaki; Yamazaki, Toshimasa; Sakakibara, Yoichi; Suiko, Masahito; Asada, Yujiro; Takahashi, Yasuhiro; Yamamoto, Ken; Fukuyama, Keiichi

    2017-01-01

    Biliverdin reductase catalyses the last step in haem degradation and produces the major lipophilic antioxidant bilirubin via reduction of biliverdin, using NAD(P)H as a cofactor. Despite the importance of biliverdin reductase in maintaining the redox balance, the molecular details of the reaction it catalyses remain unknown. Here we present the crystal structure of biliverdin reductase in complex with biliverdin and NADP+. Unexpectedly, two biliverdin molecules, which we designated the proxim...

  14. 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.

  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

    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. The mechanism of the quinone reductase reaction of pig heart lipoamide dehydrogenase.

    Science.gov (United States)

    Vienozinskis, J; Butkus, A; Cenas, N; Kulys, J

    1990-01-01

    The relationship between the NADH:lipoamide reductase and NADH:quinone reductase reactions of pig heart lipoamide dehydrogenase (EC 1.6.4.3) was investigated. At pH 7.0 the catalytic constant of the quinone reductase reaction (kcat.) is 70 s-1 and the rate constant of the active-centre reduction by NADH (kcat./Km) is 9.2 x 10(5) M-1.s-1. These constants are almost an order lower than those for the lipoamide reductase reaction. The maximal quinone reductase activity is observed at pH 6.0-5.5. The use of [4(S)-2H]NADH as substrate decreases kcat./Km for the lipoamide reductase reaction and both kcat. and kcat./Km for the quinone reductase reaction. The kcat./Km values for quinones in this case are decreased 1.85-3.0-fold. NAD+ is a more effective inhibitor in the quinone reductase reaction than in the lipoamide reductase reaction. The pattern of inhibition reflects the shift of the reaction equilibrium. Various forms of the four-electron-reduced enzyme are believed to reduce quinones. Simple and 'hybrid ping-pong' mechanisms of this reaction are discussed. The logarithms of kcat./Km for quinones are hyperbolically dependent on their single-electron reduction potentials (E1(7]. A three-step mechanism for a mixed one-electron and two-electron reduction of quinones by lipoamide dehydrogenase is proposed. PMID:2375745

  17. 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.

  18. 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.

  19. Methylenetetrahydrofolate Reductase C677T: Hypoplastic Left Heart and Thrombosis.

    Science.gov (United States)

    Spronk, Kimberly J; Olivero, Anthony D; Haw, Marcus P; Vettukattil, Joseph J

    2015-10-01

    The incidence of congenital heart defects is higher in infants with mutation of methylenetetrahydrofolate reductase (MTHFR) gene. The MTHFR C677T gene decreases the bioavailability of folate and increases plasma homocysteine, a risk factor for thrombosis. There have been no reported cases in the literature on the clinical implications of this procoagulable state in the setting of cyanotic heart disease, which itself has prothrombotic predisposition. Two patients with hypoplastic left heart syndrome developed postoperative thrombotic complications, both were homozygous for MTHFR C677T. We present these cases and highlight the implications of MTHFR mutation in the management of complex congenital heart disease. © The Author(s) 2015.

  20. 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...

  1. 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...

  2. Mutational analysis of the nor gene cluster which encodes nitric-oxide reductase from Paracoccus denitrificans

    NARCIS (Netherlands)

    de Boer, A P; van der Oost, J.; Reijnders, W N; Westerhoff, H V; Stouthamer, A.H.; van Spanning, R J

    1996-01-01

    The genes that encode the hc-type nitric-oxide reductase from Paracoccus denitrificans have been identified. They are part of a cluster of six genes (norCBQDEF) and are found near the gene cluster that encodes the cd1-type nitrite reductase, which was identified earlier [de Boer, A. P. N.,

  3. 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.

  4. Dimethyl Fumarate Induces Glutathione Recycling by Upregulation of Glutathione Reductase.

    Science.gov (United States)

    Hoffmann, Christina; Dietrich, Michael; Herrmann, Ann-Kathrin; Schacht, Teresa; Albrecht, Philipp; Methner, Axel

    2017-01-01

    Neuronal degeneration in multiple sclerosis has been linked to oxidative stress. Dimethyl fumarate (DMF) is an effective oral therapeutic option shown to reduce disease activity and progression in patients with relapsing-remitting multiple sclerosis. DMF activates the transcription factor nuclear factor erythroid 2-related factor 2 (NRF2) leading to increased synthesis of the major cellular antioxidant glutathione (GSH) and prominent neuroprotection in vitro . We previously demonstrated that DMF is capable of raising GSH levels even when glutathione synthesis is inhibited, suggesting enhanced GSH recycling. Here, we found that DMF indeed induces glutathione reductase (GSR), a homodimeric flavoprotein that catalyzes GSSG reduction to GSH by using NADPH as a reducing cofactor. Knockdown of GSR using a pool of E. coli RNase III-digested siRNAs or pharmacological inhibition of GSR, however, also induced the antioxidant response rendering it impossible to verify the suspected attenuation of DMF-mediated neuroprotection. However, in cystine-free medium, where GSH synthesis is abolished, pharmacological inhibition of GSR drastically reduced the effect of DMF on glutathione recycling. We conclude that DMF increases glutathione recycling through induction of glutathione reductase.

  5. Dimethyl Fumarate Induces Glutathione Recycling by Upregulation of Glutathione Reductase

    Directory of Open Access Journals (Sweden)

    Christina Hoffmann

    2017-01-01

    Full Text Available Neuronal degeneration in multiple sclerosis has been linked to oxidative stress. Dimethyl fumarate (DMF is an effective oral therapeutic option shown to reduce disease activity and progression in patients with relapsing-remitting multiple sclerosis. DMF activates the transcription factor nuclear factor erythroid 2-related factor 2 (NRF2 leading to increased synthesis of the major cellular antioxidant glutathione (GSH and prominent neuroprotection in vitro. We previously demonstrated that DMF is capable of raising GSH levels even when glutathione synthesis is inhibited, suggesting enhanced GSH recycling. Here, we found that DMF indeed induces glutathione reductase (GSR, a homodimeric flavoprotein that catalyzes GSSG reduction to GSH by using NADPH as a reducing cofactor. Knockdown of GSR using a pool of E. coli RNase III-digested siRNAs or pharmacological inhibition of GSR, however, also induced the antioxidant response rendering it impossible to verify the suspected attenuation of DMF-mediated neuroprotection. However, in cystine-free medium, where GSH synthesis is abolished, pharmacological inhibition of GSR drastically reduced the effect of DMF on glutathione recycling. We conclude that DMF increases glutathione recycling through induction of glutathione reductase.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. Monodehydroascorbate reductase 2 and dehydroascorbate reductase 5 are crucial for a mutualistic interaction between Piriformospora indica and Arabidopsis.

    Science.gov (United States)

    Vadassery, Jyothilakshmi; Tripathi, Swati; Prasad, Ram; Varma, Ajit; Oelmüller, Ralf

    2009-08-15

    Ascorbate is a major antioxidant and radical scavenger in plants. Monodehydroascorbate reductase (MDAR) and dehydroascorbate reductase (DHAR) are two enzymes of the ascorbate-glutathione cycle that maintain ascorbate in its reduced state. MDAR2 (At3g09940) and DHAR5 (At1g19570) expression was upregulated in the roots and shoots of Arabidopsis seedlings co-cultivated with the root-colonizing endophytic fungus Piriformospora indica, or that were exposed to a cell wall extract or a culture filtrate from the fungus. Growth and seed production were not promoted by Piriformospora indica in mdar2 (SALK_0776335C) and dhar5 (SALK_029966C) T-DNA insertion lines, while colonized wild-type plants were larger and produced more seeds compared to the uncolonized controls. After 3 weeks of drought stress, growth and seed production were reduced in Piriformospora indica-colonized plants compared to the uncolonized control, and the roots of the drought-stressed insertion lines were colonized more heavily by the fungus than were wild-type plants. Upregulation of the message for the antimicrobial PDF1.2 protein in drought-stressed insertion lines indicated that MDAR2 and DHAR5 are crucial for producing sufficient ascorbate to maintain the interaction between Piriformospora indica and Arabidopsis in a mutualistic state.

  13. 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...

  14. 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.

  15. Go Green: The Antiinflammatory Effects of Biliverdin Reductase

    Directory of Open Access Journals (Sweden)

    Barbara eWegiel

    2012-03-01

    Full Text Available Biliverdin (BV has emerged as a cytoprotective and important anti-inflammatory molecule. Conversion of BV to bilirubin (BR is catalyzed by biliverdin reductase (BVR and is required for the downstream signaling and nuclear localization of BVR. Recent data by others and us make clear that BVR is a critical regulator of innate immune responses resulting from acute insult and injury and moreover, that a lack of BVR results in an enhanced pro-inflammatory phenotype. In macrophages, BVR is regulated by its substrate BV which leads to activation of the PI3K-Akt-IL10 axis and inhibition of TLR4 expression via direct binding of BVR to the TLR4 promoter. In this review, we will summarize recent findings on the role of BVR and the bile pigments in inflammation in context with its activity as an enzyme, receptor and transcriptional regulator.

  16. 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...

  17. B-vitamins, methylenetetrahydrofolate reductase (MTHFR) and hypertension.

    Science.gov (United States)

    Ward, Mary; Wilson, Carol P; Strain, J J; Horigan, Geraldine; Scott, John M; McNulty, Helene

    2011-07-01

    Hypertension is a leading risk factor for cardiovascular disease (CVD) and stroke. A common polymorphism in the gene encoding the enzyme methylenetetrahydrofolate reductase (MTHFR), previously identified as the main genetic determinant of elevated homocysteine concentration and also recognized as a risk factor for CVD, appears to be independently associated with hypertension. The B-vitamin riboflavin is required as a cofactor by MTHFR and recent evidence suggests it may have a role in modulating blood pressure, specifically in those with the homozygous mutant MTHFR 677 TT genotype. If studies confirm that this genetic predisposition to hypertension is correctable by low-dose riboflavin, the findings could have important implications for the management of hypertension given that the frequency of this polymorphism ranges from 3 to 32 % worldwide.

  18. 5-Alpha-Reductase Inhibitors and Combination Therapy.

    Science.gov (United States)

    Füllhase, Claudius; Schneider, Marc P

    2016-08-01

    By inhibiting the conversion from testosterone to dihydrotestosterone 5-Alpha reductase inhibitors (5ARIs) are able to hinder prostatic growth, shrink prostate volumes, and improve BPH-related LUTS. 5ARIs are particularly beneficial for patients with larger prostates (>30-40ml). Generally the side effects of 5ARI treatment are mild, and according to the FORTA classification 5ARIs are suitable for frail elderly. 5ARI / alpha-blocker (AB) combination therapy showed the best symptomatic outcome and risk reduction for clinical progression. Combining Phosphodieseterase type 5 inhbibitors (PDE5Is) with 5ARIs counteracts the negative androgenic sexual side effects of 5ARIs, and simultaneously combines their synergistic effects on LUTS. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. Steroid 5β-Reductase from Leaves of Vitis vinifera: Molecular Cloning, Expression, and Modeling.

    Science.gov (United States)

    Ernst, Mona; Munkert, Jennifer; Campa, Manuela; Malnoy, Mickael; Martens, Stefan; Müller-Uri, Frieder

    2015-11-25

    A steroid 5β-reductase gene corresponding to the hypothetical protein LOC100247199 from leaves of Vitis vinifera (var. 'Chardonnay') was cloned and overexpressed in Escherichia coli. The recombinant protein showed 5β-reductase activity when progesterone was used as a substrate. The reaction was stereoselective, producing only 5β-products such as 5β-pregnane-3,20-dione. Other small substrates (terpenoids and enones) were also accepted as substrates, indicating the highly promiscuous character of the enzyme class. Our results show that the steroid 5β-reductase gene, encoding an orthologous enzyme described as a key enzyme in cardenolide biosynthesis, is also expressed in leaves of the cardenolide-free plant V. vinifera. We emphasize the fact that, on some occasions, different reductases (e.g., progesterone 5β-reductase and monoterpenoid reductase) can also use molecules that are similar to the final products as a substrate. Therefore, in planta, the different reductases may contribute to the immense number of diverse small natural products finally leading to the flavor of wine.

  20. Investigation of the antioxidant and aldose reductase inhibitory activities of extracts from Peruvian tea plant infusions.

    Science.gov (United States)

    Wang, Zhiqiang; Hwang, Seung Hwan; Guillen Quispe, Yanymee N; Gonzales Arce, Paul H; Lim, Soon Sung

    2017-09-15

    In the present study, the antioxidant and aldose reductase inhibitory activities of 24 Peruvian infusion tea plants were investigated by 2,2'-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and aldose reductase assays. Phoradendron sp. showed the highest inhibition of aldose reductase (IC 50 , 1.09±0.06μg/mL) and considerable antioxidant (IC 50 of DPPH, 58.36±1.65μg/mL; IC 50 of ABTS, 9.91±0.43μg/mL) effects. In order to identify the antioxidants and aldose reductase inhibitors of Phoradendron sp., DPPH-high performance liquid chromatography (HPLC) and ultrafiltration-HPLC assays were performed. Chlorogenic acid, 3,5-di-O-caffeoylquinic acid, and 1,3,5-tri-O-caffeoylquinic acid were identified as the antioxidants and aldose reductase inhibitors; apigenin was identified as the antioxidant. Finally, Phoradendron sp. and its aldose reductase inhibitors also showed a dose-dependent anti-inflammatory effect without cellular toxicity. These results suggested that Phoradendron sp. can be a potent functional food ingredient as an antioxidant, aldose reductase inhibitor and anti-inflammatory agent. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Multi-genome analysis identifies functional and phylogenetic diversity of basidiomycete adenylate-forming reductases.

    Science.gov (United States)

    Brandenburger, Eileen; Braga, Daniel; Kombrink, Anja; Lackner, Gerald; Gressler, Julia; Künzler, Markus; Hoffmeister, Dirk

    2016-07-22

    Among the invaluable benefits of basidiomycete genomics is the dramatically enhanced insight into the potential capacity to biosynthesize natural products. This study focuses on adenylate-forming reductases, which is a group of natural product biosynthesis enzymes that resembles non-ribosomal peptide synthetases, yet serves to modify one substrate, rather than to condense two or more building blocks. Phylogenetically, these reductases fall in four classes. The phylogeny of Heterobasidion annosum (Russulales) and Serpula lacrymans (Boletales) adenylate-forming reductases was investigated. We identified a previously unrecognized phylogenetic branch within class III adenylate-forming reductases. Three representatives were heterologously produced and their substrate preferences determined in vitro: NPS9 and NPS11 of S. lacrymans preferred l-threonine and benzoic acid, respectively, while NPS10 of H. annosum accepted phenylpyruvic acid best. We also investigated two class IV adenylate-forming reductases of Coprinopsis cinerea, which each were active with l-alanine, l-valine, and l-serine as substrates. Our results show that adenylate-forming reductases are functionally more diverse than previously recognized. As none of the natural products known from the species investigated in this study includes the identified substrates of their respective reductases, our findings may help further explore the diversity of these basidiomycete secondary metabolomes. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. 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.

  3. Nitrite and Nitrous Oxide Reductase Regulation by Nitrogen Oxides in Rhodobacter sphaeroides f. sp. denitrificans IL106

    Science.gov (United States)

    Sabaty, Monique; Schwintner, Carole; Cahors, Sandrine; Richaud, Pierre; Verméglio, Andre

    1999-01-01

    We have cloned the nap locus encoding the periplasmic nitrate reductase in Rhodobacter sphaeroides f. sp. denitrificans IL106. A mutant with this enzyme deleted is unable to grow under denitrifying conditions. Biochemical analysis of this mutant shows that in contrast to the wild-type strain, the level of synthesis of the nitrite and N2O reductases is not increased by the addition of nitrate. Growth under denitrifying conditions and induction of N oxide reductase synthesis are both restored by the presence of a plasmid containing the genes encoding the nitrate reductase. This demonstrates that R. sphaeroides f. sp. denitrificans IL106 does not possess an efficient membrane-bound nitrate reductase and that nitrate is not the direct inducer for the nitrite and N2O reductases in this species. In contrast, we show that nitrite induces the synthesis of the nitrate reductase. PMID:10498715

  4. 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...

  5. 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

  6. 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 ...

  7. Plasmodium falciparum dihydrofolate reductase alleles and pyrimethamine use in pregnant Ghanaian women

    NARCIS (Netherlands)

    Mockenhaupt, F. P.; Eggelte, T. A.; Böhme, T.; Thompson, W. N.; Bienzle, U.

    2001-01-01

    Drug resistance in Plasmodium falciparum affects prevention of malaria in pregnancy. In a cross-sectional study of 530 pregnant Ghanaian women, P. falciparum dihydrofolate reductase (DHFR) gene mutations linked with pyrimethamine resistance were assessed and associations with pyrimethamine intake

  8. 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

  9. 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...

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

    Science.gov (United States)

    2010-08-01

    aldo-keto reductase family protein AKR1B10 is highly correlated with smokers ’ non -small cell lung carcinomas. Clin Cancer Res 11: 1776-1785. Gallego...reductase family 1 B10 protein detoxifies dietary and lipid -derived alpha, beta-unsaturated carbonyls at physiological levels . Biochem Biophys Res...AKR1B10 expression in breast cancer, define the role of AKR1B10 in lipid metabolism, proliferation, and tumorigenicity of breast cancer cells using

  11. Tissue thioredoxin reductase-1 expression in astrocytomas of different grades.

    Science.gov (United States)

    Esen, Hasan; Erdi, Fatih; Kaya, Bulent; Feyzioglu, Bahadır; Keskin, Fatih; Demir, Lutfi Saltuk

    2015-02-01

    Thioredoxin (Trx) is a redox active protein that regulates several physiological and biochemical functions, such as growth, apoptosis and cellular defense. The function of Trx itself is regulated by thioredoxin reductase (TrxR). Studies performed in a variety of human primary tumors have shown that thioredoxin reductase 1 (TrxR1) is overexpressed in tumoral tissues compared with corresponding normal tissues. This study was designed to determine the expression of TrxR1 in astrocytoma tissues of different World Health Organization (WHO) grades (grade I-IV). The proliferative (Ki-67) and apoptotic indices of the specimens were also investigated for correlation analysis. Astrocytoma tissues were extracted from the histopathological specimens of 40 patients. These samples included seven histologically normal brain tissues that served as a control group and ten tumoral samples for each grade of astrocytoma (grade I-IV). The histologically normal brain tissues were obtained from the non-tumoral portions of the pathological specimens of grade I (2 cases), grade II (2 cases), grade III (2 cases) and grade IV (1 case) astrocytomas. TrxR1 expression was evaluated using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunostaining. The proliferative and apoptotic indices of the specimens were investigated by Ki-67 immunostaining and TUNEL assay, respectively. TrxR1 expression, as assessed by qRT-PCR, increased significantly with astrocytoma grade (p = 0.01). The immunostaining intensity of TrxR1 in grade IV astrocytomas was significantly greater than that in the control tissue and all other astrocytoma grades (p grade III astrocytomas was significantly greater than that in the control group and grade I astrocytomas (p grades, but the differences between grade I and the control, grade II and the control, grades II and I, grades III and II were not statistically significant (p > 0.05). Ki-67 index values increased significant in accordance with grade

  12. Physiological roles for two periplasmic nitrate reductases in Rhodobacter sphaeroides 2.4.3 (ATCC 17025).

    Science.gov (United States)

    Hartsock, Angela; Shapleigh, James P

    2011-12-01

    The metabolically versatile purple bacterium Rhodobacter sphaeroides 2.4.3 is a denitrifier whose genome contains two periplasmic nitrate reductase-encoding gene clusters. This work demonstrates nonredundant physiological roles for these two enzymes. One cluster is expressed aerobically and repressed under low oxygen while the second is maximally expressed under low oxygen. Insertional inactivation of the aerobically expressed nitrate reductase eliminated aerobic nitrate reduction, but cells of this strain could still respire nitrate anaerobically. In contrast, when the anaerobic nitrate reductase was absent, aerobic nitrate reduction was detectable, but anaerobic nitrate reduction was impaired. The aerobic nitrate reductase was expressed but not utilized in liquid culture but was utilized during growth on solid medium. Growth on a variety of carbon sources, with the exception of malate, the most oxidized substrate used, resulted in nitrite production on solid medium. This is consistent with a role for the aerobic nitrate reductase in redox homeostasis. These results show that one of the nitrate reductases is specific for respiration and denitrification while the other likely plays a role in redox homeostasis during aerobic growth.

  13. Androgen regulation of 5α-reductase isoenzymes in prostate cancer: implications for prostate cancer prevention.

    Directory of Open Access Journals (Sweden)

    Jin Li

    Full Text Available The enzyme 5α-reductase, which converts testosterone to dihydrotestosterone (DHT, performs key functions in the androgen receptor (AR signaling pathway. The three isoenzymes of 5α-reductase identified to date are encoded by different genes: SRD5A1, SRD5A2, and SRD5A3. In this study, we investigated mechanisms underlying androgen regulation of 5α-reductase isoenzyme expression in human prostate cells. We found that androgen regulates the mRNA level of 5α-reductase isoenzymes in a cell type-specific manner, that such regulation occurs at the transcriptional level, and that AR is necessary for this regulation. In addition, our results suggest that AR is recruited to a negative androgen response element (nARE on the promoter of SRD5A3 in vivo and directly binds to the nARE in vitro. The different expression levels of 5α-reductase isoenzymes may confer response or resistance to 5α-reductase inhibitors and thus may have importance in prostate cancer prevention.

  14. Purification and characterization of NADPH-cytochrome P450 reductase from filamentous fungus Rhizopus nigricans.

    Science.gov (United States)

    Makovec, T; Breskvar, K

    1998-09-15

    We report here the isolation and partial characterization of a flavoprotein, NADPH-cytochrome P450 (cytochrome c) reductase. The enzyme is a part of steroid 11 alpha-hydroxylating system and is associated with the microsomal fraction of the fungus Rhizopus nigricans. Fungal reductase was solubilized from microsomal membranes with Triton X-100 and purified to apparent homogeneity by affinity and high-performance ion-exchange chromatography. A 350-fold purification of the enzyme with specific activity of 37 mumol cytochrome c reduced/min/mg protein was achieved. A single protein band was obtained on SDS-PAGE analysis with an apparent molecular weight of 79 kDa. Purified reductase contained approximately equimolar quantities of flavin adenine dinucleotide and flavin mononucleotide per mole of the enzyme. Upon induction of the steroid hydroxylating system with progesterone the activity of microsomal NADPH-cytochrome c (P450) reductase increased 10-fold. This is in good correlation with the increase in content of fungal cytochrome P450. Purified fungal flavoprotein was active in a reconstituted system with cytochrome P450 C21 from adrenal gland but could not replace adrenodoxin reductase in the mitochondrial steroid 11 beta-hydroxylating system. We were able to confirm the role of the enzyme by reconstituting steroid 11 alpha-hydroxylating activity from the separated components NADPH-cytochrome P450 reductase and cytochrome P450, partly purified from fungal microsomes.

  15. X-ray structure of trypanothione reductase from Crithidia fasciculata at 2. 4- angstrom resolution

    Energy Technology Data Exchange (ETDEWEB)

    Kuriyan, J.; Xiangpeng Kong; Krishna, T.S.R.; Murgolo, N.J.; Field, H.; Cerami, A.; Henderson, G.B. (Rockefeller Univ., New York, NY (United States)); Sweet, R.M. (Brookhaven National Lab., Upton, NY (United States))

    1991-10-01

    Trypanosomes and related protozoan parasites lack glutathione reductase and possess instead a closely related enzyme that serves as the reductant of a bis(glutathione)-spermidien conjugate, trypanothione. The human and parasite enzymes have mutually exclusive substrate specificities, providing a route for the design of therapeutic agents by specific inhibition of the parasite enzyme. The authors report here the three-dimensional structure of trypanothione reductase from Crithidia fasciculata and show that it closely resembles the structure of human glutathione reductase. In particular, the core structure surrounding the catalytic machinery is almost identical in the two enzymes. However, significant differences are found at the substrate binding sites. A cluster of basic residues in glutathione reductase is replaced by neutral, hydrophobic, or acidic residues in trypanothione reductase, consistent with the nature of the spermidine linkage and the change in overall charge of the substrate from {minus}2 to +1, respectively. The binding site is more open in trypanothione reductase due to rotations of about 4{degree} in the domains that form in site, with relative shifts of as much as 2-3 {angstrom} in residues that can interact with potential inhibitors and complement previous modeling and mutagenesis studies on the two enzymes.

  16. Binding of Natural and Synthetic Polyphenols to Human Dihydrofolate Reductase

    Directory of Open Access Journals (Sweden)

    José Neptuno Rodríguez-López

    2009-12-01

    Full Text Available Dihydrofolate reductase (DHFR is the subject of intensive investigation since it appears to be the primary target enzyme for antifolate drugs. Fluorescence quenching experiments show that the ester bond-containing tea polyphenols (--epigallocatechin gallate (EGCG and (--epicatechin gallate (ECG are potent inhibitors of DHFR with dissociation constants (KD of 0.9 and 1.8 μM, respectively, while polyphenols lacking the ester bound gallate moiety [e.g., (--epigallocatechin (EGC and (--epicatechin (EC] did not bind to this enzyme. To avoid stability and bioavailability problems associated with tea catechins we synthesized a methylated derivative of ECG (3-O-(3,4,5-trimethoxybenzoyl-(--epicatechin; TMECG, which effectively binds to DHFR (KD = 2.1 μM. In alkaline solution, TMECG generates a stable quinone methide product that strongly binds to the enzyme with a KD of 8.2 nM. Quercetin glucuronides also bind to DHFR but its effective binding was highly dependent of the sugar residue, with quercetin-3-xyloside being the stronger inhibitor of the enzyme with a KD of 0.6 μM. The finding that natural polyphenols are good inhibitors of human DHFR could explain the epidemiological data on their prophylactic effects for certain forms of cancer and open a possibility for the use of natural and synthetic polyphenols in cancer chemotherapy.

  17. Arabidopsis thaliana dehydroascorbate reductase 2: Conformational flexibility during catalysis

    Science.gov (United States)

    Bodra, Nandita; Young, David; Astolfi Rosado, Leonardo; Pallo, Anna; Wahni, Khadija; de Proft, Frank; Huang, Jingjing; van Breusegem, Frank; Messens, Joris

    2017-02-01

    Dehydroascorbate reductase (DHAR) catalyzes the glutathione (GSH)-dependent reduction of dehydroascorbate and plays a direct role in regenerating ascorbic acid, an essential plant antioxidant vital for defense against oxidative stress. DHAR enzymes bear close structural homology to the glutathione transferase (GST) superfamily of enzymes and contain the same active site motif, but most GSTs do not exhibit DHAR activity. The presence of a cysteine at the active site is essential for the catalytic functioning of DHAR, as mutation of this cysteine abolishes the activity. Here we present the crystal structure of DHAR2 from Arabidopsis thaliana with GSH bound to the catalytic cysteine. This structure reveals localized conformational differences around the active site which distinguishes the GSH-bound DHAR2 structure from that of DHAR1. We also unraveled the enzymatic step in which DHAR releases oxidized glutathione (GSSG). To consolidate our structural and kinetic findings, we investigated potential conformational flexibility in DHAR2 by normal mode analysis and found that subdomain mobility could be linked to GSH binding or GSSG release.

  18. Reductive activation of E. coli respiratory nitrate reductase.

    Science.gov (United States)

    Ceccaldi, Pierre; Rendon, Julia; Léger, Christophe; Toci, René; Guigliarelli, Bruno; Magalon, Axel; Grimaldi, Stéphane; Fourmond, Vincent

    2015-10-01

    Over the past decades, a number of authors have reported the presence of inactive species in as-prepared samples of members of the Mo/W-bisPGD enzyme family. This greatly complicated the spectroscopic studies of these enzymes, since it is impossible to discriminate between active and inactive species on the basis of the spectroscopic signatures alone. Escherichia coli nitrate reductase A (NarGHI) is a member of the Mo/W-bisPGD family that allows anaerobic respiration using nitrate as terminal electron acceptor. Here, using protein film voltammetry on NarGH films, we show that the enzyme is purified in a functionally heterogeneous form that contains between 20 and 40% of inactive species that activate the first time they are reduced. This activation proceeds in two steps: a non-redox reversible reaction followed by an irreversible reduction. By carefully correlating electrochemical and EPR spectroscopic data, we show that neither the two major Mo(V) signals nor those of the two FeS clusters that are the closest to the Mo center are associated with the two inactive species. We also conclusively exclude the possibility that the major "low-pH" and "high-pH" Mo(V) EPR signatures correspond to species in acid-base equilibrium. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. 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.

  20. 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.

  1. 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

  2. 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.

  3. 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.

  4. DFT Based QSAR Study of Enzyme Ribonucleoside Diphosphate Reductase

    Directory of Open Access Journals (Sweden)

    Mohiuddin Ansari

    2010-01-01

    Full Text Available Quantum chemical descriptors such as heat of formation, energy of HOMO, total energy, absolute hardness and chemical potential in different combinations have been used to develop QSAR models of inhibitors of enzyme ribonucleoside diphosphate reductase, RDR. The inhibitors are mainly derivatives of 1-formylisoquinoline thiosemicarbazone and 2-formylpyridine thiosemicarbazone. The values of various descriptors have been evaluated with the help of Win MOPAC 7.21 software using DFT method. Multiple linear regression analysis has been made with the help of above mentioned descriptors using the same software. Regression equations have been found to be successful models as indicated by the regression coefficient r2 having the value as high as 0.96 and cross validation coefficient rCV2 having the value approaching 0.95. The value of these two coefficients is indicative of high order of reliability for the proposed prediction. The results obtained are also validated on account of the closeness of observed and predicted inhibitory activities. The best combination of descriptors is heat of formation, total energy and energy of HOMO. Thus the prediction of suitability of inhibitors of the enzyme RDR can be made with the help of the best regression equation.

  5. Methylenetetrahydrofolate reductase (MTHFR) deficiency enhances resistance against cytomegalovirus infection.

    Science.gov (United States)

    Fodil-Cornu, N; Kozij, N; Wu, Q; Rozen, R; Vidal, S M

    2009-10-01

    Folates provide one-carbon units for nucleotide synthesis and methylation reactions. A common polymorphism in the MTHFR gene (677C --> T) results in reduced enzymatic activity, and is associated with an increased risk for neural tube defects and cardiovascular disease. The high prevalence of this polymorphism suggests that it may have experienced a selective advantage under environmental pressure, possibly an infectious agent. To test the hypothesis that methylenetetrahydrofolate reductase (MTHFR) genotype influences the outcome of infectious disease, we examined the response of Mthfr-deficient mice against mouse cytomegalovirus (MCMV) infection. Acute MCMV infection of Mthfr(-/-) mice resulted in early control of cytokine secretion, decreased viral titer and preservation of spleen immune cells, in contrast to Mthfr wild-type littermates. The phenotype was abolished in MTHFR transgenic mice carrying an extra copy of the gene. Infection of primary fibroblasts with MCMV showed a decrease in viral replication and in the number of productively infected cells in Mthfr(+/-) fibroblasts compared with wild-type cells. These results indicate that Mthfr deficiency protects against MCMV infection in vivo and in vitro, suggesting that human genetic variants may provide an advantage in the host response against certain pathogens.

  6. Prognostic Relevance of Methylenetetrahydrofolate Reductase Polymorphisms for Prostate Cancer.

    Science.gov (United States)

    Lin, Victor C; Lu, Te-Ling; Yin, Hsin-Ling; Yang, Sheau-Fang; Lee, Yung-Chin; Liu, Chia-Chu; Huang, Chao-Yuan; Yu, Chia-Cheng; Chang, Ta-Yuan; Huang, Shu-Pin; Bao, Bo-Ying

    2016-11-29

    Folate metabolism has been associated with cancers via alterations in nucleotide synthesis, DNA methylation, and DNA repair. We hypothesized that genetic variants in methylenetetrahydrofolate reductase (MTHFR), a key enzyme of folate metabolism, would affect the prognosis of prostate cancer. Three haplotype-tagging single-nucleotide polymorphisms (SNPs) across the MTHFR gene region were genotyped in a cohort of 458 patients with clinically localized prostate cancer treated with radical prostatectomy. One SNP, rs9651118, was associated with disease recurrence, and the association persisted after multivariate analyses adjusting for known risk factors. Public dataset analyses suggested that rs9651118 affects MTHFR expression. Quantitative real-time polymerase chain reaction analysis revealed that MTHFR expression is significantly upregulated in prostate tumor tissues when compared with adjacent normal tissues. Furthermore, overexpression of MTHFR correlates with cancer recurrence and death in two independent publicly available prostate cancer datasets. In conclusion, our data provide rationale to further validate the clinical utility of MTHFR rs9651118 as a biomarker for prognosis in prostate cancer.

  7. Studies on aldose reductase inhibitors from natural products. IV. Constituents and aldose reductase inhibitory effect of Chrysanthemum morifolium, Bixa orellana and Ipomoea batatas.

    Science.gov (United States)

    Terashima, S; Shimizu, M; Horie, S; Morita, N

    1991-12-01

    The hot water extracts of Chrysanthemum morifolium, Bixa orellana and Ipomoea batatas, were found to have potent inhibitory activity towards lens aldose reductase (AR). Ellagic acid (4) was isolated from C. morifolium and I. batatas, isoscutellarein (7) from B. orellana and 3,5-dicaffeoylquinic acid (10) from I. batatas, respectively, as potent inhibitors.

  8. Seven novel mutations in the methylenetetrahydrofolate reductase gene and genotype/phenotype correlations in severe methylenetetrahydrofolate reductase deficiency

    Energy Technology Data Exchange (ETDEWEB)

    Goyette, P.; Frosst, P.; Rosenblatt, D.S.; Rozen. R. [McGill Univ., Montreal (Canada)

    1995-05-01

    5-Methyltetrahydrofolate, the major form of folate in plasma, is a carbon donor for the remethylation of homocysteine to methionine. This form of folate is generated from 5,10-methylenetetrahydrofolate through the action of 5,10-methylenetetrahydrofolate reductase (MTHFR), a cytosolic flavoprotein. Patients with an autosomal recessive severe deficiency of MTHFR have homocystinuria and a wide range of neurological and vascular disturbances. We have recently described the isolation of a cDNA for MTHFR and the identification of two mutations in patients with severe MTHFR deficiency. We report here the characterization of seven novel mutations in this gene: six missense mutations and a 5{prime} splice-site defect that activates a cryptic splice in the coding sequence. We also present a preliminary analysis of the relationship between genotype and phenotype for all nine mutations identified thus far in this gene. A nonsense mutation and two missense mutations (proline to leucine and threonine to methionine) in the homozygous state are associated with extremely low activity (0%-3%) and onset of symptoms within the 1st year of age. Other missense mutations (arginine to cysteine and arginine to glutamine) are associated with higher enzyme activity and later onset of symptoms. 19 refs., 4 figs., 2 tabs.

  9. Recombinant bovine dihydrofolate reductase produced by mutagenesis and nested PCR of murine dihydrofolate reductase cDNA.

    Science.gov (United States)

    Cody, Vivian; Mao, Qilong; Queener, Sherry F

    2008-11-01

    Recent reports of the slow-tight binding inhibition of bovine liver dihydrofolate reductase (bDHFR) in the presence of polyphenols isolated from green tea leaves has spurred renewed interest in the biochemical properties of bDHFR. Earlier studies were done with native bDHFR but in order to validate models of polyphenol binding to bDHFR, larger quantities of bDHFR are necessary to support structural studies. Bovine DHFR differs from its closest sequence homologue, murine DHFR, by 19 amino acids. To obtain the bDHFR cDNA, murineDHFR cDNA was transformed by a series of nested PCRs to reproduce the amino acid coding sequence for bovine DHFR. The bovine liver DHFR cDNA has an open reading frame of 561 base pairs encoding a protein of 187 amino acids that has a high level of conservation at the primary sequence level with other DHFR enzymes, and more so for the amino acid residues in the active site of the mammalian DHFR enzymes. Expression of the bovine DHFR cDNA in bacterial cells produced a stable recombinant protein with high enzymatic activity and kinetic properties similar to those previously reported for the native protein.

  10. Nitrous oxide reduction by members of the family Rhodospirillaceae and the nitrous oxide reductase of Rhodopseudomonas capsulata.

    OpenAIRE

    McEwan, A G; Greenfield, A J; Wetzstein, H G; Jackson, J B; Ferguson, S J

    1985-01-01

    After growth in the absence of nitrogenous oxides under anaerobic phototrophic conditions, several strains of Rhodopseudomonas capsulata were shown to possess a nitrous oxide reductase activity. The enzyme responsible for this activity had a periplasmic location and resembled a nitrous oxide reductase purified from Pseudomonas perfectomarinus. Electron flow to nitrous oxide reductase was coupled to generation of a membrane potential and inhibited by rotenone but not antimycin. It is suggested...

  11. 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

  12. Resolution and reconstitution of the NADPH-cytochrome c (P-450) reductase induced by progesterone in Rhizopus nigricans.

    Science.gov (United States)

    Cresnar, B; Breskvar, K; Hudnik-Plevnik, T

    1985-12-31

    The NADPH-cytochrome c (P-450) reductase induced in the filamentous fungus Rhizopus nigricans as a component of 11 alpha-hydroxylase of progesterone was resolved by DEAE-cellulose chromatography into two components. One of the components is an iron-sulfur protein (rhizoporedoxin), whereas the other component is a protein with reductase activity dependent on NADPH (rhizoporedoxin reductase). As shown in the reconstitution assay, the NADPH-cytochrome c (P-450) reductase activity was restored upon combination of these two proteins.

  13. Thioredoxin reductase induction coincides with melanin biosynthesis in brown and black guinea pigs and in murine melanoma cells.

    Science.gov (United States)

    Schallreuter, K U; Lemke, K R; Hill, H Z; Wood, J M

    1994-12-01

    X-rays were used to induce melanin biosynthesis in brown and black guinea pigs in vivo. During the course of pigmentation, the expression of thioredoxin reductase was increased, whereas for the other antioxidant enzymes, superoxide dismutase (cytosol Cu/Zn-enzyme), catalase, and glutathione reductase, levels and activities decreased. Isobutylmethylxanthine induced eumelanin biosynthesis in murine melanoma cells (Cloudman S-91). In these cells, thioredoxin reductase levels coincided with melanogenesis. Our results suggest that both tyrosinase and thioredoxin reductase respond to oxidative stress in the epidermis as well as in melanoma cells and react with superoxide anion radicals to stimulate melanogenesis and to prevent peroxidative damage, respectively.

  14. Diffuse multicystic encephalomalacia in a preterm baby due to homozygous methylenetetrahydrofolate reductase 677 C-->T mutation.

    Science.gov (United States)

    Aygun, Canan; Tanyeri, Bilge; Ceyhan, Meltem; Bagci, Hasan; Kucukoduk, Sukru

    2008-06-01

    Methylenetetrahydrofolate reductase catalyzes the formation of 5-methyltetrahydrofolate from 5,10-methylentetrahydrofolate and produces folate for the methylation of homocysteine to methionine. Due to insufficient conversion of homocysteine to methionine, plasma homocysteine levels increase in methylenetetrahydrofolate reductase deficiency. Homocysteine is an amino acid that contains a neurotoxic sulfur molecule and can induce neuronal apoptosis. Methylenetetrahydrofolate reductase deficiency is 1 of the etiological factors that causes neurological symptoms and signs in the newborn and childhood period. Here, we report a premature baby with prenatal onset diffuse multicystic encephalomalacia and cerebellar atrophy due to homozygous methylenetetrahydrofolate reductase mutation.

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. Cyanobacterial sulfide-quinone reductase: cloning and heterologous expression.

    Science.gov (United States)

    Bronstein, M; Schütz, M; Hauska, G; Padan, E; Shahak, Y

    2000-06-01

    The gene encoding sulfide-quinone reductase (SQR; E.C.1.8.5.'), the enzyme catalyzing the first step of anoxygenic photosynthesis in the filamentous cyanobacterium Oscillatoria limnetica, was cloned by use of amino acid sequences of tryptic peptides as well as sequences conserved in the Rhodobacter capsulatus SQR and in an open reading frame found in the genome of Aquifex aeolicus. SQR activity was also detected in the unicellular cyanobacterium Aphanothece halophytica following sulfide induction, with a V(max) of 180 micromol of plastoquinone-1 (PQ-1) reduced/mg of chlorophyll/h and apparent K(m) values of 20 and 40 microM for sulfide and quinone, respectively. Based on the conserved sequences, the gene encoding A. halophytica SQR was also cloned. The SQR polypeptides deduced from the two cyanobacterial genes consist of 436 amino acids for O. limnetica SQR and 437 amino acids for A. halophytica SQR and show 58% identity and 74% similarity. The calculated molecular mass is about 48 kDa for both proteins; the theoretical isoelectric points are 7.7 and 5.6 and the net charges at a neutral pH are 0 and -14 for O. limnetica SQR and A. halophytica SQR, respectively. A search of databases showed SQR homologs in the genomes of the cyanobacterium Anabaena PCC7120 as well as the chemolithotrophic bacteria Shewanella putrefaciens and Thiobacillus ferrooxidans. All SQR enzymes contain characteristic flavin adenine dinucleotide binding fingerprints. The cyanobacterial proteins were expressed in Escherichia coli under the control of the T7 promoter. Membranes isolated from E. coli cells expressing A. halophytica SQR performed sulfide-dependent PQ-1 reduction that was sensitive to the quinone analog inhibitor 2n-nonyl-4-hydroxyquinoline-N-oxide. The wide distribution of SQR genes emphasizes the important role of SQR in the sulfur cycle in nature.

  2. Prokaryotic arsenate reductase enhances arsenate resistance in Mammalian cells.

    Science.gov (United States)

    Wu, Dan; Tao, Xuanyu; Wu, Gaofeng; Li, Xiangkai; Liu, Pu

    2014-01-01

    Arsenic is a well-known heavy metal toxicant in the environment. Bioremediation of heavy metals has been proposed as a low-cost and eco-friendly method. This article described some of recent patents on transgenic plants with enhanced heavy metal resistance. Further, to test whether genetic modification of mammalian cells could render higher arsenic resistance, a prokaryotic arsenic reductase gene arsC was transfected into human liver cancer cell HepG2. In the stably transfected cells, the expression level of arsC gene was determined by quantitative real-time PCR. Results showed that arsC was expressed in HepG2 cells and the expression was upregulated by 3 folds upon arsenate induction. To further test whether arsC has function in HepG2 cells, the viability of HepG2-pCI-ArsC cells exposed to arsenite or arsenate was compared to that of HepG2-pCI cells without arsC gene. The results indicated that arsC increased the viability of HepG2 cells by 25% in arsenate, but not in arsenite. And the test of reducing ability of stably transfected cells revealed that the concentration of accumulated trivalent arsenic increased by 25% in HepG2-pCI-ArsC cells. To determine the intracellular localization of ArsC, a fusion vector with fluorescent marker pEGFP-N1-ArsC was constructed and transfected into.HepG2. Laser confocal microscopy showed that EGFP-ArsC fusion protein was distributed throughout the cells. Taken together, these results demonstrated that prokaryotic arsenic resistant gene arsC integrated successfully into HepG2 genome and enhanced arsenate resistance of HepG2, which brought new insights of arsenic detoxification in mammalian cells.

  3. 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.

  4. 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

  5. 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.

  6. 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.

  7. 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.

  8. A substrate-bound structure of cyanobacterial biliverdin reductase identifies stacked substrates as critical for activity.

    Science.gov (United States)

    Takao, Haruna; Hirabayashi, Kei; Nishigaya, Yuki; Kouriki, Haruna; Nakaniwa, Tetsuko; Hagiwara, Yoshinori; Harada, Jiro; Sato, Hideaki; Yamazaki, Toshimasa; Sakakibara, Yoichi; Suiko, Masahito; Asada, Yujiro; Takahashi, Yasuhiro; Yamamoto, Ken; Fukuyama, Keiichi; Sugishima, Masakazu; Wada, Kei

    2017-02-07

    Biliverdin reductase catalyses the last step in haem degradation and produces the major lipophilic antioxidant bilirubin via reduction of biliverdin, using NAD(P)H as a cofactor. Despite the importance of biliverdin reductase in maintaining the redox balance, the molecular details of the reaction it catalyses remain unknown. Here we present the crystal structure of biliverdin reductase in complex with biliverdin and NADP+. Unexpectedly, two biliverdin molecules, which we designated the proximal and distal biliverdins, bind with stacked geometry in the active site. The nicotinamide ring of the NADP+ is located close to the reaction site on the proximal biliverdin, supporting that the hydride directly attacks this position of the proximal biliverdin. The results of mutagenesis studies suggest that a conserved Arg185 is essential for the catalysis. The distal biliverdin probably acts as a conduit to deliver the proton from Arg185 to the proximal biliverdin, thus yielding bilirubin.

  9. 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

    We report the isolation and characterization of a genomic clone containing the open reading frame sequence for 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase from Trypanosoma cruzi, the causative agent of Chagas' disease. The protozoan gene encoded for a smaller polypeptide than the rest...... sensitive to proteolytic inactivation. Furthermore the enzyme can be efficiently overexpressed in a highly active form by using the expression vector pET-11c. Thus Trypanosoma cruzi HMG-CoA reductase is unique in the sense that it totally lacks the membrane-spanning sequences present in all eukaryotic HMG...... 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...

  10. Aldose reductase is involved in long-term adaptation of EUE cells to hyperosmotic stress.

    Science.gov (United States)

    Ferraretto, A; Negri, A; Giuliani, A; De Grada, L; Fuhrman Conti, A M; Ronchi, S

    1993-02-17

    Aldose reductase has been shown to be expressed in large amount by human embryonic epithelial cells (EUE) in response to osmotic stress. This conclusion is the result of studies undertaken following the purification to homogeneity of two forms of a 35-kDa protein overexpressed in EUE cells grown in hypertonic saline culture medium as compared to EUE cells grown in isoosmotic medium. Amino-acid composition, molecular weight and partial internal amino-acid sequence showed that the above proteins are two different forms of aldose reductase. These findings were confirmed by the observation that aldose reductase activity increased about 150-fold in adapted cells and returned to basal levels in de-adapted cells.

  11. Aldose reductase inhibition prevents metaplasia of airway epithelial cells.

    Directory of Open Access Journals (Sweden)

    Umesh C S Yadav

    Full Text Available BACKGROUND: Goblet cell metaplasia that causes mucus hypersecretion and obstruction in the airway lumen could be life threatening in asthma and chronic obstructive pulmonary disease patients. Inflammatory cytokines such as IL-13 mediate the transformation of airway ciliary epithelial cells to mucin-secreting goblet cells in acute as well as chronic airway inflammatory diseases. However, no effective and specific pharmacologic treatment is currently available. Here, we investigated the mechanisms by which aldose reductase (AR regulates the mucus cell metaplasia in vitro and in vivo. METHODOLOGY/FINDINGS: Metaplasia in primary human small airway epithelial cells (SAEC was induced by a Th2 cytokine, IL-13, without or with AR inhibitor, fidarestat. After 48 h of incubation with IL-13 a large number of SAEC were transformed into goblet cells as determined by periodic acid-schiff (PAS-staining and immunohistochemistry using antibodies against Mucin5AC. Further, IL-13 significantly increased the expression of Mucin5AC at mRNA and protein levels. These changes were significantly prevented by treatment of the SAEC with AR inhibitor. AR inhibition also decreased IL-13-induced expression of Muc5AC, Muc5B, and SPDEF, and phosphorylation of JAK-1, ERK1/2 and STAT-6. In a mouse model of ragweed pollen extract (RWE-induced allergic asthma treatment with fidarestat prevented the expression of IL-13, phosphorylation of STAT-6 and transformation of epithelial cells to goblet cells in the lung. Additionally, while the AR-null mice were resistant, wild-type mice showed goblet cell metaplasia after challenge with RWE. CONCLUSIONS: The results show that exposure of SAEC to IL-13 caused goblet cell metaplasia, which was significantly prevented by AR inhibition. Administration of fidarestat to mice prevented RWE-induced goblet cell metaplasia and AR null mice were largely resistant to allergen induced changes in the lung. Thus our results indicate that AR inhibitors

  12. Aldose reductase inhibition prevents metaplasia of airway epithelial cells.

    Science.gov (United States)

    Yadav, Umesh C S; Aguilera-Aguirre, Leopoldo; Ramana, Kota V; Boldogh, Istvan; Srivastava, Satish K

    2010-12-28

    Goblet cell metaplasia that causes mucus hypersecretion and obstruction in the airway lumen could be life threatening in asthma and chronic obstructive pulmonary disease patients. Inflammatory cytokines such as IL-13 mediate the transformation of airway ciliary epithelial cells to mucin-secreting goblet cells in acute as well as chronic airway inflammatory diseases. However, no effective and specific pharmacologic treatment is currently available. Here, we investigated the mechanisms by which aldose reductase (AR) regulates the mucus cell metaplasia in vitro and in vivo. Metaplasia in primary human small airway epithelial cells (SAEC) was induced by a Th2 cytokine, IL-13, without or with AR inhibitor, fidarestat. After 48 h of incubation with IL-13 a large number of SAEC were transformed into goblet cells as determined by periodic acid-schiff (PAS)-staining and immunohistochemistry using antibodies against Mucin5AC. Further, IL-13 significantly increased the expression of Mucin5AC at mRNA and protein levels. These changes were significantly prevented by treatment of the SAEC with AR inhibitor. AR inhibition also decreased IL-13-induced expression of Muc5AC, Muc5B, and SPDEF, and phosphorylation of JAK-1, ERK1/2 and STAT-6. In a mouse model of ragweed pollen extract (RWE)-induced allergic asthma treatment with fidarestat prevented the expression of IL-13, phosphorylation of STAT-6 and transformation of epithelial cells to goblet cells in the lung. Additionally, while the AR-null mice were resistant, wild-type mice showed goblet cell metaplasia after challenge with RWE. The results show that exposure of SAEC to IL-13 caused goblet cell metaplasia, which was significantly prevented by AR inhibition. Administration of fidarestat to mice prevented RWE-induced goblet cell metaplasia and AR null mice were largely resistant to allergen induced changes in the lung. Thus our results indicate that AR inhibitors such as fidarestat could be developed as therapeutic agents to

  13. drFrnE Represents a Hitherto Unknown Class of Eubacterial Cytoplasmic Disulfide Oxido-Reductases.

    Science.gov (United States)

    Bihani, Subhash C; Panicker, Lata; Rajpurohit, Yogendra S; Misra, Hari S; Kumar, Vinay

    2017-10-16

    Living cells employ thioredoxin and glutaredoxin disulfide oxido-reductases to protect thiol groups in intracellular proteins. FrnE protein of Deinococcus radiodurans (drFrnE) is a disulfide oxido-reductase that is induced in response to Cd(2+) exposure and is involved in cadmium and radiation tolerance. The aim of this study is to probe structure, function, and cellular localization of FrnE class of proteins. Here, we show drFrnE as a novel cytoplasmic oxido-reductase that could be functional in eubacteria under conditions where thioredoxin/glutaredoxin systems are inhibited or absent. Crystal structure analysis of drFrnE reveals thioredoxin fold with an alpha helical insertion domain and a unique, flexible, and functionally important C-terminal tail. The C-tail harbors a novel 239-CX4C-244 motif that interacts with the active site 22-CXXC-25 motif. Crystal structures with different active site redox states, including mixed disulfide (Cys22-Cys244), are reported here. The biochemical data show that 239-CX4C-244 motif channels electrons to the active site cysteines. drFrnE is more stable in the oxidized form, compared with the reduced form, supporting its role as a disulfide reductase. Using bioinformatics analysis and fluorescence microscopy, we show cytoplasmic localization of drFrnE. We have found "true" orthologs of drFrnE in several eubacterial phyla and, interestingly, all these groups apparently lack a functional glutaredoxin system. Innovation and Conclusion: We show that drFrnE represents a new class of hitherto unknown intracellular oxido-reductases that are abundantly present in eubacteria. Unlike other well-known oxido-reductases, FrnE harbors an additional dithiol motif that acts as a conduit to channel electrons to the active site during catalytic turnover. Antioxid. Redox Signal. 00, 000-000.

  14. 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.

  15. 5{alpha}-reductase expression by prostate cancer cell lines and benign prostatic hyperplasia in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Smith, C.M.; Masters, J.R.W. [Univ. College of London (United Kingdom)]|[Pfizer Central Research, Kent (United Kingdom); Ballard, S.A.; Worman, N. [Pfizer Central Research, Sandwich (United Kingdom)

    1996-04-01

    5{alpha}-Reductase (5{alpha}R) activity in two human prostate cancer cell lines was compared to that in benign prostatic hyperplasia (BPH) tissue and COS cells transfected with and expressing the human genes for 5{alpha}-reductase type 1 (5{alpha}R1) and type 2 (5{alpha}R2). Comparisons were based on pH profiles and sensitivities to selective inhibitors of 5{alpha}-reductase. In the cancer lines, activity was greatest over the pH range 7-8, compared to a sharp peak of activity between pH 5-5.5 in BPH tissue and COS cells expressing 5{alpha}R2. Finasteride and SKF105,657 were potent inhibitors of 5{alpha}-reductase activity in BPH tissue and COS cells expressing 5{alpha}R2, but weak inhibitors in the cancer lines and in COS cells expressing 5{alpha}R1. In contrast, LTK1 17,026 was a more potent inhibitor of 5{alpha}-reductase activity in the prostate cancer cell lines and in COS cells expressing 5{alpha}R1. These data indicate that human prostate cancer cell lines express 5{alpha}-reductase activity similar to that in COS cells transfected with 5{alpha}R1, but different from that in BPH tissue. This may be a consequence of in vitro culture. Alternatively, it may reflect a change occurring as a result of neoplastic transformation, in which case it will be important to select appropriate inhibitors in the clinic. 29 refs., 3 figs., 2 tabs.

  16. Methylenetetrahydrofolate reductase in the rat central nervous system: intracellular and regional distribution

    Energy Technology Data Exchange (ETDEWEB)

    Burton, E.G.; Sallach, H.J.

    1975-01-01

    Methylenetetrahydrofolate reductase and methyltetrahydrofolate-homocysteine methyltransferase were found to be localized in the soluble fraction of rat brain. They are clearly separated from serine hydroxymethyltransferase and the glycine cleavage complex which are localized in the mitochondria in this tissue. Hence, although the primary, if not the only, site of 5, 10-methylenetetrahydrofolate formation in brain appears to be the mitochondrion, the utilization of this compound for 5-methyltetrahydrofolate synthesis and utilization of the latter compound for methylation of homocysteine occurs in the cytosol. Parallel experiments with rat liver confirmed that the reductase and homocysteine methyltransferase of this tissue are also localized in the soluble fraction, while the hydroxymethyltransferase is about evenly divided between mitochondria and cytosol. However, in liver (but not in brain) the reductase activity of the supernatant fraction is only partially expressed unless the fraction is dialyzed. We have found that this phenomenon, which initially suggested the occurrence of an endogenous inhibitor in liver extracts is due to loss of the product (5, 10-methylenetetrahydrofolate) of the reductase assay via its oxidation by methylenetetrahydrofolate dehydrogenase, in combination with the NADP present in undialyzed extracts. All regions of the rat central nervous system tested contained methylenetetrahydrofolate reductase activity. Regional variations were observed however, with an almost threefold difference between the specific activities of the highest and lowest ranking regions. Comparison of the rank order of 12 regions tested with respect to reductase specific activity (this study) and methyltetrahydrofolate-tryptamine N-methyltransferase specific activity shows a high positive correlation (r = 0.916) between these activities in the selected regions.

  17. Involvement of the reductase domain of neuronal nitric oxide synthase in superoxide anion production.

    Science.gov (United States)

    Miller, R T; Martásek, P; Roman, L J; Nishimura, J S; Masters, B S

    1997-12-09

    Neuronal nitric oxide synthase (nNOS) is a modular enzyme which consists of a flavin-containing reductase domain and a heme-containing oxygenase domain, linked by a stretch of amino acids which contains a calmodulin (CaM) binding site. CaM binding to nNOS facilitates the transfer of NADPH-derived electrons from the reductase domain to the oxygenase domain, resulting in the conversion of L-arginine to L-citrulline with the concomitant formation of a guanylate cyclase activating factor, putatively nitric oxide. Numerous studies have established that peroxynitrite-derived nitrogen oxides are present following nNOS turnover. Since peroxynitrite is formed by the diffusion-limited reaction between the two radical species, nitric oxide and O2.-, we employed the adrenochrome assay to examine whether nNOS was capable of producing O2.- during catalytic turnover in the presence of L-arginine. To differentiate between the role played by the reductase domain and that of the oxygenase domain in O2.- production, we compared its production by nNOS against that of a nNOS mutant (CYS-331), which was unable to transfer NADPH-derived electrons efficiently to the heme iron under special conditions, and against that of a flavoprotein module construct of nNOS. We report that O2.- production by nNOS and the CYS-331 mutant is CaM-dependent and that O2.- production can be modulated by substrates and inhibitors of nNOS. O2.- was also produced by the reductase domain of nNOS; however, it did not display the same CaM dependency. We conclude that both the reductase and oxygenase domains of nNOS produce O2.-, but that the reductase domain is both necessary and sufficient for O2.- production.

  18. Inhibitory activity of Cinnamomum cassia bark-derived component against rat lens aldose reductase.

    Science.gov (United States)

    Lee, Hoi-Seon

    2002-01-01

    To evaluate the inhibitory activity of active compounds isolated from Cinnamomum cassia bark against lens aldose reductase and compare to that of three commercially available compounds (cinnamyl alcohol, trans -cinnamic acid, and eugenol) and quercitrin as aldose reductase inhibitors. The IC (50) value of cinnamaldehyde was determined. Active compound was purified on repeated silica gel column and HPLC (Waters Delta Prep 4000). Aldose reductase was prepared from lenses of Sprague-Dawley male rat eyes. The incubation mixture contained 135 mM Na, K-phosphate buffer (pH 7.0), 100 mM lithium sulfate, 0.03 mM NADPH, 0.04 mM DL-glyceraldehyde and 50 micro L of an enzyme preparation, with or without a plant extract. The reaction was initiated by adding NADPH at 37 degrees C and stopped by adding 0.5 N hydrochloric acid. Subsequently, 6 N NaOH containing 10 mM imidazole was added, and the mixture was incubated at 60 degrees C for 10 min to convert NADP into a fluorescent product. The fluorescence was measured with a spectrofluorophotometer. The biologically active constituents of C. cassia extract against lens aldose reductase were characterized as trans -cinnamaldehyde by spectral analysis. The IC (50) value of cinnamaldehyde is 0.003 mg/mL. However, cinnamyl alcohol, trans -cinnamic acid and eugenol exhibited only weak inhibition against aldose reductase. In comparison, quercitrin was 6 times more potent than cinnamaldehyde. These results suggest that cinnamaldehyde isolated from C. cassia barks may be useful as a lead compound and a medicinal foodstuff for aldose reductase inhibition.

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

    Directory of Open Access Journals (Sweden)

    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.

  20. NADPH-Thioredoxin Reductase C Mediates the Response to Oxidative Stress and Thermotolerance in the Cyanobacterium Anabaena sp PCC7120

    NARCIS (Netherlands)

    Sanchez-Riego, Ana M.; Mata-Cabana, Alejandro; Galmozzi, CarlaV.; Florencio, Francisco J.

    2016-01-01

    NADPH-thioredoxin reductase C (NTRC) is a bimodular enzyme composed of an NADPH-thioredoxin reductase and a thiioredoxin domain extension in the same protein. In plants, NTRC has been described to be involved in the protection of the chloroplast against oxidative stress damage through reduction of

  1. 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 ...

  2. 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.

  3. Subcellular localization of the five members of the human steroid 5α-reductase family

    Directory of Open Access Journals (Sweden)

    Antonella Scaglione

    2017-06-01

    We report the cloning and transient expression in HeLa cells of the five members of the human steroid 5α-reductase family as both N- and C-terminus green fluorescent protein tagged protein constructs. Following the intrinsic fluorescence of the tag, we have determined that the subcellular localization of these enzymes is in the endoplasmic reticulum, upon expression in HeLa cells. The presence of the tag at either end of the polypeptide chain can affect protein expression and, in the case of trans enoyl-CoA reductase, it induces the formation of protein aggregates.

  4. Structure of Hordeum vulgare NADPH-dependent thioredoxin reductase 2. Unwinding the reaction mechanism

    DEFF Research Database (Denmark)

    Kirkensgaard, Kristine Groth; Hägglund, Per; Finnie, Christine

    2009-01-01

    Thioredoxins (Trxs) are protein disulfide reductases that regulate the intracellular redox environment and are important for seed germination in plants. Trxs are in turn regulated by NADPH-dependent thioredoxin reductases (NTRs), which provide reducing equivalents to Trx using NADPH to recycle Trxs...... is structurally similar to the structures of AtNTR-B from Arabidopsis thaliana and other known low-molecular-weight NTRs. However, the relative position of the two NTR cofactor-binding domains, the FAD and the NADPH domains, is not the same. The NADPH domain is rotated by 25 degrees and bent by a 38% closure...

  5. Purification and characterization of maleylacetate reductase from Alcaligenes eutrophus JMP134(pJP4).

    OpenAIRE

    Seibert, V; Stadler-Fritzsche, K; Schlömann, M

    1993-01-01

    Maleylacetate reductase (EC 1.3.1.32) plays a major role in the degradation of chloroaromatic compounds by channeling maleylacetate and some of its substituted derivatives into the 3-oxoadipate pathway. The enzyme was purified to apparent homogeneity from an extract of 2,4-dichlorophenoxyacetate (2,4-D)-grown cells of Alcaligenes eutrophus JMP134. Maleylacetate reductase appears to be a dimer of two identical subunits of 35 kDa. The pI was determined to be at pH 5.4. There was no indication o...

  6. Isolation, modification, and aldose reductase inhibitory activity of rosmarinic acid derivatives from the roots of Salvia grandifolia.

    Science.gov (United States)

    Kang, Jie; Tang, Yanbo; Liu, Quan; Guo, Nan; Zhang, Jian; Xiao, Zhiyan; Chen, Ruoyun; Shen, Zhufang

    2016-07-01

    To find aldose reductase inhibitors, two previously unreported compounds, grandifolias H and I, and five known compounds, including rosmarinic acid and rosmarinic acid derivatives, were isolated from the roots of Salvia grandifolia. A series of rosmarinic acid derivatives was obtained from rosmarinic acid using simple synthetic methods. The aldose reductase inhibitory activity of the isolated and synthesized compounds was assessed. Seven of the tested compounds showed moderate aldose reductase inhibition (IC50=0.06-0.30μM). The structure-activity relationship of aldose reductase inhibitory activity of rosmarinic acid derivatives was discussed for the first time. This study provided useful information that will facilitate the development of aldose reductase inhibitors. Copyright © 2016. Published by Elsevier B.V.

  7. Hypothesis on Serenoa repens (Bartram) small extract inhibition of prostatic 5α-reductase through an in silico approach 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 5α-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 5α-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 5β-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.

  8. Hypothesis on Serenoa repens (Bartram small extract inhibition of prostatic 5α-reductase through an in silico approach on 5β-reductase x-ray structure

    Directory of Open Access Journals (Sweden)

    Paolo Governa

    2016-11-01

    Full Text Available 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 5α-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 5α-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 5β-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.

  9. Molecular cloning and expression of the human Δ7-sterol reductase

    Science.gov (United States)

    Moebius, Fabian F.; Fitzky, Barbara U.; Lee, Joon No; Paik, Young-Ki; Glossmann, Hartmut

    1998-01-01

    Inhibitors of the last steps of cholesterol biosynthesis such as AY9944 and BM15766 severely impair brain development. Their molecular target is the Δ7-sterol reductase (EC 1.3.1.21), suspected to be defective in the Smith–Lemli–Opitz syndrome, a frequent inborn disorder of sterol metabolism. Molecular cloning of the cDNA revealed that the human enzyme is a membrane-bound protein with a predicted molecular mass of 55 kDa and six to nine putative transmembrane segments. The protein is structurally related to plant and yeast sterol reductases. In adults the ubiquitously transcribed mRNA is most abundant in adrenal gland, liver, testis, and brain. The Δ7-sterol reductase is the ultimate enzyme of cholesterol biosynthesis in vertebrates and is absent from yeast. Microsomes from Saccharomyces cerevisiae strains heterologously expressing the human cDNA remove the C7–8 double bond in 7-dehydrocholesterol. The conversion to cholesterol depends on NADPH and is potently inhibited by AY9944 (IC50 0.013 μM), BM15766 (IC50 1.2 μM), and triparanol (IC50 14 μM). Our work paves the way to clarify whether a defect in the Δ7-sterol reductase gene underlies the Smith–Lemli–Opitz syndrome. PMID:9465114

  10. Inhibitory effect of polar oregano extracts on aldose reductase and soybean lipoxygenase in vitro.

    Science.gov (United States)

    Koukoulitsa, Catherine; Zika, Chariklia; Hadjipavlou-Litina, Dimitra; Demopoulos, Vassilis J; Skaltsa, Helen

    2006-07-01

    The effect of methanol and aqueous methanol extract of Origanum vulgare L. ssp. hirtum on aldose reductase and soybean lipoxygenase was investigated. The results revealed a promising potential of oregano for preventing diabetes complications in the long term and an antiinflammatory efficacy by inhibiting soybean lipoxygenase.

  11. The 5,10-methylenetetrahydrofolate reductase C677T polymorphism interacts with smoking to increase homocysteine.

    NARCIS (Netherlands)

    Brown, K.S.; Kluijtmans, L.A.J.; Young, I.S.; Murray, L.; McMaster, D.; Woodside, J.; Yarnell, J.W.; Boreham, C.A.; McNulty, H.; Strain, J.J.; McPartlin, J.; Scott, J.M.; Mitchell, L.E.; Whitehead, A.S.

    2004-01-01

    Elevated homocysteine is a risk marker for several human pathologies. Risk factors for elevated homocysteine include low folate and homozygosity for the T allele of the 5,10-methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism. Because nitric oxide may inhibit folate catabolism and

  12. A New Type of YumC-Like Ferredoxin (Flavodoxin) Reductase Is Involved in Ribonucleotide Reduction

    DEFF Research Database (Denmark)

    Chen, Jun; Shen, Jing; Solem, Christian

    2015-01-01

    . subtilis but that the addition of deoxynucleosides cannot compensate for the lethal phenotype displayed by the B. subtilis yumC knockout mutant. Ferredoxin (flavodoxin) reductase (FdR) is involved in many important reactions in both eukaryotes and prokaryotes, such as photosynthesis, nitrate reduction, etc. The recently...

  13. 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,...

  14. NMR structure of the flavin domain from soluble methane monooxygenase reductase from Methylococcus capsulatus (Bath).

    Science.gov (United States)

    Chatwood, Lisa L; Müller, Jens; Gross, John D; Wagner, Gerhard; Lippard, Stephen J

    2004-09-28

    Soluble methane monooxygenase (sMMO) catalyzes the hydroxylation of methane by dioxygen to methanol, the first step in carbon assimilation by methanotrophs. This multicomponent system transfers electrons from NADH through a reductase component to the non-heme diiron center in the hydroxylase where O(2) is activated. The reductase component comprises three distinct domains, a [2Fe-2S] ferredoxin domain along with FAD- and NADH-binding domains. We report the solution structure of the reduced 27.6 kDa FAD- and NADH-binding domains (MMOR-FAD) of the reductase from Methylococcus capsulatus (Bath). The FAD-binding domain consists of a six-stranded antiparallel beta-barrel and one alpha-helix, with the first 10 N-terminal residues unstructured. In the interface between the two domains, the FAD cofactor is tightly bound in an unprecedented extended conformation. The NADH-binding domain consists of a five-stranded parallel beta-sheet with four alpha-helices packing closely around this sheet. MMOR-FAD is structurally homologous to other FAD-containing oxidoreductases, and we expect similar structures for the FAD/NADH-binding domains of reductases that occur in other multicomponent monooxygenases.

  15. Pharmaco-economic assessment of the HMG- CoA reductase ...

    African Journals Online (AJOL)

    by a decrease in the incidence of CHD morbidity and mortality.'-3. In the pharmacological treatment of primary hypercholesterolaemia, the HMG-CoA reductase inhibitors are emerging as the preferred pharmacological therapy! The two agents in this class currently available in South Africa are simvastatin and pravastatin.

  16. HMG-coenzyme A reductase inhibition, type 2 diabetes, and bodyweight

    DEFF Research Database (Denmark)

    Swerdlow, Daniel I; Preiss, David; Kuchenbaecker, Karoline B

    2015-01-01

    BACKGROUND: Statins increase the risk of new-onset type 2 diabetes mellitus. We aimed to assess whether this increase in risk is a consequence of inhibition of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), the intended drug target. METHODS: We used single nucleotide polymorphisms in the HMGCR...

  17. Biliverdin Reductase inhibitors did not improve severe unconjugated hyperbilirubinemia in vivo

    NARCIS (Netherlands)

    van Dijk, Remco; Aronson, Sem J.; de Waart, Dirk R.; van de Graaf, Stan F.; Duijst, Suzanne; Seppen, Jurgen; Oude Elferink, Ronald; Beuers, Ulrich; Bosma, Piter J.

    2017-01-01

    We aimed to identify potent biliverdin reductase (BVRA) inhibitors as a novel concept for the treatment of severe unconjugated hyperbilirubinemia. 1280 FDA-approved compounds were screened in vitro for their ability to inhibit human and rat BVRA activity and 26 compounds were identified as BVRA

  18. [The usefulness of the nitrate reductase assay for detecting drug-resistant Mycobacterium tuberculosis].

    Science.gov (United States)

    González, Lorena; Sánchez, Ricardo; Murcia, Martha Isabel

    2014-04-01

    The early detection of resistance in Mycobacterium tuberculosis is of primary importance for both patient management and infection control. To evaluate nitrate reductase assay (NRA) performance for the testing of Mycobacterium tuberculosis drug-resistance against first-line anti-tuberculosis drugs, such as rifampicin (RIF), isoniazid (INH), streptomycin (STR) and ethambutol (EMB). Fifty isolates were tested by using both the proportion method and the nitrate reductase assay. RIF, INH, STR and EMB sensitivity was found to be 92%, 91%, 63% and 80% and 100%, respectively, and a corresponding specificity of 100%, 100%, 100% and 98% by comparing NRA results to those obtained with the gold standard (i.e., the proportion method). The positive predictive values for RIF, INH, STR and EMB were 100%, 100%, 100% and 80% and the negative predictive values were 97%, 93%, 73% and 98%, respectively. The mean time for obtaining results was shorter when using the nitrate reductase assay (10 days) compared to using the proportion method (28 days). Excellent agreement was observed between both phenotypic tests: 98%, 96%, 81% and 96% for RIF, INH, STR and EMB, respectively . The results showed that the nitrate reductase assay is suitable for the early determination of multidrug-resistant tuberculosis (MDR-TB) and is a useful tool for the quick and accurate determination of a rapid M. tuberculosis drug-sensitivity test in countries having low resources.

  19. Voltammetry and In Situ Scanning Tunneling Microscopy of Cytochrome c Nitrite Reductase on Au(111)-Electrodes

    DEFF Research Database (Denmark)

    Gwyer, James; Zhang, Jingdong; Butt, Julea

    2006-01-01

    Escherichia coli cytochrome c nitrite reductase (NrfA) catalyzes the six-electron reduction of nitrite to perform an important role in the biogeochemical cycling of nitrogen. Here we describe NrfA adsorption on single-crystal Au(111) electrodes as an electrocatalytically active film in which the ...

  20. Calorimetric and spectroscopic investigations of the thermal denaturation of wild type nitrite reductase

    NARCIS (Netherlands)

    Stirpe, A; Guzzi, R; Wijma, H; Verbeet, MP; Canters, GW; Sportelli, L

    2005-01-01

    Nitrite reductase (NiR) is a multicopper protein, with a trimeric structure containing two types of copper site: type I is present in each subunit whereas type 2 is localized at the subunits interface. The paper reports on the thermal behaviour of wild type NiR from Alcaligenes faecalis S-6. The

  1. Nitrate reductase assay using sodium nitrate for rapid detection of multidrug resistant tuberculosis

    Science.gov (United States)

    Macedo, Maíra Bidart; Groll, Andrea Von; Fissette, Krista; Palomino, Juan Carlos; da Silva, Pedro Eduardo Almeida; Martin, Anandi

    2012-01-01

    We validated the nitrate reductase assay (NRA) for the detection of multidrug-resistant Mycobacterium tuberculosis (MDR-TB) using sodium nitrate (NaNO3) in replacement of potassium nitrate (KNO3) as nitrate source. NaNO3 is cheaper than KNO3 and has no restriction on use which facilitates the implementation of NRA to detect MDR-TB. PMID:24031916

  2. Spectrophotometric activity microassay for pure and recombinant cytochrome P450-type nitric oxide reductase

    CSIR Research Space (South Africa)

    Garny, S

    2014-02-01

    Full Text Available Nitric oxide reductase (NOR) of the P450 oxidoreductase family accepts electrons directly from its cofactor, NADH, to reduce two nitric oxide (NO) molecules to one nitrous oxide molecule and water. The enzyme plays a key role in removal of radical...

  3. Cloning, expression and antigenicity of the L. donovani reductase

    DEFF Research Database (Denmark)

    Jensen, A T; Kemp, K; Theander, T G

    2001-01-01

    (K). Only 2 of 22 plasma samples from patients with visceral leishmaniasis were found to have detectable anti-reductase antibodies and peripheral blood mononuclear cells (PBMC) from one of three individuals previously infected with visceral leishmaniasis proliferated in the presence of recombinant...

  4. Cotton Benzoquinone Reductase: Up-regulation During Early Cotton Fiber Developement

    Science.gov (United States)

    Benzoquinone reductase (BR; EC 1.6.5.7) is an enzyme that catalyzes the bivalent redox reactions of quinones without the production of free radical intermediates. Using 2-D PAGE comparisons, two proteins were found to be up-regulated in wild-type cotton ovules during the fiber initiation stage but ...

  5. Redox-state-dependent complex formation between pseudoazurin and nitrite reductase.

    Science.gov (United States)

    Impagliazzo, Antonietta; Blok, Anneloes J; Cliff, Matthew J; Ladbury, John E; Ubbink, Marcellus

    2007-01-10

    Bacterial copper-containing nitrite reductase catalyzes the reduction of nitrite to nitric oxide as part of the denitrification process. Pseudoazurin interacts with nitrite reductase in a transient fashion to supply the necessary electrons. The redox-state dependence of complex formation between pseudoazurin and nitrite reductase was studied by nuclear magnetic resonance spectroscopy and isothermal titration calorimetry. Binding of pseudoazurin in the reduced state is characterized by the presence of two binding modes, a slow and a fast exchange mode, with a K(d)(app) of 100 microM. In the oxidized state of pseudoazurin, binding occurs in a single fast exchange mode with a similar affinity. Metal-substituted proteins have been used to show that the mode of binding of pseudoazurin is independent of the metal charge of nitrite reductase. Contrary to what was found for other cupredoxins, protonation of the exposed His ligand to the copper of pseudoazurin, His81, does not appear to be involved directly in the dual binding mode of the reduced form. A model assuming the presence of a minor form of pseudoazurin is proposed to explain the behavior of the complex in the reduced state.

  6. 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...

  7. On the role of fumarate reductase in anaerobic carbohydrate catabolism of Mytilus edulis L

    NARCIS (Netherlands)

    Holwerda, Dirk A.; Zwaan, Albertus de

    1980-01-01

    1. 1. The role of the fumarate:NADH oxidoreduction in the anaerobic glycolysis of the sea mussel is examined and discussed. 2. 2. Fumarate reductase activity is present in submitochondrial particles especially from adductor muscle, digestive gland and mantle. 3. 3. The pH optimum of the enzyme

  8. Methylenetetrahydrofolate reductase (MTHFR) and susceptibility for (pre)neoplastic cervical disease

    NARCIS (Netherlands)

    Zoodsma, M; Nolte, IM; Schipper, M; Oosterom, E; van der Steege, G; de Vries, E; te Meerman, GJ; van der Zee, AGJ

    Methylenetetrahydrofolate reductase (MTHFR) is a critical enzyme regulating the metabolism of folate and methionine. The potential influence of MTHFR activity on DNA methylation and on the availability of uridylates and thymidylates for DNA synthesis and repair presents MTHFR as a candidate for

  9. Protein film voltammetry of copper-containing nitrite reductase reveals reversible inactivation

    NARCIS (Netherlands)

    Wijma, Hein J.; Jeuken, Lars J. C.; Verbeet, Martin Ph.; Armstrong, Fraser A.; Canters, Gerard W.

    2007-01-01

    The Cu-containing nitrite reductase from Alcaligenes faecalis S-6 catalyzes the one-electron reduction of nitrite to nitric oxide (NO). Electrons enter the enzyme at the so-called type-1 Cu site and are then transferred internally to the catalytic type-2 Cu site. Protein film voltammetry experiments

  10. Role of aldo-keto reductase family 1 (AKR1) enzymes in human steroid metabolism.

    Science.gov (United States)

    Rižner, Tea Lanišnik; Penning, Trevor M

    2014-01-01

    Human aldo-keto reductases AKR1C1-AKR1C4 and AKR1D1 play essential roles in the metabolism of all steroid hormones, the biosynthesis of neurosteroids and bile acids, the metabolism of conjugated steroids, and synthetic therapeutic steroids. These enzymes catalyze NADPH dependent reductions at the C3, C5, C17 and C20 positions on the steroid nucleus and side-chain. AKR1C1-AKR1C4 act as 3-keto, 17-keto and 20-ketosteroid reductases to varying extents, while AKR1D1 acts as the sole Δ(4)-3-ketosteroid-5β-reductase (steroid 5β-reductase) in humans. AKR1 enzymes control the concentrations of active ligands for nuclear receptors and control their ligand occupancy and trans-activation, they also regulate the amount of neurosteroids that can modulate the activity of GABAA and NMDA receptors. As such they are involved in the pre-receptor regulation of nuclear and membrane bound receptors. Altered expression of individual AKR1C genes is related to development of prostate, breast, and endometrial cancer. Mutations in AKR1C1 and AKR1C4 are responsible for sexual development dysgenesis and mutations in AKR1D1 are causative in bile-acid deficiency. Copyright © 2013 Elsevier Inc. All rights reserved.

  11. Novel bacterial sulfur oxygenase reductases from bioreactors treating gold-bearing concentrates

    DEFF Research Database (Denmark)

    Chen, Z-W; Liu, Y-Y; Wu, J-F

    2007-01-01

    The microbial community and sulfur oxygenase reductases of metagenomic DNA from bioreactors treating gold-bearing concentrates were studied by 16S rRNA library, real-time polymerase chain reaction (RT-PCR), conventional cultivation, and molecular cloning. Results indicated that major bacterial...

  12. Characterization of a cinnamoyl-CoA reductase gene in Ginkgo ...

    African Journals Online (AJOL)

    Cinnamoyl-CoA reductase (CCR, EC 1.2.1.44) catalyzes key steps in the biosynthesis of monolignols, which serve as building blocks in the formation of plant lignin. ... The expression analysis by quantitative real-time polymerase chain reaction (QRT-PCR showed that GbCCR was seen in a tissue specific manner in Ginkgo ...

  13. Polymorphisms in catechol-O-methyltransferase and methylenetetrahydrofolate reductase in relation to the risk of schizophrenia.

    NARCIS (Netherlands)

    Muntjewerff, J.W.; Gellekink, H.; Heijer, M. den; Hoogendoorn, M.L.; Kahn, R.S.; Sinke, R.J.; Blom, H.J.

    2008-01-01

    BACKGROUND: Evidence is emerging for the association of aberrant homocysteine-methylation cycle and increased risk of schizophrenia. METHODS: We examined the prevalence of the catechol-O-methyltransferase (COMT) 324G>A (Val108/158Met) and methylenetetrahydrofolate reductase (MTHFR) 677C>T

  14. Inherited glutathione reductase deficiency and Plasmodium falciparum malaria--a case study

    NARCIS (Netherlands)

    Gallo, Valentina; Schwarzer, Evelin; Rahlfs, Stefan; Schirmer, R. Heiner; van Zwieten, Rob; Roos, Dirk; Arese, Paolo; Becker, Katja

    2009-01-01

    In Plasmodium falciparum-infected red blood cells (RBCs), the flavoenzyme glutathione reductase (GR) regenerates reduced glutathione, which is essential for antioxidant defense. GR utilizes NADPH produced in the pentose phosphate shunt by glucose-6-phosphate dehydrogenase (G6PD). Thus, conditions

  15. Folate and the methylenetetrahydrofolate reductase 677C ---> T mutation correlate with cognitive performance

    NARCIS (Netherlands)

    Durga, J.; Boxtel, van M.P.J.; Schouten, E.G.; Bots, M.L.; Kok, F.J.; Verhoef, P.

    2006-01-01

    Low folate status has been associated with cognitive decline. We investigated the association of folate status and the 5,10-methylenetetrahydrofolate reductase (MTHFR) 677C ¿ T polymorphism with performance on a battery of neuropsychological tests. Furthermore, we investigated whether the

  16. GLUTATHIONE TRANSFERASE, GLUTATHIONE REDUCTASE AND GLUTATHIONE PEROXIDASE OF CAECUM AND LIVER OF PIG

    OpenAIRE

    FEDETS O.M.

    2009-01-01

    The content of glutathione and the activity of enzymes have been investigated. The data of glutathione, glutathione transferase and glutathione peroxidase are the highest in the liver. In mucosa of caecum activity of glutathione reductase is the highest than in liver.

  17. Relationships between nitrate uptake and nitrate reductase activity in Cucumis sativus L.

    Directory of Open Access Journals (Sweden)

    Grażyna Kłobus

    2014-01-01

    Full Text Available Anti-NR IgG fragments obtained after papain digestion of polyclonal antibodies gave the positive immunological reaction with both, a soluble and plasma membrane-bound nitrate reductase. Anti-NR antibody as well as IgG fragments almost totally inhibited the nitrate reductase activity in cytosol proving a crossreaction of antibody with the catalytic site of a soluble NR. Anti-NR IgG fragments, but not undigested polyclonal antibodies affected the activity of the nitrate reductase associated with plasma membranes. Discrepancy in the action of intact antibodies and fragments obtained after they digestion were interpreted as a consequence of same differences in the ability of those molecules to the penetration through the membrane. Undigested anti-NR antibody have no effect on the nitrate uptake by intact plants, as well as by the right-side plasma membrane vesicles. On the other hand, IgG fragments of polyclonal antibodies abolished almost totally the nitrate uptake in the case of intact seedlings, but have only slight effect on the N03 uptake in plasma membranes. On the basis of above findings, some relations between nitrate uptake and its assimilation inside the cell are suggested. Since IgG fragments only slightly changed the N03 absorption in vesicles whereas the activity of plasmalemma associated nitrate reductase was strongly repressed, we concluded that the PM-NR is not structurally involved in the nitrate transport through the membrane.

  18. Nitrate reductase assay using sodium nitrate for rapid detection of multidrug resistant tuberculosis

    OpenAIRE

    Macedo, Ma?ra Bidart; Groll, Andrea Von; Fissette, Krista; Palomino, Juan Carlos; da Silva, Pedro Eduardo Almeida; Martin, Anandi

    2012-01-01

    We validated the nitrate reductase assay (NRA) for the detection of multidrug-resistant Mycobacterium tuberculosis (MDR-TB) using sodium nitrate (NaNO3) in replacement of potassium nitrate (KNO3) as nitrate source. NaNO3 is cheaper than KNO3 and has no restriction on use which facilitates the implementation of NRA to detect MDR-TB.

  19. New 5alpha-reductase inhibitors: in vitro and in vivo effects.

    Science.gov (United States)

    Pérez-Ornelas, Víctor; Cabeza, Marisa; Bratoeff, Eugene; Heuze, Ivonne; Sánchez, Mauricio; Ramírez, Elena; Naranjo-Rodríguez, Elia

    2005-03-01

    The enzyme 5alpha-reductase is responsible for the conversion of testosterone (T) to its more potent androgen dihydrotestosterone (DHT). This steroid had been implicated in androgen-dependent diseases such as: benign prostatic hyperplasia, prostate cancer, acne and androgenic alopecia. The inhibition of 5alpha-reductase enzyme offers a potentially useful treatment for these diseases. In this study, we report the synthesis and pharmacological evaluation of several new 3-substituted pregna-4, 16-diene-6, 20-dione derivatives. These compounds were prepared from the commercially available 16-dehydropregnenolone acetate. The biological activity of the new steroidal derivatives was determined in vivo as well as in vitro experiments. In vivo experiments, the anti-androgenic effect of the steroids was demonstrated by the decrease of the weight of the prostate gland of gonadectomized hamster treated with T plus finasteride or the new steroids. The IC50 value of these steroids was determined by measuring the conversion of radio labeled T to DHT. The results of this study carried out with 5alpha-reductase enzyme from hamster and human prostate showed that four of the six steroidal derivatives (5, 7, 9, 10) exhibited much higher 5alpha-reductase inhibitory activity, as indicated by the IC50 values than the presently used Proscar 3 (finasteride). The comparison of the weight of the hamster's prostate gland indicated that compound 5 had a comparable weight decrease as finasteride. The overall data of this study showed very clearly those compounds 5, 7, 9, 10 are good inhibitors for the 5alpha-reductase enzyme.

  20. Purification of glutamyl-tRNA reductase from Synechocystis sp. PCC 6803

    Energy Technology Data Exchange (ETDEWEB)

    Rieble, S.; Beale, S.I. (Brown Univ., Providence, RI (United States))

    1991-05-01

    {delta}-Aminolevulinic acid (ALA) is the universal precursor for all tetrapyrroles including hemes, chlorophylls, and bilins. In plants, algae, cyanobacteria, and many other bacteria, ALA is synthesized from glutamate in a reaction sequence that requires three enzymes, ATP, NADPH, and tRNA{sup Glu}. The three enzymes have been characterized as glutamyl-tRNA synthetase, glutamyl-tRNA reductase, and glutamate-1-semialdehyde (GSA) aminotransferase. All three enzymes have been separated and partially characterized from plants and algae. In prokaryotic phototrophs, only the glutamyl-tRNA synthetase and GSA aminotransferase have been described. The authors report here the purification and some properties of the glutamyl-tRNA reductase from extracts of the unicellular cyanobacterium, Synechocystis sp. PCC 6803. The glutamyl-tRNA reductase has been purified over 370 fold to apparent homogeneity. Its native molecular mass was determined to be 350 kDa by SDS-PAGE. The N-terminal amino acid sequence was determined for 42 residues. Much higher activity occurred with NADPH than with NADH as the reduced pyridine nucleotide substrate. Half-maximal rates occurred at 5 {mu}M NADPH, whereas saturation was not reached even at 10 mM NADH. Purified Synechocystis glutamyl-tRNA reductase was inhibited 50% by 5 {mu}M heme. Activity was unaffected by 10 {mu}M gabaculine. No flavin, pyridine nucleotide, or other light-absorbing prosthetic group was detected on the purified enzyme. The catalytic turnover number of purified Synechocystis glutamyl-tRNA reductase is comparable to those of prokaryotic and plastidic glutamyl-tRNA synthetases.

  1. The Fungal Pathogen Candida glabrata Does Not Depend on Surface Ferric Reductases for Iron Acquisition

    Directory of Open Access Journals (Sweden)

    Franziska Gerwien

    2017-06-01

    Full Text Available Iron acquisition is a crucial virulence determinant for many bacteria and fungi, including the opportunistic fungal pathogens Candida albicans and C. glabrata. While the diverse strategies used by C. albicans for obtaining iron from the host are well-described, much less is known about the acquisition of this micronutrient from host sources by C. glabrata – a distant relative of C. albicans with closer evolutionary ties to Saccharomyces cerevisiae, which nonetheless causes severe clinical symptoms in humans. Here we show that C. glabrata is much more restricted than C. albicans in using host iron sources, lacking, for example, the ability to grow on transferrin and hemin/hemoglobin. Instead, C. glabrata is able to use ferritin and non-protein-bound iron (FeCl3 as iron sources in a pH-dependent manner. As in other fungal pathogens, iron-dependent growth requires the reductive high affinity (HA iron uptake system. Typically highly conserved, this uptake mechanism normally relies on initial ferric reduction by cell-surface ferric reductases. The C. glabrata genome contains only three such putative ferric reductases, which were found to be dispensable for iron-dependent growth. In addition and in contrast to C. albicans and S. cerevisiae, we also detected no surface ferric reductase activity in C. glabrata. Instead, extracellular ferric reduction was found in this and the two other fungal species, which was largely dependent on an excreted low-molecular weight, non-protein ferric reductant. We therefore propose an iron acquisition strategy of C. glabrata which differs from other pathogenic fungi, such as C. albicans, in that it depends on a limited set of host iron sources and that it lacks the need for surface ferric reductases. Extracellular ferric reduction by a secreted molecule possibly compensates for the loss of surface ferric reductase activity in the HA iron uptake system.

  2. 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.

  3. Molecular simulation to investigate the cofactor specificity for pichia stipitis Xylose reductase.

    Science.gov (United States)

    Xia, Xiao-Le; Cong, Shan; Weng, Xiao-Rong; Chen, Jin-Hua; Wang, Jing-Fang; Chou, Kuo-Chen

    2013-11-01

    Xylose is one of the most abundant carbohydrates in nature, and widely used to produce bioethanol via fermentation in industry. Xylulose can produce two key enzymes: xylose reductase and xylitol dehydrogenase. Owing to the disparate cofactor specificities of xylose reductase and xylitol dehydrogenase, intracellular redox imbalance is detected during the xylose fermentation, resulting in low ethanol yields. To overcome this barrier, a common strategy is applied to artificially modify the cofactor specificity of xylose reductase. In this study, we utilized molecular simulation approaches to construct a 3D (three-dimensional) structural model for the NADP-dependent Pichia stipitis xylose reductase (PsXR). Based on the 3D model, the favourable binding modes for both cofactors NAD and NADP were obtained using the flexible docking procedure and molecular dynamics simulation. Structural analysis of the favourable binding modes showed that the cofactor binding site of PsXR was composed of 3 major components: a hydrophilic pocket, a hydrophobic pocket as well as a linker channel between the aforementioned two pockets. The hydrophilic pocket could recognize the nicotinamide moiety of the cofactors by hydrogen bonding networks, while the hydrophobic pocket functioned to position the adenine moiety of the cofactors by hydrophobic and Π-Π stacking interactions. The linker channel contained some key residues for ligand-binding; their mutation could have impact to the specificity of PsXR. Finally, it was found that any of the two single mutations, K21A and K270N, might reverse the cofactor specificity of PsXR from major NADP- to NADdependent, which was further confirmed by the additional experiments. Our findings may provide useful insights into the cofactor specificity of PsXR, stimulating new strategies for better designing xylose reductase and improving ethanol production in industry.

  4. Identification of Multiple Soluble Fe(III Reductases in Gram-Positive Thermophilic Bacterium Thermoanaerobacter indiensis BSB-33

    Directory of Open Access Journals (Sweden)

    Subrata Pal

    2014-01-01

    Full Text Available Thermoanaerobacter indiensis BSB-33 has been earlier shown to reduce Fe(III and Cr(VI anaerobically at 60°C optimally. Further, the Gram-positive thermophilic bacterium contains Cr(VI reduction activity in both the membrane and cytoplasm. The soluble fraction prepared from T. indiensis cells grown at 60°C was found to contain the majority of Fe(III reduction activity of the microorganism and produced four distinct bands in nondenaturing Fe(III reductase activity gel. Proteins from each of these bands were partially purified by chromatography and identified by mass spectrometry (MS with the help of T. indiensis proteome sequences. Two paralogous dihydrolipoamide dehydrogenases (LPDs, thioredoxin reductase (Trx, NADP(H-nitrite reductase (Ntr, and thioredoxin disulfide reductase (Tdr were determined to be responsible for Fe(III reductase activity. Amino acid sequence and three-dimensional (3D structural similarity analyses of the T. indiensis Fe(III reductases were carried out with Cr(VI reducing proteins from other bacteria. The two LPDs and Tdr showed very significant sequence and structural identity, respectively, with Cr(VI reducing dihydrolipoamide dehydrogenase from Thermus scotoductus and thioredoxin disulfide reductase from Desulfovibrio desulfuricans. It appears that in addition to their iron reducing activity T. indiensis LPDs and Tdr are possibly involved in Cr(VI reduction as well.

  5. Inhibition of aldose reductase activity by Cannabis sativa chemotypes extracts with high content of cannabidiol or cannabigerol.

    Science.gov (United States)

    Smeriglio, Antonella; Giofrè, Salvatore V; Galati, Enza M; Monforte, Maria T; Cicero, Nicola; D'Angelo, Valeria; Grassi, Gianpaolo; Circosta, Clara

    2018-02-07

    Aldose reductase (ALR2) is a key enzyme involved in diabetic complications and the search for new aldose reductase inhibitors (ARIs) is currently very important. The synthetic ARIs are often associated with deleterious side effects and medicinal and edible plants, containing compounds with aldose reductase inhibitory activity, could be useful for prevention and therapy of diabetic complications. Non-psychotropic phytocannabinoids exert multiple pharmacological effects with therapeutic potential in many diseases such as inflammation, cancer, diabetes. Here, we have investigated the inhibitory effects of extracts and their fractions from two Cannabis sativa L. chemotypes with high content of cannabidiol (CBD)/cannabidiolic acid (CBDA) and cannabigerol (CBG)/cannabigerolic acid (CBGA), respectively, on human recombinant and pig kidney aldose reductase activity in vitro. A molecular docking study was performed to evaluate the interaction of these cannabinoids with the active site of ALR2 compared to known ARIs. The extracts showed significant dose-dependent aldose reductase inhibitory activity (>70%) and higher than fractions. The inhibitory activity of the fractions was greater for acidic cannabinoid-rich fractions. Comparative molecular docking results have shown a higher stability of the ALR2-cannabinoid acids complex than the other inhibitors. The extracts of Cannabis with high content of non-psychotropic cannabinoids CBD/CBDA or CBG/CBGA significantly inhibit aldose reductase activity. These results may have some relevance for the possible use of C. sativa chemotypes based preparations as aldose reductase inhibitors. Copyright © 2018 Elsevier B.V. All rights reserved.

  6. FQR1, a Novel Primary Auxin-Response Gene, Encodes a Flavin Mononucleotide-Binding Quinone Reductase1

    Science.gov (United States)

    Laskowski, Marta J.; Dreher, Kate A.; Gehring, Mary A.; Abel, Steffen; Gensler, Arminda L.; Sussex, Ian M.

    2002-01-01

    FQR1 is a novel primary auxin-response gene that codes for a flavin mononucleotide-binding flavodoxin-like quinone reductase. Accumulation of FQR1 mRNA begins within 10 min of indole-3-acetic acid application and reaches a maximum of approximately 10-fold induction 30 min after treatment. This increase in FQR1 mRNA abundance is not diminished by the protein synthesis inhibitor cycloheximide, demonstrating that FQR1 is a primary auxin-response gene. Sequence analysis reveals that FQR1 belongs to a family of flavin mononucleotide-binding quinone reductases. Partially purified His-tagged FQR1 isolated from Escherichia coli catalyzes the transfer of electrons from NADH and NADPH to several substrates and exhibits in vitro quinone reductase activity. Overexpression of FQR1 in plants leads to increased levels of FQR1 protein and quinone reductase activity, indicating that FQR1 functions as a quinone reductase in vivo. In mammalian systems, glutathione S-transferases and quinone reductases are classified as phase II detoxification enzymes. We hypothesize that the auxin-inducible glutathione S-transferases and quinone reductases found in plants also act as detoxification enzymes, possibly to protect against auxin-induced oxidative stress. PMID:11842161

  7. FQR1, a novel primary auxin-response gene, encodes a flavin mononucleotide-binding quinone reductase.

    Science.gov (United States)

    Laskowski, Marta J; Dreher, Kate A; Gehring, Mary A; Abel, Steffen; Gensler, Arminda L; Sussex, Ian M

    2002-02-01

    FQR1 is a novel primary auxin-response gene that codes for a flavin mononucleotide-binding flavodoxin-like quinone reductase. Accumulation of FQR1 mRNA begins within 10 min of indole-3-acetic acid application and reaches a maximum of approximately 10-fold induction 30 min after treatment. This increase in FQR1 mRNA abundance is not diminished by the protein synthesis inhibitor cycloheximide, demonstrating that FQR1 is a primary auxin-response gene. Sequence analysis reveals that FQR1 belongs to a family of flavin mononucleotide-binding quinone reductases. Partially purified His-tagged FQR1 isolated from Escherichia coli catalyzes the transfer of electrons from NADH and NADPH to several substrates and exhibits in vitro quinone reductase activity. Overexpression of FQR1 in plants leads to increased levels of FQR1 protein and quinone reductase activity, indicating that FQR1 functions as a quinone reductase in vivo. In mammalian systems, glutathione S-transferases and quinone reductases are classified as phase II detoxification enzymes. We hypothesize that the auxin-inducible glutathione S-transferases and quinone reductases found in plants also act as detoxification enzymes, possibly to protect against auxin-induced oxidative stress.

  8. FAD-induced in vitro activation of glutathione reductase in the lens of B2 deficient rats.

    Science.gov (United States)

    Ono, S; Hirano, H

    1984-04-01

    We studied the FAD-induced in vitro stimulation of lenticular glutathione reductase in riboflavin-deficient rats. The stimulatory effect of FAD on lenticular glutathione reductase in rats fed a B2-deficient diet for 4 weeks was remarkably higher than in paired control rats fed a B2-supplemented basal diet and control rats had ad libitum access to a B2-supplemented basal diet. The in vitro FAD stimulation effect on rat lenticular glutathione reductase represents a sensitive indicator of the B2 deficient status.

  9. The N-terminal region of mature mitochondrial aspartate aminotransferase can direct cytosolic dihydrofolate reductase into mitochondria in vitro.

    Science.gov (United States)

    Giannattasio, S; Azzariti, A; Marra, E; Quagliariello, E

    1994-06-30

    Two fused genes were constructed which encode for two chimeric proteins in which either 10 or 191 N-terminal amino acids of mature mitochondrial aspartate aminotransferase had been attached to the entire polypeptide chain of cytosolic dihydrofolate reductase. The precursor and mature form of mitochondrial aspartate aminotransferase, dihydrofolate reductase and both chimeric proteins were synthesized in vitro and their import into isolated mitochondria was studied. Both chimeric proteins were taken up by isolated organelles, where they became protease resistant, thus indicating the ability of the N-terminal portion of the mature moiety of the precursor of mitochondrial aspartate aminotransferase to direct cytosolic dihydrofolate reductase into mitochondria.

  10. Glutathione Reductase of Vacuole. Comparison of Glutathione Reductase Activity of Vacuole and Tissue Extract of Red Beet Root (Beta vulgaris L.

    Directory of Open Access Journals (Sweden)

    E.V. Pradedova

    2016-02-01

    Full Text Available Glutathione reductase (GR, EC 1.8.1.7 is the enzyme that reduces oxidized glutathione (GSSG and thus regulates the redox state of glutathione (GSH/GSSG. GR has been studied in most plants. This enzyme has been identified in chloroplasts and cytosol, so these cellular compartments are considered to be the main place of the enzyme localization. In the same time, just a little is known about GR vacuoles. There are no conclusive evidences to prove the presence or absence of this enzyme in the vacuoles. GR activity was found in the vacuoles of red beet root cells (Beta vulgaris L.. The level of activity, the optimum pH and isoenzyme composition of GR were compared in the vacuoles and tissue extract of beet root. Vacuolar GR activity was quite high, it was 1.5-2 times higher than the activity of the tissue extract. Enzyme pH optimum of all the objects were identical. pH-optimum depend on the pyridine nucleotide nature: pH 7.0-8.0 was an optimal range with NADPH; pH 5.0 – with NADH. GR activity of the vacuoles and tissue extracts decreased in the presence of a noncompetitive inhibitor 1-chloro-2.4-dinitrobenzene (CDNB, indicating the specificity of this enzymatic reaction. Two bands with glutathione reductase activity have been identified in the vacuoles and tissue extracts using zymography method to determine the enzymatic activity in PAAG after electrophoresis of proteins. Belonging to the GR isoforms of these bands was confirmed by enzyme immunoassay (Western blotting. The electric mobility of isoforms of the study objects did not differ significantly. It is concluded that the biochemical characteristics of vacuolar glutathione reductase were substantially identical to the biochemical characteristics of other localization GR.

  11. Subcellular distribution of nitroblue tetrazolium reductase (NBT-R) in human polymorphonuclear leukocytes (PMN).

    Science.gov (United States)

    Baehner, R L

    1975-11-01

    Subcellular distribution study of cytoplasmic organelles was performed on human polymorphonuclear leukocytes after homogenization in 0.34 molar sucrose by differential centrifugation and sucrose density gradient centrifugation of the homogenate. The whole homogenate and each fraction was assayed for nitroblue tetrazolium (NBT)-reductase with and without 1 mM potassium cyanide, and the distribution of this enzyme was compared to the distribution of lysozyme, peroxidase, beta-glucuronidase, and acid and alkaline phosphatase. Enzyme recovery was 97 per cent and ranged between 74 and 124 per cent. Latent activity of all enzymes except NBT-reductase, acid, and alkaline phosphatase was demonstrated by observing a four- to sixfold increase in activity after the addition of Triton-X 100. Maximal relative specific activity using either DPNH or without cyanide for NBT-reductase was found in the 100,000 x g differential centrifugation fraction and was concentrated in the less dense top fraction of the sucrose density gradient. The distribution pattern was similar to acid and alkaline phosphatase. In contrast, the maximal concentration of beta-glucuronidase and peroxidase was found in the heavier 7,200 x g granule fraction and in the more dense bottom fractions of the sucrose density gradient. Maximal lysozyme activity was concentrated in the 30,000 x g granule fraction and in the fractions located between the heaviest and lightest fractions of the sucrose density gradient. The lack of latent activity and the similarity of subcellular distribution of NBT-reductase to acid and alkaline phosphatase, two enzymes associated with microsomes and plasmalemal membranes in human polymorphonuclear leukocytes (PMN), indicates that NBT-reductase is also a nonlysosomal enzyme located in microsomes or in plasmalemal membranes. These findings support the previously described histochemical observations that initial reduction of NBT to formazan occurs on the PMN plasmalemal surface membrane at

  12. Colour formation in fermented sausages by meat-associated staphylococci with different nitrite- and nitrate-reductase activities

    DEFF Research Database (Denmark)

    Gøtterup, Jacob; Olsen, Karsten; Knøchel, Susanne

    2008-01-01

    nitrate depended on the specific Staphylococcus strain. Strains with high nitrate-reductase activity showed a significantly faster rate of pigment formation, but other factors were of influence as well. Product stability for the sliced, packaged sausage was evaluated as surface colour and oxidation......Three Staphylococcus strains, S. carnosus, S. simulans and S. saprophyticus, selected due to their varying nitrite and/or nitrate-reductase activities, were used to initiate colour formation during sausage fermentation. During fermentation of sausages with either nitrite or nitrate added, colour...... with hexanal content, and may be used as predictive tools. Overall, nitrite- and nitrate-reductase activities of Staphylococcus strains in nitrite-cured sausages were of limited importance regarding colour development, while in nitrate-cured sausages strains with higher nitrate reductase activity were crucial...

  13. 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

    Leishmania lacks the membrane domain characteristic of eukaryotic cells but exhibits sequence similarity with eukaryotic reductases. Highly purified protein was achieved by ammonium sulphate precipitation followed by chromatography on hydroxyapatite. Kinetic parameters were determined for the protozoan...... reductase, obtaining K(m) values for the overall reaction of 40.3+/-5.8 microM for (R,S)-HMG-CoA and 81.4+/-5.3 microM for NADPH; V(max) was 33.55+/-1.8 units x mg(-1). Gel-filtration experiments suggested an apparent molecular mass of 184 kDa with subunits of 46 kDa. Finally, in order to achieve a better...... understanding of the role of this enzyme in trypanosomatids, the effect of possible regulators of isoprenoid biosynthesis in cultured promastigote cells was studied. Neither mevalonic acid nor serum sterols appear to modulate enzyme activity whereas incubation with lovastatin results in significant increases...

  14. Inhibitor-bound complexes of dihydrofolate reductase-thymidylate synthase from Babesia bovis.

    Science.gov (United States)

    Begley, Darren W; Edwards, Thomas E; Raymond, Amy C; Smith, Eric R; Hartley, Robert C; Abendroth, Jan; Sankaran, Banumathi; Lorimer, Donald D; Myler, Peter J; Staker, Bart L; Stewart, Lance J

    2011-09-01

    Babesiosis is a tick-borne disease caused by eukaryotic Babesia parasites which are morphologically similar to Plasmodium falciparum, the causative agent of malaria in humans. Like Plasmodium, different species of Babesia are tuned to infect different mammalian hosts, including rats, dogs, horses and cattle. Most species of Plasmodium and Babesia possess an essential bifunctional enzyme for nucleotide synthesis and folate metabolism: dihydrofolate reductase-thymidylate synthase. Although thymidylate synthase is highly conserved across organisms, the bifunctional form of this enzyme is relatively uncommon in nature. The structural characterization of dihydrofolate reductase-thymidylate synthase in Babesia bovis, the causative agent of babesiosis in livestock cattle, is reported here. The apo state is compared with structures that contain dUMP, NADP and two different antifolate inhibitors: pemetrexed and raltitrexed. The complexes reveal modes of binding similar to that seen in drug-resistant malaria strains and point to the utility of applying structural studies with proven cancer chemotherapies towards infectious disease research.

  15. The fate of the carboxyl oxygens during D-proline reduction by clostridial proline reductase.

    Science.gov (United States)

    Arkowitz, R A; Dhe-Paganon, S; Abeles, R H

    1994-06-01

    D-Proline is converted to 5-amino valeric acid by D-proline reductase. This conversion involves the reductive cleavage of the alpha-carbon-nitrogen bond. We have examined the fate of the carboxyl oxygen atoms during conversion of D-proline to delta-NH2-valeric acid. 18O atoms from the carboxyl group of D-proline are not lost during conversion to product. In contrast, in the conversion of glycine to acetyl phosphate by glycine reductase a carboxyl oxygen atom is lost to solvent. An intermediate acyl-enzyme is found during the reduction of glycine. We conclude that the reduction of proline proceeds without the formation of an acyl enzyme intermediate.

  16. Peroxisomal trans-2-enoyl-CoA reductase is involved in phytol degradation.

    Science.gov (United States)

    Gloerich, J; Ruiter, J P N; van den Brink, D M; Ofman, R; Ferdinandusse, S; Wanders, R J A

    2006-04-03

    Phytol is a naturally occurring precursor of phytanic acid. The last step in the conversion of phytol to phytanoyl-CoA is the reduction of phytenoyl-CoA mediated by an, as yet, unidentified enzyme. A candidate for this reaction is a previously described peroxisomal trans-2-enoyl-CoA reductase (TER). To investigate this, human TER was expressed in E. coli as an MBP-fusion protein. The purified recombinant protein was shown to have high reductase activity towards trans-phytenoyl-CoA, but not towards the peroxisomal beta-oxidation intermediates C24:1-CoA and pristenoyl-CoA. In conclusion, our results show that human TER is responsible for the reduction of phytenoyl-CoA to phytanoyl-CoA in peroxisomes.

  17. A novel prokaryotic trans-2-enoyl-CoA reductase from the spirochete Treponema denticola.

    Science.gov (United States)

    Tucci, Sara; Martin, William

    2007-04-17

    An NADH-dependent trans-2-enoyl-CoA reductase (EC1.1.1.36) from the Gram negative spirochete Treponema denticola was identified, expressed and biochemically characterized. The recombinant protein is a monomeric enzyme with a molecular mass of 44 kDa with a specific activity of 43+/-4.8 U/mg (micromol mg(-1)min(-1)) and K(m) value of 2.7 microM for crotonoyl-CoA. This NADH-dependent trans-2-enoyl-CoA reductase represents the first enzymatically characterized member of a prokaryotic protein family involved in a fatty acid synthesis pathway that is distinct from the familiar fatty acid synthase system.

  18. The use of 5-alpha reductase inhibitors in the treatment of benign prostatic hyperplasia

    Directory of Open Access Journals (Sweden)

    Eric H. Kim

    2018-01-01

    Full Text Available Benign prostatic hyperplasia (BPH is characterized by an enlarged prostate, lower urinary tract symptoms (LUTS, and a decreased urinary flow rate. Common in older men, BPH is a progressive disease that can eventually lead to complications including acute urinary retention (AUR and the need for BPH-related surgery. Both normal and abnormal prostate growth is driven by the androgen dihydrotestosterone (DHT, which is formed from testosterone under the influence of 5-alpha reductase. Thus, 5-alpha reductase inhibitors (5-ARIs effectively reduce the serum and intraprostatic concentration of DHT, causing an involution of prostate tissue. Two 5-ARIs are currently available for the treatment of BPH—finasteride and dutasteride. Both have been demonstrated to decrease prostate volume, improve LUTS and urinary flow rates, which ultimately reduces the risk of AUR and BPH-related surgery. Therefore, either alone or in combination with other BPH medications, 5-ARIs are a mainstay of BPH management.

  19. The structure of Lactococcus lactis thioredoxin reductase reveals molecular features of photo-oxidative damage

    DEFF Research Database (Denmark)

    Skjoldager, Nicklas; Bang, Maria Blanner; Rykær, Martin

    2017-01-01

    The NADPH-dependent homodimeric flavoenzyme thioredoxin reductase (TrxR) provides reducing equivalents to thioredoxin, a key regulator of various cellular redox processes. Crystal structures of photo-inactivated thioredoxin reductase (TrxR) from the Gram-positive bacterium Lactococcus lactis have...... been determined. These structures reveal novel molecular features that provide further insight into the mechanisms behind the sensitivity of this enzyme toward visible light. We propose that a pocket on the si-face of the isoalloxazine ring accommodates oxygen that reacts with photo-excited FAD...... thus be a widespread feature among bacterial TrxR with the described characteristics, which affords applications in clinical photo-therapy of drug-resistant bacteria....

  20. Co-Expression of Monodehydroascorbate Reductase and Dehydroascorbate Reductase from Brassica rapa Effectively Confers Tolerance to Freezing-Induced Oxidative Stress

    Science.gov (United States)

    Shin, Sun-Young; Kim, Myung-Hee; Kim, Yul-Ho; Park, Hyang-Mi; Yoon, Ho-Sung

    2013-01-01

    Plants are exposed to various environmental stresses and have therefore developed antioxidant enzymes and molecules to protect their cellular components against toxicity derived from reactive oxygen species (ROS). Ascorbate is a very important antioxidant molecule in plants, and monodehydroascorbate reductase (MDHAR; EC 1.6.5.4) and dehydroascorbate reductase (DHAR; EC 1.8.5.1) are essential to regeneration of ascorbate for maintenance of ROS scavenging ability. The MDHAR and DHAR genes from Brassica rapa were cloned, transgenic plants overexpressing either BrMDHAR and BrDHAR were established, and then, each transgenic plant was hybridized to examine the effects of co-expression of both genes conferring tolerance to freezing. Transgenic plants co-overexpressing BrMDHAR and BrDHAR showed activated expression of relative antioxidant enzymes, and enhanced levels of glutathione and phenolics under freezing condition. Then, these alteration caused by co-expression led to alleviated redox status and lipid peroxidation and consequently conferred improved tolerance against severe freezing stress compared to transgenic plants overexpressing single gene. The results of this study suggested that although each expression of BrMDHAR or BrDHAR was available to according tolerance to freezing, the simultaneous expression of two genes generated synergistic effects conferring improved tolerance more effectively even severe freezing. PMID:24170089

  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. Efforts toward the direct experimental characterization of enzyme microenvironments: tyrosine100 in dihydrofolate reductase.

    Science.gov (United States)

    Groff, Dan; Thielges, Megan C; Cellitti, Susan; Schultz, Peter G; Romesberg, Floyd E

    2009-01-01

    State secrets: Site-specific deuteration and FTIR studies reveal that Tyr100 in dihydrofolate reductase plays an important role in catalysis, with a strong electrostatic coupling occurring between Tyr100 and the charge that develops in the hydride-transfer transition state (see picture, NADP(+) purple, Tyr100 green). However, relaying correlated motions that facilitate catalysis from distal sites of the protein to the hydride donor may also be involved.

  3. HMG-CoA Reductase Inhibitors from Monascus-Fermented Rice

    Directory of Open Access Journals (Sweden)

    Xuemei Li

    2013-01-01

    Full Text Available Seven compounds were isolated from Monascus-fermented rice by column chromatography with silica gel and semiprep HPLC. Their structures were elucidated by extensive spectroscopic methods. All compounds displayed HMG-CoA reductase inhibitory potential, among them compound 7 exhibited strong inhibition with IC50 value comparable with lovastatin. In this study, two compounds (1 and 2 were obtained from natural source for the first time.

  4. Immunological Characterization of Nitrate Reductase in Different Tissues of Spinach Seedlings

    OpenAIRE

    Hiroki, Nakagawa; Kenji, Yamagishi; Naoko, Yamashita; Takahide, Sato; Nagao, Ogura; Ann, Oaks; Department of Agricultural Chemistry, Faculty of Horticulture, Chiba University; Department of Biology, McMaster University

    1986-01-01

    In spinach seedlings and roots, NADH-nitrate reductase (NR) activity (per g fresh weight) decreased as the seedlings aged. Experiments using double immunodiffusion analysis and immunotitration showed no differences in the immunological properties of NR from spinach seedlings at various stages of aging. Comparison of spinach leaf to the spinach root enzyme using the Ouchterlony double diffusion technique revealed a high degree of similarity between them.

  5. Functional Characterization of Four Putative δ1-Pyrroline-5-Carboxylate Reductases from Bacillus subtilis

    Energy Technology Data Exchange (ETDEWEB)

    Forlani, Giuseppe; Nocek, Boguslaw; Chakravarthy, Srinivas; Joachimiak, Andrzej

    2017-08-02

    In most living organisms, the amino acid proline is synthesized starting from both glutamate and ornithine. In prokaryotes, in the absence of an ornithine cyclodeaminase that has been identified to date only in a small number of soil and plant bacteria, these pathways share the last step, the reduction of delta(1)-pyrroline-5-carboxylate (P5C) catalyzed by P5C reductase (EC 1.5.1.2). In several species, multiple forms of P5C reductase have been reported, possibly reflecting the dual function of proline. Aside from its common role as a building block of proteins, proline is indeed also involved in the cellular response to osmotic and oxidative stress conditions. Genome analysis of Bacillus subtilis identifies the presence of four genes (ProH, ProI, ProG, and ComER) that, based on bioinformatic and phylogenic studies, were defined as respectively coding a putative P5C reductase. Here we describe the cloning, heterologous expression, functional analysis and small-angle X-ray scattering studies of the four affinity-purified proteins. Results showed that two of them, namely ProI and ComER, lost their catalytic efficiency or underwent subfunctionalization. In the case of ComER, this could be likely explained by the loss of the ability to form a dimer, which has been previously shown to be an essential structural feature of the catalytically active P5C reductase. The properties of the two active enzymes are consistent with a constitutive role for ProG, and suggest that ProH expression may be beneficial to satisfy an increased need for proline.

  6. Functional Characterization of Four Putative δ1-Pyrroline-5-Carboxylate Reductases from Bacillus subtilis

    Energy Technology Data Exchange (ETDEWEB)

    Forlani, Giuseppe; Nocek, Boguslaw; Chakravarthy, Srinivas; Joachimiak, Andrzej

    2017-08-02

    In most living organisms, the amino acid proline is synthesized starting from both glutamate and ornithine. In prokaryotes, in the absence of an ornithine cyclodeaminase that has been identified to date only in a small number of soil and plant bacteria, these pathways share the last step, the reduction of δ1-pyrroline-5-carboxylate (P5C) catalyzed by P5C reductase (EC 1.5.1.2). In several species, multiple forms of P5C reductase have been reported, possibly reflecting the dual function of proline. Aside from its common role as a building block of proteins, proline is indeed also involved in the cellular response to osmotic and oxidative stress conditions. Genome analysis of Bacillus subtilis identifies the presence of four genes (ProH, ProI, ProG, and ComER) that, based on bioinformatic and phylogenic studies, were defined as respectively coding a putative P5C reductase. Here we describe the cloning, heterologous expression, functional analysis and small-angle X-ray scattering studies of the four affinity-purified proteins. Results showed that two of them, namely ProI and ComER, lost their catalytic efficiency or underwent subfunctionalization. In the case of ComER, this could be likely explained by the loss of the ability to form a dimer, which has been previously shown to be an essential structural feature of the catalytically active P5C reductase. The properties of the two active enzymes are consistent with a constitutive role for ProG, and suggest that ProH expression may be beneficial to satisfy an increased need for proline.

  7. Biochemical and structural characterization of the trans-enoyl-CoA reductase from Treponema denticola.

    Science.gov (United States)

    Bond-Watts, Brooks B; Weeks, Amy M; Chang, Michelle C Y

    2012-08-28

    The production of fatty acids is an important cellular pathway for both cellular function and the development of engineered pathways for the synthesis of advanced biofuels. Despite the conserved reaction chemistry of various fatty acid synthase systems, the individual isozymes that catalyze these steps are quite diverse in their structural and biochemical features and are important for controlling differences at the cellular level. One of the key steps in the fatty acid elongation cycle is the enoyl-ACP (CoA) reductase function that drives the equilibrium forward toward chain extension. In this work, we report the structural and biochemical characterization of the trans-enoyl-CoA reductase from Treponema denticola (tdTer), which has been utilized for the engineering of synthetic biofuel pathways with an order of magnitude increase in product titers compared to those of pathways constructed with other enoyl-CoA reductase components. The crystal structure of tdTer was determined to 2.00 Å resolution and shows that the Ter enzymes are distinct from members of the FabI, FabK, and FabL families but are highly similar to members of the FabV family. Further biochemical studies show that tdTer uses an ordered bi-bi mechanism initiated by binding of the NADH redox cofactor, which is consistent with the behavior of other enoyl-ACP (CoA) reductases. Mutagenesis of the substrate binding loop, characterization of enzyme activity with respect to crotonyl-CoA, hexenoyl-CoA, and dodecenoyl-CoA substrates, and product inhibition by lauroyl-CoA suggest that this region is important for controlling chain length specificity, with the major portal playing a more important role for longer chain length substrates.

  8. Identification of new potent inhibitor of aldose reductase from Ocimum basilicum.

    Science.gov (United States)

    Bhatti, Huma Aslam; Tehseen, Yildiz; Maryam, Kiran; Uroos, Maliha; Siddiqui, Bina S; Hameed, Abdul; Iqbal, Jamshed

    2017-12-01

    Recent efforts to develop cure for chronic diabetic complications have led to the discovery of potent inhibitors against aldose reductase (AKR1B1, EC 1.1.1.21) whose role in diabetes is well-evident. In the present work, two new natural products were isolated from the ariel part of Ocimum basilicum; 7-(3-hydroxypropyl)-3-methyl-8-β-O-d-glucoside-2H-chromen-2-one (1) and E-4-(6'-hydroxyhex-3'-en-1-yl)phenyl propionate (2) and confirmed their structures with different spectroscopic techniques including NMR spectroscopy etc. The isolated compounds (1, 2) were evaluated for in vitro inhibitory activity against aldose reductase (AKR1B1) and aldehyde reductase (AKR1A1). The natural product (1) showed better inhibitory activity for AKR1B1 with IC50 value of 2.095±0.77µM compare to standard sorbinil (IC50=3.14±0.02µM). Moreover, the compound (1) also showed multifolds higher activity (IC50=0.783±0.07µM) against AKR1A1 as compared to standard valproic acid (IC50=57.4±0.89µM). However, the natural product (2) showed slightly lower activity for AKR1B1 (IC50=4.324±1.25µM). Moreover, the molecular docking studies of the potent inhibitors were also performed to identify the putative binding modes within the active site of aldose/aldehyde reductases. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Microbial production of branched-chain dicarboxylate 2-methylsuccinic acid via enoate reductase-mediated bioreduction.

    Science.gov (United States)

    Wang, Jian; Yang, Yaping; Zhang, Ruihua; Shen, Xiaolin; Chen, Zhenya; Wang, Jia; Yuan, Qipeng; Yan, Yajun

    2018-01-01

    2-Methylsuccinic acid (2-MSA) is a C5 branched-chain dicarboxylate that serves as an attractive synthon for the synthesis of polymers with extensive applications in coatings, cosmetic solvents and bioplastics. However, the lack of natural pathways for 2-MSA biosynthesis has limited its application as a promising bio-replacement. Herein, we conceived a non-natural three-step biosynthetic route for 2-MSA, via employing the citramalate pathway in combination with enoate reductase-mediated bioreduction of the pathway intermediate citraconate. First, over-expression of codon-optimized citramalate synthase variant CimA* from Methanococcus jannaschii, endogenous isopropylmalate isomerase EcLeuCD and enoate reductase YqjM from Bacillus subtilis allowed the production of 2-MSA in Escherichia coli for the first time, with a titer of 0.35g/L in shake flask experiments. Subsequent screening of YqjM-like enoate reductases of different bacterial origins enabled identification and characterization of a new NAD(P)H-dependent enoate reductase KpnER from Klebsiella pneumoniae, which exhibited higher activity towards citraconate than YqjM. Incorporation of KpnER into the 2-MSA biosynthetic pathway led to 2-MSA production improvement to a titer of 0.96g/L in aerobic condition. Subsequent optimizations including cofactor regeneration, microaerobic cultivation and host strain engineering, boosted 2-MSA titer to 3.61g/L with a molar yield of 0.36 in shake flask experiments. This work established a promising platform for 2-MSA bioproduction, which enabled the highest titer of 2-MSA production in microbial hosts so far. Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  10. Profiling patterns of glutathione reductase inhibition by the natural product illudin S and its acylfulvene analogues

    OpenAIRE

    LIU, XIAODAN; Sturla, Shana J.

    2009-01-01

    Acylfulvenes (AFs) are a class of antitumor agents with favorable cytotoxic selectivity profiles compared to their natural product precursor, illudin S. Like many alkylating agents, illudin S and AFs readily react with thiol-containing small molecules such as cysteine, glutathione and cysteine-containing peptides; reduced cellular glutathione levels can affect illudin S toxicity. Glutathione reductase (GR) is a critical cellular anti-oxidant enzyme that regulates the intracellular ratio of re...

  11. Regulation of schistosome egg production by HMG CoA reductase

    Energy Technology Data Exchange (ETDEWEB)

    VandeWaa, E.A.; Bennett, J.L.

    1986-03-05

    Hydroxymethylglutaryl coenzyme A reductase (HMG CoA reductase) catalyzes the conversion of HMG CoA to mevalonate in the synthesis of steroids, isoprenoids and terpenes. Mevinolin, an inhibitor of this enzyme, decreased egg production in Schistosoma mansoni during in vitro incubations. This was associated with a reduction in the incorporation of /sup 14/C-acetate into polyisoprenoids and a reduction in the formation of a lipid-linked oligosaccharide. In vivo, mevinolin in daily doses of 50 mg/kg (p.o., from days 30-48 post-infection) caused no change in gross liver pathology in S. mansoni infected mice. However, when parasites exposed to mevinolin or its vehicle in vivo were cultured in vitro, worms from mevinolin-treated mice produced six times more eggs than control parasites. When infected mice were dosed with 250 mg/kg mevinolin daily (p.o., from days 35-45 post-infection), liver pathology was reduced in comparison to control mice. Thus, during in vivo exposure to a high dose of the drug egg production is decreased, while at a lower dose it appears unaffected until the parasites are cultured in a drug-free in vitro system wherein egg production is stimulated to extraordinarily high levels. It may be that at low doses mevinolin, by inhibiting the enzyme, is blocking the formation of a product (such as an isoprenoid) which normally acts to down-regulate enzyme synthesis, resulting in enzyme induction. Induction of HMG CoA reductase is then expressed as increased egg production when the worms are removed from the drug. These data suggest that HMG CoA reductase plays a role in schistosome egg production.

  12. Thymol, a monoterpene, inhibits aldose reductase and high-glucose-induced cataract on isolated goat lens

    OpenAIRE

    Divya M Kanchan; Kale, Smita S.; Gauresh S Somani; Aakruti A Kaikini; Sadhana Sathaye

    2016-01-01

    Background: Overactivation of aldose reductase (AR) enzyme has been implicated in the development of various diabetic complications. In the present study, the inhibitory effect of thymol was investigated on AR enzyme and its anti-cataract activity was also examined on isolated goat lens. Materials and Methods: Various concentrations of thymol were incubated with AR enzyme prepared from isolated goat lens. Molecular docking studies were carried out using Schrodinger software to verify the bind...

  13. Valproic acid increases expression of methylenetetrahydrofolate reductase (MTHFR) and induces lower teratogenicity in MTHFR deficiency

    OpenAIRE

    Roy, Marc; Leclerc, Daniel; Wu, Qing; Gupta, Sapna; Kruger, Warren D.; Rozen, Rima

    2008-01-01

    Valproate (VPA) treatment in pregnancy leads to congenital anomalies, possibly by disrupting folate or homocysteine metabolism. Since methylenetetrahydrofolate reductase (MTHFR) is a key enzyme of folate interconversion and homocysteine metabolism, we addressed the possibility that VPA might have different teratogenicity in Mthfr+/+ and Mthfr+/− mice and that VPA might interfere with folate metabolism through MTHFR modulation. Mthfr+/+ and Mthfr+/− pregnant mice were injected with VPA on gest...

  14. Structure of the Membrane-intrinsic Nitric Oxide Reductase from Roseobacter denitrificans

    OpenAIRE

    Crow, Allister; Matsuda, Yuji; Arata, Hiroyuki; Oubrie, Arthur

    2016-01-01

    This is the author accepted manuscript. The final version is available from the American Chemical Society via http://dx.doi.org/10.1021/acs.biochem.6b00332 Membrane-intrinsic nitric oxide reductases (NORs) are key components of bacterial denitrification pathways with a close evolutionary relationship to the cytochrome oxidase (COX) complex found in aerobic respiratory chains. A key distinction between COX and NOR is the identity of the metal directly opposite heme b$_3$ within the active s...

  15. The stability of the three transmembrane and the four transmembrane human vitamin K epoxide reductase models

    Science.gov (United States)

    Wu, Sangwook

    2016-04-01

    The three transmembrane and the four transmembrane helix models are suggested for human vitamin K epoxide reductase (VKOR). In this study, we investigate the stability of the human three transmembrane/four transmembrane VKOR models by employing a coarse-grained normal mode analysis and molecular dynamics simulation. Based on the analysis of the mobility of each transmembrane domain, we suggest that the three transmembrane human VKOR model is more stable than the four transmembrane human VKOR model.

  16. CLINICAL SIGNIFICANCE OF 5αα-REDUCTASE AND ANDROGEN RECEPTOR GENE POLYMORPHISMS IN PROSTATE CANCER

    Directory of Open Access Journals (Sweden)

    O. B. Loran

    2014-07-01

    Full Text Available The development of prostate cancer is inseparably linked with the effect of androgens on the fundamental prostatic intracellular processes,such as proliferation, apoptosis, which is realized through a number of second messengers. Major of them are the AR gene encoding androgenreceptors and the SRD5A2 gene encoding 5α-reductase enzyme. This paper deals with the study of the role of these genes in prostate cancer.  

  17. CLINICAL SIGNIFICANCE OF 5αα-REDUCTASE AND ANDROGEN RECEPTOR GENE POLYMORPHISMS IN PROSTATE CANCER

    Directory of Open Access Journals (Sweden)

    O. B. Loran

    2009-01-01

    Full Text Available The development of prostate cancer is inseparably linked with the effect of androgens on the fundamental prostatic intracellular processes,such as proliferation, apoptosis, which is realized through a number of second messengers. Major of them are the AR gene encoding androgenreceptors and the SRD5A2 gene encoding 5α-reductase enzyme. This paper deals with the study of the role of these genes in prostate cancer.  

  18. Physiological Roles for Two Periplasmic Nitrate Reductases in Rhodobacter sphaeroides 2.4.3 (ATCC 17025)▿

    Science.gov (United States)

    Hartsock, Angela; Shapleigh, James P.

    2011-01-01

    The metabolically versatile purple bacterium Rhodobacter sphaeroides 2.4.3 is a denitrifier whose genome contains two periplasmic nitrate reductase-encoding gene clusters. This work demonstrates nonredundant physiological roles for these two enzymes. One cluster is expressed aerobically and repressed under low oxygen while the second is maximally expressed under low oxygen. Insertional inactivation of the aerobically expressed nitrate reductase eliminated aerobic nitrate reduction, but cells of this strain could still respire nitrate anaerobically. In contrast, when the anaerobic nitrate reductase was absent, aerobic nitrate reduction was detectable, but anaerobic nitrate reduction was impaired. The aerobic nitrate reductase was expressed but not utilized in liquid culture but was utilized during growth on solid medium. Growth on a variety of carbon sources, with the exception of malate, the most oxidized substrate used, resulted in nitrite production on solid medium. This is consistent with a role for the aerobic nitrate reductase in redox homeostasis. These results show that one of the nitrate reductases is specific for respiration and denitrification while the other likely plays a role in redox homeostasis during aerobic growth. PMID:21949073

  19. [Manganese-dependent ribonucleotide reductase of Propionibacterium freudenreichii subsp. shermanii: partial purification, characterization, and role in DNA biosynthesis].

    Science.gov (United States)

    Iordan, E P; Bryukhanov, A L; Dunaevskiĭ, Ia E; Pryanishnikova, N I; Danilova, I V

    2000-01-01

    Like Lactobacillus leichmanii, Rhizobium meliloti, and Euglena gracilis, P. freudenreichii implicates cobalamin in DNA anabolism via adenosylcobalamin-dependent ribonucleotide reductase. However, in the absence of corrinoids, P. freudenreichii is able to synthesize DNA with the involvement of an alternative ribonucleotide reductase, which is independent of adenosylcobalamin. This enzyme is localized in both the cytoplasm (80% of activity) and the cytoplasmic membrane (20% of activity), being loosely bound to the latter. Experiments with crude ribonucleotide reductase isolated from extracts of corrinoid-deficient cells showed that manganese specifically stimulates this enzyme and that it is composed of two protein subunits, a feature that is typical of all metal-containing reductases activated by molecular oxygen. Low concentrations of manganese ions enhanced DNA synthesis in corrinoid-deficient manganese-limited cells. This effect was prevented by the addition of 80 mM hydroxyurea, a specific inhibitor of metal-containing aerobic ribonucleotide reductases. It was concluded that, in adenosylcobalamin-deficient P. freudenreichii cells, DNA synthesis is provided with deoxyribosyl precursors through the functioning of manganese-dependent aerobic ribonucleotide reductase composed of two subunits.

  20. Overexpression of Soybean Isoflavone Reductase (GmIFR) Enhances Resistance to Phytophthora sojae in Soybean.

    Science.gov (United States)

    Cheng, Qun; Li, Ninghui; Dong, Lidong; Zhang, Dayong; Fan, Sujie; Jiang, Liangyu; Wang, Xin; Xu, Pengfei; Zhang, Shuzhen

    2015-01-01

    Isoflavone reductase (IFR) is an enzyme involved in the biosynthetic pathway of isoflavonoid phytoalexin in plants. IFRs are unique to the plant kingdom and are considered to have crucial roles in plant response to various biotic and abiotic environmental stresses. Here, we report the characterization of a novel member of the soybean isoflavone reductase gene family GmIFR. Overexpression of GmIFR transgenic soybean exhibited enhanced resistance to Phytophthora sojae. Following stress treatments, GmIFR was significantly induced by P. sojae, ethephon (ET), abscisic acid (placeCityABA), salicylic acid (SA). It is located in the cytoplasm when transiently expressed in soybean protoplasts. The daidzein levels reduced greatly for the seeds of transgenic plants, while the relative content of glyceollins in transgenic plants was significantly higher than that of non-transgenic plants. Furthermore, we found that the relative expression levels of reactive oxygen species (ROS) of transgenic soybean plants were significantly lower than those of non-transgenic plants after incubation with P. sojae, suggesting an important role of GmIFR might function as an antioxidant to reduce ROS in soybean. The enzyme activity assay suggested that GmIFR has isoflavone reductase activity.

  1. Major Peptides from Amaranth (Amaranthus cruentus Protein Inhibit HMG-CoA Reductase Activity

    Directory of Open Access Journals (Sweden)

    Rosana Aparecida Manólio Soares

    2015-02-01

    Full Text Available The objective of this study was to identify the major peptides generated by the in vitro hydrolysis of Amaranthus cruentus protein and to verify the effect of these peptides on the activity of 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG-CoA reductase, a key enzyme in cholesterol biosynthesis. A protein isolate was prepared, and an enzymatic hydrolysis that simulated the in vivo digestion of the protein was performed. After hydrolysis, the peptide mixture was filtered through a 3 kDa membrane. The peptide profile of this mixture was determined by reversed phase high performance chromatography (RP-HPLC, and the peptide identification was performed by LC-ESI MS/MS. Three major peptides under 3 kDa were detected, corresponding to more than 90% of the peptides of similar size produced by enzymatic hydrolysis. The sequences identified were GGV, IVG or LVG and VGVI or VGVL. These peptides had not yet been described for amaranth protein nor are they present in known sequences of amaranth grain protein, except LVG, which can be found in amaranth α‑amylase. Their ability to inhibit the activity of HMG-CoA reductase was determined, and we found that the sequences GGV, IVG, and VGVL, significantly inhibited this enzyme, suggesting a possible hypocholesterolemic effect.

  2. Enhancing survival of Escherichia coli by expression of azoreductase AZR possessing quinone reductase activity.

    Science.gov (United States)

    Liu, Guangfei; Zhou, Jiti; Jin, Ruofei; Zhou, Mi; Wang, Jing; Lu, Hong; Qu, Yuanyuan

    2008-09-01

    Quinone reductase activity of azoreductase AZR from Rhodobacter sphaeroides was reported. High homologies were found in the cofactor/substrate-binding regions of quinone reductases from different domains. 3D structure comparison revealed that AZR shared a common overall topology with mammal NAD(P)H/quinone oxidoreductase NQO1. With menadione as substrate, the optimal pH value and temperature were pH 8-9 and 50 degrees C, respectively. Following the ping-pong kinetics, AZR transferred two electrons from NADPH to quinone substrate. It could reduce naphthoquinones and anthraquinones, such as menadione, lawsone, anthraquinone-2-sulfonate, and anthraquinone-2,6-disulfonate. However, no activity was detected with 1,4-benzoquinone. Dicoumarol competitively inhibited AZR's quinone reductase activity with respect to NADPH, with an obtained K (i) value of 87.6 microM. Significantly higher survival rates were obtained in Escherichia coli YB overexpressing AZR than in the control strain when treated by heat shock and oxidative stressors such as H(2)O(2) and menadione.

  3. Expression of steroid 5α-reductase isozymes in prostate of adult rats after environmental stress.

    Science.gov (United States)

    Sánchez, Pilar; Torres, Jesús M; Castro, Beatriz; Olmo, Asunción; del Moral, Raimundo G; Ortega, Esperanza

    2013-01-01

    The elevated incidence of prostate cancer and benign prostatic hypertrophy is a cause of increasing public health concern in the Western world. The normal and pathological growth of the prostate are both dependent on stimulation by dihydrotestosterone, which is synthesized from circulating testosterone by two 5α-reductase (5α-R) isozymes, 5α-reductase type 1 (5α-R1) and 5α-reductase type 2 (5α-R2). Both isozymes have been implicated in prostate disease. We used quantitative RT-PCR and immunohistochemistry, respectively, to quantify mRNA and protein levels of 5α-R isozymes in the ventral prostate of adult rats under environmental stress conditions analogous to those found in some common workplace situations, i.e. artificial light, excessive heat, and the sensation of immobility in a small space. Transcription and expression levels of both 5α-R isozymes were significantly higher in environmentally stressed rats than in unstressed rats. Increased 5α-R isozyme levels may play a role in the development or maintenance of prostate disease. Further research is warranted to explore these effects of environmental stress on human health and their implications for environmental and occupational health policies. © 2012 The Authors Journal compilation © 2012 FEBS.

  4. The occurence of a nitrate reductase inactivating factor in extracts of Spirodela polyrrhiza

    Directory of Open Access Journals (Sweden)

    Józef Buczek

    2014-01-01

    Full Text Available NADH-nitrate reductase (NR had a low activity immediately after extraction from 14 day-old cultures of Spirodela polyrrhiza with the basic extraction solution (50 mM K-phosphate buffer, pH 7.5, 5 mM cysteine and 3 mM EDTA whereas nitrite reductase (NiR activity in the same extract was very high. Increasing the EDTA or cysteine concentration or replacing cysteine by dithiothreitol had no effect on the level of NR activity. The addition of 3% bovine serum albumine (BSA and 0.4 mM phenylmethyl sulfonyfluoride (PMSF or 3% BSA and 1.5% polyvinylpolypyrrolidone (PVPP to the extraction solution greatly increased the NR activity. The enzyme activity increased 40 times in the presence of 1.5% PVPP with 3% BSA and 0.4 mM PMSF. The highest NR activity (over 100-fold was found in the presence of 3% caseine. The probable nature of the factor inactivating nitrate reductase in S. polyrrhiza extracts is discussed.

  5. Screening for inhibitors of dihydrofolate reductase using pulsed ultrafiltration mass spectrometry.

    Science.gov (United States)

    Nikolic, D; van Breemen, R B

    1998-04-01

    A method of screening combinatorial libraries for inhibitors of eukaryotic dihydrofolate reductase has been developed using pulsed ultra-filtration electrospray mass spectrometry, which is a continuous-flow affinity separation system for extracting and identifying high affinity ligands in combinatorial libraries. In this application, pulsed ultrafiltration conditions were optimized for the isolation and identification of inhibitors of dihydrofolate reductase from a 22 compound library containing six known inhibitors of the enzyme including trimethoprim, aminopterin, methotrexate, pyrimethamine, folic acid, and folinic acid, and 16 compounds without known affinity. In order to optimize the screening method, sources of non-specific binding were identified and minimized. A significant source of non-specific binding for this set of library compounds was hydrophobic interaction with the surfaces of the ultrafiltration chamber. After affinity separation of bound (high affinity) versus free (low affinity) library compounds during pulsed ultrafiltration, receptor-bound ligands were released and eluted using either organic solvent or acidified mobile phase. Although 80% methanol easily disrupted the receptor-ligand complexes, organic solvent had the undesirable effect of releasing non-specifically bound compounds from the chamber and thereby increasing the background noise. Interference from non-specific binding was minimized by releasing bound ligands using a low pH mobile phase eluent instead of organic solvent. Under the conditions used, pulsed ultrafiltration mass spectrometry selectively identified the two library compounds with the highest affinity for dihydrofolate reductase, methotrexate and aminopterin.

  6. Modulating the molybdenum coordination sphere of Escherichia coli trimethylamine N-oxide reductase.

    Science.gov (United States)

    Kaufmann, Paul; Duffus, Benjamin R; Mitrova, Biljana; Iobbi-Nivol, Chantal; Teutloff, Christian; Nimtz, Manfred; Jänsch, Lothar; Wollenberger, Ulla; Leimkühler, Silke

    2018-01-15

    The well-studied enterobacterium Escherichia coli present in the human gut is able to reduce TMAO to trimethylamine (TMA) during anaerobic respiration. The TMAO reductase TorA is a monomeric, bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor-containing enzyme belonging to the dimethylsulfoxide (DMSO) reductase family of molybdoenzymes. We report on a system for the in vitro reconstitution of TorA with molybdenum cofactors (Moco) from different sources. Higher TMAO reductase activities for TorA were obtained when using Moco-sources containing a sulfido ligand at the molybdenum atom. For the first time, we were able to isolate functional bis-MGD from Rhodobacter capsulatus formate dehydrogenase (FDH), which remained intact in its isolated state and after insertion into apo-TorA yielded a highly active enzyme. Combined characterizations of the reconstituted TorA enzymes by EPR spectroscopy and direct electrochemistry emphasize that TorA activity can be modified by changes in the Mo-coordination sphere. The combination of these results together with studies on amino acid exchanges at the active site led us to propose a novel model for substrate binding to the molybdenum atom of TorA.

  7. A DFT-based QSAR study on inhibition of human dihydrofolate reductase.

    Science.gov (United States)

    Karabulut, Sedat; Sizochenko, Natalia; Orhan, Adnan; Leszczynski, Jerzy

    2016-11-01

    Diaminopyrimidine derivatives are frequently used as inhibitors of human dihydrofolate reductase, for example in treatment of patients whose immune system are affected by human immunodeficiency virus. Forty-seven dicyclic and tricyclic potential inhibitors of human dihydrofolate reductase were analyzed using the quantitative structure-activity analysis supported by DFT-based and DRAGON-based descriptors. The developed model yielded an RMSE deviation of 1.1 a correlation coefficient of 0.81. The prediction set was characterized by R 2 =0.60 and RMSE=3.59. Factors responsible for inhibition process were identified and discussed. The resulting model was validated via cross validation and Y-scrambling procedure. From the best model, we found several mass-related descriptors and Sanderson electronegativity-related descriptors that have the best correlations with the investigated inhibitory concentration. These descriptors reflect results from QSAR studies based on characteristics of human dihydrofolate reductase inhibitors. Copyright © 2016. Published by Elsevier Inc.

  8. Inhibition of 3-Hydroxy-3-methylglutaryl Coenzyme A Reductase (Ex Vivo by Morus indica (Mulberry

    Directory of Open Access Journals (Sweden)

    Vanitha Reddy Palvai

    2014-01-01

    Full Text Available Phytochemicals are the bioactive components that contribute to the prevention of cardiovascular and other degenerative diseases. Inhibition of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA reductase would be an effective means of lowering plasma cholesterol in humans. The present study explores the HMG CoA reductase inhibitory effect of extracts from leaves of Morus indica varieties, M5, V1, and S36, compared with the statin, using an ex vivo method. The assay is based on the stoichiometric formation of coenzyme A during the reduction of microsomal HMG CoA to mevalonate. Dechlorophyllised extract of three varieties was studied at 300 µg. The coenzyme A released at the end of assay in control (100.31 nmoles and statins (94.46 nm was higher than the dechlorphyllised extracts of the samples. The coenzyme A released during the reduction of HMG CoA to mevalonate in dechlorophyllised extracts of the samples was as follows: S36 < M5 < V1. The results indicated that the samples were highly effective in inhibiting the enzyme compared to statins (standard drug. The results indicate the role of Morus varieties extracts in modulating the cholesterol metabolism by inhibiting the activity of HMG CoA reductase. These results provide scope for designing in vivo animal studies to confirm their effect.

  9. Crystal structure of quinone reductase 2 in complex with cancer prodrug CB1954.

    Science.gov (United States)

    Fu, Yue; Buryanovskyy, Leonid; Zhang, Zhongtao

    2005-10-14

    CB1954 is a cancer pro-drug that can be activated through reduction by Escherichia coli nitro-reductases and quinone reductases. Human quinone reductase 2 is very efficient in the activation of CB1954, approximately 3000 times more efficient than human QR1 in terms of k(cat)/K(m). We have solved the three-dimensional structure of QR2 in complex with CB1954 to a nominal resolution of 1.5A. The complex structure indicates the essentiality of the two nitro groups: one nitro group forms hydrogen bonds with the side-chain of Asn161 of QR2 to hold the other nitro group in position for the reduction. We further conclude that residue 161, an Asn in QR2 and a His in QR1, is critical in differentiating the substrate specificities of these two enzymes. Mutation of Asn161 to His161 in QR2 resulted in the total loss of the enzymatic activity towards activation of CB1954, whereas the rates of reduction towards menadione are not altered.

  10. Differential regulation of wheat quinone reductases in response to powdery mildew infection.

    Science.gov (United States)

    Greenshields, David L; Liu, Guosheng; Selvaraj, Gopalan; Wei, Yangdou

    2005-11-01

    At least two types of quinone reductases are present in plants: (1) the zeta-crystallin-like quinone reductases (QR1, EC 1.6.5.5) that catalyze the univalent reduction of quinones to semiquinone radicals, and (2) the DT-diaphorase-like quinone reductases (QR2, EC 1.6.99.2) that catalyze the divalent reduction of quinones to hydroquinones. QR2s protect cells from oxidative stress by making the quinones available for conjugation, thereby releasing them from the superoxide-generating one electron redox cycling, catalyzed by QR1s. Two genes, putatively encoding a QR1 and a QR2, respectively, were isolated from an expressed sequence tag collection derived from the epidermis of a diploid wheat Triticum monococcum L. 24 h after inoculation with the powdery mildew fungus Blumeria graminis (DC) EO Speer f. sp. tritici Em. Marchal. Northern analysis and tissue-specific RT-PCR showed that TmQR1 was repressed while TmQR2 was induced in the epidermis during powdery mildew infection. Heterologous expression of TmQR2 in Escherichia coli confirmed that the gene encoded a functional, dicumarol-inhibitable QR2 that could use either NADH or NADPH as an electron donor. The localization of dicumarol-inhibitable QR2 activity around powdery mildew infection sites was accomplished using a histochemical technique, based on tetrazolium dye reduction.

  11. Aldose Reductase Inhibitory Activity of Compounds from  Zea mays L.

    Science.gov (United States)

    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 reductase (rhAR) were performed, and intracellular galactitol levels were measured. Hirsutrin, one of 12 isolated compounds, showed the most potent RLAR inhibitory activity (IC50, 4.78 μM). In the kinetic analyses using Lineweaver-Burk plots of 1/velocity and 1/substrate concentration, hirsutrin showed competitive inhibition against rhAR. Furthermore, hirsutrin inhibited galactitol formation in rat lens and erythrocytes sample incubated with a high concentration of galactose; this finding indicates that hirsutrin may effectively prevent osmotic stress in hyperglycemia. Therefore, hirsutrin derived from Zea mays L. may be a potential therapeutic agent against diabetes complications. PMID:23586057

  12. Atomic Structure of Salutaridine Reductase from the Opium Poppy (Papaver somniferum)

    Energy Technology Data Exchange (ETDEWEB)

    Higashi, Yasuhiro; Kutchan, Toni M.; Smith, Thomas J. (Danforth)

    2011-11-18

    The opium poppy (Papaver somniferum L.) is one of the oldest known medicinal plants. In the biosynthetic pathway for morphine and codeine, salutaridine is reduced to salutaridinol by salutaridine reductase (SalR; EC 1.1.1.248) using NADPH as coenzyme. Here, we report the atomic structure of SalR to a resolution of {approx}1.9 {angstrom} in the presence of NADPH. The core structure is highly homologous to other members of the short chain dehydrogenase/reductase family. The major difference is that the nicotinamide moiety and the substrate-binding pocket are covered by a loop (residues 265-279), on top of which lies a large 'flap'-like domain (residues 105-140). This configuration appears to be a combination of the two common structural themes found in other members of the short chain dehydrogenase/reductase family. Previous modeling studies suggested that substrate inhibition is due to mutually exclusive productive and nonproductive modes of substrate binding in the active site. This model was tested via site-directed mutagenesis, and a number of these mutations abrogated substrate inhibition. However, the atomic structure of SalR shows that these mutated residues are instead distributed over a wide area of the enzyme, and many are not in the active site. To explain how residues distal to the active site might affect catalysis, a model is presented whereby SalR may undergo significant conformational changes during catalytic turnover.

  13. A preliminary study on estimating extra-cellular nitrate reductase activities in estuarine systems

    Directory of Open Access Journals (Sweden)

    Pant H. K.

    2009-07-01

    Full Text Available Enzymes catalyzing ammonium (NH4+/nitrate (NO3– into nitrous oxide (N2O/molecular nitrogen (N2, play critical roles in water quality management. The objective of this paper was to investigate the role of extra-cellular enzymes in cycling of nitrogen (N in aquatic systems. It appears that N in estuaries, salt marshes, etc., does not stay long enough to be available for uptake, thus, creating N limited conditions. This study showed that indigenous extra-cellular nitrate reductase along with others involved in N transformations in the waters/sediments of estuarine systems can cause complete removal of NH4+ and NO3– from the waters and available NH4+ and NO3– from the sediments. These results indicate that due to high extra-cellular nitrate reductase and other enzymes associated with N transformations in sediments/waters, substantial amounts of NH4+ and NO3– can be quickly lost from the systems as N2O and/or nitric oxide (NO, in turn, creating N limited conditions in estuarine systems. Such high activities of indigenous nitrate reductase and others are useful in removing readily bioavailable N from the systems, thereby avoidance of eutrophic conditions. However, they might contribute in increasing the N2O, a potent greenhouse gas with global warming potential (GWP of 296, in the atmosphere.

  14. An overview of the non-mevalonate pathway for terpenoid ...

    Indian Academy of Sciences (India)

    Unknown

    methyl-2-(E)-butenyl-4-diphosphate; HMG-CoA, 3-hydroxy-3-methylglutaryl-CoA; HMGR, 3-hydroxy-3-methylglutaryl-CoA reductase ... functional roles in plants, human health and commerce. ... done in E. coli and demonstration of orthologous genes in various plants, encoding such enzymes that catalyze the same reaction.

  15. Olea europaea Linn (Oleaceae) Fruit Pulp Exhibits ...

    African Journals Online (AJOL)

    as hydroxymethylglutaryl-CoA reductase (HMGR), acyl-CoA:cholesterol acyltransferase (ACAT), cytochrome P450 7A1 (CYP7A1) and peroxisome proliferation-activated receptor alpha (PPAR-á) in rat livers were evaluated using Western blotting. Results: OFP-EA-extract markedly altered the increased plasma TC, TG, LDL ...

  16. Effect of a novel steroid (PM-9) on the inhibition of 5alpha-reductase present in Penicillium crustosum broths.

    Science.gov (United States)

    Flores, Eugenio; Cabeza, Marisa; Quiroz, Alexandra; Bratoeff, Eugene; García, Genoveva; Ramírez, Elena

    2003-03-01

    The conversion of testosterone (T) to 5alpha-dihydrotestosterone (DHT) has been demonstrated in Penicillium crustosum broth obtained from fermented pistachios, lemons and corn tortillas. Furthermore, the presence of 5alpha-reductase enzyme, which is responsible for this conversion, has been established by electrophoretical techniques in these cultures.5alpha-Reductase enzyme is also present in animal and human androgen-dependent tissues as well as in prostate and seminal vesicles. The increase of the conversion of T to DHT in prostate gland, has been related to some illnesses such as benign prostate hyperplasia and prostate cancer. Furthermore, treatment with 5alpha-reductase inhibitors such as finasteride reduces the prostate growth. These data have stimulated research for the synthesis of new molecules with antiandrogenic activity, whose biological effect needs to be demonstrated. The purpose of this study is to determine the inhibition pattern of 5alpha-reductase in P. crustosum by finasteride and the new steroidal compound PM-9. K(m) and V(max) values for T, were determined in the broths by Lineweaver-Burk plots using different testosterone concentrations. The inhibition pattern of finasteride and PM-9 was also determined by Lineweaver-Burk using different concentrations of T and inhibitors. Results show that finasteride and PM-9 inhibit 5alpha-reductase present in the broth in a competitive manner.

  17. HMG-CoA reductase inhibitory activity and phytocomponent investigation of Basella alba leaf extract as a treatment for hypercholesterolemia

    Directory of Open Access Journals (Sweden)

    Baskaran G

    2015-01-01

    Full Text Available Gunasekaran Baskaran,1 Shamala Salvamani,1 Siti Aqlima Ahmad,1 Noor Azmi Shaharuddin,1 Parveen Devi Pattiram,2 Mohd Yunus Shukor1 1Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, 2Department of Food Technology, Faculty of Food Science and Technology, Universiti Putra Malaysia, Selangor, Malaysia Abstract: The enzyme 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA reductase is the key enzyme of the mevalonate pathway that produces cholesterol. Inhibition of HMG-CoA reductase reduces cholesterol biosynthesis in the liver. Synthetic drugs, statins, are commonly used for the treatment of hypercholesterolemia. Due to the side effects of statins, natural HMG-CoA reductase inhibitors of plant origin are needed. In this study, 25 medicinal plant methanol extracts were screened for anti-HMG-CoA reductase activity. Basella alba leaf extract showed the highest inhibitory effect at about 74%. Thus, B. alba was examined in order to investigate its phytochemical components. Gas chromatography with tandem mass spectrometry and reversed phase high-performance liquid chromatography analysis revealed the presence of phenol 2,6-bis(1,1-dimethylethyl, 1-heptatriacotanol, oleic acid, eicosyl ester, naringin, apigenin, luteolin, ascorbic acid, and a-tocopherol, which have been reported to possess antihypercholesterolemic effects. Further investigation of in vivo models should be performed in order to confirm its potential as an alternative treatment for hypercholesterolemia and related cardiovascular diseases. Keywords: HMG-CoA reductase, Basella alba, phytochemical, GC-MS/MS, RP-HPLC, hypercholesterolemia

  18. Identification of a fish short-chain dehydrogenase/reductase associated with bone metabolism.

    Science.gov (United States)

    Rosa, Joana; Cox, Cymon J; Leonor Cancela, M; Laizé, Vincent

    2017-12-14

    Although human and mouse genetics have largely contributed to the better understanding of the mechanisms underlying skeletogenesis, much more remains to be uncovered. In this regard alternative and complementary systems have been sought and cell systems capable of in vitro calcification have been developed to study the mechanisms underlying bone formation. In gilthead seabream (Sparus aurata), a gene coding for an unknown protein that is strongly up-regulated during extracellular matrix (ECM) mineralization of a pre-osteoblast cell line was recently identified as a potentially important player in bone formation. In silico analysis of the deduced protein revealed the presence of domains typical of short-chain dehydrogenase/reductases (SDR). Closely related to carbonyl reductase 1, seabream protein belongs to a novel subfamily of SDR proteins with no orthologs in mammals. Analysis of gene expression by qPCR confirmed the strong up-regulation of sdr-like expression during in vitro mineralization but also revealed high expression levels in calcified tissues. A possible role for Sdr-like in osteoblast and bone metabolism was further evidenced through (i) the localization by in situ hybridization of sdr-like transcript in pre-osteoblasts of the operculum and (ii) the regulation of sdr-like gene transcription by Runx2 and retinoic acid receptor, two regulators of osteoblast differentiation and mineralization. Expression data also indicated a role for Sdr-like in gastrointestinal tract homeostasis and during gilthead seabream development at gastrulation and metamorphosis. This study reports a new subfamily of short-chain dehydrogenases/reductases in vertebrates and, for the first time, provides evidence of a role for SDRs in bone metabolism, osteoblast differentiation and/or tissue mineralization. Copyright © 2017. Published by Elsevier B.V.

  19. Selective non-steroidal inhibitors of 5 alpha-reductase type 1.

    Science.gov (United States)

    Occhiato, Ernesto G; Guarna, Antonio; Danza, Giovanna; Serio, Mario

    2004-01-01

    The enzyme 5 alpha-reductase (5 alpha R) catalyses the reduction of testosterone (T) into the more potent androgen dihydrotestosterone (DHT). The abnormal production of DHT is associated to pathologies of the main target organs of this hormone: the prostate and the skin. Benign prostatic hyperplasia (BPH), prostate cancer, acne, androgenetic alopecia in men, and hirsutism in women appear related to the DHT production. Two isozymes of 5 alpha-reductase have been cloned, expressed and characterized (5 alpha R-1 and 5 alpha R-2). They share a poor homology, have different chromosomal localization, enzyme kinetic parameters, and tissue expression patterns. Since 5 alpha R-1 and 5 alpha R-2 are differently distributed in the androgen target organs, a different involvement of the two isozymes in the pathogenesis of prostate and skin disorders can be hypothesized. High interest has been paid to the synthesis of inhibitors of 5 alpha-reductase for the treatment of DHT related pathologies, and the selective inhibition of any single isozyme represents a great challenge for medical and pharmaceutical research in order to have more specific drugs. At present, no 5 alpha R-1 inhibitor is marketed for the treatment of 5 alpha R-1 related pathologies but pharmaceutical research is very active in this field. This paper will review the major classes of 5 alpha R inhibitors focusing in particular on non-steroidal inhibitors and on structural features that enhance the selectivity versus the type 1 isozyme. Biological tests to assess the inhibitory activity towards the two 5 alpha R isozymes will be also discussed.

  20. Computational and experimental studies on the catalytic mechanism of biliverdin-IXbeta reductase.

    Science.gov (United States)

    Smith, Liam J; Browne, Seamus; Mulholland, Adrian J; Mantle, Timothy J

    2008-05-01

    BVR-B (biliverdin-IXbeta reductase) also known as FR (flavin reductase) is a promiscuous enzyme catalysing the pyridine-nucleotide-dependent reduction of a variety of flavins, biliverdins, PQQ (pyrroloquinoline quinone) and ferric ion. Mechanistically it is a good model for BVR-A (biliverdin-IXalpha reductase), a potential pharmacological target for neonatal jaundice and also a potential target for adjunct therapy to maintain protective levels of biliverdin-IXalpha during organ transplantation. In a commentary on the structure of BVR-B it was noted that one outstanding issue remained: whether the mechanism was a concerted hydride transfer followed by protonation of a pyrrolic anion or protonation of the pyrrole followed by hydride transfer. In the present study we have attempted to address this question using QM/MM (quantum mechanics/molecular mechanics) calculations. QM/MM potential energy surfaces show that the lowest energy pathway proceeds with a positively charged pyrrole intermediate via two transition states. These initial calculations were performed with His(153) as the source of the proton. However site-directed mutagenesis studies with both the H153A and the H153N mutant reveal that His(153) is not required for catalytic activity. We have repeated the calculation with a solvent hydroxonium donor and obtain a similar energy landscape indicating that protonation of the pyrrole is the most likely first step followed by hydride transfer and that the required proton may come from bulk solvent. The implications of the present study for the design of inhibitors of BVR-A are discussed.

  1. Direct antioxidant properties of bilirubin andbiliverdin. Is there a role for biliverdin reductase?

    Directory of Open Access Journals (Sweden)

    Thomas eJansen

    2012-03-01

    Full Text Available Reactive oxygen species (ROS and signaling events are involved in the pathogenesis of endothelial dysfunction and represent a major contribution to vascular regulation. Molecular signaling is highly dependent on reactive oxygen species. But depending on the amount of ROS production it might have toxic or protective effects. Despite a large number of negative outcomes in large clinical trials (e.g. HOPE, HOPE-TOO, antioxidant molecules and agents are important players to influence the critical balance between production and elimination of RONS. However, chronic systemic antioxidant therapy lacks clinical efficacy, probably by interfering with important physiological redox signaling pathways. Therefore, it may be a much more promising attempt to induce intrinsic antioxidant pathways in order to increase the antioxidants not systemically but at the place of oxidative stress and complications. Among others, heme oxygenase (HO has been shown to be important for attenuating the overall production of ROS in a broad range of disease states through its ability to degrade heme and to produce carbon monoxide (CO, biliverdin/bilirubin, and the release of free iron with subsequent ferritin induction. With the present review we would like to highlight the important antioxidant role of the heme oxygenase system and especially discuss the contribution of the biliverdin, bilirubin and biliverdin reductase to these beneficial effects. The bilierdin reductase was reported to confer an antioxidant redox amplification cycle by which low, physiological bilirubin concentrations confer potent antioxidant protection via recycling of biliverdin from oxidized bilirubin by the biliverdin reductase, linking this sink for oxidants to the NADPH pool. To date the existence and role of this antioxidant redox cycle is still under debate and we present and discuss the pros and cons as well as our own findings on this topic.

  2. Cloning the putative gene of vinyl phenol reductase of Dekkera bruxellensis in Saccharomyces cerevisiae.

    Science.gov (United States)

    Romano, Diego; Valdetara, Federica; Zambelli, Paolo; Galafassi, Silvia; De Vitis, Valerio; Molinari, Francesco; Compagno, Concetta; Foschino, Roberto; Vigentini, Ileana

    2017-05-01

    Vinylphenol reductase of Dekkera bruxellensis, the characteristic enzyme liable for "Brett" sensory modification of wine, has been recently recognized to belong to the short chain dehydrogenases/reductases family. Indeed, a preliminary biochemical characterisation has conferred to the purified protein a dual significance acting as superoxide dismutase and as a NADH-dependent reductase. The present study aimed for providing a certain identification of the enzyme by cloning the VPR gene in S. cerevisiae, a species not producing ethyl phenols. Transformed clones of S. cerevisiae resulted capable of expressing a biologically active form of the heterologous protein, proving its role in the conversion of 4-vinyl guaiacol to 4-ethyl guaiacol. A VPR specific protein activity of 9 ± 0.6 mU/mg was found in crude extracts of S. cerevisiae recombinant strain. This result was confirmed in activity trials carried out with the protein purified from transformant cells of S. cerevisiae by a his-tag purification approach; in particular, VPR-enriched fractions showed a specific activity of 1.83 ± 0.03 U/mg at pH 6.0. Furthermore, in agreement with literature, the purified protein behaves like a SOD, with a calculated specific activity of approximatively 3.41 U/mg. The comparative genetic analysis of the partial VPR gene sequences from 17 different D. bruxellesis strains suggested that the observed polymorphism (2.3%) and the allelic heterozygosity state of the gene do not justify the well described strain-dependent character in producing volatile phenols of this species. Actually, no correlation exists between genotype membership of the analysed strains and their capability to release off-flavours. This work adds valuable knowledge to the study of D. bruxellensis wine spoilage and prepare the ground for interesting future industrial applications. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. The catalytic cycle of nitrous oxide reductase - The enzyme that catalyzes the last step of denitrification.

    Science.gov (United States)

    Carreira, Cíntia; Pauleta, Sofia R; Moura, Isabel

    2017-12-01

    The reduction of the potent greenhouse gas nitrous oxide requires a catalyst to overcome the large activation energy barrier of this reaction. Its biological decomposition to the inert dinitrogen can be accomplished by denitrifiers through nitrous oxide reductase, the enzyme that catalyzes the last step of the denitrification, a pathway of the biogeochemical nitrogen cycle. Nitrous oxide reductase is a multicopper enzyme containing a mixed valence CuA center that can accept electrons from small electron shuttle proteins, triggering electron flow to the catalytic sulfide-bridged tetranuclear copper "CuZ center". This enzyme has been isolated with its catalytic center in two forms, CuZ*(4Cu1S) and CuZ(4Cu2S), proven to be spectroscopic and structurally different. In the last decades, it has been a challenge to characterize the properties of this complex enzyme, due to the different oxidation states observed for each of its centers and the heterogeneity of its preparations. The substrate binding site in those two "CuZ center" forms and which is the active form of the enzyme is still a matter of debate. However, in the last years the application of different spectroscopies, together with theoretical calculations have been useful in answering these questions and in identifying intermediate species of the catalytic cycle. An overview of the spectroscopic, kinetics and structural properties of the two forms of the catalytic "CuZ center" is given here, together with the current knowledge on nitrous oxide reduction mechanism by nitrous oxide reductase and its intermediate species. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. A novel arsenate reductase from the bacterium Thermus thermophilus HB27: its role in arsenic detoxification.

    Science.gov (United States)

    Del Giudice, Immacolata; Limauro, Danila; Pedone, Emilia; Bartolucci, Simonetta; Fiorentino, Gabriella

    2013-10-01

    Microorganisms living in arsenic-rich geothermal environments act on arsenic with different biochemical strategies, but the molecular mechanisms responsible for the resistance to the harmful effects of the metalloid have only partially been examined. In this study, we investigated the mechanisms of arsenic resistance in the thermophilic bacterium Thermus thermophilus HB27. This strain, originally isolated from a Japanese hot spring, exhibited tolerance to concentrations of arsenate and arsenite up to 20mM and 15mM, respectively; it owns in its genome a putative chromosomal arsenate reductase (TtarsC) gene encoding a protein homologous to the one well characterized from the plasmid pI258 of the Gram+bacterium Staphylococcus aureus. Differently from the majority of microorganisms, TtarsC is part of an operon including genes not related to arsenic resistance; qRT-PCR showed that its expression was four-fold increased when arsenate was added to the growth medium. The gene cloning and expression in Escherichia coli, followed by purification of the recombinant protein, proved that TtArsC was indeed a thioredoxin-coupled arsenate reductase with a kcat/KM value of 1.2×10(4)M(-1)s(-1). It also exhibited weak phosphatase activity with a kcat/KM value of 2.7×10(-4)M(-1)s(-1). The catalytic role of the first cysteine (Cys7) was ascertained by site-directed mutagenesis. These results identify TtArsC as an important component in the arsenic resistance in T. thermophilus giving the first structural-functional characterization of a thermophilic arsenate reductase. Copyright © 2013 Elsevier B.V. All rights reserved.

  5. Auranofin inactivates Trichomonas vaginalis thioredoxin reductase and is effective against trichomonads in vitro and in vivo.

    Science.gov (United States)

    Hopper, Melissa; Yun, Jeong-Fil; Zhou, Bianhua; Le, Christine; Kehoe, Katelin; Le, Ryan; Hill, Ryan; Jongeward, Gregg; Debnath, Anjan; Zhang, Liangfang; Miyamoto, Yukiko; Eckmann, Lars; Land, Kirkwood M; Wrischnik, Lisa A

    2016-12-01

    Trichomoniasis, caused by the protozoan parasite Trichomonas vaginalis, is the most common, non-viral, sexually transmitted infection in the world, but only two closely related nitro drugs are approved for its treatment. New antimicrobials against trichomoniasis remain an urgent need. Several organic gold compounds were tested for activity against T. vaginalis thioredoxin reductase (TrxR) in cell-free systems as well as for activity against different trichomonads in vitro and in a murine infection model. The organic gold(I) compounds auranofin and chloro(diethylphenylphosphine)gold(I) inhibited TrxR in a concentration-dependent manner in assays with recombinant purified reductase and in cytoplasmic extracts of T. vaginalis transfected with a haemagglutinin epitope-tagged form of the reductase. Auranofin potently suppressed the growth of three independent clinical T. vaginalis isolates as well as several strains of another trichomonad (Tritrichomonas foetus) in a 24 h-assay, with 50% inhibitory concentrations of 0.7-2.5 µM and minimum lethal concentrations of 2-6 µM. The drug also compromised the ability of the parasite to overcome oxidant stress, supporting the notion that auranofin acts, in part, by inactivating TrxR-dependent antioxidant defences. Chloro(diethylphenylphosphine)gold(I) was 10-fold less effective against T. vaginalis in vitro than auranofin. Oral administration of auranofin for 4 days cleared the parasites in a murine model of vaginal T. foetus infection without displaying any apparent adverse effects. The approved human drug auranofin may be a promising agent as an alternative treatment of trichomoniasis in cases when standard nitro drug therapies have failed. Copyright © 2016 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

  6. Evidence that the intra-amoebal Legionella drancourtii acquired a sterol reductase gene from eukaryotes

    Directory of Open Access Journals (Sweden)

    Fournier Pierre-Edouard

    2009-03-01

    Full Text Available Abstract Background Free-living amoebae serve as a natural reservoir for some bacteria that have evolved into «amoeba-resistant» bacteria. Among these, some are strictly intra-amoebal, such as Candidatus "Protochlamydia amoebophila" (Candidatus "P. amoebophila", whose genomic sequence is available. We sequenced the genome of Legionella drancourtii (L. drancourtii, another recently described intra-amoebal bacterium. By comparing these two genomes with those of their closely related species, we were able to study the genetic characteristics specific to their amoebal lifestyle. Findings We identified a sterol delta-7 reductase-encoding gene common to these two bacteria and absent in their relatives. This gene encodes an enzyme which catalyses the last step of cholesterol biosynthesis in eukaryotes, and is probably functional within L. drancourtii since it is transcribed. The phylogenetic analysis of this protein suggests that it was acquired horizontally by a few bacteria from viridiplantae. This gene was also found in the Acanthamoeba polyphaga Mimivirus genome, a virus that grows in amoebae and possesses the largest viral genome known to date. Conclusion L. drancourtii acquired a sterol delta-7 reductase-encoding gene of viridiplantae origin. The most parsimonious hypothesis is that this gene was initially acquired by a Chlamydiales ancestor parasite of plants. Subsequently, its descendents transmitted this gene in amoebae to other intra-amoebal microorganisms, including L. drancourtii and Coxiella burnetii. The role of the sterol delta-7 reductase in prokaryotes is as yet unknown but we speculate that it is involved in host cholesterol parasitism.

  7. X-ray structural studies of quinone reductase 2 nanomolar range inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Pegan, Scott D.; Sturdy, Megan; Ferry, Gilles; Delagrange, Philippe; Boutin, Jean A.; Mesecar, Andrew D. (IdRS); (Purdue); (Colorado); (UIC)

    2011-09-06

    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 identified as the third melatonin receptor (MT3) through in cellulo and in vitro inhibition of QR2 by traditional MT3 ligands, and through recent X-ray structures of human QR2 (hQR2) in complex with melatonin and 2-iodomelatonin. Several MT3 specific ligands have been developed that exhibit both potent in cellulo inhibition of hQR2 nanomolar, affinity for MT3. The potency of these ligands suggest their use as molecular probes for hQR2. However, no definitive correlation between traditionally obtained MT3 ligand affinity and hQR2 inhibition exists limiting our understanding of how these ligands are accommodated in the hQR2 active site. To obtain a clearer relationship between the structures of developed MT3 ligands and their inhibitory properties, in cellulo and in vitro IC{sub 50} values were determined for a representative set of MT3 ligands (MCA-NAT, 2-I-MCANAT, prazosin, S26695, S32797, and S29434). Furthermore, X-ray structures for each of these ligands in complex with hQR2 were determined allowing for a structural evaluation of the binding modes of these ligands in relation to the potency of MT3 ligands.

  8. X-ray structural studies of quinone reductase 2 nanomolar range inhibitors.

    Science.gov (United States)

    Pegan, Scott D; Sturdy, Megan; Ferry, Gilles; Delagrange, Philippe; Boutin, Jean A; Mesecar, Andrew D

    2011-07-01

    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 identified as the third melatonin receptor (MT3) through in cellulo and in vitro inhibition of QR2 by traditional MT3 ligands, and through recent X-ray structures of human QR2 (hQR2) in complex with melatonin and 2-iodomelatonin. Several MT3 specific ligands have been developed that exhibit both potent in cellulo inhibition of hQR2 nanomolar, affinity for MT3. The potency of these ligands suggest their use as molecular probes for hQR2. However, no definitive correlation between traditionally obtained MT3 ligand affinity and hQR2 inhibition exists limiting our understanding of how these ligands are accommodated in the hQR2 active site. To obtain a clearer relationship between the structures of developed MT3 ligands and their inhibitory properties, in cellulo and in vitro IC₅₀ values were determined for a representative set of MT3 ligands (MCA-NAT, 2-I-MCANAT, prazosin, S26695, S32797, and S29434). Furthermore, X-ray structures for each of these ligands in complex with hQR2 were determined allowing for a structural evaluation of the binding modes of these ligands in relation to the potency of MT3 ligands. Copyright © 2011 The Protein Society.

  9. Thioredoxin glutathione reductase as a novel drug target: evidence from Schistosoma japonicum.

    Directory of Open Access Journals (Sweden)

    LiJun Song

    Full Text Available BACKGROUND: Schistosomiasis remains a major public health concern affecting billions of people around the world. Currently, praziquantel is the only drug of choice for treatment of human schistosomiasis. The emergence of drug resistance to praziquantel in schistosomes makes the development of novel drugs an urgent task. Thioredoxin glutathione reductase (TGR enzymes in Schistosoma mansoni and some other platyhelminths have been identified as alternative targets. The present study was designed to confirm the existense and the potential value of TGR as a target for development of novel antischistosomal agents in Schistosoma japonicum, a platyhelminth endemic in Asia. METHODS AND FINDINGS: After cloning the S. japonicum TGR (SjTGR gene, the recombinant SjTGR selenoprotein was purified and characterized in enzymatic assays as a multifunctional enzyme with thioredoxin reductase (TrxR, glutathione reductase (GR and glutaredoxin (Grx activities. Immunological and bioinformatic analyses confirmed that instead of having separate TrxR and GR proteins in mammalian, S. japonicum only encodes TGR, which performs the functions of both enzymes and plays a critical role in maintaining the redox balance in this parasite. These results were in good agreement with previous findings in Schistosoma mansoni and some other platyhelminths. Auranofin, a known inhibitor against TGR, caused fatal toxicity in S. japonicum adult worms in vitro and reduced worm and egg burdens in S. japonicum infected mice. CONCLUSIONS: Collectively, our study confirms that a multifunctional enzyme SjTGR selenoprotein, instead of separate TrxR and GR enzymes, exists in S. japonicum. Furthermore, TGR may be a potential target for development of novel agents against schistosomes. This assumption is strengthened by our demonstration that the SjTGR is an essential enzyme for maintaining the thiol-disulfide redox homeostasis of S. japonicum.

  10. Dicarbonyl L-xylulose reductase (DCXR, a "moonlighting protein" in the bovine epididymis.

    Directory of Open Access Journals (Sweden)

    Ayodélé Akintayo

    Full Text Available During maturation and the acquisition of their fertilization potential, male germ cells are subjected to various sequential modifications that occur in the epididymis. Protein addition, reorganization or withdrawal, comprise some of these modifications. Dicarbonyl L-xylulose reductase (DCXR, a multifunctional protein involved in various enzymatic and protein interaction processes in different physiological systems, is one of the proteins added to spermatozoa in the epididymis. DCXR is a well-conserved protein with multiple characteristics including enzymatic activities and mediation of cell-cell interaction. In this study, we characterized the DCXR gene and protein expression in the bovine epididymis. Dicarbonyl L-xylulose reductase mRNA is differentially expressed in the caput, corpus, and cauda epididymide epithelial cells with a higher level observed in the cauda region. Tissue protein expression follows the same pattern as the corresponding mRNA expression with a cytoplasmic and apical distribution in the corpus and cauda epithelial cells, respectively. The protein can also be found with a nuclear localization in cauda epididymidis epithelial cells. Dicarbonyl L-xylulose reductase is secreted in the epididymis luminal compartment in the soluble fraction and is associated with microvesicular elements named epididymosomes. In spermatozoa, the DCXR protein was found in the cytoplasmic and membranous fractions. Expression of the DCXR protein is higher on caput spermatozoa but finally shows a weak detection in semen. These data describe DCXR in the bovine epididymis and reveal that its behavior differs from that found in humans. It seems that, in this model, the DCXR protein might have a questionable involvement in the fertilization process.

  11. Progesterone 5β-reductase genes of the Brassicaceae family as function-associated molecular markers.

    Science.gov (United States)

    Munkert, J; Costa, C; Budeanu, O; Petersen, J; Bertolucci, S; Fischer, G; Müller-Uri, F; Kreis, W

    2015-11-01

    This study aimed to define progesterone 5β-reductases (P5βR, EC 1.3.99.6, enone 1,4-reductases) as function-associated molecular markers at the plant family level. Therefore cDNAs were isolated from 25 Brassicaceae species, including two species, Erysimum crepidifolium and Draba aizoides, known to produce cardiac glycosides. The sequences were used in a molecular phylogeny study. The cladogram created is congruent to the existing molecular analyses. Recombinant His-tagged forms of the P5βR cDNAs from Aethionema grandiflorum, Draba aizoides, Nasturtium officinale, Raphanus sativus and Sisymbrium officinale were expressed in E. coli. Enone 1,4-reductase activity was demonstrated in vitro using progesterone and 2-cyclohexen-1-one as substrates. Evidence is provided that functional P5βRs are ubiquitous in the Brassicaceae. The recombinant P5βR enzymes showed different substrate preferences towards progesterone and 2-cyclohexen-1-one. Sequence comparison of the catalytic pocket of the P5βR enzymes and homology modelling using Digitalis lanata P5βR (PDB ID: 2V6G) as template highlighted the importance of the hydrophobicity of the binding pocket for substrate discrimination. It is concluded that P5βR genes or P5βR proteins can be used as valuable function-associated molecular markers to infer taxonomic relationship and evolutionary diversification from a metabolic/catalytic perspective. © 2015 German Botanical Society and The Royal Botanical Society of the Netherlands.

  12. 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.

  13. Sunflower (Helianthus annuus) fatty acid synthase complex: enoyl-[acyl carrier protein]-reductase genes.

    Science.gov (United States)

    González-Thuillier, Irene; Venegas-Calerón, Mónica; Garcés, Rafael; von Wettstein-Knowles, Penny; Martínez-Force, Enrique

    2015-01-01

    Enoyl-[acyl carrier protein]-reductases from sunflower. A major factor contributing to the amount of fatty acids in plant oils are the first steps of their synthesis. The intraplastidic fatty acid biosynthetic pathway in plants is catalysed by type II fatty acid synthase (FAS). The last step in each elongation cycle is carried out by the enoyl-[ACP]-reductase, which reduces the dehydrated product of β-hydroxyacyl-[ACP] dehydrase using NADPH or NADH. To determine the mechanisms involved in the biosynthesis of fatty acids in sunflower (Helianthus annuus) seeds, two enoyl-[ACP]-reductase genes have been identified and cloned from developing seeds with 75 % identity: HaENR1 (GenBank HM021137) and HaENR2 (HM021138). The two genes belong to the ENRA and ENRB families in dicotyledons, respectively. The genetic duplication most likely originated after the separation of di- and monocotyledons. RT-qPCR revealed distinct tissue-specific expression patterns. Highest expression of HaENR1 was in roots, stems and developing cotyledons whereas that of H a ENR2 was in leaves and early stages of seed development. Genomic DNA gel blot analyses suggest that both are single-copy genes. In vivo activity of the ENR enzymes was tested by complementation experiments with the JP1111 fabI(ts) E. coli strain. Both enzymes were functional demonstrating that they interacted with the bacterial FAS components. That different fatty acid profiles resulted infers that the two Helianthus proteins have different structures, substrate specificities and/or reaction rates. The latter possibility was confirmed by in vitro analysis with affinity-purified heterologous-expressed enzymes that reduced the crotonyl-CoA substrate using NADH with different V max.

  14. Methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism and age at onset of schizophrenia

    DEFF Research Database (Denmark)

    Saetre, Peter; Grove, Jakob; Børglum, Anders

    2012-01-01

    Methylenetetrahydrofolate reductase (MTHFR) is an enzyme involved in metabolic pathways of importance for nucleotide synthesis and methylation of DNA, membranes, proteins and lipids. The MTHFR gene includes a common polymorphism (rs1801133 or C677T), which is associated with enzyme activity. The T...... the original Scandinavian samples, there was no significant association between MTHFR C677T polymorphism and age at onset in schizophrenia. The present results do not suggest that the investigated MTHFR polymorphism has any significant influence on age at onset of schizophrenia in the Nordic population. © 2012...

  15. 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....

  16. Virtual screening reveals allosteric inhibitors of the Toxoplasma gondii thymidylate synthase-dihydrofolate reductase.

    Science.gov (United States)

    Sharma, Hitesh; Landau, Mark J; Sullivan, Todd J; Kumar, Vidya P; Dahlgren, Markus K; Jorgensen, William L; Anderson, Karen S

    2014-02-15

    The parasite Toxoplasma gondii can lead to toxoplasmosis in those who are immunocompromised. To combat the infection, the enzyme responsible for nucleotide synthesis thymidylate synthase-dihydrofolate reductase (TS-DHFR) is a suitable drug target. We have used virtual screening to determine novel allosteric inhibitors at the interface between the two TS domains. Selected compounds from virtual screening inhibited TS activity. Thus, these results show that allosteric inhibition by small drug-like molecules can occur in T. gondii TS-DHFR and pave the way for new and potent species-specific inhibitors. Copyright © 2014 Elsevier Ltd. All rights reserved.

  17. Role of methylenetetrahydrofolate reductase A1298C polymorphism in cerebral venous thrombosis.

    Science.gov (United States)

    Fekih-Mrissa, Najiba; Klai, Sarra; Mrad, Meriem; Zaouali, Jamel; Sayeh, Aycha; Nsiri, Brahim; Gritli, Nasreddine; Mrissa, Ridha

    2013-03-01

    The association between the methylenetetrahydrofolate reductase (MTHFR) gene and cerebral venous thrombosis (CVT) remains controversial. This study principally investigated the potential role of the MTHFR A1298C variant and CVT. The genotyping of the A1298C variant of the MTHFR gene was performed in 35 CVT patients and 200 healthy controls. The frequency of A1298C genotype among CVT patients was significantly higher compared with controls (P MTHFR A1298C variant and CVT. Large study populations would be required to understand the contribution of this marker in the risk of CVT.

  18. Influence of methylene tetrahydrofolate reductase polymorphisms and coadministration of antimetabolites on toxicity after high dose methotrexate

    DEFF Research Database (Denmark)

    Niekerk, P.B. van Kooten; Schmiegelow, K.; Schroeder, H.

    2008-01-01

    .006-0.027), fever (OR = 2.65; P = 0.037) and interruption of maintenance treatment (OR = 3.04; P = 0.032). No convincing associations were found between the MTHFR C677T or A1298C polymorphisms and toxicity. CONCLUSION: Our findings demonstrate that toxicity after HDMTX is influenced by coadministrated...... in the methylene tetrahydrofolate reductase (MTHFR) gene and coadministration of antimetabolites on post-HDMTX toxicity. METHODS: Toxicity was retrospectively analysed after 656 HDMTX courses administered to 88 paediatric ALL patients at a single treatment centre. RESULTS: High-dose methotrexate with high...

  19. A structural account of substrate and inhibitor specificity differences between two Naphthol reductases

    Energy Technology Data Exchange (ETDEWEB)

    Liao, D.-I.; Thompson, J.E.; Fahnestock, S.; Valent, B.; Jordan, D.B. (DuPont)

    2010-03-08

    Two short chain dehydrogenase/reductases mediate naphthol reduction reactions in fungal melanin biosynthesis. An X-ray structure of 1,3,6,8-tetrahydroxynaphthalene reductase (4HNR) complexed with NADPH and pyroquilon was determined for examining substrate and inhibitor specificities that differ from those of 1,3,8-trihydroxynaphthalene reductase (3HNR). The 1.5 {angstrom} resolution structure allows for comparisons with the 1.7 {angstrom} resolution structure of 3HNR complexed with the same ligands. The sequences of the two proteins are 46% identical, and they have the same fold. The 30-fold lower affinity of the 4HNR-NADPH complex for pyroquilon (a commercial fungicide that targets 3HNR) in comparison to that of the 3HNR-NADPH complex can be explained by unfavorable interactions between the anionic carboxyl group of the C-terminal Ile282 of 4HNR and CH and CH{sub 2} groups of the inhibitor that are countered by favorable inhibitor interactions with 3HNR. 1,3,8-Trihydroxynaphthalene (3HN) and 1,3,6,8-tetrahydroxynaphthalene (4HN) were modeled onto the cyclic structure of pyroquilon in the 4HNR-NADPH-pyroquilon complex to examine the 300-fold preference of the enzyme for 4HN over 3HN. The models suggest that the C-terminal carboxyl group of Ile282 has a favorable hydrogen bonding interaction with the C6 hydroxyl group of 4HN and an unfavorable interaction with the C6 CH group of 3HN. Models of 3HN and 4HN in the 3HNR active site suggest a favorable interaction of the sulfur atom of the C-terminal Met283 with the C6 CH group of 3HN and an unfavorable one with the C6 hydroxyl group of 4HN, accounting for the 4-fold difference in substrate specificities. Thus, the C-terminal residues of the two naphthol reductase are determinants of inhibitor and substrate specificities.

  20. Inhibition of HMG-CoA reductase induces the UPR pathway in C. elegans

    DEFF Research Database (Denmark)

    Olsen, Louise Cathrine Braun; Hansen, Nadia Jin Storm; Pilon, Marc

    -requiring enzyme-1 (IRE-1), and activating transcription factor-6 (ATF-6). Using a transgenic GFP reporter strain of the model organism C. elegans, we have recently identified that inhibition of the enzyme HMG-CoA reductase (HMG-CoAR) with Fluvastatin and knock down of HMG-CoAR using RNA interference (RNAi) both...... including farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) which are necessary for posttranslational prenylation of several small G proteins. C. elegans are cholesterol auxotrophs, which enable us to investigate the isoprenoid branch and its role in UPR induction. We found...

  1. Sulfur globule oxidation in green sulfur bacteria is dependent on the dissimilatory sulfite reductase system

    DEFF Research Database (Denmark)

    Holkenbrink, Carina; Ocón Barbas, Santiago; Mellerup, Anders

    2011-01-01

    Green sulfur bacteria oxidize sulfide and thiosulfate to sulfate with extracellular globules of elemental sulfur as intermediate. Here we investigated which genes are involved in the formation and consumption of these sulfur globules in the green sulfur bacterium Chlorobaculum tepidum. We show...... that sulfur globule oxidation is strictly dependent on the dissimilatory sulfite reductase (DSR) system. Deletion of dsrM/CT2244 or dsrT/CT2245 or the two dsrCABL clusters (CT0851-CT0854, CT2247-2250) abolished sulfur globule oxidation and prevented formation of sulfate from sulfide, whereas deletion of dsr...

  2. Allosteric control of internal electron transfer in cytochrome cd1 nitrite reductase

    DEFF Research Database (Denmark)

    Farver, Ole; Kroneck, Peter M H; Zumft, Walter G

    2003-01-01

    Cytochrome cd1 nitrite reductase is a bifunctional multiheme enzyme catalyzing the one-electron reduction of nitrite to nitric oxide and the four-electron reduction of dioxygen to water. Kinetics and thermodynamics of the internal electron transfer process in the Pseudomonas stutzeri enzyme have...... been studied and found to be dominated by pronounced interactions between the c and the d1 hemes. The interactions are expressed both in dramatic changes in the internal electron-transfer rates between these sites and in marked cooperativity in their electron affinity. The results constitute a prime...... example of intraprotein control of the electron-transfer rates by allosteric interactions....

  3. Differential expression of 5-alpha reductase isozymes in the prostate and its clinical implications

    Directory of Open Access Journals (Sweden)

    Kai Wang

    2014-04-01

    Full Text Available The development of human benign or malignant prostatic diseases is closely associated with androgens, primarily testosterone (T and dihydrotestosterone (DHT. T is converted to DHT by 5-alpha reductase (5-AR isozymes. Differential expression of 5-AR isozymes is observed in both human benign and malignant prostatic tissues. 5-AR inhibitors (5-ARI are commonly used for the treatment of benign prostatic hyperplasia (BPH and were once promoted as chemopreventive agents for prostate cancer (PCa. This review discusses the role of the differential expression of 5-AR in the normal development of the human prostate and in the pathogenesis and progression of BPH and PCa.

  4. Ammonium Inhibits Chromomethylase 3-Mediated Methylation of the Arabidopsis Nitrate Reductase Gene NIA2

    OpenAIRE

    Kim, Joo Yong; Kwon, Ye Jin; Kim, Sung-Il; Kim, Do Youn; Song, Jong Tae; Seo, Hak Soo

    2016-01-01

    Gene methylation is an important mechanism regulating gene expression and genome stability. Our previous work showed that methylation of the nitrate reductase (NR) gene NIA2 was dependent on chromomethylase 3 (CMT3). Here, we show that CMT3-mediated NIA2 methylation is regulated by ammonium in Arabidopsis thaliana. CHG sequences (where H can be A, T, or C) were methylated in NIA2 but not in NIA1, and ammonium [(NH4)2SO4] treatment completely blocked CHG methylation in NIA2. By contrast, ammon...

  5. Multicenter evaluation of the nitrate reductase assay for drug resistance detection of Mycobacterium tuberculosis.

    Science.gov (United States)

    Martin, Anandi; Montoro, Ernesto; Lemus, Dihadenys; Simboli, Norberto; Morcillo, Nora; Velasco, Maritza; Chauca, José; Barrera, Lucía; Ritacco, Viviana; Portaels, Françoise; Palomino, Juan Carlos

    2005-11-01

    The performance of the nitrate reductase assay was evaluated in a multicenter laboratory study to detect resistance of Mycobacterium tuberculosis to the first-line anti-tuberculosis drugs rifampicin, isoniazid, ethambutol and streptomycin using a set of coded isolates. Compared with the gold standard proportion method on Löwenstein-Jensen medium, the assay was highly accurate in detecting resistance to rifampicin, isoniazid and ethambutol with an accuracy of 98%, 96.6% and 97.9%, respectively. For streptomycin, discrepant results were obtained with an overall accuracy of 85.3%. The assay proved easy to be implemented in countries with limited laboratory facilities.

  6. A founder mutation causing a severe methylenetetrahydrofolate reductase (MTHFR) deficiency in Bukharian Jews.

    Science.gov (United States)

    Ben-Shachar, Shay; Zvi, Tal; Rolfs, Arndt; Breda Klobus, Andrea; Yaron, Yuval; Bar-Shira, Anat; Orr-Urtreger, Avi

    2012-11-01

    Methylenetetrahydrofolate reductase (MTHFR) deficiency is a rare autosomal recessive disorder. A novel homozygous MTHFR c.474A>T (p.G158G) mutation was detected in two unrelated children of Jewish Bukharian origin. This mutation generates an abnormal splicing and early termination codon. A carrier frequency of 1:39 (5/196) was determined among unrelated healthy Bukharian Jews. Given the disease severity and allele frequency, a population screening for individuals of this ancestry is warranted in order to allow prenatal, or preimplantation diagnosis. Copyright © 2012 Elsevier Inc. All rights reserved.

  7. Voltammetry and Electrocatalysis of Achrornobacter Xylosoxidans Copper Nitrite Reductase on Functionalized Au(111)-Electrode Surfaces

    DEFF Research Database (Denmark)

    Welinder, Anna C.; Zhang, Jingdong; Hansen, Allan G.

    2007-01-01

    planar electrode surfaces is a step towards the resolution of this central issue. We report here the voltammetry of copper nitrite reductase (CNiR, Achromobacter xylosoxidons) on Au(111)-electrode surfaces modified by monolayers of a broad variety of thiol-based linker molecules. These represent......NiR thus shows highly efficient, close to ideal reversible electrocatalytic voltammetry on cysteamine-covered Au(111)-electrode surfaces, most likely due to two cysteamine orientations previously disclosed by in situ scanning tunnelling microscopy. Such a dual orientation exposes both a hydrophobic...

  8. Quantum chemical study of the mechanism of action of vitamin K epoxide reductase (VKOR)

    Science.gov (United States)

    Deerfield, David, II; Davis, Charles H.; Wymore, Troy; Stafford, Darrel W.; Pedersen, Lee G.

    Possible model, but simplistic, mechanisms for the action of vitamin K epoxide reductase (VKOR) are investigated with quantum mechanical methods (B3LYP/6-311G**). The geometries of proposed model intermediates in the mechanisms are energy optimized. Finally, the energetics of the proposed (pseudo-enzymatic) pathways are compared. We find that the several pathways are all energetically feasible. These results will be useful for designing quantum mechanical/molecular mechanical method (QM/MM) studies of the enzymatic pathway once three-dimensional structural data are determined and available for VKOR.

  9. In vitro evaluation of 5-arylidene-2-thioxo-4-thiazolidinones active as aldose reductase inhibitors.

    Science.gov (United States)

    Maccari, Rosanna; Del Corso, Antonella; Giglio, Marco; Moschini, Roberta; Mura, Umberto; Ottanà, Rosaria

    2011-01-01

    2-Thioxo-4-thiazolidinone derivatives were evaluated as aldose reductase inhibitors (ARIs) and most of them exhibited good or excellent in vitro efficacy. Out of the tested compounds, most N-unsubstituted analogues were found to possess inhibitory effects at low micromolar doses and two of them exhibited higher potency than sorbinil, used as a reference drug. The insertion of an acetic chain on N-3 of the thiazolidinone scaffold led to analogues with submicromolar affinity for ALR2 and IC(50) values very similar to that of epalrestat, the only ARI currently used in therapy. Copyright © 2010 Elsevier Ltd. All rights reserved.

  10. A fast virtual screening approach to identify structurally diverse inhibitors of trypanothione reductase.

    Science.gov (United States)

    Maccari, Giorgio; Jaeger, Timo; Moraca, Francesca; Biava, Mariangela; Flohé, Leopold; Botta, Maurizio

    2011-09-15

    Trypanothione reductase (TryR) is one of the favorite targets for those designing drugs for the treatment of Chagas disease. We present the application of a fast virtual screening approach for designing hit compounds active against TryR. Our protocol combines information derived from structurally known inhibitors and from the TryR receptor structure. Five structurally diverse hit compounds active against TryR and holding promise for the treatment of Chagas disease are reported. Copyright © 2011 Elsevier Ltd. All rights reserved.

  11. Molecular cloning and characterization of a novel Dehydrogenase/reductase (SDR family) member 1 genea from human fetal brain.

    Science.gov (United States)

    Wu, Q; Xu, M; Cheng, C; Zhou, Z; Huang, Y; Zhao, W; Zeng, L; Xu, J; Fu, X; Ying, K; Xie, Y; Mao, Y

    2001-01-01

    Short-chain dehydrogenases/reductases (SDR) constitute a large protein family of NAD(P)(H)-dependent oxidoreductase. They are defined by distinct, common sequence motifs and show a wide range of substrate specialisms. By large-scale sequencing analysis of a human fetal brain cDNA library, we isolated a novel human SDR-type dehydrogenase/reductase gene named Dehydrogenase/reductase (SDR family) member 1 (DHRS1). The DHRS1 cDNA is 1411 base pair in length, encoding a 314-amino-acid polypeptide which has a SDR motif. Northern blot reveals two bands, of about 0.9 and 1.4 kb in size. These two forms are expressed in many tissues. The DHRS1 gene is localized on chromosome 14q21.3. It has 9 exons and spans 9.2 kb of the genomic DNA.

  12. Crystal structure of the YffB protein from Pseudomonas aeruginosa suggests a glutathione-dependent thiol reductase function

    Directory of Open Access Journals (Sweden)

    Dauter Zbigniew

    2004-03-01

    Full Text Available Abstract Background The yffB (PA3664 gene of Pseudomonas aeruginosa encodes an uncharacterized protein of 13 kDa molecular weight with a marginal sequence similarity to arsenate reductase from Escherichia coli. The crystal structure determination of YffB was undertaken as part of a structural genomics effort in order to assist with the functional assignment of the protein. Results The structure was determined at 1.0 Å resolution by single-wavelength anomalous diffraction. The fold is very similar to that of arsenate reductase, which is an extension of the thioredoxin fold. Conclusion Given the conservation of the functionally important residues and the ability to bind glutathione, YffB is likely to function as a GSH-dependent thiol reductase.

  13. HMG-CoA reductase, cholesterol 7alpha-hydroxylase, LCAT, ACAT, LDL receptor, and SRB-1 in hereditary analbuminemia.

    Science.gov (United States)

    Liang, Kaihui; Vaziri, Nosratola D

    2003-07-01

    Hereditary analbuminemia is associated with hypercholesterolemia, which has been shown to be primarily caused by increased extrahepatic production of cholesterol. Nagase rats with hereditary analbuminemia (NAR) have been used as a model to dissect the effect of primary hypoalbuminemia from that caused by proteinuria in nephrotic syndrome. The present study was undertaken to explore the effect of hereditary analbuminemia on protein expression of the key factors involved in cholesterol metabolism. Hepatic tissue protein abundance of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, cholesterol 7alpha-hydroxylase (a rate-limiting enzyme in cholesterol catabolism), low density lipoprotein (LDL) receptor, high density lipoprotein (HDL) receptor (SRB-1), acyl-coA cholesterol acyltransferase-2 (ACAT-2), and plasma concentration of lecithin cholesterol acyltransferase (LCAT), as well as HMG-CoA reductase, ACAT, and LCAT activities were determined in fasting male NAR and Sprague-Dawley control rats. The NAR group exhibited significant up-regulation of HMG-CoA reductase protein abundance but normal HMG-CoA reductase enzymatic activity. This was coupled with a significant up-regulation of cholesterol 7alpha-hydroxylase and a mild up-regulation of ACAT protein abundance and activity. However, hepatic LDL receptor and HDL receptor and plasma LCAT protein concentration and activity were normal in NAR. Hypercholesterolemia in NAR is associated with elevated hepatic HMG-CoA reductase protein abundance, but normal HMG-CoA reductase activity. These findings point to post-translational regulation of this enzyme and favor an extrahepatic origin of hypercholesterolemia in NAR. The observed up-regulation of cholesterol 7alpha-hydroxylase represents a compensatory response to the associated hypercholesterolemia. Unlike nephrotic syndrome, which causes severe LDL receptor, HDL receptor, and LCAT deficiencies, hereditary analbuminemia does not affect these proteins.

  14. Inhibition of NADPH cytochrome P450 reductase by the model sulfur mustard vesicant 2-chloroethyl ethyl sulfide is associated with increased production of reactive oxygen species.

    Science.gov (United States)

    Gray, Joshua P; Mishin, Vladimir; Heck, Diane E; Laskin, Debra L; Laskin, Jeffrey D

    2010-09-01

    Inhalation of vesicants including sulfur mustard can cause significant damage to the upper airways. This is the result of vesicant-induced modifications of proteins important in maintaining the integrity of the lung. Cytochrome P450s are the major enzymes in the lung mediating detoxification of sulfur mustard and its metabolites. NADPH cytochrome P450 reductase is a flavin-containing electron donor for cytochrome P450. The present studies demonstrate that the sulfur mustard analog, 2-chloroethyl ethyl sulfide (CEES), is a potent inhibitor of human recombinant cytochrome P450 reductase, as well as native cytochrome P450 reductase from liver microsomes of saline and beta-naphthoflavone-treated rats, and cytochrome P450 reductase from type II lung epithelial cells. Using rat liver microsomes from beta-naphthoflavone-treated rats, CEES was found to inhibit CYP 1A1 activity. This inhibition was overcome by microsomal cytochrome P450 reductase from saline-treated rats, which lack CYP 1A1 activity, demonstrating that the CEES inhibitory activity was selective for cytochrome P450 reductase. Cytochrome P450 reductase also generates reactive oxygen species (ROS) via oxidation of NADPH. In contrast to its inhibitory effects on the reduction of cytochrome c and CYP1A1 activity, CEES was found to stimulate ROS formation. Taken together, these data demonstrate that sulfur mustard vesicants target cytochrome P450 reductase and that this effect may be an important mechanism mediating oxidative stress and lung injury. 2010. Published by Elsevier Inc.

  15. Potency of a novel saw palmetto ethanol extract, SPET-085, for inhibition of 5alpha-reductase II.

    Science.gov (United States)

    Pais, Pilar

    2010-08-01

    The nicotinamide adenine dinucleotide phosphate (NADPH)-dependent membrane protein 5alpha-reductase irreversibly catalyses the conversion of testosterone to the most potent androgen, 5alpha-dihydrotestosterone (DHT). In humans, two 5alpha-reductase isoenyzmes are expressed: type I and type II. Type II is found primarily in prostate tissue. Saw palmetto extract (SPE) has been widely used for the treatment of lower urinary tract symptoms secondary to benign prostatic hyperplasia (BPH). The mechanisms of the pharmacological effects of SPE include the inhibition of 5alpha-reductase, among other actions. Clinical studies of SPE have been equivocal, with some showing significant results and others not. These inconsistent results may be due, in part, to varying bioactivities of the SPE used in the studies. The aim of the present study was to determine the in vitro potency of a novel saw palmetto ethanol extract (SPET-085), an inhibitor of the 5alpha-reductase isoenzyme type II, in a cell-free test system. On the basis of the enzymatic conversion of the substrate androstenedione to the 5alpha-reduced product 5alpha-androstanedione, the inhibitory potency was measured and compared to those of finasteride, an approved 5alpha-reductase inhibitor. SPET-085 concentration-dependently inhibited 5alpha-reductase type II in vitro (IC(50)=2.88+/-0.45 microg/mL). The approved 5alpha-reductase inhibitor, finasteride, tested as positive control, led to 61% inhibition of 5alpha-reductase type II. SPET-085 effectively inhibits the enzyme that has been linked to BPH, and the amount of extract required for activity is very low compared to data reported for other extracts. It can be concluded from data in the literature that SPET-085 is as effective as a hexane extract of saw palmetto that exhibited the highest levels of bioactivity, and is more effective than other SPEs tested. This study confirmed that SPET-085 has prostate health-promoting bioactivity that also corresponds favorably to

  16. The flavoheme reductase Ncb5or protects cells against endoplasmic reticulum stress-induced lipotoxicity[S

    OpenAIRE

    Zhang, Yongzhao; Larade, Kevin; Jiang, Zhi-Gang; Ito, Susumu; Wang, Wenfang; Zhu, Hao; Bunn, H. Franklin

    2010-01-01

    NCB5OR is a novel flavoheme reductase with a cytochrome b5-like domain at the N-terminus and a cytochrome b5 reductase-like domain at the C terminus. Ncb5or knock-out mice develop insulin deficient diabetes and loss of white adipose tissue. Ncb5or−/− mice have impairment of Δ9 fatty acid desaturation with elevated ratios of palmitate to palmitoleate and stearate to oleate. In this study we assess the role of the endoplasmic reticulum (ER) stress response in mediating lipotoxicity in Ncb5or−/−...

  17. Targeting aldose reductase for the treatment of diabetes complications and inflammatory diseases: new insights and future directions.

    Science.gov (United States)

    Maccari, Rosanna; Ottanà, Rosaria

    2015-03-12

    Aldose reductase (AR) is an aldo-keto reductase that has been widely investigated as an enzyme crucially involved in the pathogenesis of chronic complications associated with diabetes mellitus. Recently it was established that AR also acts as a key mediator of certain oxidative and inflammatory signaling pathways that are involved in the development of different human pathologies, such as cardiovascular disorders, sepsis, and cancer. These findings have renewed interest in the search for new AR inhibitors (ARIs) with improved profiles as potential therapeutic agents. In this review, recent advances in the field and promising future directions for developing ARIs are discussed.

  18. The roles of tissue nitrate reductase activity and myoglobin in securing nitric oxide availability in deeply hypoxic crucian carp

    DEFF Research Database (Denmark)

    Hansen, Marie Niemann; Lundberg, Jon O; Filice, Mariacristina

    2016-01-01

    In mammals, treatment with low doses of nitrite has a cytoprotective effect in ischemia/reperfusion events, as a result of nitric oxide formation and S-nitrosation of proteins. Interestingly, anoxia-tolerant lower vertebrates possess an intrinsic ability to increase intracellular nitrite concentr...... and myoglobin levels. Finally, we found a low but significant nitrate reductase activity in liver and white muscle, but not in cardiomyocytes. Nitrate reduction was inhibited by allopurinol, showing that it was partly catalyzed by xanthine oxidoreductase........ We also tested whether liver, muscle and heart tissue possess nitrate reductase activity that supplies nitrite to the tissues during severe hypoxia. Crucian carp exposed to deep hypoxia (1

  19. Robotic inhibition assay for determination of HMG-CoA reductase inhibitors in human plasma.

    Science.gov (United States)

    Fang, Wei; Liu, Lida; Hsieh, John Y-K; Zhao, Jamie; Matuszewski, Bogdan K; Rogers, John D; Dobrinska, Michael R

    2002-01-01

    The cholesterol-lowering drug simvastatin (SIMV, Zocor reduced heart attacks by 42% in patients who had high cholesterol levels and suffered from heart disease. Upon oral administration, SIMV is quickly hydrolyzed to its beta-hydroxyacid and other acid metabolites, which are potent inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase. A Tecan-based enzyme inhibition assay has been developed to improve the existing Zymark-based assay for the determination of both active and total concentrations of HMG-CoA reductase inhibitors in human plasma. A Tecan Genesis 200 robotic workstation equipped with eight probes and customized hardware was utilized to achieve higher sample throughput and improve assay reproducibility and mechanical stability. The developed enzyme inhibition assay was validated over two concentration ranges of 0.4-20 ng equivalent/mL, and 2-50 ng equivalent/mL. Intra- and interday precision data (coefficient of variation (CV)) for both concentration ranges were less than 9%, with an accuracy of 93-107%. The interday precision for the determination of quality control (QC) samples was less than 2% and 8%, respectively. The respective interday QC accuracy values were 93-103% and 97-104%. Good linearity across the two concentration ranges was observed, with acceptable reproducibility. This improved enzyme inhibition assay has been utilized to analyze human plasma samples from several clinical studies. Copyright 2002 Wiley-Liss, Inc.

  20. Nodule and Leaf Nitrate Reductases and Nitrogen Fixation in Medicago sativa L. under Water Stress

    Science.gov (United States)

    Aparicio-Tejo, P.; Sánchez-Díaz, Manuel

    1982-01-01

    The effect of water stress on patterns of nitrate reductase activity in the leaves and nodules and on nitrogen fixation were investigated in Medicago sativa L. plants watered 1 week before drought with or without NO3−. Nitrogen fixation was decreased by water stress and also inhibited strongly by the presence of NO3−. During drought, leaf nitrate reductase activity (NRA) decreased significantly particularly in plants watered with NO3−, while with rewatering, leaf NRA recovery was quite important especially in the NO3−-watered plants. As water stress progressed, the nodular NRA increased both in plants watered with NO3− and in those without NO3− contrary to the behavior of the leaves. Beyond −15.105 pascal, nodular NRA began to decrease in plants watered with NO3−. This phenomenon was not observed in nodules of plants given water only. Upon rewatering, it was observed that in plants watered with NO3− the nodular NRA increased again, while in plants watered but not given NO3−, such activity began to decrease. Nitrogen fixation increased only in plants without NO3−. PMID:16662233

  1. Manipulating the Bacterial Cell Cycle and Cell Size by Titrating the Expression of Ribonucleotide Reductase

    Directory of Open Access Journals (Sweden)

    Manlu Zhu

    2017-11-01

    Full Text Available Understanding how bacteria coordinate growth with cell cycle events to maintain cell size homeostasis remains a grand challenge in biology. The period of chromosome replication (C period is a key stage in the bacterial cell cycle. However, the mechanism of in vivo regulation of the C period remains unclear. In this study, we found that titration of the expression of ribonucleotide reductase (RNR, which changes the intracellular deoxynucleoside triphosphate (dNTP pools, enables significant perturbations of the C period, leading to a substantial change in cell size and DNA content. Our work demonstrates that the intracellular dNTP pool is indeed an important parameter that controls the progression of chromosome replication. Specially, RNR overexpression leads to a shortened C period compared with that of a wild-type strain growing under different nutrient conditions, indicating that the dNTP substrate levels are subsaturated under physiological conditions. In addition, perturbing the C period does not significantly change the D period, indicating that these two processes are largely independent from each other. Overall, titration of ribonucleotide reductase expression can serve as a standard model system for studying the coordination between chromosome replication, cell division, and cell size.

  2. Testosterone 5alpha-reductase inhibitory active constituents of Piper nigrum leaf.

    Science.gov (United States)

    Hirata, Noriko; Tokunaga, Masashi; Naruto, Shunsuke; Iinuma, Munekazu; Matsuda, Hideaki

    2007-12-01

    Previously we reported that Piper nigrum leaf extract showed a potent stimulation effect on melanogenesis and that (-)-cubebin (1) and (-)-3,4-dimethoxy-3,4-desmethylenedioxycubebin (2) were isolated as active constituents. As a part of our continuous studies on Piper species for the development of cosmetic hair-care agents, testosterone 5alpha-reductase inhibitory activity of aqueous ethanolic extracts obtained from several different parts of six Piper species, namely Piper nigrum, P. methysticum, P. betle, P. kadsura, P. longum, and P. cubeba, were examined. Among them, the extracts of P. nigrum leaf, P. nigrum fruit and P. cubeba fruit showed potent inhibitory activity. Activity-guided fractionation of P. nigrum leaf extract led to the isolation of 1 and 2. Fruits of P. cubeba contain 1 as a major lignan, thus inhibitory activity of the fruit may be attributable to 1. As a result of further assay on other known constituents of the cited Piper species, it was found that piperine, a major alkaloid amide of P. nigrum fruit, showed potent inhibitory activity, thus a part of the inhibitory activity of P. nigrum fruit may depend on piperine. The 5alpha-reductase inhibitory activities of 1 and piperine were found for the first time. In addition, the P. nigrum leaf extract showed in vivo anti-androgenic activity using the hair regrowth assay in testosterone sensitive male C57Black/6CrSlc strain mice.

  3. Structural and functional insights into Saccharomyces cerevisiae riboflavin biosynthesis reductase RIB7.

    Directory of Open Access Journals (Sweden)

    Zongyang Lv

    Full Text Available Saccharomyces cerevisiae RIB7 (ScRIB7 is a potent target for anti-fungal agents because of its involvement in the riboflavin biosynthesis pathway as a NADPH-dependent reductase. However, the catalytic mechanism of riboflavin biosynthesis reductase (RBSRs is controversial, and enzyme structure information is still lacking in eukaryotes. Here we report the crystal structure of Saccharomyces cerevisiae RIB7 at 2.10 Å resolution and its complex with NADPH at 2.35 Å resolution. ScRIB7 exists as a stable homodimer, and each subunit consists of nine central β-sheets flanked by five helices, resembling the structure of RIB7 homologues. A conserved G(76-X-G(78-Xn-G(181-G(182 motif is present at the NADPH pyrophosphate group binding site. Activity assays confirmed the necessity of Thr79, Asp83, Glu180 and Gly182 for the activity of ScRIB7. Substrate preference of ScRIB7 was altered by mutating one residue (Thr35 to a Lysine, implying that ScRIB7 Thr35 and its corresponding residue, a lysine in bacteria, are important in substrate-specific recognition.

  4. Metabolic derangement of methionine and folate metabolism in mice deficient in methionine synthase reductase

    Science.gov (United States)

    Elmore, C. Lee; Wu, Xuchu; Leclerc, Daniel; Watson, Erica D.; Bottiglieri, Teodoro; Krupenko, Natalia I.; Krupenko, Sergey A.; Cross, James C.; Rozen, Rima; Gravel, Roy A.; Matthews, Rowena G.

    2007-01-01

    Hyperhomocyst(e)inemia is a metabolic derangement that is linked to the distribution of folate pools, which provide one-carbon units for biosynthesis of purines and thymidylate and for remethylation of homocysteine to form methionine. In humans, methionine synthase deficiency results in the accumulation of methyltetrahydrofolate at the expense of folate derivatives required for purine and thymidylate biosynthesis. Complete ablation of methionine synthase activity in mice results in embryonic lethality. Other mouse models for hyperhomocyst(e)inemia have normal or reduced levels of methyltetrahydrofolate and are not embryonic lethal, although they have decreased ratios of AdoMet/AdoHcy and impaired methylation. We have constructed a mouse model with a gene trap insertion in the Mtrr gene specifying methionine synthase reductase, an enzyme essential for the activity of methionine synthase. This model is a hypomorph, with reduced methionine synthase reductase activity, thus avoiding the lethality associated with the absence of methionine synthase activity. Mtrrgt/gt mice have increased plasma homocyst(e)ine, decreased plasma methionine, and increased tissue methyltetrahydrofolate. Unexpectedly, Mtrrgt/gt mice do not show decreases in the AdoMet/AdoHcy ratio in most tissues. The different metabolite profiles in the various genetic mouse models for hyperhomocysteinemia may be useful in understanding biological effects of elevated homocyst(e)ine. PMID:17369066

  5. Identification of thioredoxin glutathione reductase inhibitors that kill cestode and trematode parasites.

    Directory of Open Access Journals (Sweden)

    Fabiana Ross

    Full Text Available Parasitic flatworms are responsible for serious infectious diseases that affect humans as well as livestock animals in vast regions of the world. Yet, the drug armamentarium available for treatment of these infections is limited: praziquantel is the single drug currently available for 200 million people infected with Schistosoma spp. and there is justified concern about emergence of drug resistance. Thioredoxin glutathione reductase (TGR is an essential core enzyme for redox homeostasis in flatworm parasites. In this work, we searched for flatworm TGR inhibitors testing compounds belonging to various families known to inhibit thioredoxin reductase or TGR and also additional electrophilic compounds. Several furoxans and one thiadiazole potently inhibited TGRs from both classes of parasitic flatworms: cestoda (tapeworms and trematoda (flukes, while several benzofuroxans and a quinoxaline moderately inhibited TGRs. Remarkably, five active compounds from diverse families possessed a phenylsulfonyl group, strongly suggesting that this moiety is a new pharmacophore. The most active inhibitors were further characterized and displayed slow and nearly irreversible binding to TGR. These compounds efficiently killed Echinococcus granulosus larval worms and Fasciola hepatica newly excysted juveniles in vitro at a 20 µM concentration. Our results support the concept that the redox metabolism of flatworm parasites is precarious and particularly susceptible to destabilization, show that furoxans can be used to target both flukes and tapeworms, and identified phenylsulfonyl as a new drug-hit moiety for both classes of flatworm parasites.

  6. Glutathione Reductase/Glutathione Is Responsible for Cytotoxic Elemental Sulfur Tolerance via Polysulfide Shuttle in Fungi*

    Science.gov (United States)

    Sato, Ikuo; Shimatani, Kanami; Fujita, Kensaku; Abe, Tsuyoshi; Shimizu, Motoyuki; Fujii, Tatsuya; Hoshino, Takayuki; Takaya, Naoki

    2011-01-01

    Fungi that can reduce elemental sulfur to sulfide are widely distributed, but the mechanism and physiological significance of the reaction have been poorly characterized. Here, we purified elemental sulfur-reductase (SR) and cloned its gene from the elemental sulfur-reducing fungus Fusarium oxysporum. We found that NADPH-glutathione reductase (GR) reduces elemental sulfur via glutathione as an intermediate. A loss-of-function mutant of the SR/GR gene generated less sulfide from elemental sulfur than the wild-type strain. Its growth was hypersensitive to elemental sulfur, and it accumulated higher levels of oxidized glutathione, indicating that the GR/glutathione system confers tolerance to cytotoxic elemental sulfur by reducing it to less harmful sulfide. The SR/GR reduced polysulfide as efficiently as elemental sulfur, which implies that soluble polysulfide shuttles reducing equivalents to exocellular insoluble elemental sulfur and generates sulfide. The ubiquitous distribution of the GR/glutathione system together with our findings that GR-deficient mutants derived from Saccharomyces cerevisiae and Aspergillus nidulans reduced less sulfur and that their growth was hypersensitive to elemental sulfur indicated a wide distribution of the system among fungi. These results indicate a novel biological function of the GR/glutathione system in elemental sulfur reduction, which is distinguishable from bacterial and archaeal mechanisms of glutathione- independent sulfur reduction. PMID:21474441

  7. Glutathione reductase/glutathione is responsible for cytotoxic elemental sulfur tolerance via polysulfide shuttle in fungi.

    Science.gov (United States)

    Sato, Ikuo; Shimatani, Kanami; Fujita, Kensaku; Abe, Tsuyoshi; Shimizu, Motoyuki; Fujii, Tatsuya; Hoshino, Takayuki; Takaya, Naoki

    2011-06-10

    Fungi that can reduce elemental sulfur to sulfide are widely distributed, but the mechanism and physiological significance of the reaction have been poorly characterized. Here, we purified elemental sulfur-reductase (SR) and cloned its gene from the elemental sulfur-reducing fungus Fusarium oxysporum. We found that NADPH-glutathione reductase (GR) reduces elemental sulfur via glutathione as an intermediate. A loss-of-function mutant of the SR/GR gene generated less sulfide from elemental sulfur than the wild-type strain. Its growth was hypersensitive to elemental sulfur, and it accumulated higher levels of oxidized glutathione, indicating that the GR/glutathione system confers tolerance to cytotoxic elemental sulfur by reducing it to less harmful sulfide. The SR/GR reduced polysulfide as efficiently as elemental sulfur, which implies that soluble polysulfide shuttles reducing equivalents to exocellular insoluble elemental sulfur and generates sulfide. The ubiquitous distribution of the GR/glutathione system together with our findings that GR-deficient mutants derived from Saccharomyces cerevisiae and Aspergillus nidulans reduced less sulfur and that their growth was hypersensitive to elemental sulfur indicated a wide distribution of the system among fungi. These results indicate a novel biological function of the GR/glutathione system in elemental sulfur reduction, which is distinguishable from bacterial and archaeal mechanisms of glutathione- independent sulfur reduction.

  8. Functional Annotation of a Presumed Nitronate Monoxygenase Reveals a New Class of NADH:Quinone Reductases*

    Science.gov (United States)

    Salvi, Francesca; Gadda, Giovanni

    2016-01-01

    The protein PA1024 from Pseudomonas aeruginosa PAO1 is currently classified as 2-nitropropane dioxygenase, the previous name for nitronate monooxygenase in the GenBankTM and PDB databases, but the enzyme was not kinetically characterized. In this study, PA1024 was purified to high levels, and the enzymatic activity was investigated by spectroscopic and polarographic techniques. Purified PA1024 did not exhibit nitronate monooxygenase activity; however, it displayed NADH:quinone reductase and a small NADH:oxidase activity. The enzyme preferred NADH to NADPH as a reducing substrate. PA1024 could reduce a broad spectrum of quinone substrates via a Ping Pong Bi Bi steady-state kinetic mechanism, generating the corresponding hydroquinones. The reductive half-reaction with NADH showed a kred value of 24 s−1 and an apparent Kd value estimated in the low micromolar range. The enzyme was not able to reduce the azo dye methyl red, routinely used in the kinetic characterization of azoreductases. Finally, we revisited and modified the existing six conserved motifs of PA1024, which define a new class of NADH:quinone reductases and are present in more than 490 hypothetical proteins in the GenBankTM, the vast majority of which are currently misannotated as nitronate monooxygenase. PMID:27502282

  9. New drug target in protozoan parasites: the role of thioredoxin reductase

    Directory of Open Access Journals (Sweden)

    Rosa M. Andrade

    2015-09-01

    Full Text Available Amebiasis causes approximately 70,000 deaths annually and is the third cause of death due to parasites worldwide. It is treated primarily with metronidazole, which has adverse side effects, is mutagenic and carcinogenic, and emergence of resistance is an increasing concern. Unfortunately, better therapeutic alternatives are lacking. Re-purposing of older FDA approved drugs is advantageous to drug discovery since safety and pharmacokinetic effects in humans are already known. In high throughput screening studies, we recently demonstrated that auranofin, a gold containing compound originally approved to treat rheumatoid arthritis, has activity against trophozoites of E. histolytica, the causative agent of amebiasis. Auranofin’s anti-parasitic activity is attributed to its monovalent gold molecule that readily inhibits E.histolytica thioredoxin reductase. This anti-oxidant enzyme is the only thiol-dependent flavo-reductase present in E.histolytica. Auranofin has also shown promising activity against other protozoans of significant public health importance. Altogether, this evidence suggests that auranofin has the potential to become a broad spectrum alternative therapeutic agent for diseases with a large global burden.

  10. Association of the methylenetetrahydrofolate reductase polymorphism in Korean patients with childhood acute lymphoblastic leukemia.

    Science.gov (United States)

    Kim, Nam Keun; Chong, So Young; Jang, Moon Ju; Hong, Seung Ho; Kim, Heung Sik; Cho, Eun Kyung; Lee, Jung Ae; Ahn, Myung Ju; Kim, Chul Soo; Oh, Doyeun

    2006-01-01

    Methylenetetrahydrofolate reductase plays a central role in converting folate to methyl donor for DNA methylation. Recently, methylenetetrahydrofolate reductase (MTHFR C677T and A1298C) mutations were discovered to be associated with childhood acute lymphoblastic leukemia (ALL), as well as colon cancer, lymphoma, esophageal and stomach cancer. Therefore, it was hypothesized that the MTHFR polymorphisms are associated with the risk of childhood ALL in the Korean population. DNA samples taken from 66 patients with ALL and 100 age-matched controls were analyzed using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay for detection of MTHFR C677T and A1298C mutations. The frequency of the AC genotype for MTHFR A1298C polymorphism was significantly different between the controls and the cases (OR, 2.22; CI, 95% 1.09-4.51, p=0.03). The 1298AC+CC genotype was also significantly different (OR, 2.11; 95% CI, 1.06-4.22; p=0.049). There was, however, no significant difference for MTHFR C677T polymorphism and combined genotype frequencies between the two groups. Although no consistent results on associations between MTHFR A 1298C polymorphism and ALL in the populations studied were obtained, the A1298C polymorphism, at least in Koreans, may be a genetic determinant among childhood ALL patients.

  11. Biliverdin reductase: new features of an old enzyme and its potential therapeutic significance.

    Science.gov (United States)

    Florczyk, Urszula M; Jozkowicz, Alicja; Dulak, Jozef

    2008-01-01

    Biliverdin reductase (BVR) was known for a long time solely as an enzyme converting biliverdin to bilirubin, the major physiological antioxidant. Recent years revealed unique features of this protein which are not related to its reductase activity. The most intriguing and surprising finding is its dual-specificity kinase character. As such serine/threonine/tyrosine kinase BVR is involved in regulation of glucose metabolism or in control of cell growth and apoptosis. In consequence, it may play a role in pathogenesis of many diseases, such as diabetes or cancers. Moreover, in the nucleus BVR, being a leucine zipper-like DNA binding protein, can act as a transcription factor for activator protein 1 (AP-1)-regulated genes. It has been shown that BVR modulates ATF-2 and HO-1 expression, what suggests its potential role in control of AP-1 and cAMP-regulated genes. In conclusion, BVR together with its substrate, biliverdin, and product, bilirubin, are revealed to be important players in cellular signal transduction pathways, gene expression and oxidative response. These features make BVR unusually interesting and unique among all enzymes characterized to date.

  12. Human heme oxygenase-1 efficiently catabolizes heme in the absence of biliverdin reductase.

    Science.gov (United States)

    Reed, James R; Huber, Warren J; Backes, Wayne L

    2010-11-01

    Heme oxygenase 1 (HO-1) uses molecular oxygen and electrons from NADPH cytochrome P450 reductase to convert heme to CO, ferrous iron, and biliverdin (BV). Enzymatic studies with the purified 30-kDa form of HO-1 routinely use a coupled assay containing biliverdin reductase (BVR), which converts BV to bilirubin (BR). BVR is believed to be required for optimal HO-1 activity. The goal of this study was to determine whether HO-1 activity could be monitored directly by following BV generation or iron release (using the ferrous iron chelator, ferrozine) in the absence of BVR. Using assays for each of the three end products, we found that HO-1 activity was stimulated in the presence of catalase and comparable rates were measured with each assay. Absorbance scans revealed characteristic spectra for BR, BV, and/or the ferrozine-iron complex. The optimal conditions were slightly different for the direct and coupled assays. BSA activated the coupled but inhibited the direct assays, and the assays had different pH optima. By measuring the activity of BVR directly using BV as a substrate, these differences were attributed to different enzymatic properties of BVR and HO-1. Thus, BVR is not needed to measure the activity of HO-1 when catalase is present. In fact, the factors affecting catalysis by HO-1 are better understood using the direct assays because the coupled assay can be influenced by properties of BVR.

  13. New progesterone derivatives as inhibitors of 5alpha-reductase enzyme and prostate cancer cell growth.

    Science.gov (United States)

    Cabeza, Marisa; Bratoeff, Eugene; Heuze, Ivonne; Rojas, Arely; Terán, Nayely; Ochoa, Martha'; Ramírez-Apan, Teresa; Ramírez, Elena; Pérez, Victor; Gracia, Isabel

    2006-08-01

    In this study we report the synthesis and pharmacological evaluation, in vivo as well as in vitro, of four new progesterone derivatives 4-7. The evaluation in vivo was carried out on gonadectomized male hamsters that were injected subcutaneously daily with 1 mg/Kg of testosterone (T) and/or 1 mg/Kg of finasteride, or with 2 mg/Kg of the novel compounds. It was observed that when testosterone (T) and finasteride or compound 4 were injected together, the weight of the prostate decreased significantly as compared to that oftestosterone-treated animals. Compounds 5-7 did not show any in vivo activity. The 5alpha-reductase inhibitory activity of the novel compounds was determined in vitro using human prostate homogenates; the steroids 4-7 inhibited the 5alpha-reductase activity with IC50 values lower than that for the reference compound finasteride. 3. The effect of compounds 4-7 on the growth of lymphocytes and prostate cancer culture cells line was that steroid 4 inhibited the growth of both cells lines at a concentration of 50 microM and showed a cytotoxic effect whereas compounds 5-7 showed a much lower inhibition. Nevertheless steroids 4-7 didn't exhibit any toxic effects in vivo since the animals remained alive during the six days of treatment.

  14. Characterization and expression patterns of nitrate reductase from Dunaliella bardawil under osmotic stress and dilution shock.

    Science.gov (United States)

    Lao, Yong-Min; Jiang, Jian-Guo; Luo, Li-Xin

    2014-07-01

    A complementary DNA (cDNA) of nitrate reductase (NR) from Dunaliella bardawil was isolated using RT-PCR and RACEs techniques. The full-length D. bardawil NR (DbNR) cDNA is 3,107 bp containing a putative open reading frame of 2,670 bp in length which encodes 889 amino acids with a calculated molecular weight (MW) of 98.37 kDa, a 34-bp 5'-untranslated region, and a 3'-untranslated region of 403 bp with a poly (A) tail. BLAST search showed that the nucleotide and putative protein sequence exhibit sequence identities of 92 and 79% with the corresponding gene from Dunaliella tertiolecta, respectively. Protein structural analysis showed a typical NR structure of DbNR with five structural distinctive domains which form three common subparts of eukaryotic NR (Euk-NR). Phylogenetic analysis based on the holo-DbNR and sulfite oxidase (SO) and cytochrome b reductase (CbR) subparts manifested that (1) DbNR has a closer relationship with those counterparts from algae and higher plants than from other species and (2) DbNR might have evolved from ancient SO and CbR in a "domain shuffling" pattern. The glycerol contents and transcriptional expression patterns of DbNR under salt stress and dilution shock treatments were also traced. The results implied an indirect role of NaCl on the induction of DbNR through an osmoregulation pathway.

  15. Aldose reductase (AKR1B) deficiency promotes phagocytosis in bone marrow derived mouse macrophages.

    Science.gov (United States)

    Singh, Mahavir; Kapoor, Aniruddh; McCracken, James; Hill, Bradford; Bhatnagar, Aruni

    2017-03-01

    Macrophages are critical drivers of the immune response during infection and inflammation. The pathogenesis of several inflammatory conditions, such as diabetes, cancer and sepsis has been linked with aldose reductase (AR), a member of the aldo-keto reductase (AKR) superfamily. However, the role of AR in the early stages of innate immunity such as phagocytosis remains unclear. In this study, we examined the role of AR in regulating the growth and the phagocytic activity of bone marrow-derived mouse macrophages (BMMs) from AR-null and wild-type (WT) mice. We found that macrophages derived from AR-null mice were larger in size and had a slower growth rate than those derived from WT mice. The AR-null macrophages also displayed higher basal, and lipopolysaccharide (LPS) stimulated phagocytic activity than WT macrophages. Moreover, absence of AR led to a marked increase in cellular levels of both ATP and NADPH. These data suggest that metabolic pathways involving AR suppress macrophage energy production, and that inhibition of AR could induce a favorable metabolic state that promotes macrophage phagocytosis. Hence, modulation of macrophage metabolism by inhibition of AR might represent a novel strategy to modulate host defense responses and to modify metabolism to promote macrophage hypertrophy and phagocytosis under inflammatory conditions. Copyright © 2017. Published by Elsevier B.V.

  16. Xanthones with quinone reductase-inducing activity from the fruits of Garcinia mangostana (Mangosteen).

    Science.gov (United States)

    Chin, Young-Won; Jung, Hyun-Ah; Chai, Heebyung; Keller, William J; Kinghorn, A Douglas

    2008-02-01

    Bioactivity-guided fractionation of a dichloromethane-soluble extract of Garcinia mangostana fruits has led to the isolation and identification of five compounds, including two xanthones, 1,2-dihydro-1,8,10-trihydroxy-2-(2-hydroxypropan-2-yl)-9-(3-methylbut-2-enyl)furo[3,2-a]xanthen-11-one (1) and 6-deoxy-7-demethylmangostanin (2), along with three known compounds, 1,3,7-trihydroxy-2,8-di-(3-methylbut-2-enyl)xanthone (3), mangostanin (4), and alpha-mangostin (5). The structures of compounds 1 and 2 were determined from analysis of their spectroscopic data. All isolated compounds in the present study together with eleven other compounds previously isolated from the pericarp of mangosteen, were tested in an in vitro quinone reductase-induction assay using murine hepatoma cells (Hepa 1c1c7) and an in vitro hydroxyl radical antioxidant assay. Of these, compounds 1-4 induced quinone reductase (concentration to double enzyme induction, 0.68-2.2microg/mL) in Hepa 1c1c7 cells and gamma-mangostin (6) exhibited hydroxyl radical-scavenging activity (IC50, 0.20microg/mL).

  17. Catalytic and immunochemical properties of NADPH-cytochrome P450 reductase from fungus Rhizopus nigricans.

    Science.gov (United States)

    Makovec, Tomaz; Breskvar, Katja

    2002-09-01

    Flavoprotein NADPH-cytochrome P450 reductase (CPR, EC 1.6.2.4) from filamentous fungus Rhizopus nigricans is a membrane bound enzyme which is involved in the reduction of cytochrome P450 during the hydroxylation of progesterone at 11alpha position. After purification of the enzyme from induced mycelia three forms of fungal CPR were detected on SDS-PAGE: a predominant form with an apparent molecular mass of 78kDa and two truncated forms. N-terminal sequences of all three forms were determined as well as some internal sequences of 78kDa form. Dose-dependent immunoinhibition of NADPH-cytochrome c reductase and progesterone 11alpha-hydroxylase activities was observed with mouse anti-CPR antisera. No cross-reactions were obtained on Western blots between mouse anti-CPR antisera and protein preparations from noninduced mycelia and microsomal fraction from fungus Pleurotus osteatus, plant Ginkgo biloba or chicken liver. The kinetic mechanism of CPR was proposed on the basis of model reaction with cytochrome c(3+). Results obtained at high ionic strength suggest a nonclassical two-site ping pong mechanism and at low ionic strength a sequential mechanism of bisubstrate reaction.

  18. Synthesis and characterization of potent inhibitors of Trypanosoma cruzi dihydrofolate reductase

    Energy Technology Data Exchange (ETDEWEB)

    Schormann, Norbert; Velu, Sadanandan E.; Murugesan, Srinivasan; Senkovich, Olga; Walker, Kiera; Chenna, Bala C.; Shinkre, Bidhan; Desai, Amar; Chattopadhyay, Debasish (UAB)

    2010-09-17

    Dihydrofolate reductase (DHFR) of the parasite Trypanosoma cruzi (T. cruzi) is a potential target for developing drugs to treat Chagas disease. We have undertaken a detailed structure-activity study of this enzyme. We report here synthesis and characterization of six potent inhibitors of the parasitic enzyme. Inhibitory activity of each compound was determined against T. cruzi and human DHFR. One of these compounds, ethyl 4-(5-[(2,4-diamino-6-quinazolinyl)methyl]amino-2-methoxyphenoxy)butanoate (6b) was co-crystallized with the bifunctional dihydrofolate reductase-thymidylate synthase enzyme of T. cruzi and the crystal structure of the ternary enzyme:cofactor:inhibitor complex was determined. Molecular docking was used to analyze the potential interactions of all inhibitors with T. cruzi DHFR and human DHFR. Inhibitory activities of these compounds are discussed in the light of enzyme-ligand interactions. Binding affinities of each inhibitor for the respective enzymes were calculated based on the experimental or docked binding mode. An estimated 60-70% of the total binding energy is contributed by the 2,4-diaminoquinazoline scaffold.

  19. Cloning and expression of koala (Phascolarctos cinereus) liver cytochrome P450 reductase.

    Science.gov (United States)

    Kong, Sandra; Ngo, Suong N T; McKinnon, Ross A; Stupans, Ieva

    2009-07-01

    The cloning, expression and characterization of hepatic NADPH-cytochrome P450 reductase (CPR) from koala (Phascolarctos cinereus) is described. Two 2059 bp koala liver CPR cDNAs, designated CPR1 and CPR2, were cloned by reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends. The koala CPR cDNAs encode proteins of 678 amino acids and share 85% amino acid sequence identity to human CPR. Transfection of the koala CPR cDNAs into Cos-7 cells resulted in the expression of proteins, which were recognized by a goat-antihuman CPR antibody. The koala CPR1 and 2 cDNA-expressed enzymes catalysed cytochrome c reductase at the rates of 4.9 +/- 0.5 and 2.6 +/- 0.4 nmol/min/mg protein (mean +/- SD, n = 3), respectively which were comparable to that of rat CPR cDNA-expressed enzyme. The apparent Km value for CPR activity in koala liver microsomes was 11.61 +/- 6.01 microM, which is consistent with that reported for rat CPR enzyme. Northern analysis detected a CPR mRNA band of approximately 2.6 kb. Southern analysis suggested a single PCR gene across species. The present study provides primary molecular data regarding koala CPR1 and CPR2 genes in this unique marsupial species.

  20. Induction of quinone reductase (QR) by withanolides isolated from Physalis pubescens L. (Solanaceae).

    Science.gov (United States)

    Ji, Long; Yuan, Yonglei; Ma, Zhongjun; Chen, Zhe; Gan, Lishe; Ma, Xiaoqiong; Huang, Dongsheng

    2013-09-01

    In the present study, it was demonstrated that the dichloromethane extract of Physalis pubescens L. (DEPP) had weak potential quinone reductase (QR) inducing activity, but an UPLC-ESI-MS method with glutathione (GSH) as the substrate revealed that the DEPP had electrophiles (with an α,β-unsaturated ketone moiety). These electrophiles could induce quinone reductase (QR) activity, which might be attributed to the modification of the highly reactive cysteine residues in Keap1. Herein, four withanolides, including three new compounds physapubescin B (2), physapubescin C (3), physapubescin D (4), together with one known steroidal compound physapubescin (1) were isolated. Structures of these compounds were determined by spectroscopic analysis and that of physapubescin C (3) was confirmed by a combination of molecular modeling and quantum chemical DFT-GIAO calculations. Evaluation of the QR inducing activities of all withanolides indicated potent activities of compounds 1 and 2, which had a common α,β-unsaturated ketone moiety. Copyright © 2013 Elsevier Ltd. All rights reserved.

  1. Induction of quinone reductase (QR) by withanolides isolated from Physalis angulata L. var. villosa Bonati (Solanaceae).

    Science.gov (United States)

    Ding, Hui; Hu, Zhijuan; Yu, Liyan; Ma, Zhongjun; Ma, Xiaoqiong; Chen, Zhe; Wang, Dan; Zhao, Xiaofeng

    2014-08-01

    In the present study, the EtOAc extract of the persistent calyx of Physalis angulata L. var. villosa Bonati (PA) was tested for its potential quinone reductase (QR) inducing activity with glutathione (GSH) as the substrate using an UPLC-ESI-MS method. The result revealed that the PA had electrophiles that could induce quinone reductase (QR) activity, which might be attributed to the modification of the highly reactive cysteine residues in Keap1. Herein, three new withanolides, compounds 3, 6 and 7, together with four known withanolides, compounds 1, 2, 4 and 5 were isolated from PA extract. Their structures were determined by spectroscopic techniques, including (1)H-, (13)C NMR (DEPT), and 2D-NMR (HMBC, HMQC, (1)H, (1)H-COSY, NOESY) experiments, as well as by HR-MS. All the seven compounds were tested for their QR induction activities towards mouse hepa 1c1c7 cells. Copyright © 2014 Elsevier Inc. All rights reserved.

  2. Methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism: epidemiology, metabolism and the associated diseases.

    Science.gov (United States)

    Liew, Siaw-Cheok; Gupta, Esha Das

    2015-01-01

    The Methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism is associated with various diseases (vascular, cancers, neurology, diabetes, psoriasis, etc) with the epidemiology of the polymorphism of the C677T that varies dependent on the geography and ethnicity. The 5,10-Methylenetetrahydrofolate reductase (MTHFR) locus is mapped on chromosome 1 at the end of the short arm (1p36.6). This enzyme is important for the folate metabolism which is an integral process for cell metabolism in the DNA, RNA and protein methylation. The mutation of the MTHFR gene which causes the C677T polymorphism is located at exon 4 which results in the conversion of valine to alanine at codon 222, a common polymorphism that reduces the activity of this enzyme. The homozygous mutated subjects have higher homocysteine levels while the heterozygous mutated subjects have mildly raised homocysteine levels compared with the normal, non-mutated controls. Hyperhomocysteinemia is an emerging risk factor for various cardiovascular diseases and with the increasing significance of this polymorphism in view of the morbidity and mortality impact on the patients, further prevention strategies and nutritional recommendations with the supplementation of vitamin B12 and folic acid which reduces plasma homocysteine level would be necessary as part of future health education. This literature review therefore focuses on the recent evidence-based reports on the associations of the MTHFR C677T polymorphism and the various diseases globally. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  3. Methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism among Gaddi tribe of Indian state of Himachal Pradesh.

    Science.gov (United States)

    Khan, Simi; Thakur, Sunil; Kallur, Saraswathy Nava; Ghosh, Pradeep Kumar; Sachdeva, Mohinder Pal; Vadlamudi, Raghavendra Rao

    2014-01-01

    Methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism has been suggested to be positively associated with several disorders. Distribution of the mutant T-allele varies in ethnic and geographical populations of the world. The aim of the present study was to investigate the distribution of methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism in a transhumant (Gaddi) tribal population of Himachal Pradesh dwelling at high and middle altitude and exposed to strong ultraviolet radiation. A total of 486 samples (141 males and 345 females) were randomly enrolled from the individuals aged 25-75 years who were unrelated up to first cousin. Among Gaddis, genotype frequencies of CC, CT and TT were 67.90%, 27.78% and 4.32%, respectively. Among males and females distribution of genotype frequencies also followed a similar trend. The studied population was in Hardy-Weinberg equilibrium (χ(2 )= 2.213, df = 1, p = 0.136). Frequency of mutant T-allele in the Gaddi population was found to be 0.183, which might be due to European ancestry, endogamous nature and selection.

  4. Expression of mercuric ion reductase in Eastern cottonwood (Populus deltoides) confers mercuric ion reduction and resistance.

    Science.gov (United States)

    Che, Dongsheng; Meagher, Richard B; Heaton, Andrew C P; Lima, Amparo; Rugh, Clayton L; Merkle, Scott A

    2003-07-01

    Mercury is one of the most hazardous heavy metals and is a particular problem in aquatic ecosystems, where organic mercury is biomagnified in the food chain. Previous studies demonstrated that transgenic model plants expressing a modified mercuric ion reductase gene from bacteria could detoxify mercury by converting the more toxic and reductive ionic form [Hg(II)] to less toxic elemental mercury [Hg(0)]. To further investigate if a genetic engineering approach for mercury phytoremediation can be effective in trees with a greater potential in riparian ecosystems, we generated transgenic Eastern cottonwood (Populus deltoides) trees expressing modified merA9 and merA18 genes. Leaf sections from transgenic plantlets produced adventitious shoots in the presence of 50 microm Hg(II) supplied as HgCl2, which inhibited shoot induction from leaf explants of wild-type plantlets. Transgenic shoots cultured in a medium containing 25 microm Hg(II) showed normal growth and rooted, while wild-type shoots were killed. When the transgenic cottonwood plantlets were exposed to Hg(II), they evolved 2-4-fold the amount of Hg(0) relative to wild-type plantlets. Transgenic merA9 and merA18 plants accumulated significantly higher biomass than control plants on a Georgia Piedmont soil contaminated with 40 p.p.m. Hg(II). Our results indicate that Eastern cottonwood plants expressing the bacterial mercuric ion reductase gene have potential as candidates for in situ remediation of mercury-contaminated soils or wastewater.

  5. Protective Role of Aldose Reductase Deletion in an Animal Model of Oxygen-Induced Retinopathy

    Directory of Open Access Journals (Sweden)

    Zhongjie Fu

    2011-05-01

    Full Text Available Retinopathy of prematurity (ROP is a common disease occurred in premature babies. Both vascular abnormality and neural dysfunction of the retina were reported, and oxidative stress was involved. Previously, it has been showed that deficiency of aldose reductase (AR, the rate-limiting enzyme in polyol pathway, lowered oxidative stress. Here, the effect of AR deletion on neonatal retinal injury was investigated by using a mouse model of ROP (oxygen-induced retinopathy, OIR. Seven-day-old pups were exposed to 75% oxygen for 5 days and then returned to room air. The vascular changes and neuronal/glial responses were examined and compared between wild-type and AR-deficient OIR mice. Significantly reduced vaso-obliterated area, blood vessel leakage, and early revascularization were observed in AR-deficient OIR mice. Moreover, reduced amacrine cells and less distorted strata were observed in AR-deficient OIR mice. Less astrocytic immunoreactivity and reduced Müller cell gliosis were also observed in AR-deficient mice. After OIR, nitrotyrosine immunoreactivity and poly (ADP-ribose (PAR translocation, which are two oxidative stress markers, were decreased in AR-deficient mice. Significant decrease in VEGF, pho-Erk1/2, pho-Akt, and pho-I?B expression was found in AR-deficient OIR retinae. Thus, these observations suggest that the deficiency of aldose reductase may protect the retina in the OIR model.

  6. Crystallization and preliminary X-ray studies of ferredoxin-NAD(P)+ reductase from Chlorobium tepidum.

    Science.gov (United States)

    Muraki, Norifumi; Seo, Daisuke; Shiba, Tomoo; Sakurai, Takeshi; Kurisu, Genji

    2008-03-01

    Ferredoxin-NAD(P)(+) reductase (FNR) is a key enzyme that catalyzes the photoreduction of NAD(P)(+) to generate NAD(P)H during the final step of the photosynthetic electron-transport chain. FNR from the green sulfur bacterium Chlorobium tepidum is a homodimeric enzyme with a molecular weight of 90 kDa; it shares a high level of amino-acid sequence identity to thioredoxin reductase rather than to conventional plant-type FNRs. In order to understand the structural basis of the ferredoxin-dependency of this unique photosynthetic FNR, C. tepidum FNR has been heterologously expressed, purified and crystallized in two forms. Form I crystals belong to space group C222(1) and contain one dimer in the asymmetric unit, while form II crystals belong to space group P4(1)22 or P4(3)22. Diffraction data were collected from a form I crystal to 2.4 A resolution on the synchrotron-radiation beamline NW12 at the Photon Factory.

  7. Crystal structure of the Trypanosoma cruzi trypanothione reductase.mepacrine complex.

    Science.gov (United States)

    Jacoby, E M; Schlichting, I; Lantwin, C B; Kabsch, W; Krauth-Siegel, R L

    1996-01-01

    The three-dimensional structure of the complex between Trypanosoma cruzi trypanothione reductase (TR) (EC 1.6.4.8) and the antiparasitic drug mepacrine (quinacrine) has been solved at 2.9 angstoms resolution. Mepacrine is a competitive inhibitor of TR but does not affect human glutathione reductase (GR), a closely related host enzyme. Of particular importance for inhibitor binding are four amino acid residues in the disulfide substrate-binding site of TR that are not conserved in human GR, namely, Glu-18 (Ala-34 in GR), Trp-21 (Arg-37), Ser-109 (Ile-113), and Met-113 (Asn-117). The acridine ring of mepacrine is fixed at the active site close to the hydrophobic wall formed by Trp-21 and Met-113. Specific pairwise interactions between functional groups of the drug and amino acid side chains include the ring nitrogen and Met-113, the chlorine atom and Trp-21, and the oxymethyl group and Ser-109. The alkylamino chain of mepacrine points into the inner region of the active site and is held in position by a solvent-mediated hydrogen bond to Glu-18. The structure of the complex shows for the first time the atomic interactions between TR and an inhibitory ligand. This is a crucial step towards the rational design of inhibitors that might be suited as drugs against Chagas' disease.

  8. Molecular dynamics simulations reveal proton transfer pathways in cytochrome C-dependent nitric oxide reductase.

    Directory of Open Access Journals (Sweden)

    Andrei V Pisliakov

    Full Text Available Nitric oxide reductases (NORs are membrane proteins that catalyze the reduction of nitric oxide (NO to nitrous oxide (N(2O, which is a critical step of the nitrate respiration process in denitrifying bacteria. Using the recently determined first crystal structure of the cytochrome c-dependent NOR (cNOR [Hino T, Matsumoto Y, Nagano S, Sugimoto H, Fukumori Y, et al. (2010 Structural basis of biological N2O generation by bacterial nitric oxide reductase. Science 330: 1666-70.], we performed extensive all-atom molecular dynamics (MD simulations of cNOR within an explicit membrane/solvent environment to fully characterize water distribution and dynamics as well as hydrogen-bonded networks inside the protein, yielding the atomic details of functionally important proton channels. Simulations reveal two possible proton transfer pathways leading from the periplasm to the active site, while no pathways from the cytoplasmic side were found, consistently with the experimental observations that cNOR is not a proton pump. One of the pathways, which was newly identified in the MD simulation, is blocked in the crystal structure and requires small structural rearrangements to allow for water channel formation. That pathway is equivalent to the functional periplasmic cavity postulated in cbb(3 oxidase, which illustrates that the two enzymes share some elements of the proton transfer mechanisms and confirms a close evolutionary relation between NORs and C-type oxidases. Several mechanisms of the critical proton transfer steps near the catalytic center are proposed.

  9. Deletion of thioredoxin reductase and effects of selenite and selenate toxicity in Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Christopher J Boehler

    Full Text Available Thioredoxin reductase-1 (TRXR-1 is the sole selenoprotein in C. elegans, and selenite is a substrate for thioredoxin reductase, so TRXR-1 may play a role in metabolism of selenium (Se to toxic forms. To study the role of TRXR in Se toxicity, we cultured C. elegans with deletions of trxr-1, trxr-2, and both in axenic media with increasing concentrations of inorganic Se. Wild-type C. elegans cultured for 12 days in Se-deficient axenic media grow and reproduce equivalent to Se-supplemented media. Supplementation with 0-2 mM Se as selenite results in inverse, sigmoidal response curves with an LC50 of 0.20 mM Se, due to impaired growth rather than reproduction. Deletion of trxr-1, trxr-2 or both does not modulate growth or Se toxicity in C. elegans grown axenically, and (75Se labeling showed that TRXR-1 arises from the trxr-1 gene and not from bacterial genes. Se response curves for selenide (LC50 0.23 mM Se were identical to selenite, but selenate was 1/4(th as toxic (LC50 0.95 mM Se as selenite and not modulated by TRXR deletion. These nutritional and genetic studies in axenic media show that Se and TRXR are not essential for C. elegans, and that TRXR alone is not essential for metabolism of inorganic Se to toxic species.

  10. Molecular characterization of genes encoding leucoanthocyanidin reductase involved in proanthocyanidin biosynthesis in apple

    Directory of Open Access Journals (Sweden)

    Yuepeng eHan

    2015-04-01

    Full Text Available Proanthocyanidins (PAs are the major component of phenolics in apple, but mechanisms involved in PA biosynthesis remain unclear. Here, the relationship between the PA biosynthesis and the expression of genes encoding leucoanthocyanidin reductase (LAR and anthocyanidin reductase (ANR was investigated in fruit skin of one apple cultivar and three crabapples. Transcript levels of LAR1 and ANR2 genes were significantly correlated with the contents of catechin and epicatechin, respectively, which suggests their active roles in PA synthesis. Surprisingly, transcript levels for both LAR1 and LAR2 genes were almost undetectable in two crabapples that accumulated both flavan-3-ols and PAs. This contradicts the previous finding that LAR1 gene is a strong candidate regulating the accumulation of metabolites such as epicatechin and PAs in apple. Ectopic expression of apple MdLAR1 gene in tobacco suppresses expression of the late genes in anthocyanin biosynthetic pathway, resulting in loss of anthocyanin in flowers. Interestingly, a decrease in PA biosynthesis was also observed in flowers of transgenic tobacco plants overexpressing the MdLAR1 gene, which could be attributed to decreased expression of both the NtANR1 and NtANR2 genes. Our study not only confirms the in vivo function of apple LAR1 gene, but it is also helpful for understanding the mechanism of PA biosynthesis.

  11. Targeting Thioredoxin Reductase by Parthenolide Contributes to Inducing Apoptosis of HeLa Cells*

    Science.gov (United States)

    Duan, Dongzhu; Zhang, Junmin; Yao, Juan; Liu, Yaping; Fang, Jianguo

    2016-01-01

    Parthenolide (PTL), a major active sesquiterpene lactone from the herbal plant Tanacetum parthenium, has been applied in traditional Chinese medicine for centuries. Although PTL demonstrates potent anticancer efficacy in numerous types of malignant cells, the cellular targets of PTL have not been well defined. We reported here that PTL interacts with both cytosolic thioredoxin reductase (TrxR1) and mitochondrial thioredoxin reductase (TrxR2), two ubiquitous selenocysteine-containing antioxidant enzymes, to elicit reactive oxygen species-mediated apoptosis in HeLa cells. PTL selectively targets the selenocysteine residue in TrxR1 to inhibit the enzyme function, and further shifts the enzyme to an NADPH oxidase to generate superoxide anions, leading to reactive oxygen species accumulation and oxidized thioredoxin. Under the conditions of inhibition of TrxRs in cells, PTL does not cause significant alteration of cellular thiol homeostasis, supporting selective target of TrxRs by PTL. Importantly, overexpression of functional TrxR1 or Trx1 confers protection, whereas knockdown of the enzymes sensitizes cells to PTL treatment. Targeting TrxRs by PTL thus discloses an unprecedented mechanism underlying the biological activity of PTL, and provides deep insights to understand the action of PTL in treatment of cancer. PMID:27002142

  12. Nuclear inelastic scattering at the diiron center of ribonucleotide reductase from Escherichia coli

    Science.gov (United States)

    Marx, J.; Srinivas, V.; Faus, I.; Auerbach, H.; Scherthan, L.; Jenni, K.; Chumakov, A. I.; Rüffer, R.; Högbom, M.; Haumann, M.; Schünemann, V.

    2017-11-01

    The enzyme ribonucleotide reductase R2 catalyzes an important step in the synthesis of the building blocks of DNA, and harbors a dinuclear iron center required for activity. Not only the iron valence states but also the protonation of the iron ligands govern the enzymatic activity of the enzyme. We have performed Nuclear Inelastic Scattering (NIS) experiments on the 57Fe reconstituted ribonucleotide reductase R2 subunit from Escherichia coli ( Ec R2a). Accompanying Mössbauer spectroscopic investigations show that the partial density of vibrational states (pDOS) of the 57Fe reconstituted Ec R2a sample contained contributions from both 57Fe- Ec R2a protein as well as unspecifically bound 57Fe. Subtraction of a featureless pDOS as obtained from protein-coated iron oxide particles allowed modeling of the contribution of non-specifically bound iron and thus the pDOS of 57Fe- Ec R2a could be obtained. Quantum-mechanics/molecular-mechanics (QM/MM) calculations of the whole 57Fe- Ec R2a protein with variations of the cofactor protonation were performed in order to assign characteristic bands to their corresponding molecular vibrational modes.

  13. Molecular Dynamics Simulations Reveal Proton Transfer Pathways in Cytochrome C-Dependent Nitric Oxide Reductase

    Science.gov (United States)

    Pisliakov, Andrei V.; Hino, Tomoya; Shiro, Yoshitsugu; Sugita, Yuji

    2012-01-01

    Nitric oxide reductases (NORs) are membrane proteins that catalyze the reduction of nitric oxide (NO) to nitrous oxide (N2O), which is a critical step of the nitrate respiration process in denitrifying bacteria. Using the recently determined first crystal structure of the cytochrome c-dependent NOR (cNOR) [Hino T, Matsumoto Y, Nagano S, Sugimoto H, Fukumori Y, et al. (2010) Structural basis of biological N2O generation by bacterial nitric oxide reductase. Science 330: 1666–70.], we performed extensive all-atom molecular dynamics (MD) simulations of cNOR within an explicit membrane/solvent environment to fully characterize water distribution and dynamics as well as hydrogen-bonded networks inside the protein, yielding the atomic details of functionally important proton channels. Simulations reveal two possible proton transfer pathways leading from the periplasm to the active site, while no pathways from the cytoplasmic side were found, consistently with the experimental observations that cNOR is not a proton pump. One of the pathways, which was newly identified in the MD simulation, is blocked in the crystal structure and requires small structural rearrangements to allow for water channel formation. That pathway is equivalent to the functional periplasmic cavity postulated in cbb 3 oxidase, which illustrates that the two enzymes share some elements of the proton transfer mechanisms and confirms a close evolutionary relation between NORs and C-type oxidases. Several mechanisms of the critical proton transfer steps near the catalytic center are proposed. PMID:22956904

  14. Reduction of azo dyes by flavin reductase from Citrobacter freundii A1

    Directory of Open Access Journals (Sweden)

    Mohd Firdaus Abdul-Wahab

    2012-12-01

    Full Text Available Citrobacter freundii A1 isolated from a sewage treatment facility was demonstrated to be able to effectively decolorize azo dyes as pure and mixed culture. This study reports on the investigation on the enzymatic systems involved. An assay performed suggested the possible involvement of flavin reductase (Fre as an azo reductase. A heterologouslyexpressed recombinant Fre from C. freundii A1 was used to investigate its involvement in the azo reduction process. Three model dyes were used, namely Acid Red 27 (AR27, Direct Blue 15 (DB15 and Reactive Black 5 (RB5. AR27 was found to be reduced the fastest by Fre, followed by RB5, and lastly DB15. Redox mediators nicotinamide adenine dinucleotide (NADH and riboflavin enhance the reduction, suggesting the redox activity of the enzyme. The rate and extent of reduction of the model dyes correlate well with the reduction potentials (Ep. The data presented here strongly suggest that Fre is one of the enzymes responsible for azo reduction in C. freundii A1, acting via an oxidation-reduction reaction.

  15. Mercuric reductase activity of multiple heavy metal-resistant Lysinibacillus sphaericus G1.

    Science.gov (United States)

    Bafana, Amit; Chakrabarti, Tapan; Krishnamurthi, Kannan

    2015-03-01

    A culture was isolated from an industrial mercuric salt-contaminated soil, which could tolerate Cd, Co, Zn, Cr, and Hg up to 190, 525, 350, 935, and 370 μM, respectively. The isolate was identified as Lysinibacillus sphaericus by 16S rRNA gene sequencing. It bioaccumulated Cd, Co, and Zn, and reductively detoxified Cr and Hg. Chromate reductase and mercuric reductase (MerA) activities in the cell extract were 2.4 and 0.13 units mg(-1) protein, respectively. The study also describes designing of broad-specificity primers based on firmicute merA genes. These primers were successfully used to amplify a 440 bp merA fragment from the current isolate. Based on the partial sequence, complete merA ORF of 1641 bp was amplified. It showed 99% similarity to a putative merA gene from distantly related Streptococcus agalactiae, but only 72% identity with the well-characterized merA from a more closely related Bacillus cereus RC607. The gene sequence possessed all the features required for the functioning of MerA enzyme, and its function was confirmed by recombinant expression in E. coli. To the best of our knowledge, this is the first report of full length merA gene from L. sphaericus. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. 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

  17. Inhibition of nitrate transport by anti-nitrate reductase IgG fragments and the identification of plasma membrane associated nitrate reductase in roots of barley seedlings

    Science.gov (United States)

    Ward, M. R.; Tischner, R.; Huffaker, R. C.

    1988-01-01

    Membrane associated nitrate reductase (NR) was detected in plasma membrane (PM) fractions isolated by aqueous two-phase partitioning from barley (Hordeum vulgare L. var CM 72) roots. The PM associated NR was not removed by washing vesicles with 500 millimolar NaCl and 1 millimolar EDTA and represented up to 4% of the total root NR activity. PM associated NR was stimulated up to 20-fold by Triton X-100 whereas soluble NR was only increased 1.7-fold. The latency was a function of the solubilization of NR from the membrane. NR, solubilized from the PM fraction by Triton X-100 was inactivated by antiserum to Chlorella sorokiniana NR. Anti-NR immunoglobulin G fragments purified from the anti-NR serum inhibited NO3- uptake by more than 90% but had no effect on NO2- uptake. The inhibitory effect was only partially reversible; uptake recovered to 50% of the control after thorough rinsing of roots. Preimmune serum immunoglobulin G fragments inhibited NO3- uptake 36% but the effect was completely reversible by rinsing. Intact NR antiserum had no effect on NO3- uptake. The results present the possibility that NO3- uptake and NO3- reduction in the PM of barley roots may be related.

  18. Biliverdin reductase: more than a namesakeThe reductase, its peptide fragments and biliverdin regulate activity of the three classes of protein kinase C.

    Directory of Open Access Journals (Sweden)

    Peter E.M. Gibbs

    2012-03-01

    Full Text Available The expanse of human biliverdin reductase (hBVR functions in the cells is arguably umatched by any single protein. hBVR is a Ser/Thr/Tyr kinase, a scaffold protein, a transcription factor and an intracellular transporter of gene regulators. hBVR is an upstream activator of the insulin/IGF-1 signaling pathway and of PKC kinases in the two major arms of the pathway. In addition, it is the sole means for generating the antioxidant bilirubin-IXα. hBVR is essential for activation of ERK1/2 kinases by upstream MAPKK-MEK and by PKCδ, as well as the nuclear import and export of ERK1/2. Small fragments of hBVR are potent activators and inhibitors of the ERK kinases and PKCs: as such, they suggest the potential application of BVR-based technology in therapeutic settings. Presently, we have reviewed the function of hBVR in cell signaling with an emphasis on regulation of PKCδ activity.

  19. The Effect of Folinic Acid on Methylenetetrahydrofolate Reductase Polymorphisms in Methotrexate-Treated Allogeneic Hematopoietic Stem Cell Transplants

    NARCIS (Netherlands)

    Murphy, Nicholas M.; Diviney, Mary; Szer, Jeff; Bardy, Peter; Grigg, Andrew; Hoyt, Rosemary; King-Kalimanis, Bellinda; Holdsworth, Rhonda; McCluskey, James; Tait, Brian D.

    2012-01-01

    This study examined the contribution single nucleotide polymorphisms (SNPs) of the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene have on clinical outcomes in hematopoietic stem cell transplant patients treated with the antiproliferative drug methotrexate. Two common SNPs, 677C>T and 1298A>C,

  20. The methylenetetrahydrofolate reductase C677T genotype and the risk of obesity in three large population-based cohorts

    DEFF Research Database (Denmark)

    Lewis, S.J.; Lawlor, D.A.; Nordestgaard, B.G.

    2008-01-01

    of this relationship. The methylenetetrahydrofolate reductase (MTHFR) C677T TT genotype is associated with reduced folate availability and may be a surrogate for measuring folate levels. We sought to determine whether MTHFR C677T genotype was associated with obesity. Design: We carried out our study on four...

  1. No evidence for a preferential transmission of the methylenetetrahydrofolate reductase 677T allele in families with schizophrenia offspring

    NARCIS (Netherlands)

    Muntjewerff, J.W.; Hoogendoorn, M.L.; Aukes, M.F.; Kahn, R.S.; Sinke, R.J.; Blom, H.J.; Heijer, M. den

    2007-01-01

    The methylenetetrahydrofolate reductase (MTHFR) 677C > T polymorphism has been associated with an increased risk of schizophrenia in various case-control studies. However, case-control studies are sensitive to population stratification, which is not an issue in family-based studies. We conducted a

  2. The FRO2 ferric reductase is required for glycine betaine's effect on chilling tolerance in Arabidopsis roots.

    Science.gov (United States)

    Einset, John; Winge, Per; Bones, Atle M; Connolly, Erin L

    2008-10-01

    FRO2 (At1g01580) codes for an NADPH-dependent ferric reductase in plasma membranes of root epidermal cells with a demonstrated role in iron uptake by plants. Ferric reductase activity has been shown to be the rate-limiting step for iron uptake in strategy I plants like Arabidopsis and in rice, but it has been unclear whether FRO genes have other physiological functions. We hypothesized that FRO2 was involved in chilling stress tolerance because its expression was upregulated by treatment of plants with glycine betaine (GB), a chemical that prevents reactive oxygen species (ROS) signaling in chilling stress. This idea was confirmed by showing that the FRO2 null mutant frd1-1 failed to respond to GB in chilling assays either in relation to root growth recovery or inhibition of ROS accumulation. Measurements of ferric reductase activity in wild-type plants treated with GB before chilling showed no significant GB effect compared with controls. In addition, 35S-FRO2 transgenics with elevated mRNA levels did not have improved chilling tolerance. However, ferric reductase activity in wild-type plants or 35S-FRO2 transgenics pretreated with GB was several-fold higher after chilling compared with non-pretreated controls. These experiments identify a new physiological function for FRO2, i.e. blocking ROS accumulation during chilling. They also suggest that GB has a major effect on FRO2 activity posttranscriptionally in the cold.

  3. Prognostic significance of numeric aberrations of genes for thymidylate synthase, thymidine phosphorylase and dihydrofolate reductase in colorectal cancer

    DEFF Research Database (Denmark)

    Jensen, Søren Astrup; Vainer, B.; Witton, C.J.

    2008-01-01

    BACKGROUND: Most human cancer cells have structural aberrations of chromosomal regions leading to loss or gain of gene specific alleles. This study aimed to assess the range of gene copies per nucleus of thymidylate synthase (TYMS), thymidine phosphorylase (TP) and dihydrofolate reductase (DHFR...

  4. Identification of acetyl phosphate as the product of clostridial glycine reductase: Evidence for an acyl enzyme intermediate.

    Science.gov (United States)

    Arkowitz, R A; Abeles, R H

    1989-05-30

    It has been reported [Tanaka, H., & Stadtman, T. C. (1979) J. Biol. Chem. 254, 447-452] that glycine reductase from Clostridium sticklandii catalyzes the reaction glycine + ADP + P(i) + 2(e)- - acetate + ATP + NH(4)+. Glycine reductase consists of three proteins, designated A, B, and C. Only A has been purified to homogeneity. A dithiol serves as an electron donor. We find that ADP is not essential for the reaction and that in its absence acetyl phosphate is formed. Upon further purification of components B and C, an acetate kinase activity can be separated from both proteins. This observation establishes that acetate kinase activity is not an intrinsic property of glycine reductase, and therefore the reaction catalyzed by glycine reductase is glycine + P(i) + 2(e)- - acetyl phosphate + NH(4)+. Experiments with [(14)C]glycine and unlabeled acetate show that free acetate is not a precursor of acetyl phosphate. When glycine labeled with l8(O) is converted to product, l8(O) is lost. The l 8 (O) content of unreacted glycine remains unchanged after approximately 50% is converted to product. We propose that an acyl enzyme, most probably an acetyl enzyme,is an intermediate in the reaction and that the acetyl enzyme reacts with P(i) to form acetyl phosphate. A mechanism is proposed for the formation of the acetyl enzyme.

  5. HMG-coenzyme A reductase inhibition, type 2 diabetes, and bodyweight : evidence from genetic analysis and randomised trials

    NARCIS (Netherlands)

    Swerdlow, Daniel I; Preiss, David; Kuchenbaecker, Karoline B; Holmes, Michael V; Engmann, Jorgen E L; Shah, Tina; Sofat, Reecha; Stender, Stefan; Johnson, Paul C D; Scott, Robert A; Leusink, Maarten|info:eu-repo/dai/nl/357581164; Verweij, Niek; Sharp, Stephen J; Guo, Yiran; Giambartolomei, Claudia; Chung, Christina; Peasey, Anne; Amuzu, Antoinette; Li, KaWah; Palmen, Jutta; Howard, Philip; Cooper, Jackie A; Drenos, Fotios; Li, Yun R; Lowe, Gordon; Gallacher, John; Stewart, Marlene C W; Tzoulaki, Ioanna; Buxbaum, Sarah G; van der A, Daphne L; Forouhi, Nita G; Onland-Moret, N Charlotte; van der Schouw, Yvonne T; Schnabel, Renate B; Hubacek, Jaroslav A; Kubinova, Ruzena; Baceviciene, Migle; Tamosiunas, Abdonas; Pajak, Andrzej; Topor-Madry, Romanvan; Stepaniak, Urszula; Malyutina, Sofia; Baldassarre, Damiano; Sennblad, Bengt; Tremoli, Elena; de Faire, Ulf; Veglia, Fabrizio; Ford, Ian; Jukema, J Wouter; Westendorp, Rudi G J; de Borst, Gert Jan; de Jong, Pim A; Algra, Ale; Spiering, Wilko; der Zee, Anke H Maitland-van|info:eu-repo/dai/nl/255164688; Klungel, Olaf H|info:eu-repo/dai/nl/181447649; de Boer, Anthonius|info:eu-repo/dai/nl/075097346; Doevendans, Pieter A; Eaton, Charles B; Robinson, Jennifer G; Duggan, David; Kjekshus, John; Downs, John R; Gotto, Antonio M; Keech, Anthony C; Marchioli, Roberto; Tognoni, Gianni; Sever, Peter S; Poulter, Neil R; Waters, David D; Pedersen, Terje R; Amarenco, Pierre; Nakamura, Haruo; McMurray, John J V; Lewsey, James D; Chasman, Daniel I; Ridker, Paul M; Maggioni, Aldo P; Tavazzi, Luigi; Ray, Kausik K; Seshasai, Sreenivasa Rao Kondapally; Manson, JoAnn E; Price, Jackie F; Whincup, Peter H; Morris, Richard W; Lawlor, Debbie A; Smith, George Davey; Ben-Shlomo, Yoav; Schreiner, Pamela J; Fornage, Myriam; Siscovick, David S; Cushman, Mary; Kumari, Meena; Wareham, Nick J; Verschuren, W M Monique; Redline, Susan; Patel, Sanjay R; Whittaker, John C; Hamsten, Anders; Delaney, Joseph A; Dale, Caroline; Gaunt, Tom R; Wong, Andrew; Kuh, Diana; Hardy, Rebecca; Kathiresan, Sekar; Castillo, Berta A; van der Harst, Pim; Brunner, Eric J; Tybjaerg-Hansen, Anne; Marmot, Michael G; Krauss, Ronald M; Tsai, Michael; Coresh, Josef; Hoogeveen, Ronald C; Psaty, Bruce M; Lange, Leslie A; Hakonarson, Hakon; Dudbridge, Frank; Humphries, Steve E; Talmud, Philippa J; Kivimäki, Mika; Timpson, Nicholas J; Langenberg, Claudia; Asselbergs, Folkert W; Voevoda, Mikhail; Bobak, Martin; Pikhart, Hynek; Wilson, James G; Reiner, Alex P; Keating, Brendan J; Hingorani, Aroon D; Sattar, Naveed; DIAGRAM Consortium, MAGIC Consortium, InterAct Consortium

    2014-01-01

    BACKGROUND: Statins increase the risk of new-onset type 2 diabetes mellitus. We aimed to assess whether this increase in risk is a consequence of inhibition of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), the intended drug target. METHODS: We used single nucleotide polymorphisms in the HMGCR

  6. HMG-coenzyme A reductase inhibition, type 2 diabetes, and bodyweight : evidence from genetic analysis and randomised trials

    NARCIS (Netherlands)

    Swerdlow, Daniel I.; Preiss, David; Kuchenbaecker, Karoline B.; Holmes, Michael V.; Engmann, Jorgen E. L.; Shah, Tina; Sofat, Reecha; Stender, Stefan; Johnson, Paul C. D.; Scott, Robert A.; Leusink, Maarten; Verweij, Niek; Sharp, Stephen J.; Guo, Yiran; Giambartolomei, Claudia; Chung, Christina; Peasey, Anne; Amuzu, Antoinette; Li, Kawah; Palmen, Jutta; Howard, Philip; Cooper, Jackie A.; Drenos, Fotios; Li, Yun R.; Lowe, Gordon; Gallacher, John; Stewart, Marlene C. W.; Tzoulaki, Ioanna; Buxbaum, Sarah G.; Daphne, L. van der A.; Forouhi, Nita G.; Onland-Moret, N. Charlotte; van der Schouw, Yvonne T.; Schnabel, Renate B.; Hubacek, Jaroslav A.; Kubinova, Ruzena; Baceviciene, Migle; Tamosiunas, Abdonas; Pajak, Andrzej; Topor-Madry, Roman; Stepaniak, Urszula; Malyutina, Sofi A.; Baldassarre, Damiano; Sennblad, Bengt; Tremoli, Elena; de Faire, Ulf; Veglia, Fabrizio; Ford, Ian; Jukema, J. Wouter; Westendorp, Rudi G. J.; de Borst, Gert Jan; de Jong, Pim A.; Algra, Ale; Spiering, Wilko; Maitland-van der Zee, Anke H.; Klungel, Olaf H.; de Boer, Anthonius; Doevendans, Pieter A.; Eaton, Charles B.; Robinson, Jennifer G.; Duggan, David; Kjekshus, John; Downs, John R.; Gotto, Antonio M.; Keech, Anthony C.; Marchioli, Roberto; Tognoni, Gianni; Sever, Peter S.; Poulter, Neil R.; Waters, David D.; Pedersen, Terje R.; Amarenco, Pierre; Nakamura, Haruo; McMurray, John J. V.; Lewsey, James D.; Chasman, Daniel I.; Ridker, Paul M.; Maggioni, Aldo P.; Tavazzi, Luigi; Ray, Kausik K.; Seshasai, Sreenivasa Rao Kondapally; Manson, Joann E.; Price, Jackie F.; Whincup, Peter H.; Morris, Richard W.; Lawlor, Debbie A.; Smith, George Davey; Ben-Shlomo, Yoav; Schreiner, Pamela J.; Fornage, Myriam; Siscovick, David S.; Cushman, Mary; Kumari, Meena; Wareham, Nick J.; Verschuren, W. M. Monique; Redline, Susan; Patel, Sanjay R.; Whittaker, John C.; Hamsten, Anders; Delaney, Joseph A.; Dale, Caroline; Gaunt, Tom R.; Wong, Andrew; Kuh, Diana; Hardy, Rebecca; Kathiresan, Sekar; Castillo, Berta A.; van der Harst, Pim; Brunner, Eric J.; Tybjaerg-Hansen, Anne; Marmot, Michael G.; Krauss, Ronald M.; Tsai, Michael; Coresh, Josef; Hoogeveen, Ronald C.; Psaty, Bruce M.; Lange, Leslie A.; Hakonarson, Hakon; Dudbridge, Frank; Humphries, Steve E.; Talmud, Philippa J.; Kivimaeki, Mika; Timpson, Nicholas J.; Langenberg, Claudia; Asselbergs, Folkert W.; Voevoda, Mikhail; Bobak, Martin; Pikhart, Hynek; Wilson, James G.; Reiner, Alex P.; Keating, Brendan J.; Hingorani, Aroon D.; Sattar, Naveed; Wijmenga, T. N.

    2015-01-01

    BACKGROUND: Statins increase the risk of new-onset type 2 diabetes mellitus. We aimed to assess whether this increase in risk is a consequence of inhibition of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), the intended drug target. METHODS: We used single nucleotide polymorphisms in the HMGCR

  7. HMG-coenzyme A reductase inhibition, type 2 diabetes, and bodyweight : Evidence from genetic analysis and randomised trials

    NARCIS (Netherlands)

    Swerdlow, Daniel I.; Preiss, David; Kuchenbaecker, Karoline B.; Holmes, Michael V.; Engmann, Jorgen E L; Shah, Tina; Sofat, Reecha; Stender, Stefan; Johnson, Paul C D; Scott, Robert A.; Leusink, Maarten; Verweij, Niek; Sharp, Stephen J.; Guo, Yiran; Giambartolomei, Claudia; Chung, Christina; Peasey, Anne; Amuzu, Antoinette; Li, Kawah; Palmen, Jutta; Howard, Philip; Cooper, Jackie A.; Drenos, Fotios; Li, Yun R.; Lowe, Gordon; Gallacher, John; Stewart, Marlene C W; Tzoulaki, Ioanna; Buxbaum, Sarah G.; Van Der A, Daphne L.; Forouhi, Nita G.; Onland-Moret, N. Charlotte|info:eu-repo/dai/nl/26504362X; Van Der Schouw, Yvonne T.|info:eu-repo/dai/nl/073449253; Schnabel, Renate B.; Hubacek, Jaroslav A.; Kubinova, Ruzena; Baceviciene, Migle; Tamosiunas, Abdonas; Pajak, Andrzej; Topor-Madry, Romanvan; Stepaniak, Urszula; Malyutina, Sofia; Baldassarre, Damiano; Sennblad, Bengt; Tremoli, Elena; De Faire, Ulf; Veglia, Fabrizio; Ford, Ian; Jukema, J. Wouter; Westendorp, Rudi G J; De Borst, Gert Jan|info:eu-repo/dai/nl/237108151; De Jong, Pim A.|info:eu-repo/dai/nl/287955672; Algra, Ale|info:eu-repo/dai/nl/07483472X; Spiering, Wilko|info:eu-repo/dai/nl/269114173; Der Zee, Anke H Maitland Van; Klungel, Olaf H.|info:eu-repo/dai/nl/181447649; De Boer, Anthonius; Doevendans, Pieter A.|info:eu-repo/dai/nl/164248366; Eaton, Charles B.; Robinson, Jennifer G.; Duggan, David; Kjekshus, John; Downs, John R.; Gotto, Antonio M.; Keech, Anthony C.; Marchioli, Roberto; Tognoni, Gianni; Sever, Peter S.; Poulter, Neil R.; Waters, David D.; Pedersen, Terje R.; Amarenco, Pierre; Nakamura, Haruo; McMurray, John J V; Lewsey, James D.; Chasman, Daniel I.; Ridker, Paul M.; Maggioni, Aldo P.; Tavazzi, Luigi; Ray, Kausik K.; Seshasai, Sreenivasa Rao Kondapally; Manson, Joann E.; Price, Jackie F.; Whincup, Peter H.; Morris, Richard W.; Lawlor, Debbie A.; Smith, George Davey; Ben-Shlomo, Yoav; Schreiner, Pamela J.; Fornage, Myriam; Siscovick, David S.; Cushman, Mary; Kumari, Meena; Wareham, Nick J.; Verschuren, W. M Monique|info:eu-repo/dai/nl/071858849; Redline, Susan; Patel, Sanjay R.; Whittaker, John C.; Hamsten, Anders; Delaney, Joseph A.; Dale, Caroline; Gaunt, Tom R.; Wong, Andrew; Kuh, Diana; Hardy, Rebecca; Kathiresan, Sekar; Castillo, Berta A.; Van Der Harst, Pim; Brunner, Eric J.; Tybjaerg-Hansen, Anne; Marmot, Michael G.; Krauss, Ronald M.; Tsai, Michael; Coresh, Josef; Hoogeveen, Ronald C.; Psaty, Bruce M.; Lange, Leslie A.; Hakonarson, Hakon; Dudbridge, Frank; Humphries, Steve E.; Talmud, Philippa J.; Kivimäki, Mika; Timpson, Nicholas J.; Langenberg, Claudia; Asselbergs, Folkert W.|info:eu-repo/dai/nl/270752137; Voevoda, Mikhail; Bobak, Martin; Pikhart, Hynek; Wilson, James G.; Reiner, Alex P.; Keating, Brendan J.; Hingorani, Aroon D.; Sattar, Naveed

    2015-01-01

    Background Statins increase the risk of new-onset type 2 diabetes mellitus. We aimed to assess whether this increase in risk is a consequence of inhibition of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), the intended drug target. Methods We used single nucleotide polymorphisms in the HMGCR

  8. A random-sequential mechanism for nitrite binding and active site reduction in copper-containing nitrite reductase

    NARCIS (Netherlands)

    Wijma, HJ; Jeuken, LJC; Verbeet, MP; Armstrong, FA; Canters, GW

    2006-01-01

    The homotrimeric copper-containing nitrite reductase ( NiR) contains one type-1 and one type-2 copper center per monomer. Electrons enter through the type-1 site and are shuttled to the type-2 site where nitrite is reduced to nitric oxide. To investigate the catalytic mechanism of NiR the effects of

  9. The synergistic interaction of gemcitabine and cytosine arabinoside with the ribonucleotide reductase inhibitor triapine is schedule dependent

    NARCIS (Netherlands)

    Sigmond, J.; Kamphuis, J. A. E.; Laan, A. C.; Hoebe, E. K.; Bergman, A. M.; Peters, G. J.

    2007-01-01

    Gemcitabine and ara-C have multiple mechanisms of action: DNA incorporation and for gemcitabine also ribonucleotide reductase (RNR) inhibition. Since dCTP competes with their incorporation into DNA, dCTP depletion can potentiate their cytotoxicity. We investigated whether additional RNR inhibition

  10. Met144Ala mutation of the copper-containing nitrite reductase from Alcaligenes xylosoxidans reverses the intramolecular electron transfer

    DEFF Research Database (Denmark)

    Farver, Ole; Eady, Robert R; Sawers, Gary

    2004-01-01

    Pulse radiolysis has been employed to investigate the intramolecular electron transfer (ET) between the type 1 (T1) and type 2 (T2) copper sites in the Met144Ala Alcaligenes xylosoxidans nitrite reductase (AxCuNiR) mutant. This mutation increases the reduction potential of the T1 copper center...

  11. Biliverdin amides reveal roles for propionate side chains in bilin reductase recognition and in holophytochrome assembly and photoconversion.

    Science.gov (United States)

    Shang, Lixia; Rockwell, Nathan C; Martin, Shelley S; Lagarias, J Clark

    2010-07-27

    Linear tetrapyrroles (bilins) perform important antioxidant and light-harvesting functions in cells from bacteria to humans. To explore the role of the propionate moieties in bilin metabolism, we report the semisynthesis of mono- and diamides of biliverdin IXalpha and those of its non-natural XIIIalpha isomer. Initially, these were examined as substrates of two types of NADPH-dependent biliverdin reductase, BVR and BvdR, and of the representative ferredoxin-dependent bilin reductase, phycocyanobilin:ferredoxin oxidoreductase (PcyA). Our studies indicate that the NADPH-dependent biliverdin reductases are less accommodating to amidation of the propionic acid side chains of biliverdin IXalpha than PcyA, which does not require free carboxylic acid side chains to yield its phytobilin product, phycocyanobilin. Bilin amides were also assembled with BV-type and phytobilin-type apophytochromes, demonstrating a role for the 8-propionate in the formation of the spectroscopically native P(r) dark states of these biliprotein photosensors. Neither ionizable propionate side chain proved to be essential to primary photoisomerization for both classes of phytochromes, but an unsubstituted 12-propionate was required for full photointerconversion of phytobilin-type phytochrome Cph1. Taken together, these studies provide insight into the roles of the ionizable propionate side chains in substrate discrimination by two bilin reductase families while further underscoring the mechanistic differences between the photoconversions of BV-type and phytobilin-type phytochromes.

  12. Biliverdin amides reveal roles for propionate side chains in bilin reductase recognition and in holophytochrome assembly and photoconversion†

    Science.gov (United States)

    Shang, Lixia; Rockwell, Nathan C.; Martin, Shelley S.; Lagarias, J. Clark

    2010-01-01

    Linear tetrapyrroles (bilins) perform important antioxidant and light harvesting functions in cells from bacteria to humans. To explore the role of the propionate moieties in bilin metabolism, we report the semisynthesis of mono- and di-amides of biliverdin IXα and those of its non-natural XIIIα isomer. Initially, these were examined as substrates of two types of NADPH-dependent biliverdin reductase, BVR and BvdR, and of the representative ferredoxin-dependent bilin reductase, phycocyanobilin:ferredoxin oxidoreductase (PcyA). Our studies indicate that the NADPH-dependent biliverdin reductases are less accommodating to amidation of the propionic acid sidechains of biliverdin IXα than PcyA, which does not require free carboxylic acid sidechains to yield its phytobilin product, phycocyanobilin. Bilin amides were also assembled with BV-type and phytobilin-type apophytochromes, demonstrating a role for the 8-propionate in formation of the spectroscopically native Pr dark states of these biliprotein photosensors. Neither ionizable propionate sidechain proved essential to primary photoisomerization for both classes of phytochromes, but an unsubstituted 12-propionate was required for full photointerconversion of phytobilin-type phytochrome Cph1. Taken together, these studies provide insight into the roles of the ionizable propionate sidechains in substrate discrimination by two bilin reductase families while further underscoring the mechanistic differences between the photoconversions of BV-type and phytobilin-type phytochromes. PMID:20565135

  13. Effect of the methionine ligand on the reorganization energy of the type-1 copper site of nitrite Reductase

    DEFF Research Database (Denmark)

    Farver, Ole; Wijma, Hein J.; MacPherson, Iain

    2007-01-01

    Copper-containing nitrite reductase harbors a type-1 and a type-2 Cu site. The former acts as the electron acceptor site of the enzyme, and the latter is the site of catalytic action. The effect of the methionine ligand on the reorganization energy of the type-1 site was explored by studying...

  14. Inhibition of carnitine palmitoyltransferase leads to induction of 3-hydroxymethylglutaryl coenzyme A reductase activity in rat liver

    NARCIS (Netherlands)

    H. Jansen (Hans); N. Hoogerbrugge van der Linden (N.); W.C. Hülsmann (William)

    1990-01-01

    markdownabstractAbstract The relation between carnitine palmitoyltransferase (CPT) activity and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity was investigated. Rats were treated with aminocarnitine or 1-carnitine overnight. In rats, in which CPT activity was inhibited by

  15. 5 alpha-reductase inhibition by a new synthetic steroid (PM-9) in cultures of Penicillium crustosum.

    Science.gov (United States)

    Cabeza, Marisa; Quiroz, Alexandra; Bratoeff, Eugene; García, Genoveva; Ramírez, Elena; Flores, Eugenio

    2002-01-01

    The conversion of testosterone (T) to 5 alpha-dihydrotestosterone (DHT), plus the presence of 5 alpha-reductase enzyme, which is responsible for this reduction, had been demonstrated in P. crustosum broth. This enzyme is also present in androgen-dependent animal and human tissues such as prostate and seminal vesicles. The increase in the conversion of T to DHT has been implicated in androgen-dependent diseases such as benign prostate hyperplasia and prostate cancer. The use of 5 alpha-reductase inhibitors could mitigate these illnesses by inhibiting the DHT-receptor complex formation. The purpose of this study is to determine the inhibition pattern of 5 alpha-reductase by finasteride and PM-9 in P. crustosum broth. Km and Vmax values were determined in the broth by Lineweaver-Burk plots using different testosterone concentrations. The Km value was 0.22 microM and Vmax 0.833 pmol of DHT/mg of mycelium/day. The inhibition pattern of finasteride and PM-9 was also determined by Lineweaver-Burk plot, using different concentrations of T and inhibitors. The results of this study show that both finasteride and PM-9 inhibit 5 alpha-reductase in a competitive manner.

  16. Peroxo-Type Intermediates in Class I Ribonucleotide Reductase and Related Binuclear Non-Heme Iron Enzymes

    DEFF Research Database (Denmark)

    Kepp, Kasper Planeta; Bell, Caleb B.; Clay, MIchael D.

    2009-01-01

    We have performed a systematic study of chemically possible peroxo-type intermediates occurring in the non-heme di-iron enzyme class la ribonucleotide reductase, using spectroscopically calibrated computational chemistry. Density functional computations of equilibrium structures, Fe-O and O-O str...

  17. Aldo-keto reductase 1B10 and its role in proliferation capacity of drug-resistant cancers

    Directory of Open Access Journals (Sweden)

    Toshiyuki eMatsunaga

    2012-01-01

    Full Text Available The human aldo-keto reductase AKR1B10, originally identified as an aldose reductase-like protein and human small intestine aldose reductase, is a cytosolic NADPH-dependent reductase that metabolizes a variety of endogenous compounds, such as aromatic and aliphatic aldehydes and dicarbonyl compounds, and some drug ketones. The enzyme is highly expressed in solid tumors of several tissues including lung and liver, and as such has received considerable interest as a relevant biomarker for the development of those tumors. In addition, AKR1B10 has been recently reported to be significantly up-regulated in some cancer cell lines (medulloblastoma D341 and colon cancer HT29 acquiring resistance towards chemotherapeutic agents (cyclophosphamide and mitomycin c, suggesting the validity of the enzyme as a chemoresistance marker. Although the detailed information on the AKR1B10-mediated mechanisms leading to the drug resistance process is not well understood so far, the enzyme has been proposed to be involved in functional regulations of cell proliferation and metabolism of drugs and endogenous lipids during the development of chemoresistance. This article reviews the current literature focusing mainly on expression profile and roles of AKR1B10 in the drug resistance of cancer cells. Recent developments of AKR1B10 inhibitors and their usefulness in restoring sensitivity to anticancer drugs are also reviewed.

  18. Effect of pharmaceutical potential endocrine disruptor compounds on protein disulfide isomerase reductase activity using di-eosin-oxidized-glutathione.

    Directory of Open Access Journals (Sweden)

    Danièle Klett

    Full Text Available BACKGROUND: Protein Disulfide Isomerase (PDI in the endoplasmic reticulum of all cells catalyzes the rearrangement of disulfide bridges during folding of membrane and secreted proteins. As PDI is also known to bind various molecules including hormones such as estradiol and thyroxin, we considered the hypothesis that adverse effects of endocrine-disrupter compounds (EDC could be mediated through their interaction with PDI leading to defects in membrane or secreted proteins. METHODOLOGY/PRINCIPAL FINDINGS: Taking advantage of the recent description of the fluorescence self quenched substrate di-eosin-oxidized-glutathione (DiE-GSSG, we determined kinetically the effects of various potential pharmaceutical EDCs on the in-vitro reductase activity of bovine liver PDI by measuring the fluorescence of the reaction product (E-GSH. Our data show that estrogens (ethynylestradiol and bisphenol-A as well as indomethacin exert an inhibition whereas medroxyprogesteroneacetate and nortestosterone exert a potentiation of bovine PDI reductase activity. CONCLUSIONS: The present data indicate that the tested EDCs could not only affect endocrine target cells through nuclear receptors as previously shown, but could also affect these and all other cells by positively or negatively affecting PDI activity. The substrate DiE-GSSG has been demonstrated to be a convenient substrate to measure PDI reductase activity in the presence of various potential EDCs. It will certainly be usefull for the screening of potential effect of all kinds of chemicals on PDI reductase activity.

  19. Changing flux of xylose metabolites by altering expression of xylose reductase and xylitol dehydrogenase in recombinant Saccharomyces cerevisiae

    Science.gov (United States)

    Yong-Su Jin; Thomas W. Jeffries

    2003-01-01

    We changed the fluxes of xylose metabolites in recombinant Saccharomyces cerevisiae by manipulating expression of Pichia stipitis genes(XYL1 and XYL2) coding for xylose reductase (XR) and xylitol dehydrogenase (XDH), respectively. XYL1 copy number was kept constant by integrating it into the chromosome. Copy numbers of XYL2 were varied either by integrating XYL2 into...

  20. Alteration of the alkaloid profile in genetically modified tobacco reveals a role of methylenetetrahydrofolate reductase in nicotine N-demethylation

    Science.gov (United States)

    Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme of the tetrahydrofolate (THF)-mediated one-carbon (C1) metabolic network. This enzyme catalyzes reduction of 5,10-methylene-THF to 5-methyl-THF. The latter donates its methyl group to homocysteine forming Met, which is then used for the syn...

  1. Dihydroflavonol 4-Reductase Genes from Freesia hybrida Play Important and Partially Overlapping Roles in the Biosynthesis of Flavonoids

    National Research Council Canada - National Science Library

    Li, Yueqing; Liu, Xingxue; Cai, Xinquan; Shan, Xiaotong; Gao, Ruifang; Yang, Song; Han, Taotao; Wang, Shucai; Wang, Li; Gao, Xiang

    2017-01-01

    ... were firstly cloned from Freesia hybrida. Phylogenetic analysis showed that they were classified into different branches, and FhDFR1, FhDFR2 and FhDFR3 were clustered into DFR subgroup, whereas others fell into the group with cinnamoyl-CoA reductase (CCR) proteins. Then, the functions of the three FhDFR genes were further characterized. Different...

  2. The role of 5α-reductase type 1 associated with intratumoral dihydrotestosterone concentrations in human endometrial carcinoma.

    Science.gov (United States)

    Tanaka, Sota; Miki, Yasuhiro; Hashimoto, Chiaki; Takagi, Kiyoshi; Doe, Zhulanqiqige; Li, Bin; Yaegashi, Nobuo; Suzuki, Takashi; Ito, Kiyoshi

    2015-02-05

    Endometrial carcinoma, especially endometrioid endometrial adenocarcinoma, is an estrogen-dependent tumor that is similar to breast cancer. Androgen is closely associated with other steroid hormones, but its correlation with endometrioid endometrial adenocarcinoma remains largely unclear. We previously demonstrated the expression of the androgen receptor, 5α-reductase type 1, and 5α-reductase type 2 in endometrioid endometrial adenocarcinoma tissue, but androgen action and its correlation with prognosis are unknown. In this study, we measured the tissue and serum concentrations of androgen and performed immunohistochemical analyses of androgen-associated factors in 41 patients. In 86 additional patients, we performed the same immunohistochemical analyses to identify correlations associated with prognosis. We found that 5α-reductase type 1 was associated with intratumoral dihydrotestosterone concentrations, and it was an independent prognostic factor in endometrioid endometrial adenocarcinoma. The poor prognosis of patients negative for both androgen receptor and 5α-reductase type 1 suggests that androgens have inhibitory effects on tumor growth. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  3. Identification of the bchP gene, encoding geranylgeranyl reductase in Chlorobaculum tepidum

    DEFF Research Database (Denmark)

    Gomez Maqueo Chew, Aline; Frigaard, Niels-Ulrik; Bryant, Donald A

    2008-01-01

    The Chlorobaculum tepidum genome contains two paralogous genes, CT2256 and CT1232, whose products are members of the FixC dehydrogenase superfamily and have sequence similarity to geranylgeranyl reductases. Each gene was insertionally inactivated, and the resulting mutants were characterized. CT2...

  4. /sup 13/C NMR evidence of the slow exchange of tryptophans in dihydrofolate reductase between stable conformations

    Energy Technology Data Exchange (ETDEWEB)

    London, R.E. (Univ. of California, Los Alamos, NM); Groff, J.P.; Blakley, R.L.

    1979-01-01

    /sup 13/C NMR spectra are reported for dihydrofolate reductase of Streptococcus faecium labeled with (..gamma..-/sup 13/C)tryptophan. Two of the four tryptophans generate unusual resonances indicating slow exchange of the residues between alternative stable conformations. Since 3', 5'-dichloromethotrexate sharpens two of the resonances, it apparently locks the corresponding residues into one conformation.

  5. DISRUPTION OF THE SACCHAROMYCES CEREVISIAE GENE FOR NADPH-CYTOCHROME P450-REDUCTASE CAUSES INCREASED SENSITIVITY TO KETOCONAZOLE

    Science.gov (United States)

    Strains of Saccharomyces cerevisiae deleted in the NADPH-cytochrome P450 reductase gene by transplacement are 200-fold more sensitive to ketoconazole, an inhibitor of the cytochrome P450 lanosterol 14-demethylase. Resistance is restored through complementation by the plasmid-born...

  6. Long-term Use of 5α-Reductase Inhibitors and the Risk of Male Breast Cancer.

    NARCIS (Netherlands)

    Duijnhoven, R.G.; Straus, S.M.J.M.; Souverein, P.C.; de Boer, A.; Bosch, J.L.H.R.; Hoes, A.W.; De Bruin, M.L.; Sub Pharmacoepidemiology; Dep Farmaceutische wetenschappen; Sub Pharmacotherapy, Theoretical

    2014-01-01

    Background The 5α-reductase inhibitors (5-ARI) finasteride and dutasteride are indicated for the treatment of lower urinary tract symptoms caused by benign prostatic hyperplasia. Case reports have suggested that 5-ARIs increase the risk for male breast cancer, with no conclusive evidence. The

  7. Long-term use of 5α-reductase inhibitors and the risk of male breast cancer

    DEFF Research Database (Denmark)

    Duijnhoven, Ruben G; Straus, Sabine M J M; Souverein, Patrick C

    2014-01-01

    BACKGROUND: The 5α-reductase inhibitors (5-ARI) finasteride and dutasteride are indicated for the treatment of lower urinary tract symptoms caused by benign prostatic hyperplasia. Case reports have suggested that 5-ARIs increase the risk for male breast cancer, with no conclusive evidence...

  8. Contribution of thermolabile methylenetetrahydrofolate reductase variant to total plasma homocysteine levels in healthy men and women. Inter99 (2)

    DEFF Research Database (Denmark)

    Husemoen, Lise Lotte N; Thomsen, Troels F; Fenger, Mogens

    2003-01-01

    Elevation in plasma total homocysteine (tHcy) is believed to be causally related to cardiovascular disease. Like age and sex, the thermolabile variant of methylenetetrahydrofolate reductase (MTHFR(C677T)) is an important nonmodifiable determinant of tHcy, which may be considered when describing...

  9. Unexpected ethical dilemmas in sex assignment in 46,XY DSD due to 5-alpha reductase type 2 deficiency.

    Science.gov (United States)

    Byers, Heather M; Mohnach, Lauren H; Fechner, Patricia Y; Chen, Ming; Thomas, Inas H; Ramsdell, Linda A; Shnorhavorian, Margarett; McCauley, Elizabeth A; Amies Oelschlager, Anne-Marie E; Park, John M; Sandberg, David E; Adam, Margaret P; Keegan, Catherine E

    2017-06-01

    Sex assignment at birth remains one of the most clinically challenging and controversial topics in 46,XY disorders of sexual development (DSD). This is particularly challenging in deficiency of 5-alpha reductase type 2 given that external genitalia are typically undervirilized at birth but typically virilize at puberty to a variable degree. Historically, most individuals with 5-alpha reductase deficiency were raised females. However, reports that over half of patients who underwent a virilizing puberty adopted an adult male gender identity have challenged this practice. Consensus guidelines on assignment of sex of rearing at birth are equivocal or favor male assignment in the most virilized cases. While a male sex of rearing assignment may avoid lifelong hormonal therapy and/or allow the potential for fertility, female sex assignment may be more consistent with external anatomy in the most severely undervirilized cases. Herein, we describe five patients with 46,XY DSD due 5-alpha-reductase type 2 deficiency, all with a severe phenotype. An inter-disciplinary DSD medical team at one of two academic centers evaluated each patient. This case series illustrates the complicated decision-making process of assignment of sex of rearing at birth in 5-alpha reductase type 2 deficiency and the challenges that arise when the interests of the child, parental wishes, recommendations of the medical team, and state law collide. © 2017 Wiley Periodicals, Inc.

  10. Mutations in the gene for methylenetetrahydrofolate reductase, homocysteine levels, and vitamin status in women with a history of preeclampsia

    NARCIS (Netherlands)

    Lachmeijer, AMA; Arngrimsson, R; Bastiaans, EJ; Pals, G; ten Kate, LP; de Vries, JIP; Kostense, PJ; Aarnoudse, JG; Dekker, GA

    OBJECTIVE: This study was undertaken to assess frequencies of the methylenetetrahydrofolate reductase gene mutations cytosine-to-thymine substitution at base 677 (C677T) and adenine-to-cytosine substitution at base 1298 (A1298C) and their interactions with homocysteine and vitamin levels among Dutch

  11. Comparative molecular modeling study of Arabidopsis NADPH-dependent thioredoxin reductase and its hybrid protein.

    Directory of Open Access Journals (Sweden)

    Yuno Lee

    Full Text Available 2-Cys peroxiredoxins (Prxs play important roles in the protection of chloroplast proteins from oxidative damage. Arabidopsis NADPH-dependent thioredoxin reductase isotype C (AtNTRC was identified as efficient electron donor for chloroplastic 2-Cys Prx-A. There are three isotypes (A, B, and C of thioredoxin reductase (TrxR in Arabidopsis. AtNTRA contains only TrxR domain, but AtNTRC consists of N-terminal TrxR and C-terminal thioredoxin (Trx domains. AtNTRC has various oligomer structures, and Trx domain is important for chaperone activity. Our previous experimental study has reported that the hybrid protein (AtNTRA-(Trx-D, which was a fusion of AtNTRA and Trx domain from AtNTRC, has formed variety of structures and shown strong chaperone activity. But, electron transfer mechanism was not detected at all. To find out the reason of this problem with structural basis, we performed two different molecular dynamics (MD simulations on AtNTRC and AtNTRA-(Trx-D proteins with same cofactors such as NADPH and flavin adenine dinucleotide (FAD for 50 ns. Structural difference has found from superimposition of two structures that were taken relatively close to average structure. The main reason that AtNTRA-(Trx-D cannot transfer the electron from TrxR domain to Trx domain is due to the difference of key catalytic residues in active site. The long distance between TrxR C153 and disulfide bond of Trx C387-C390 has been observed in AtNTRA-(Trx-D because of following reasons: i unstable and unfavorable interaction of the linker region, ii shifted Trx domain, and iii different or weak interface interaction of Trx domains. This study is one of the good examples for understanding the relationship between structure formation and reaction activity in hybrid protein. In addition, this study would be helpful for further study on the mechanism of electron transfer reaction in NADPH-dependent thioredoxin reductase proteins.

  12. Heterotrimeric NADH-oxidizing methylenetetrahydrofolate reductase from the acetogenic bacterium Acetobacterium woodii.

    Science.gov (United States)

    Bertsch, Johannes; Öppinger, Christian; Hess, Verena; Langer, Julian D; Müller, Volker

    2015-05-01

    The methylenetetrahydrofolate reductase (MTHFR) of acetogenic bacteria catalyzes the reduction of methylene-THF, which is highly exergonic with NADH as the reductant. Therefore, the enzyme was suggested to be involved in energy conservation by reducing ferredoxin via electron bifurcation, followed by Na(+) translocation by the Rnf complex. The enzyme was purified from Acetobacterium woodii and shown to have an unprecedented subunit composition containing the three subunits RnfC2, MetF, and MetV. The stable complex contained 2 flavin mononucleotides (FMN), 23.5 ± 1.2 Fe and 24.5 ± 1.5 S, which fits well to the predicted six [4Fe4S] clusters in MetV and RnfC2. The enzyme catalyzed NADH:methylviologen and NADH:ferricyanide oxidoreductase activity but also methylene-tetrahydrofolate (THF) reduction with NADH as the reductant. The NADH:methylene-THF reductase activity was high (248 U/mg) and not stimulated by ferredoxin. Furthermore, reduction of ferredoxin, alone or in the presence of methylene-THF and NADH, was never observed. MetF or MetVF was not able to catalyze the methylene-THF-dependent oxidation of NADH, but MetVF could reduce methylene-THF using methyl viologen as the electron donor. The purified MTHFR complex did not catalyze the reverse reaction, the endergonic oxidation of methyl-THF with NAD(+) as the acceptor, and this reaction could not be driven by reduced ferredoxin. However, addition of protein fractions made the oxidation of methyl-THF to methylene-THF coupled to NAD(+) reduction possible. Our data demonstrate that the MTHFR of A. woodii catalyzes methylene-THF reduction according to the following reaction: NADH + methylene-THF → methyl-THF + NAD(+). The differences in the subunit compositions of MTHFRs of bacteria are discussed in the light of their different functions. Energy conservation in the acetogenic bacterium Acetobacterium woodii involves ferredoxin reduction followed by a chemiosmotic mechanism involving Na(+)-translocating ferredoxin

  13. Role of Lysine-54 in determining cofactor specificity and binding in human dihydrofolate reductase

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Shaoming; Tan, Xuehai; Thompson, P.D.; Freisheim, J.H. (Medical College of Ohio, Todedo (USA)); Appleman, J.R.; Blakley, R.L. (St. Jude Children' s Research Hospital, Memphis, TN (USA)); Sheridan, R.P.; Venkataraghavan, R. (Lederle Laboratories, Pearl River, NY (USA))

    1990-09-04

    Lysine-54 of human dihydrofolate reductase (hDHFR) appears to be involved in the interaction with the 2{prime}-phosphate of NADPH and is conserved as a basic residue in other species. Studies have suggested that in Lactobacillus casei dihydrofolate reductase Arg-43, the homologous residue at this position, plays an important role in the binding of NADPH and in the differentiation of K{sub m} values for NADPH and NADH. A Lys-54 to Gln-54 mutant (K54Q) of hDHFR has been constructed by oligodeoxynucleotide-directed mutagenesis in order to study the role of Lys-54 in differentiating K{sub m} and k{sub cat} values for NADPH and NADH as well as in other functions of hDHFR. The purpose of this paper is to delineate in quantitative terms the magnitude of the effect of the Lys-54 to Gln-54 replacement on the various kinetic parameters of hDHFR. Such quantitative effects cannot be predicted solely on the basis of X-ray structures. The ratio of K{sub m}(NADH)/K{sub m}(NADPH) decreases from 69 in the wild-type enzyme to 4.7 in the K54Q enzyme, suggesting that Lys-54, among other interactions between protein side-chain residues and the 2{prime}-phosphate, makes a major contribution in terms of binding energy and differentiation of K{sub m} values for NADPH and NADH. Agents at concentrations that show activating effects on the wild-type enzyme such as potassium chloride and urea all inactivate the K54Q enzyme. There appear to be no gross conformational differences between wild-type and K54Q enzyme molecules as judged by competitive ELISA using peptide-specific antibodies against human dihydrofolate reductase and from protease susceptibility studies on both wild-type and K54Q mutant enzymes. The pH-rate profiles using NADPH for K54Q and wild-type enzymes show divergences at certain pH values, suggesting the possibility of alteration(s) in the steps of the catalytic pathway for the K54Q enzyme.

  14. Recent advances in the chemistry and pharmacological activity of new steroidal antiandrogens and 5 alpha-reductase inhibitors.

    Science.gov (United States)

    Bratoeff, Eugene; Cabeza, Marisa; Ramirez, Elena; Heuze, Yvonne; Flores, Eugenio

    2005-01-01

    The object of this paper is to summarize for the past two years the most recent development in the field of prostate cancer and 5 alpha-reductase inhibitors. In addition we are also including some results on the synthesis and pharmacological evaluation of new steroidal compounds developed in our laboratory. Most of the new steroidal derivatives are based on the progesterone skeleton and showed a high inhibitory activity for the enzyme 5 alpha-reductase. Presently, similar compounds are used for the treatment of androgen dependent diseases such as: hirsutism, androgenic alopecia, benign prostatic hyperplasia and prostate cancer. Dihydrotestosterone 2 (Fig. 1) a 5 alpha-reduced metabolite of testosterone 1 has been implicated as a causative factor for the progression of these diseases, largely through the clinical evaluation of males who are genetically deficient of the enzyme steroid 5 alpha-reductase. As a result of this study, the inhibition of this enzyme has become a pharmacological strategy for the design and synthesis of new antiandrogenic drugs. The advent of finasteride 7 (Fig. 3) a 5 alpha-reductase inhibitor has greatly alleviated the symptoms associated with benign prostatic hyperplasia. In our laboratory, we recently synthesized several new 16 beta-methylpregnadiene-3,20-diones: 40, 41 (Fig. 8), 16 beta-phenylpregnadiene-3,17a-dione derivatives 46 and 47 (Fig. 9) and 49 (C-4 bromoderivative) (Fig. 11), 52-56 (Fig. 13). The analogue pregnatriene derivatives were also prepared: 44, 45 (Fig. 9) 50, 51 (Fig. 11) and 57-60 (Fig. 13) These compounds were evaluated as 5 alpha-reductase inhibitors in the following biological models: Penicillium crustosum broths, the flank organs of gonadectomized male hamsters, the incorporation of radiolabeled sodium acetate into lipids, the effect of the new steroids on the reduction of the weight of the seminal vesicles and on the in vitro metabolism of [3H]T to [3H]DHT in seminal vesicles of homogenates of gonadectomized

  15. The impact of low molecular weight heparin on obstetric outcomes among unexplained recurrent miscarriages complicated with methylenetetrahydrofolate reductase gene polymorphism.

    Science.gov (United States)

    Cetin, Orkun; Karaman, Erbil; Cim, Numan; Dirik, Deniz; Sahin, Hanim Guler; Kara, Erdal; Esen, Ramazan

    2017-01-01

    The association between methylenetetrahydrofolate reductase gene polymorphisms and unexplained recurrent miscarriage is elusive. The recommendations for improving pregnancy outcomes in these patients keep changing based on the available evidence. The aim of this study is to analyze the impact of low molecular weight heparin on obstetric outcomes of recurrent miscarriage patients complicated with methylenetetrahydrofolate reductase gene polymorphism. We reviewed medical records of 121 patients with a history of recurrent miscarriage complicated by methylenetetrahydrofolate reductase gene polymorphisms, retrospectively. From among them, 68 patients were treated only with folic acid and iron. The remaining 53 patients were treated with folic acid, iron and prophylactic doses of low molecular weight heparin. The subsequent pregnancy outcomes of these patients were noted. The live birth rate was higher in patients with anticoagulant therapy than in patients without anticoagulant therapy (48.5% vs. 69.8%, respectively, p: 0.015) and the congenital anomaly rate was lower in anticoagulant therapy group (17.6% vs. 3.8%, respectively, p: 0.022). The other obstetric outcomes were found to be similar between the two groups. The current study demonstrated that low molecular weight heparin improved the live birth rates among unex-plained recurrent miscarriage patients complicated with methylenetetrahydrofolate reductase gene polymorphisms. How-ever, the routine use of low molecular weight heparin did not improve the late pregnancy complications in these selected patients in the eastern region of our country. Further studies are needed to discriminate the effect of anticoagulation on the live birth rate of each of methylenetetrahydrofolate reductase gene polymorphism type.

  16. Evaluation of constitutive iron reductase (AtFRO2 expression on mineral accumulation and distribution in soybean (Glycine max. L

    Directory of Open Access Journals (Sweden)

    Marta Wilton Vasconcelos

    2014-04-01

    Full Text Available Iron is an important micronutrient in human and plant nutrition. Adequate iron nutrition during crop production is central for assuring appropriate iron concentrations in the harvestable organs, for human food or animal feed. The whole-plant movement of iron involves several processes, including the reduction of ferric to ferrous iron at several locations throughout the plant, prior to transmembrane trafficking of ferrous iron. In this study, soybean plants that constitutively expressed the AtFRO2 iron reductase gene were analyzed for leaf iron reductase activity, as well as the effect of this transgene's expression on root, leaf, pod wall, and seed mineral concentrations. High Fe supply, in combination with the constitutive expression of AtFRO2, resulted in significantly higher concentrations of different minerals in roots (K, P, Zn, Ca, Ni, Mg and Mo, pod walls (Fe, K, P, Cu and Ni, leaves (Fe, P, Cu, Ca, Ni and Mg and seeds (Fe, Zn, Cu and Ni. Leaf and pod wall iron concentrations increased as much as 500% in transgenic plants, while seed iron concentrations only increased by 10%, suggesting that factors other than leaf and pod wall reductase activity were limiting the translocation of iron to seeds. Protoplasts isolated from transgenic leaves had three-fold higher reductase activity than controls. Expression levels of the iron storage protein, ferritin, were higher in the transgenic leaves than in wild-type, suggesting that the excess iron may be stored as ferritin in the leaves and therefore unavailable for phloem loading and delivery to the seeds. Also, citrate and malate levels in the roots and leaves of transgenic plants were significantly higher than in wild-type, suggesting that organic acid production could be related to the increased accumulation of minerals in roots, leaves and pod walls, but not in the seeds. All together, these results suggest a more ubiquitous role for the iron reductase in whole-plant mineral accumulation and

  17. Insights into Enzyme Catalysis and Thyroid Hormone Regulation of Cerebral Ketimine Reductase/μ-Crystallin Under Physiological Conditions.

    Science.gov (United States)

    Hallen, André; Cooper, Arthur J L; Jamie, Joanne F; Karuso, Peter

    2015-06-01

    Mammalian ketimine reductase is identical to μ-crystallin (CRYM)-a protein that is also an important thyroid hormone binding protein. This dual functionality implies a role for thyroid hormones in ketimine reductase regulation and also a reciprocal role for enzyme catalysis in thyroid hormone bioavailability. In this research we demonstrate potent sub-nanomolar inhibition of enzyme catalysis at neutral pH by the thyroid hormones L-thyroxine and 3,5,3'-triiodothyronine, whereas other thyroid hormone analogues were shown to be far weaker inhibitors. We also investigated (a) enzyme inhibition by the substrate analogues pyrrole-2-carboxylate, 4,5-dibromopyrrole-2-carboxylate and picolinate, and (b) enzyme catalysis at neutral pH of the cyclic ketimines S-(2-aminoethyl)-L-cysteine ketimine (owing to the complex nomenclature trivial names are used for the sulfur-containing cyclic ketimines as per the original authors' descriptions) (AECK), Δ(1)-piperideine-2-carboxylate (P2C), Δ(1)-pyrroline-2-carboxylate (Pyr2C) and Δ(2)-thiazoline-2-carboxylate. Kinetic data obtained at neutral pH suggests that ketimine reductase/CRYM plays a major role as a P2C/Pyr2C reductase and that AECK is not a major substrate at this pH. Thus, ketimine reductase is a key enzyme in the pipecolate pathway, which is the main lysine degradation pathway in the brain. In silico docking of various ligands into the active site of the X-ray structure of the enzyme suggests an unusual catalytic mechanism involving an arginine residue as a proton donor. Given the critical importance of thyroid hormones in brain function this research further expands on our knowledge of the connection between amino acid metabolism and regulation of thyroid hormone levels.

  18. Aromatic Halogenation by Using Bifunctional Flavin Reductase-Halogenase Fusion Enzymes.

    Science.gov (United States)

    Andorfer, Mary C; Belsare, Ketaki D; Girlich, Anna M; Lewis, Jared C

    2017-09-06

    The remarkable site selectivity and broad substrate scope of flavin-dependent halogenases (FDHs) has led to much interest in their potential as biocatalysts. Multiple engineering efforts have demonstrated that FDHs can be tuned for non-native substrate scope and site selectivity. FDHs have also proven useful as in vivo biocatalysts and have been successfully incorporated into biosynthetic pathways to build new chlorinated aromatic compounds in several heterologous organisms. In both cases, reduced flavin cofactor, usually supplied by a separate flavin reductase (FR), is required. Herein, we report functional synthetic, fused FDH-FR proteins containing various FDHs and FRs joined by different linkers. We show that FDH-FR fusion proteins can increase product titers compared to the individual components for in vivo biocatalysis in Escherichia coli. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Evidence for a plasma-membrane-bound nitrate reductase involved in nitrate uptake of Chlorella sorokiniana

    Science.gov (United States)

    Tischner, R.; Ward, M. R.; Huffaker, R. C.

    1989-01-01

    Anti-nitrate-reductase (NR) immunoglobulin-G (IgG) fragments inhibited nitrate uptake into Chlorella cells but had no affect on nitrate uptake. Intact anti-NR serum and preimmune IgG fragments had no affect on nitrate uptake. Membrane-associated NR was detected in plasma-membrane (PM) fractions isolated by aqueous two-phase partitioning. The PM-associated NR was not removed by sonicating PM vesicles in 500 mM NaCl and 1 mM ethylenediaminetetraacetic acid and represented up to 0.8% of the total Chlorella NR activity. The PM NR was solubilized by Triton X-100 and inactivated by Chlorella NR antiserum. Plasma-membrane NR was present in ammonium-grown Chlorella cells that completely lacked soluble NR activity. The subunit sizes of the PM and soluble NRs were 60 and 95 kDa, respectively, as determined by sodium-dodecyl-sulfate electrophoresis and western blotting.

  20. Molecular characterization of five patients with homocystinuria due to severe methylenetetrahydrofolate reductase deficiency

    DEFF Research Database (Denmark)

    Urreizti, R; Moya-García, A A; Pino-Ángeles, A

    2010-01-01

    with homocystinuria due to severe MTHFR deficiency. Methylenetetrahydrofolate reductase (MTHFR) plays a major role in folate metabolism. Disturbed function of the enzyme results in hyperhomocysteinemia and causes severe vascular and neurological disorders and developmental delay. Five patients suspected of having non......-classical homocystinuria due to MTHFR deficiency were examined with respect to their symptoms, MTHFR enzyme activity and genotypes of the MTHFR gene. All patients presented symptoms of severe central nervous system disease. Two patients died, at the ages of 15 months and 14 years. One patient is currently 32 years old......, and is being treated with betaine and folinic acid. The other two patients, with an early diagnosis and a severe course of the disease, are currently improving under treatment. MTHFR enzyme activity in the fibroblasts of four of the patients was practically undetectable. We found four novel mutations, three...

  1. Chemoenzymatic synthesis of (S)-duloxetine using carbonyl reductase from Rhodosporidium toruloides.

    Science.gov (United States)

    Chen, Xiang; Liu, Zhi-Qiang; Lin, Chao-Ping; Zheng, Yu-Guo

    2016-04-01

    A chemoenzymatic strategy was developed for (S)-duloxetine production employing carbonyl reductases from newly isolated Rhodosporidium toruloides into the enantiodetermining step. Amongst the ten most permissive enzymes identified, cloned, and overexpressed in Escherichia coli, RtSCR9 exhibited excellent activity and enantioselectivity. Using co-expressed E. coli harboring both RtSCR9 and glucose dehydrogenase, (S)-3-(dimethylamino)-1-(2-thienyl)-1-propanol 3a was fabricated with so far the highest substrate loading (1000mM) in a space-time yield per gram of biomass (DCW) of 22.9mmolL(-1)h(-1)gDCW(-1) at a 200-g scale. The subsequent synthetic steps from RtSCR9-catalyzed (S)-3a were further performed, affording (S)-duloxetine with 60.2% overall yield from 2-acethylthiophene in >98.5% ee. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. 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.

  3. The role of thioredoxin reductase 1 in melanoma metabolism and metastasis.

    Science.gov (United States)

    Cassidy, Pamela B; Honeggar, Matthew; Poerschke, Robyn L; White, Karen; Florell, Scott R; Andtbacka, Robert H I; Tross, Joycelyn; Anderson, Madeleine; Leachman, Sancy A; Moos, Philip J

    2015-11-01

    Although significant progress has been made in targeted and immunologic therapeutics for melanoma, many tumors fail to respond, and most eventually progress when treated with the most efficacious targeted combination therapies thus far identified. Therefore, alternative approaches that exploit distinct melanoma phenotypes are necessary to develop new approaches for therapeutic intervention. Tissue microarrays containing human nevi and melanomas were used to evaluate levels of the antioxidant protein thioredoxin reductase 1 (TR1), which was found to increase as a function of disease progression. Melanoma cell lines revealed metabolic differences that correlated with TR1 levels. We used this new insight to design a model treatment strategy that creates a synthetic lethal interaction wherein targeting TR1 sensitizes melanoma to inhibition of glycolytic metabolism, resulting in a decrease in metastases in vivo. This approach holds the promise of a new clinical therapeutic strategy, distinct from oncoprotein inhibition. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  4. Cloning and heterologous expression of Plasmodium ovale dihydrofolate reductase-thymidylate synthase gene.

    Science.gov (United States)

    Tirakarn, Srisuda; Riangrungroj, Pinpunya; Kongsaeree, Palangpon; Imwong, Mallika; Yuthavong, Yongyuth; Leartsakulpanich, Ubolsree

    2012-06-01

    Plasmodial bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) is a validated antimalarial drug target. In this study, expression of the putative dhfr-ts of Plasmodium ovale rescued the DHFR chemical knockout and a TS null bacterial strain, demonstrating its DHFR and TS catalytic functions. PoDHFR-TS was expressed in Escherichia coli BL21 (DE3) and affinity purified by Methotrexate Sepharose column. Biochemical and enzyme kinetics characterizations indicated that PoDHFR-TS is similar to other plasmodial enzymes, albeit with lower catalytic activity but better tolerance of acidic pH. Importantly, the PoDHFR from Thai isolate EU266602 remains sensitive to the antimalarials pyrimethamine and cycloguanil, in contrast to P. falciparum and P. vivax isolates where resistance to these drugs is widespread. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  5. The radical mechanism of biological methane synthesis by methyl-coenzyme M reductase

    Energy Technology Data Exchange (ETDEWEB)

    Wongnate, T.; Sliwa, D.; Ginovska, B.; Smith, D.; Wolf, M. W.; Lehnert, N.; Raugei, S.; Ragsdale, S. W.

    2016-05-19

    Methyl-coenzyme M reductase (MCR), the rate-limiting enzyme in methanogenesis and anaerobic methane oxidation, is responsible for the production of over one billion tons of methane per year. The mechanism of methane synthesis is unknown, with the two leading proposals involving either a methyl-nickel(III) (Mechanism I) or methyl radical/Ni(II)-thiolate (Mechanism II) intermediate(s). When the reaction between the active Ni(I) enzyme with substrates was studied by transient kinetic, spectroscopic and computational methods, formation of an EPR-silent Ni(II)-thiolate intermediate was positively identified by magnetic circular dichroism spectroscopy. There was no evidence for an EPR-active methyl-Ni(III) species. Temperature-dependent transient kinetic studies revealed that the activation energy for the initial catalytic step closely matched the value computed by density functional theory for Mechanism II. Thus, our results demonstrate that biological methane synthesis occurs by generation of a methyl radical.

  6. The crystal structure of Candida glabrata dihydrofolate reductase drives new inhibitor design toward efficacious antifungal agents

    Science.gov (United States)

    Liu, Jieying; Bolstad, David B.; Smith, Adrienne E.; Priestley, Nigel D.; Wright, Dennis L.; Anderson, Amy C.

    2008-01-01

    SUMMARY Candida glabrata is a lethal fungal pathogen resistant to many antifungal agents and has emerged as a critical target for drug discovery. Over the past several years we have been developing a class of propargyl-linked antifolates as new antimicrobials and hypothesized that these compounds could be effective inhibitors of dihydrofolate reductase (DHFR) from C. glabrata. We initially screened a small collection of these inhibitors and found modest levels of potency. Subsequently, we determined the crystal structure of C. glabrata DHFR bound to a representative inhibitor with data to 1.6 Å resolution. Using this structure, we designed and synthesized second generation inhibitors. These new inhibitors bind the C. glabrata DHFR enzyme with subnanomolar potency, display greater than 2,000-fold levels of selectivity over the human enzyme and inhibit the growth of C. glabrata at levels observed with clinically employed antifungal therapeutics. PMID:18804036

  7. The use of dimethylsulfoxide as a solvent in enzyme inhibition studies: the case of aldose reductase.

    Science.gov (United States)

    Misuri, Livia; Cappiello, Mario; Balestri, Francesco; Moschini, Roberta; Barracco, Vito; Mura, Umberto; Del-Corso, Antonella

    2017-12-01

    Aldose reductase (AR) is an enzyme devoted to cell detoxification and at the same time is strongly involved in the aetiology of secondary diabetic complications and the amplification of inflammatory phenomena. AR is subjected to intense inhibition studies and dimethyl sulfoxide (DMSO) is often present in the assay mixture to keep the inhibitors in solution. DMSO was revealed to act as a weak but well detectable AR differential inhibitor, acting as a competitive inhibitor of the L-idose reduction, as a mixed type of non-competitive inhibitor of HNE reduction and being inactive towards 3-glutathionyl-4-hydroxynonanal transformation. A kinetic model of DMSO action with respect to differently acting inhibitors was analysed. Three AR inhibitors, namely the flavonoids neohesperidin dihydrochalcone, rutin and phloretin, were used to evaluate the effects of DMSO on the inhibition studies on the reduction of L-idose and HNE.

  8. Acceleration of azo dye decolorization by using quinone reductase activity of azoreductase and quinone redox mediator.

    Science.gov (United States)

    Liu, Guangfei; Zhou, Jiti; Wang, Jing; Zhou, Mi; Lu, Hong; Jin, Ruofei

    2009-06-01

    This study demonstrated the effective application of intracellular azoreductase in mediated decolorization of azo dyes. Using the quinone reductase activity of overexpressed azoreductase AZR and quinone redox mediators, the decolorization performance of the recombinant strain Escherichia coli YB was significantly enhanced. In the presence of 0.2 mM lawsone, 75% acid red 27 (1 mM) was decolorized by E. coli YB in only 2 h, which was the highest bacterial decolorization rate ever reported. Compared to lawsone, menadione was a less effective redox mediator. Glucose was found to be the best carbon source for mediated decolorization by E. coli YB. The recombinant strain could complete four rounds of mediated decolorization repeatedly in 12 h. In addition, a 10-min pre-incubation of E. coli JM109 and activated sludge with 2-methylhydroquinone resulted in great improvement of mediated decolorization performance, which may be applied in practical treatment.

  9. Association between methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism and age of onset in schizophrenia

    DEFF Research Database (Denmark)

    Vares, Maria; Saetre, Peter; Deng, Hong

    2010-01-01

    ) significantly affected age at onset of schizophrenia in a dose-dependent manner (P = 0.0015), with lower age of onset with increasing numbers of the mutant T-allele. There was no evidence of rs1801131 (A1298C) affecting age of onset in schizophrenia. Within the Chinese high-risk families carriers of the MTHFR......Different lines of evidence indicate that methylenetetrahydrofolate reductase (MTHFR) functional gene polymorphisms, causative in aberrant folate-homocysteine metabolism, are associated with increased vulnerability to several heritable developmental disorders. Opposing views are expressed...... considering the possible association between MTHFR and susceptibility for schizophrenia. In order to evaluate if age of onset could explain some of this discrepancy we investigated the relationship between two functional MTHFR gene polymorphisms and age at onset in this disorder. Scandinavian patients (n...

  10. Rv2074 is a novel F420 H2 -dependent biliverdin reductase in Mycobacterium tuberculosis.

    Science.gov (United States)

    Ahmed, F Hafna; Mohamed, A Elaaf; Carr, Paul D; Lee, Brendon M; Condic-Jurkic, Karmen; O'Mara, Megan L; Jackson, Colin J

    2016-09-01

    Bilirubin is a potent antioxidant that is produced from the reduction of the heme degradation product biliverdin. In mammalian cells and Cyanobacteria, NADH/NADPH-dependent biliverdin reductases (BVRs) of the Rossmann-fold have been shown to catalyze this reaction. Here, we describe the characterization of Rv2074 from Mycobacterium tuberculosis, which belongs to a structurally and mechanistically distinct family of F420 H2 -dependent BVRs (F-BVRs) that are exclusively found in Actinobacteria. We have solved the crystal structure of Rv2074 bound to its cofactor, F420 , and used this alongside molecular dynamics simulations, site-directed mutagenesis and NMR spectroscopy to elucidate its catalytic mechanism. The production of bilirubin by Rv2074 could exploit the anti-oxidative properties of bilirubin and contribute to the range of immuno-evasive mechanisms that have evolved in M. tuberculosis to allow persistent infection. © 2016 The Protein Society.

  11. Prevalence of methylenetetrahydrofolate reductase C677T polymorphism in eastern Uttar Pradesh

    Science.gov (United States)

    Rai, Vandana; Yadav, Upendra; Kumar, Pradeep

    2012-01-01

    AIM: This study was aimed to evaluate the 5, 10-methylenetetrahydrofolate reductase (MTHFR) C677T mutation in eastern Uttar Pradesh population. MATERIALS AND METHODS: Polymerase chain reaction (PCR) using specific primers followed by amplicon digestion by Hinf I restriction enzyme was used for MTHFR C677T polymorphism analysis. Total 250 subjects were analyzed. RESULTS: The CC genotype was found in 192 subjects, followed by CT in 56 subjects and TT in 2 subject. Genotype frequencies of CC, CT and TT were 0.768, 0.224 and 0.008, respectively. The frequency of C allele was found to be 0.88 and that of T allele was 0.12. CONCLUSION: It is evident from the results of the present study that the percentage of homozygous genotype (CC) is highest in the target population. PMID:22754220

  12. Inhibitors of Human Dihydrofolate Reductase: A Computational Design and Docking Studies Using Glide

    Directory of Open Access Journals (Sweden)

    Lingala Yamini

    2008-01-01

    Full Text Available Dihydrofolate reductase (DHFR plays a vital role in the DNA synthesis by reducing dihydrofolic acid to tetrahydrofolic acid which is an essential component. Synthetic ligands like methotrexate (MTX, aminopterin (AMP and their analogues act as potential anti metabolites by mimicking the coenzyme dihydrofolic acid (DHFA they inhibit the activity of DHFR antagonistically. Several ligands which are similar to MTX analogues and 6, 8 substituted 2 – naphthyls (NAP which can mimic the pteridyl group of DHFA have been computationally designed. These ligands were proposed to hinder the formation N5, N10 methylene tetrahydrofolic acid (THFA coenzyme, which is essential for the DNA synthesis. The docking studies were done using grid, generated with the 0.9 Vander Waals scaling for non polar bonds in the active site of the receptor. These newly designed ligands such as 14 -21 ,23 and 28 have shown good docking scores and predicted activities when compared to already existing ligands MTX and its analogues.

  13. NADPH-dependent D-aldose reductases and xylose fermentation in Fusarium oxysporum

    DEFF Research Database (Denmark)

    Panagiotou, Gianni; Christakopoulos, P.

    2004-01-01

    Two aldose (xylose) reductases (ARI and ARII) from Fusarium oxysporum were purified and characterized. The native ARI was a monomer with M-r 41000, pI 5.2 and showed a 52-fold preference for NADPH over NADH, while ARII was homodimeric with a subunit of M-r 37000, pI 3.6 and a 60-fold preference...... for NADPH over NADH. In this study, the influence of aeration and the response to the addition of electron acceptors on xylose fermentation by F. oxysporum were also studied. The batch cultivation of F. oxysporum on xylose was performed under aerobic, anaerobic and oxygen-limited conditions in stirred tank...

  14. Response to arsenate treatment in Schizosaccharomyces pombe and the role of its arsenate reductase activity.

    Directory of Open Access Journals (Sweden)

    Alejandro Salgado

    Full Text Available Arsenic toxicity has been studied for a long time due to its effects in humans. Although epidemiological studies have demonstrated multiple effects in human physiology, there are many open questions about the cellular targets and the mechanisms of response to arsenic. Using the fission yeast Schizosaccharomyces pombe as model system, we have been able to demonstrate a strong activation of the MAPK Spc1/Sty1 in response to arsenate. This activation is dependent on Wis1 activation and Pyp2 phosphatase inactivation. Using arsenic speciation analysis we have also demonstrated the previously unknown capacity of S. pombe cells to reduce As (V to As (III. Genetic analysis of several fission yeast mutants point towards the cell cycle phosphatase Cdc25 as a possible candidate to carry out this arsenate reductase activity. We propose that arsenate reduction and intracellular accumulation of arsenite are the key mechanisms of arsenate tolerance in fission yeast.

  15. A protective role of methionine-R-sulfoxide reductase against cadmium in Schizosaccharomyces pombe.

    Science.gov (United States)

    Lim, Chang-Jin; Jo, Hannah; Kim, Kyunghoon

    2014-11-01

    The Schizosaccharomyces pombe cells harboring the methionine- R-sulfoxide reductase (MsrB)-overexpressing recombinant plasmid pFMetSO exhibited better growth than vector control cells, when shifted into fresh medium containing cadmium chloride (abbreviated as Cd). Although both groups of cells contained enhanced reactive oxygen species (ROS) and nitric oxide (NO) levels in the presence of Cd, ROS and NO levels were significantly lower in the S. pombe cells harboring pFMetSO than in vector control cells. Conversely, the S. pombe cells harboring pFMetSO possessed higher total glutathione (GSH) levels and a greater reduced/oxidized GSH ratio than vector control cells under the same conditions.

  16. Enhanced succinic acid production in Aspergillus saccharolyticus by heterologous expression of fumarate reductase from Trypanosoma brucei

    DEFF Research Database (Denmark)

    Yang, Lei; Lübeck, Mette; Ahring, Birgitte K.

    2015-01-01

    production medium as well as the complete medium, but the measured enzyme activities were different depending on the media. Furthermore, a soluble NADH-dependent fumarate reductase gene (frd) from Trypanosoma brucei was inserted and expressed in A. saccharolyticus. The expression of the frd gene led......Aspergillus saccharolyticus exhibits great potential as a cell factory for industrial production of dicarboxylic acids. In the analysis of the organic acid profile, A. saccharolyticus was cultivated in an acid production medium using two different pH conditions. The specific activities...... on the pattern and the amount of organic acids produced by A. saccharolyticus. The wild-type strain produced higher amount of malic acid and succinic acid in the pH buffered condition (pH 6.5) compared with the pH non-buffered condition. The enzyme assays showed that the rTCA branch was active in the acid...

  17. Association between methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism and age of onset in schizophrenia

    DEFF Research Database (Denmark)

    Vares, Maria; Saetre, Peter; Deng, Hong

    2010-01-01

    = 820) diagnosed with schizophrenia, schizoaffective disorder, and schizophreniform disorder were investigated. Two functional MTHFR single nucleotide polymorphisms (SNPs; rs1801131 and rs1801133) were genotyped and the effect of MTHFR polymorphisms on the age of onset was examined with survival......Different lines of evidence indicate that methylenetetrahydrofolate reductase (MTHFR) functional gene polymorphisms, causative in aberrant folate-homocysteine metabolism, are associated with increased vulnerability to several heritable developmental disorders. Opposing views are expressed...... considering the possible association between MTHFR and susceptibility for schizophrenia. In order to evaluate if age of onset could explain some of this discrepancy we investigated the relationship between two functional MTHFR gene polymorphisms and age at onset in this disorder. Scandinavian patients (n...

  18. Effect of Punicalagin and Resveratrol on Methionine Sulfoxide Reductase: A Possible Protective Contribution against Alzheimer's Disease.

    Science.gov (United States)

    Clementi, M E; Sampaolese, B; Lazzarino, G; Giardina, B

    2015-01-01

    Methionine sulfoxide reductase A (MsrA) has been postulated to act as a catalytic antioxidant system involved in the protection of oxidative stress-induced cell injury. MsrA has recently turned attention in coupling with the neurodegenerative disorders and in particular with Alzheimer disease. In fact this neurodegenerative disorder depends to a deposit of beta amyloid a peptide with an oxidizable methionine in position 35 which is proved able to modulate the expression to MsrA in neuronal cells. Here, we firstly provided evidence that pretreatment with Resveratrol and Punicalagin (a potent antioxidant extracted from pomegranate), up-regulate the expression and enzymatic activity of MsrA in human neuroblastoma IMR-32 cells with beta amyloid peptides. This effect determines a lowering of oxidative potential of the cells as demonstrated by the ROS measurement and a protective effect on cellular availability. Therefore we hypothesize a possible prevent role for these molecules in Alzheimer and in other neurodegenerative diseases.

  19. Controversial roles of methylenetetrahydrofolate reductase polymorphisms and folate in breast cancer disease.

    Science.gov (United States)

    Bravatà, Valentina

    2015-02-01

    Breast cancer (BC) represents a highly heterogeneous tumour at both the clinical and molecular levels. Single-nucleotide polymorphisms (SNPs) of the folate-metabolising enzyme methylenetetrahydrofolate-reductase (MTHFR) may modify the association between folate intake and BC and influence plasma folate concentration. The role of folate in BC is equivocal, association studies between the common MTHFR SNPs C677T and A1298C and BC risk are controversial. In this study, I have reviewed observed associations between folate intake, as well as its blood levels, and BC. The purpose of this review is to analyse the role of folate and the two SNPs associated with reduced enzyme activity in BC. I explored the most relevant and updated work that emphasises positive and negative associations among these variables. My findings indicate that no definitive conclusions can be drawn from the studies on this topic. However, this manuscript highlights variables that could be useful to explore in further association analyses.

  20. Osmolarity and Glucose Differentially Regulate Aldose Reductase Activity in Cultured Mouse Podocytes

    Directory of Open Access Journals (Sweden)

    Barbara Lewko

    2011-01-01

    Full Text Available Podocyte injury is associated with progression of many renal diseases, including diabetic nephropathy. In this study we examined whether aldose reductase (AR, the enzyme implicated in diabetic complications in different tissues, is modulated by high glucose and osmolarity in podocyte cells. AR mRNA, protein expression, and activity were measured in mouse podocytes cultured in both normal and high glucose and osmolarity for 6 hours to 5 days. Hyperosmolarity acutely stimulated AR expression and activity, with subsequent increase of AR expression but decrease of activity. High glucose also elevated AR protein level; however, this was not accompanied by respective enzyme activation. Furthermore, high glucose appeared to counteract the osmolarity-dependent activation of AR. In conclusion, in podocytes AR is modulated by high glucose and increased osmolarity in a different manner. Posttranslational events may affect AR activity independent of enzyme protein amount. Activation of AR in podocytes may be implicated in diabetic podocytopathy.

  1. The potential behavioral and economic impacts of widespread HMG-CoA reductase inhibitor (statin) use.

    Science.gov (United States)

    Gendle, Mathew H

    2016-12-01

    Dyslipidemia is a common pathology throughout the industrialized world, and HMG-CoA reductase inhibitors (statins) are often administered to treat elevated lipid levels. Substantial concern has been raised regarding the aggressive clinical lowering of cholesterol, particularly in light of a growing body of research linking low circulating lipid levels with negative behavioral outcomes in both human samples and non-human primate models. In 2009, Goldstein and colleagues tentatively speculated that the greed, impulsiveness, and lack of foresight that lead to the worldwide economic collapse in 2007-2008 could have been caused (in part) by depressed population cholesterol levels resulting from the widespread use of statins by workers in the financial services industry. This paper reviews the literature that links low circulating lipid levels with neurobehavioral dysfunction, develops Goldstein and colleagues' initial speculation into a formal hypothesis, and proposes several specific studies that could rigorously empirically evaluate this hypothesis. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  3. Phenolic derivatives from soy flour ethanol extract are potent in vitro quinone reductase (QR) inducing agents.

    Science.gov (United States)

    Bolling, Bradley W; Parkin, Kirk L

    2008-11-26

    The fractionation of soy flour directed by a cellular bioassay for induction of phase 2 detoxification enzymes was used to identify quinone reductase (QR) inducing agents. A phospholipid-depleted, 80% methanol-partitioned isolate from a crude ethanol extract of soy flour was resolved using normal phase medium-pressure liquid chromatography (MPLC). Early eluting fractions were found to be the most potent QR inducing agents among the separated fractions. Fraction 2 was the most potent, doubling QR at QR inducers. Benzofuran-3-carbaldehyde, 4-hydroxybenzaldeyde, 4-ethoxybenzoic acid, 4-ethoxycinnamic acid, benzofuran-2-carboxylic ethyl ester, and ferulic acid ethyl ester (FAEE) were also identified as QR inducing constituents of this fraction. FAEE was the most potent of the identified constituents, doubling QR specific activity at 3.2 muM in the cellular bioassay.

  4. β-carboline derivatives and diphenols from soy sauce are in vitro quinone reductase (QR) inducers.

    Science.gov (United States)

    Li, Ying; Zhao, Mouming; Parkin, Kirk L

    2011-03-23

    A murine hepatoma (Hepa 1c1c7) cellular bioassay was used to guide the isolation of phase II enzyme inducers from fermented soy sauce, using quinone reductase (QR) as a biomarker. A crude ethyl acetate extract, accounting for 8.7% of nonsalt soluble solids of soy sauce, was found to double relative QR specific activity at 25 μg/mL (concentration required to double was defined as a "CD value"). Further silica gel column fractionation yielded 17 fractions, 16 of which exhibited CD values for QR induction of QR activity corresponded to dose-dependent increases in cellular levels of NAD[P]H:quinone oxidoreductase 1 protein by these four QR inducers. To the authors' knowledge, this is the first report on the ability of β-carboline-derived alkaloids to induce phase II enzymes.

  5. Anti-inflammatory and Quinone Reductase Inducing Compounds from Fermented Noni (Morinda citrifolia) Juice Exudates.

    Science.gov (United States)

    Youn, Ui Joung; Park, Eun-Jung; Kondratyuk, Tamara P; Sang-Ngern, Mayuramas; Wall, Marisa M; Wei, Yanzhang; Pezzuto, John M; Chang, Leng Chee

    2016-06-24

    A new fatty acid ester disaccharide, 2-O-(β-d-glucopyranosyl)-1-O-(2E,4Z,7Z)-deca-2,4,7-trienoyl-β-d-glucopyranose (1), a new ascorbic acid derivative, 2-caffeoyl-3-ketohexulofuranosonic acid γ-lactone (2), and a new iridoid glycoside, 10-dimethoxyfermiloside (3), were isolated along with 13 known compounds (4-16) from fermented noni fruit juice (Morinda citrifolia). The structures of the new compounds, together with 4 and 5, were determined by 1D and 2D NMR experiments, as well as comparison with published values. Compounds 2 and 7 showed moderate inhibitory activities in a TNF-α-induced NF-κB assay, and compounds 4 and 6 exhibited considerable quinone reductase-1 (QR1) inducing effects.

  6. Evaluation of Enoyl-Acyl Carrier Protein Reductase Inhibitors as Pseudomonas aeruginosa Quorum-Quenching Reagents

    Directory of Open Access Journals (Sweden)

    Søren Molin

    2010-02-01

    Full Text Available Pseudomonas aeruginosa is an opportunistic pathogen which is responsible for a wide range of infections. Production of virulence factors and biofilm formation by P. aeruginosa are partly regulated by cell-to-cell communication quorum-sensing systems. Identification of quorum-quenching reagents which block the quorum-sensing process can facilitate development of novel treatment strategies for P. aeruginosa infections. We have used molecular dynamics simulation and experimental studies to elucidate the efficiencies of two potential quorum-quenching reagents, triclosan and green tea epigallocatechin gallate (EGCG, which both function as inhibitors of the enoyl-acyl carrier protein (ACP reductase (ENR from the bacterial type II fatty acid synthesis pathway. Our studies suggest that EGCG has a higher binding affinity towards ENR of P. aeruginosa and is an efficient quorum-quenching reagent. EGCG treatment was further shown to be able to attenuate the production of virulence factors and biofilm formation of P. aeruginosa.

  7. Role of methylenetetrahydrofolate reductase gene (MTHFR) 677C>T polymorphism in pediatric cerebrovascular disorders.

    Science.gov (United States)

    Alsayouf, Hamza; Zamel, Khaled M; Heyer, Geoffrey L; Khuhro, A Latif; Kahwash, Samir B; de los Reyes, Emily C

    2011-03-01

    Homozygosity for the methylenetetrahydrofolate reductase (MTHFR) 677C>T mutation (MTHFR TT) has been linked to an increased risk for stroke, coronary artery disease, and migraine headaches. The authors analyzed the potential link between MTHFR 677C>T homozygosity and childhood stroke. A true association might facilitate screening, recurrence risk stratification, and treatment in patients with cerebrovascular disease. They performed a retrospective chart review of children tested for the MTHFR 677C>/T mutation; 533 patients underwent MTHFR testing, and 8% were homozygous for the MTHFR 677C>T mutation. There was no difference in the cohort compared with the prevalence in the general population. This suggests that the MTHFR 677 C>T polymorphism played a minimal role or no role in stroke risk. However, the data suggest that the MTHFR TT genotype may influence migraine susceptibility in children because there was a higher proportion of migraine patients (28.6%) with the MTHFR TT homozygous genotype.

  8. Trypanothione Reductase: A Target for the Development of Anti- Trypanosoma cruzi Drugs.

    Science.gov (United States)

    Vázquez, Karina; Paulino, Margot; Salas, Cristian O; Zarate-Ramos, Juan J; Vera, Brenda; Rivera, Gildardo

    2017-01-01

    Chagas disease or American trypanosomiasis is a major parasitic disease in Latin America with restricted available treatment: nifurtimox and benznidazole. These two drugs are ineffective in the chronic phase of the disease; therefore, there is a need for the development of new, efficient and safe drugs for the treatment of this pathology. With this goal, one of the promising targets is trypanothione reductase (TR), a key enzyme in the metabolism of Trypanosoma cruzi. In this review, we analyse the importance of TR as a drug target, as well as the well-known and new inhibitors reported in the last decade as potential therapeutic agents for Chagas disease. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  9. Membrane composition influences the activity of in vitro refolded human vitamin K epoxide reductase.

    Science.gov (United States)

    Jaenecke, Frank; Friedrich-Epler, Beatrice; Parthier, Christoph; Stubbs, Milton T

    2015-10-27

    Human vitamin K epoxide reductase (hVKOR) is an integral membrane protein responsible for the maintenance of reduced vitamin K pools, a prerequisite for the action of γ-glutamyl carboxylase and hence for hemostasis. Here we describe the recombinant expression of hVKOR as an insoluble fusion protein in Escherichia coli, followed by purification and chemical cleavage under denaturing conditions. In vitro renaturation and reconstitution of purified solubilized hVKOR in phospholipids could be established to yield active protein. Crucially, the renatured enzyme is inhibited by the powerful coumarin anticoagulant warfarin, and we demonstrate that enzyme activity depends on lipid composition. The completely synthetic system for protein production allows a rational investigation of the multiple variables in membrane protein folding and paves the way for the provision of pure, active membrane protein for structural studies.

  10. Evaluation of Enoyl-Acyl Carrier Protein Reductase Inhibitors as Pseudomonas aeruginosa Quorum-Quenching Reagents

    DEFF Research Database (Denmark)

    Yang, Liang; Liu, Yang; Sternberg, Claus

    2010-01-01

    Pseudomonas aeruginosa is an opportunistic pathogen which is responsible for a wide range of infections. Production of virulence factors and biofilm formation by P. aeruginosa are partly regulated by cell-to-cell communication quorum-sensing systems. Identification of quorum-quenching reagents...... which block the quorum-sensing process can facilitate development of novel treatment strategies for P. aeruginosa infections. We have used molecular dynamics simulation and experimental studies to elucidate the efficiencies of two potential quorum-quenching reagents, triclosan and green tea...... epigallocatechin gallate (EGCG), which both function as inhibitors of the enoyl-acyl carrier protein (ACP) reductase (ENR) from the bacterial type II fatty acid synthesis pathway. Our studies suggest that EGCG has a higher binding affinity towards ENR of P. aeruginosa and is an efficient quorum-quenching reagent...

  11. Identification of new non-carboxylic acid containing inhibitors of aldose reductase.

    Science.gov (United States)

    Maccari, Rosanna; Ciurleo, Rosella; Giglio, Marco; Cappiello, Mario; Moschini, Roberta; Corso, Antonella Del; Mura, Umberto; Ottanà, Rosaria

    2010-06-01

    Non-carboxylic acid containing bioisosteres of (5-arylidene-2,4-dioxothiazolidin-3-yl)acetic acids, which are active as aldose reductase (ALR2) inhibitors, were designed by replacing the carboxylic group with the trifluoromethyl ketone moiety. The in vitro evaluation of the ALR2 inhibitory effects of these trifluoromethyl substituted derivatives led to the identification of two inhibitors effective at low micromolar doses. It was further confirmed that a carboxylic chain on N-3 of the thiazolidinedione scaffold is a determining requisite to obtain the highest efficacy levels; however, it is not essential for the interaction with the target enzyme and it can be replaced by different polar groups, thus obtaining less ionised or unionised inhibitors.

  12. Caracemide, a site-specific irreversible inhibitor of protein R1 of Escherichia coli ribonucleotide reductase

    DEFF Research Database (Denmark)

    Larsen, I. K.; Cornett, Claus; Karlsson, M.

    1992-01-01

    The anticancer drug caracemide, N-acetyl-N,O-di(methylcarbamoyl)hydroxylamine, and one of its degradation products, N-acetyl-O-methylcarbamoyl-hydroxylamine, were found to inhibit the enzyme ribonucleotide reductase of Escherichia coli by specific interaction with its larger component protein R1....... No effect on the smaller protein R2 was observed. The effect of the degradation product was about 30 times lower than that of caracemide itself. The caracemide inactivation of R1 is irreversible, with an apparent second-order rate constant of 150 M-1 s-1. The R1R2 holoenzyme was approximately 30 times more...... inactivation. These results indicate that caracemide inactivates R1 by covalent modification at the substrate-binding site. By analogy with the known interaction between caracemide and acetylcholinesterase or choline acetyltransferase, we propose that the modification of R1 occurs at an activated cysteine...

  13. Comparative inhibition of tetrameric carbonyl reductase activity in pig heart cytosol by alkyl 4-pyridyl ketones.

    Science.gov (United States)

    Shimada, Hideaki; Tanigawa, Takahiro; Matayoshi, Kazunori; Katakura, Kazufumi; Babazono, Ken; Takayama, Hiroyuki; Murahashi, Tsuyoshi; Akita, Hiroyuki; Higuchi, Toshiyuki; Eto, Masashi; Imamura, Yorishige

    2014-06-01

    The present study is to elucidate the comparative inhibition of tetrameric carbonyl reductase (TCBR) activity by alkyl 4-pyridyl ketones, and to characterize its substrate-binding domain. The inhibitory effects of alkyl 4-pyridyl ketones on the stereoselective reduction of 4-benzoylpyridine (4-BP) catalyzed by TCBR were examined in the cytosolic fraction of pig heart. Of alkyl 4-pyridyl ketones, 4-hexanoylpyridine, which has a straight-chain alkyl group of five carbon atoms, inhibited most potently TCBR activity and was a competitive inhibitor. Furthermore, cyclohexyl pentyl ketone, which is substituted by cyclohexyl group instead of phenyl group of hexanophenone, had much lower ability to be reduced than hexanophenone. These results suggest that in addition to a hydrophobic cleft corresponding to a straight-chain alkyl group of five carbon atoms, a hydrophobic pocket with affinity for an aromatic group is located in the substrate-binding domain of TCBR.

  14. Conditional gene expression and promoter replacement in Zymoseptoria tritici using fungal nitrate reductase promoters.

    Science.gov (United States)

    Marchegiani, Elisabetta; Sidhu, Yaadwinder; Haynes, Ken; Lebrun, Marc-Henri

    2015-06-01

    Studying essential genes in haploid fungi requires specific tools. Conditional promoter replacement (CPR) is an efficient method for testing gene essentiality. However, this tool requires promoters that can be strongly down-regulated. To this end, we tested the nitrate reductase promoters of Magnaporthe oryzae (pMoNIA1) and Zymoseptoria tritici (pZtNIA1) for their conditional expression in Z. tritici. Expression of EGFP driven by pMoNIA1 or pZtNIA1 was induced on nitrate and down-regulated on glutamate (10-fold less than nitrate). Levels of differential expression were similar for both promoters, demonstrating that the Z. tritici nitrogen regulatory network functions with a heterologous promoter similarly to a native promoter. To establish CPR, the promoter of Z. tritici BGS1, encoding a β-1,3-glucan synthase, was replaced by pZtNIA1 using targeted sequence replacement. Growth of pZtNIA1::BGS1 CPR transformants was strongly reduced in conditions repressing pZtNIA1, while their growth was similar to wild type in conditions inducing pZtNIA1. This differential phenotype demonstrates that BGS1 is important for growth in Z. tritici. In addition, in inducing conditions, pZtNIA1::BGS1 CPR transformants were hyper-sensitive to Calcofluor white, a cell wall disorganizing agent. Nitrate reductase promoters are therefore suitable for conditional promoter replacement in Z. tritici. This tool is a major step toward identifying novel fungicide targets. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. 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.

  16. Exogenous auxin-induced NO synthesis is nitrate reductase-associated in Arabidopsis thaliana root primordia.

    Science.gov (United States)

    Kolbert, Zsuzsanna; Bartha, Bernadett; Erdei, László

    2008-06-16

    Nitric oxide (NO) functions in various physiological and developmental processes in plants. However, the source of this signaling molecule in the diversity of plant responses is not well understood. It is known that NO mediates auxin-induced adventitious and lateral root (LR) formation. In this paper, we provide genetic and pharmacological evidence that the production of NO is associated with the nitrate reductase (NR) enzyme during indole-3-butyric acid (IBA)-induced lateral root development in Arabidopsis thaliana L. NO production was detected using 4,5-diaminofluorescein diacetate (DAF-2DA) in the NR-deficient nia1, nia2 and Atnoa1 (former Atnos1) mutants of A. thaliana. An inhibitor for nitric oxide synthase (NOS) N(G)-monomethyl-l-arginine (l-NMMA) was applied. Our data clearly show that IBA increased LR frequency in the wild-type plant and the LR initials emitted intensive NO-dependent fluorescence of the triazol product of NO and DAF-2DA. Increased levels of NO were restricted only to the LR initials in contrast to primary root (PR) sections, where NO remained at the control level. The mutants had different NO levels in their control state (i.e. without IBA treatment): nia1, nia2 showed lower NO fluorescence than Atnoa1 or the wild-type plant. The role of NR in IBA-induced NO formation in the wild type was shown by the zero effects of the NOS inhibitors l-NMMA. Finally, it was clearly demonstrated that IBA was able to induce NO generation in both the wild-type and Atnoa1 plants, but failed to induce NO in the NR-deficient mutant. It is concluded that the IBA-induced NO production is nitrate reductase-associated during lateral root development in A. thaliana.

  17. A transgenic Neospora caninum strain based on mutations of the dihydrofolate reductase-thymidylate synthase gene.

    Science.gov (United States)

    Pereira, Luiz Miguel; Baroni, Luciana; Yatsuda, Ana Patrícia

    2014-03-01

    Neospora caninum is an Apicomplexa parasite related to abortion and losses of fertility in cattle. The amenability of Toxoplasma gondii and Plasmodium to genetic manipulation offers several tools to determine the invasion and replication processes, which support posterior strategies related to the combat of these diseases. For Plasmodium the use of pyrimethamine as an auxiliary drug on malaria treatment has been affected by the rise of resistant strains and the analyses on Dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene indicated several point mutations. In this work we developed a method for stable insertion of genes based on resistance to pyrimethamine. For that, the coding sequence of NcDHFR-TS (Dihydrofolate reductase-thymidylate synthase) was point mutated in two amino acids, generating DHFRM2M3. The DHFRM2M3 flanked by the promoter and 3'UTR of Ncdhfr-ts (Ncdhfr-DHFRM2M3) conferred resistance to pyrimethamine after transfection. For illustration of stability and expression, the cassette Ncdhfr-DHFRM2M3 was ligated to the reporter gene Lac-Z (β-galactosidase enzyme) controlled by the N. caninum tubulin promoter and was transfected and selected in N. caninum. The cassette was integrated into the genome and the selected tachyzoites expressed Lac-Z, allowing the detection of tachyzoites by the CPRG reaction and X-gal precipitation. The obtainment of transgenic N. caninum resistant to pyrimethamine confirms the effects on DHFR-TS among the Apicomplexa members and will support future approaches on pholate inhibitors for N. caninum prophylaxis. The construction of stable tachyzoites based on vectors with N. caninum promoters initiates the molecular manipulation of this parasite independently of T. gondii. Copyright © 2014. Published by Elsevier Inc.

  18. Aldo-keto reductases in retinoid metabolism: search for substrate specificity and inhibitor selectivity.

    Science.gov (United States)

    Porté, Sergio; Xavier Ruiz, F; Giménez, Joan; Molist, Iago; Alvarez, Susana; Domínguez, Marta; Alvarez, Rosana; de Lera, Angel R; Parés, Xavier; Farrés, Jaume

    2013-02-25

    Biological activity of natural retinoids requires the oxidation of retinol to retinoic acid (RA) and its binding to specific nuclear receptors in target tissues. The first step of this pathway, the reversible oxidoreduction of retinol to retinaldehyde, is essential to control RA levels. The enzymes of retinol oxidation are NAD-dependent dehydrogenases of the cytosolic medium-chain (MDR) and the membrane-bound short-chain (SDR) dehydrogenases/reductases. Retinaldehyde reduction can be performed by SDR and aldo-keto reductases (AKR), while its oxidation to RA is carried out by aldehyde dehydrogenases (ALDH). In contrast to SDR, AKR and ALDH are cytosolic. A common property of these enzymes is that they only use free retinoid, but not retinoid bound to cellular retinol binding protein (CRBP). The relative contribution of each enzyme type in retinoid metabolism is discussed in terms of the different subcellular localization, topology of membrane-bound enzymes, kinetic constants, binding affinity of CRBP for retinol and retinaldehyde, and partition of retinoid pools between membranes and cytoplasm. The development of selective inhibitors for AKR enzymes 1B1 and 1B10, of clinical relevance in diabetes and cancer, granted the investigation of some structure-activity relationships. Kinetics with the 4-methyl derivatives of retinaldehyde isomers was performed to identify structural features for substrate specificity. Hydrophilic derivatives were better substrates than the more hydrophobic compounds. We also explored the inhibitory properties of some synthetic retinoids, known for binding to retinoic acid receptors (RAR) and retinoid X receptors (RXR). Consistent with its substrate specificity towards retinaldehyde, AKR1B10 was more effectively inhibited by synthetic retinoids than AKR1B1. A RARβ/γ agonist (UVI2008) inhibited AKR1B10 with the highest potency and selectivity, and docking simulations predicted that its carboxyl group binds to the anion-binding pocket

  19. Domain engineering of the reductase component of soluble methane monooxygenase from Methylococcus capsulatus (Bath).

    Science.gov (United States)

    Blazyk, Jessica L; Lippard, Stephen J

    2004-02-13

    Soluble methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath) is a three-component enzyme system that catalyzes the conversion of methane to methanol. A reductase (MMOR), which contains [2Fe-2S] and FAD cofactors, facilitates electron transfer from NADH to the hydroxylase diiron active sites where dioxygen activation and substrate hydroxylation take place. By separately expressing the ferredoxin (MMORFd, MMOR residues 1-98) and FAD/NADH (MMOR-FAD, MMOR residues 99-348) domains of the reductase, nearly all biochemical properties of full-length MMOR are retained, except for interdomain electron transfer rates. To investigate the extent to which rapid electron transfer between domains might be restored and further to explore the modularity of MMOR, MMOR-Fd and MMOR-FAD were connected in a non-native fashion. Four different linker sequences were employed to create MMOR reversed-domain (MMOR-RD) constructs, MMOR(99-342)-linker-MMOR(2-98), with a domain connectivity observed in other homologous oxidoreductases. The optical, redox, and electron transfer properties of the four MMOR-RD proteins were characterized and compared with those of wild-type MMOR. The linker sequence plays a key role in controlling solvent accessibility to the FAD cofactor, as evidenced by perturbed flavin optical spectra, decreased FADox/FADsq redox potentials, and increased steady-state oxidase activities in three of the constructs. Stopped-flow optical spectroscopy revealed slow interdomain electron transfer (k < 0.04 s(-1) at 4 degrees C, compared with 90 s(-1) for wild-type MMOR) for all three MMOR-RD proteins with 7-residue linkers. A long (14-residue), flexible linker afforded much faster electron transfer between the FAD and [2Fe-2S] cofactors (k = 0.9 s(-1) at 4 degrees C).

  20. 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.

  1. Functional profiling of mercuric reductase (mer A genes in biofilm communities of a technical scale biocatalyzer

    Directory of Open Access Journals (Sweden)

    von Canstein Harald

    2003-10-01

    Full Text Available Abstract Background Bacterial mercury resistance is based on enzymatic reduction of ionic mercury to elemental mercury and has recently been demonstrated to be applicable for industrial wastewater clean-up. The long-term monitoring of such biocatalyser systems requires a cultivation independent functional community profiling method targeting the key enzyme of the process, the merA gene coding for the mercuric reductase. We report on the development of a profiling method for merA and its application to monitor changes in the functional diversity of the biofilm community of a technical scale biocatalyzer over 8 months of on-site operation. Results Based on an alignment of 30 merA sequences from Gram negative bacteria, conserved primers were designed for amplification of merA fragments with an optimized PCR protocol. The resulting amplicons of approximately 280 bp were separated by thermogradient gelelectrophoresis (TGGE, resulting in strain specific fingerprints for mercury resistant Gram negative isolates with different merA sequences. The merA profiling of the biofilm community from a technical biocatalyzer showed persistence of some and loss of other inoculum strains as well as the appearance of new bands, resulting in an overall increase of the functional diversity of the biofilm community. One predominant new band of the merA community profile was also detected in a biocatalyzer effluent isolate, which was identified as Pseudomonas aeruginosa. The isolated strain showed lower mercury reduction rates in liquid culture than the inoculum strains but was apparently highly competitive in the biofilm environment of the biocatalyzer where moderate mercury levels were prevailing. Conclusions The merA profiling technique allowed to monitor the ongoing selection for better adapted strains during the operation of a biocatalyzer and to direct their subsequent isolation. In such a way, a predominant mercury reducing Ps. aeruginosa strain was identified by

  2. The Importance of Homozygous Polymorphisms of Methylenetetrahydrofolate Reductase Gene in Romanian Patients with Idiopathic Venous Thromboembolism

    Directory of Open Access Journals (Sweden)

    Cristina Hotoleanu

    2013-06-01

    Full Text Available 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- and age-matched controls. Methods: MTHFR C677T and A1298C polymorphisms were detected using PCR-RFLP method. Results: The homozygous MTHFR 677TT genotype, present in 18.8% of patients with VTE versus 6.6% of controls, was significantly associated with VTE (p= 0.021, OR= 3.26, 95%CI (1.141-9.313. The heterozygous MTHFR A1298C genotype, presenting the highest prevalence in the VTE group (34.4% as well as in controls (37.3%, was not associated with VTE (p=0.7. No associations were found for heterozygous MTHFR C677T (with a frequency of 32.2% in VTE and 37.3% in controls, p=0.492, respective homozygous MTHFR A1298C genotype (with a frequency of 1.1% in VTE and 2.6% in controls, p=0.456. Conclusion: Among MTHFR polymorphisms, only homozygosity for MTHFR 677TT may be considered a risk factor for VTE; the MTHFR A1298C polymorphism is not significantly associated with an increased risk of VTE.

  3. Punicalagin, an active pomegranate component, is a new inhibitor of PDIA3 reductase activity.

    Science.gov (United States)

    Giamogante, Flavia; Marrocco, Ilaria; Cervoni, Laura; Eufemi, Margherita; Chichiarelli, Silvia; Altieri, Fabio

    2018-02-06

    Polyphenolic compounds isolated from pomegranate fruit possess several pharmacological activities including anti-inflammatory, hepatoprotective, antigenotoxic and anticoagulant activities. The present work focuses the attention on PDIA3 interaction with punicalagin and ellagic acid, the most predominant components of pomegranate extracts. PDIA3, a member of the protein disulfide isomerase family involved in several cellular functions, is associated with different human diseases and it has the potential to be a pharmacological target. The interaction of polyphenols with PDIA3 purified protein was explored by fluorescence quenching and calorimetric techniques and their effect on PDIA3 activity was investigated. A higher affinity was observed for punicalagin which also strongly affects PDIA3 reductase activity in vitro as a non-competitive inhibitor. Isothermal titration calorimetry confirmed the high affinity of punicalagin for PDIA3. Considering the PDIA3 involvement in oxidative cellular stress response observed in neuroblastoma cells after treatment with hydrogen peroxide, a comparative study was conducted to evaluate the effect of punicalagin on wild type and PDIA3-silenced cells. Punicalagin increases the cell sensitivity to hydrogen peroxide in neuroblastoma cells, but this effect is drastically reduced in PDIA3-silenced cells treated in the same experimental conditions. Punicalagin binds PDIA3 and inhibits its redox activity. Comparative experiments conducted on unsilenced and PDIA3-silenced neuroblastoma cells suggest the potential of punicalagin to modulate PDIA3 reductase activity also in a biological model. Punicalagin can be used as a new PDIA3 inhibitor and this can provide information on the molecular mechanisms underlying the biological activities of PDIA3 and punicalagin. Copyright © 2018. Published by Elsevier B.V.

  4. Effect of the protein ligand in DMSO reductase studied by computational methods.

    Science.gov (United States)

    Dong, Geng; Ryde, Ulf

    2017-06-01

    The DMSO reductase family is the largest and most diverse family of mononuclear molybdenum oxygen-atom-transfer proteins. Their active sites contain a Mo ion coordinated to two molybdopterin ligands, one oxo group in the oxidised state, and one additional, often protein-derived ligand. We have used density-functional theory to evaluate how the fourth ligand (serine, cysteine, selenocysteine, OH(-), O(2-), SH(-), or S(2-)) affects the geometries, reaction mechanism, reaction energies, and reduction potentials of intermediates in the DMSO reductase reaction. Our results show that there are only small changes in the geometries of the reactant and product states, except from the elongation of the MoX bond as the ionic radius of XO, S, Se increases. The five ligands with a single negative charge gave an identical two-step reaction mechanism, in which DMSO first binds to the reduced active site, after which the SO bond is cleaved, concomitantly with the transfer of two electrons from Mo in a rate-determining second transition state. The five models gave similar activation energies of 69-85kJ/mol, with SH(-) giving the lowest barrier. In contrast, the O(2-) and S(2-) ligands gave much higher activation energies (212 and 168kJ/mol) and differing mechanisms (a more symmetric intermediate for O(2-) and a one-step reaction without any intermediate for S(2-)). The high activation energies are caused by a less exothermic reaction energy, 13-25kJ/mol, and by a more stable reactant state owing to the strong MoO(2-) or MoS(2-) bonds. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Polyethylene glycol conjugation enhances the nitrite reductase activity of native and cross-linked hemoglobin.

    Science.gov (United States)

    Lui, Francine E; Dong, Pengcheng; Kluger, Ronald

    2008-10-07

    Although stabilized hemoglobins have been evaluated as oxygen-carrying replacements for red cells in transfusions, in vivo evaluations have noted that these materials are associated with vasoactivity, a serious complication. Scavenging of endogenous nitric oxide by the deoxyheme sites of the stabilized proteins is one likely source of vasoactivity. Recent reports indicate that modification of cell-free hemoglobin derivatives with multiple chains of polyethylene glycol (PEG) suppresses vasoactivity. Gladwin and co-workers observed that the nitrite reductase activity of hemoglobin serves as a major endogenous source of nitric oxide. If PEG conjugation leads to enhanced nitrite reductase activity, this could compensate for scavenged endogenous nitric oxide. To test this possibility, the rates of conversion of nitrite ion to nitric oxide by altered hemoglobins with and without PEG were measured at 25 degrees C. Fumaryl (alpha99-alpha99) cross-linked hemoglobin reacts with nitrite with a bimolecular rate constant of 0.52 M (-1) s (-1), which is comparable to that associated with native hemoglobin (0.25 M (-1) s (-1)). Addition of PEG chains to the cross-linked hemoglobin at beta-Cys93 (alphaalpha-Hb-PEG5K 2) results in a material that produces nitric oxide much more rapidly ( k = 1.41 M (-1) s (-1)). R-State-stabilized hemoglobins with multiple PEG chains (Hb-PEG5K 2 and Hb-PEG5K 6) react 10 times faster with nitrite to produce nitric oxide than does native hemoglobin ( k = 2.5 and 2.4 M (-1) s (-1), respectively). These results, showing enhanced production of nitric oxide resulting from an increased proportion of the protein residing in the R-state, are consistent with the decrease in vasoactivity associated with PEG conjugation.

  6. Expression, purification and characterization of enoyl-ACP reductase II, FabK, from Porphyromonas gingivalis

    Energy Technology Data Exchange (ETDEWEB)

    Hevener, Kirk E.; Mehboob, Shahila; Boci, Teuta; Truong, Kent; Santarsiero, Bernard D.; Johnson, Michael E. (UIC)

    2012-10-25

    The rapid rise in bacterial drug resistance coupled with the low number of novel antimicrobial compounds in the discovery pipeline has led to a critical situation requiring the expedient discovery and characterization of new antimicrobial drug targets. Enzymes in the bacterial fatty acid synthesis pathway, FAS-II, are distinct from their mammalian counterparts, FAS-I, in terms of both structure and mechanism. As such, they represent attractive targets for the design of novel antimicrobial compounds. Enoyl-acyl carrier protein reductase II, FabK, is a key, rate-limiting enzyme in the FAS-II pathway for several bacterial pathogens. The organism, Porphyromonas gingivalis, is a causative agent of chronic periodontitis that affects up to 25% of the US population and incurs a high national burden in terms of cost of treatment. P. gingivalis expresses FabK as the sole enoyl reductase enzyme in its FAS-II cycle, which makes this a particularly appealing target with potential for selective antimicrobial therapy. Herein we report the molecular cloning, expression, purification and characterization of the FabK enzyme from P. gingivalis, only the second organism from which this enzyme has been isolated. Characterization studies have shown that the enzyme is a flavoprotein, the reaction dependent upon FMN and NADPH and proceeding via a Ping-Pong Bi-Bi mechanism to reduce the enoyl substrate. A sensitive assay measuring the fluorescence decrease of NADPH as it is converted to NADP{sup +} during the reaction has been optimized for high-throughput screening. Finally, protein crystallization conditions have been identified which led to protein crystals that diffract x-rays to high resolution.

  7. Inhibition of Fumarate Reductase in Leishmania major and L. donovani by Chalcones

    Science.gov (United States)

    Chen, Ming; Zhai, Lin; Christensen, Søren Brøgger; Theander, Thor G.; Kharazmi, Arsalan

    2001-01-01

    Our previous studies have shown that chalcones exhibit potent antileishmanial and antimalarial activities in vitro and in vivo. Preliminary studies showed that these compounds destroyed the ultrastructure of Leishmania parasite mitochondria and inhibited the respiration and the activity of mitochondrial dehydrogenases of Leishmania parasites. The present study was designed to further investigate the mechanism of action of chalcones, focusing on the parasite respiratory chain. The data show that licochalcone A inhibited the activity of fumarate reductase (FRD) in the permeabilized Leishmania major promastigote and in the parasite mitochondria, and it also inhibited solubilized FRD and a purified FRD from L. donovani. Two other chalcones, 2,4-dimethoxy-4′-allyloxychalcone (24m4ac) and 2,4-dimethoxy-4′-butoxychalcone (24mbc), also exhibited inhibitory effects on the activity of solubilized FRD in L. major promastigotes. Although licochalcone A inhibited the activities of succinate dehydrogenase (SDH), NADH dehydrogenase (NDH), and succinate- and NADH-cytochrome c reductases in the parasite mitochondria, the 50% inhibitory concentrations (IC50) of licochalcone A for these enzymes were at least 20 times higher than that for FRD. The IC50 of licochalcone A for SDH and NDH in human peripheral blood mononuclear cells were at least 70 times higher than that for FRD. These findings indicate that FRD, one of the enzymes of the parasite respiratory chain, might be the specific target for the chalcones tested. Since FRD exists in the Leishmania parasite and does not exist in mammalian cells, it could be an excellent target for antiprotozoal drugs. PMID:11408218

  8. Gene Cloning and Expression of the Pyrroline-5-carboxylate Reductase Gene of Perennial Ryegrass (Lolium perenne

    Directory of Open Access Journals (Sweden)

    Cao Li

    2015-09-01

    Full Text Available Salt and drought limit the range of applications of perennial ryegrass (Lolium perenne L., which is one of the important turf and forage grasses. Previous studies have suggested that pyrroline-5-carboxylate reductase (P5CR might play a central role in proline accumulation in plants that are responsive to stresses. In the present study, the Lolium perenne L. pyrroline-5-carboxylate reductase (LpP5CR gene was cloned from leaves of the cultivar ‘Derby’ using the RACE technique. The full-length LpP5CR gene was 1 047 bp in length, which comprised an open reading frame (ORF of 840 bp in size. Sequence alignment revealed that the putative LpP5CR had a 94.3% similarity to TaP5CR. qRT-PCR displayed that the mRNA levels of the LpP5CR gene were almost the same as that in the roots, stems, and leaves of perennial ryegrass seedlings subjected to normal condition or NaCl treatment for 1 h. Moreover, the transcription level of LpP5CR was up- or down-regulated with NaCl, polyethylene glycol (PEG, cold, or abscisic acid (ABA treatment for 3 to 48 h. In addition, confocal microscopy localized the GFP-LpP5CR fusion protein to the cytoplasm of onion epidermal cells. These findings suggest that LpP5CR encodes a cytoplasmic P5CR protein that plays an important role in the response of perennial ryegrass to various stresses.

  9. Antimicrobial activity and physical characterization of silver nanoparticles green synthesized using nitrate reductase from Fusarium oxysporum.

    Science.gov (United States)

    Gholami-Shabani, Mohammadhassan; Akbarzadeh, Azim; Norouzian, Dariush; Amini, Abdolhossein; Gholami-Shabani, Zeynab; Imani, Afshin; Chiani, Mohsen; Riazi, Gholamhossein; Shams-Ghahfarokhi, Masoomeh; Razzaghi-Abyaneh, Mehdi

    2014-04-01

    Nanostructures from natural sources have received major attention due to wide array of biological activities and less toxicity for humans, animals, and the environment. In the present study, silver nanoparticles were successfully synthesized using a fungal nitrate reductase, and their biological activity was assessed against human pathogenic fungi and bacteria. The enzyme was isolated from Fusarium oxysporum IRAN 31C after culturing on malt extract-glucose-yeast extract-peptone (MGYP) medium. The enzyme was purified by a combination of ultrafiltration and ion exchange chromatography on DEAE Sephadex and its molecular weight was estimated by gel filtration on Sephacryl S-300. The purified enzyme had a maximum yield of 50.84 % with a final purification of 70 folds. With a molecular weight of 214 KDa, it is composed of three subunits of 125, 60, and 25 KDa. The purified enzyme was successfully used for synthesis of silver nanoparticles in a way dependent upon NADPH using gelatin as a capping agent. The synthesized silver nanoparticles were characterized by X-ray diffraction, dynamic light scattering spectroscopy, and transmission and scanning electron microscopy. These stable nonaggregating nanoparticles were spherical in shape with an average size of 50 nm and a zeta potential of -34.3. Evaluation of the antimicrobial effects of synthesized nanoparticles by disk diffusion method showed strong growth inhibitory activity against all tested human pathogenic fungi and bacteria as evident from inhibition zones that ranged from 14 to 25 mm. Successful green synthesis of biologically active silver nanoparticles by a nitrate reductase from F. oxysporum in the present work not only reduces laborious downstream steps such as purification of nanoparticle from interfering cellular components, but also provides a constant source of safe biologically-active nanomaterials with potential application in agriculture and medicine.

  10. Methyl-coenzyme M reductase A as an indicator to estimate methane production from dairy cows.

    Science.gov (United States)

    Aguinaga Casañas, M A; Rangkasenee, N; Krattenmacher, N; Thaller, G; Metges, C C; Kuhla, B

    2015-06-01

    The evaluation of greenhouse gas mitigation strategies requires the quantitative assessment of individual methane production. Because methane measurement in respiration chambers is highly accurate, but also comprises various disadvantages such as limited capacity and high costs, the establishment of an indicator for estimating methane production of individual ruminants would provide an alternative to direct methane measurement. Methyl-coenzyme M reductase is involved in methanogenesis and the subunit α of methyl-coenzyme M reductase is encoded by the mcrA gene of rumen archaea. We therefore examined the relationship between methane emissions of Holstein dairy cows measured in respiration chambers with 2 different diets (high- and medium-concentrate diet) and the mcrA DNA and mcrA cDNA abundance determined from corresponding rumen fluid samples. Whole-body methane production per kilogram of dry matter intake and mcrA DNA normalized to the abundance of the rrs gene coding for 16S rRNA correlated significantly when using qmcrA primers. Use of qmcrA primers also revealed linear correlation between mcrA DNA copy number and methane yield. Regression analyses based on normalized mcrA cDNA abundances revealed no significant linear correlation with methane production per kilogram of dry matter intake. Furthermore, the correlations between normalized mcrA DNA abundance and the rumen fluid concentration of acetic and isobutyric acid were positive, whereas the correlations with propionic and lactic acid were negative. These data suggest that the mcrA DNA approach based on qmcrA primers could potentially be a molecular proxy for methane yield after further refinement. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  11. Cynoglossus semilaevis thioredoxin: a reductase and an antioxidant with immunostimulatory property.

    Science.gov (United States)

    Sun, Jin-sheng; Li, Yong-xin; Sun, Li

    2012-07-01

    Thioredoxin (Trx) is a small redox protein existing ubiquitously in all living organisms and plays an important role in multiple cellular processes, including transcriptional regulation and immune response. To date very few studies have been carried out to examine the function of piscine Trx. In this study, we identified and analyzed the function of a Trx homologue, CsTrx1, from half-smooth tongue sole (Cynoglossus semilaevis). The deduced amino acid sequence of CsTrx1 is composed of 107 residues and shares 54.1-60.8% overall identities with the Trx of other teleosts. CsTrx1 contains the highly conserved CXXC motif, which in mammals is known to be the active site, in the form of CQPC. Expression of CsTrx1 as determined by quantitative real-time reverse transcriptase PCR was highest in liver and upregulated in time-dependent manners by bacterial infection and by exposure to iron, copper, and hydrogen peroxide. Purified recombinant CsTrx1 (rCsTrx1) exhibited insulin disulfide reductase activity and antioxidant activity, both which, however, were lost when the two cysteine residues in the CQPC motif were mutated to serine. Further analysis showed that rCsTrx1 was able to stimulate the proliferation of head kidney leukocytes, upregulate the expression of immune relevant genes, and enhance the resistance of leukocytes against bacterial infection. Taken together, these results indicate that CsTrx1 is a biologically active reductase and an antioxidant that requires the CXXC motif for activity and that CsTrx1 possesses cytokine-like immunoregulatory property. These results suggest a role for CsTrx1 in protecting cells against oxidative stress caused by oxidant exposure and pathogen infection.

  12. A dual role for plant quinone reductases in host-fungus interaction.

    Science.gov (United States)

    Heyno, Eiri; Alkan, Noam; Fluhr, Robert

    2013-11-01

    Quinone reductases (QR, EC 1.5.6.2) are flavoproteins that protect organisms from oxidative stress. The function of plant QRs has not as yet been addressed in vivo despite biochemical evidence for their involvement in redox reactions. Here, using knock-out (KO) and overexpressing lines, we studied the protective role of two groups of Arabidopsis thaliana cytosolic QRs, Nqr (NAD(P)H:quinone oxidoreductase) and Fqr (flavodoxin-like quinone reductase), in response to infection by necrotrophic fungi. The KO lines nqr(-) and fqr1(-) displayed significantly slower development of lesions of Botrytis cinerea and Sclerotinia sclerotium in comparison to the wild type (WT). Consistent with this observation, the overexpressing line FQR1(+) was hypersensitive to the pathogens. Both the nqr(-) and fqr1(-) displayed increased fluorescence of 2',7'-dichlorofluorescein,‬ a reporter for reactive oxygen species in response to B. cinerea. Infection by B. cinerea was accompanied with increased Nqr and Fqr1 protein levels in the WT as revealed by western blotting. In addition, a marked stimulation of salicylic acid-sensitive transcripts and suppression of jasmonate-sensitive transcripts was observed in moderately wounded QR KO mutant leaves, a condition mimicking the early stage of infection. In contrast to the above observations, germination of conidia was accelerated on leaves of QR KO mutants in comparison with the WT and FQR1(+). The same effect was observed in water-soluble leaf surface extracts. It is proposed that the altered interaction between B. cinerea and the QR mutants is a consequence of subtly altered redox state of the host, which perturbs host gene expression in response to environmental stress such as fungal growth.‬‬‬‬‬‬ © 2013 Scandinavian Plant Physiology Society.

  13. Callitriche cophocarpa (water starwort) proteome under chromate stress: evidence for induction of a quinone reductase.

    Science.gov (United States)

    Kaszycki, Paweł; Dubicka-Lisowska, Aleksandra; Augustynowicz, Joanna; Piwowarczyk, Barbara; Wesołowski, Wojciech

    2018-01-13

    Chromate-induced physiological stress in a water-submerged macrophyte Callitriche cophocarpa Sendtn. (water starwort) was tested at the proteomic level. The oxidative stress status of the plant treated with 1 mM Cr(VI) for 3 days revealed stimulation of peroxidases whereas catalase and superoxide dismutase activities were similar to the control levels. Employing two-dimensional electrophoresis, comparative proteomics enabled to detect five differentiating proteins subjected to identification with mass spectrometry followed by an NCBI database search. Cr(VI) incubation led to induction of light harvesting chlorophyll a/b binding protein with a concomitant decrease of accumulation of ribulose bisphosphate carboxylase (RuBisCO). The main finding was, however, the identification of an NAD(P)H-dependent dehydrogenase FQR1, detectable only in Cr(VI)-treated plants. The FQR1 flavoenzyme is known to be responsive to oxidative stress and to act as a detoxification protein by protecting the cells against oxidative damage. It exhibits the in vitro quinone reductase activity and is capable of catalyzing two-electron transfer from NAD(P)H to several substrates, presumably including Cr(VI). The enhanced accumulation of FQR1 was chromate-specific since other stressful conditions, such as salt, temperature, and oxidative stresses, all failed to induce the protein. Zymographic analysis of chromate-treated Callitriche shoots showed a novel enzymatic protein band whose activity was attributed to the newly identified enzyme. We suggest that Cr(VI) phytoremediation with C. cophocarpa can be promoted by chromate reductase activity produced by the induced quinone oxidoreductase which might take part in Cr(VI) → Cr(III) bioreduction process and thus enable the plant to cope with the chromate-generated oxidative stress.

  14. Role of Ribonucleotide Reductase in Bacillus subtilis Stress-Associated Mutagenesis.

    Science.gov (United States)

    Castro-Cerritos, Karla Viridiana; Yasbin, Ronald E; Robleto, Eduardo A; Pedraza-Reyes, Mario

    2017-02-15

    The Gram-positive microorganism Bacillus subtilis relies on a single class Ib ribonucleotide reductase (RNR) to generate 2'-deoxyribonucleotides (dNDPs) for DNA replication and repair. In this work, we investigated the influence of RNR levels on B. subtilis stationary-phase-associated mutagenesis (SPM). Since RNR is essential in this bacterium, we engineered a conditional mutant of strain B. subtilis YB955 (hisC952 metB5 leu427) in which expression of the nrdEF operon was modulated by isopropyl-β-d-thiogalactopyranoside (IPTG). Moreover, genetic inactivation of ytcG, predicted to encode a repressor (NrdR) of nrdEF in this strain, dramatically increased the expression levels of a transcriptional nrdE-lacZ fusion. The frequencies of mutations conferring amino acid prototrophy in three genes were measured in cultures under conditions that repressed or induced RNR-encoding genes. The results revealed that RNR was necessary for SPM and overexpression of nrdEF promoted growth-dependent mutagenesis and SPM. We also found that nrdEF expression was induced by H2O2 and such induction was dependent on the master regulator PerR. These observations strongly suggest that the metabolic conditions operating in starved B. subtilis cells increase the levels of RNR, which have a direct impact on SPM. Results presented in this study support the concept that the adverse metabolic conditions prevailing in nutritionally stressed bacteria activate an oxidative stress response that disturbs ribonucleotide reductase (RNR) levels. Such an alteration of RNR levels promotes mutagenic events that allow Bacillus subtilis to escape from growth-limited conditions. Copyright © 2017 American Society for Microbiology.

  15. Evaluation of in vitro aldose reductase inhibitory potential of alkaloidal fractions of Piper nigrum, Murraya koenigii, Argemone mexicana, and Nelumbo nucifera.

    Science.gov (United States)

    Gupta, Sakshi; Singh, Nirmal; Jaggi, Amteshwar Singh

    2014-05-01

    Aldose reductase is primarily involved in development of long-term diabetic complications due to increased polyol pathway activity. The synthetic aldose reductase inhibitors are not very successful clinically. Therefore, the natural sources may be exploited for safer and effective aldose reductase inhibitors. In the present study, the aldose reductase inhibitory potential of hydroalcoholic and alkaloidal extracts of Piper nigrum, Murraya koenigii, Argemone mexicana, and Nelumbo nucifera was evaluated. The hydroalcoholic and alkaloidal extracts of the selected plants were prepared. The different concentrations of hydroalcoholic and alkaloidal extracts of these plants were evaluated for their goat lens aldose reductase inhibitory activity using dl-glyceraldehyde as substrate. The aldose reductase inhibitory potential of extracts was assessed in terms of their IC50 value. Amongst the hydroalcoholic extracts, the highest aldose reductase inhibitory activity was shown by P. nigrum (IC50 value 35.64±2.7 μg/mL) followed by M. koenigii (IC50 value 45.67±2.57 μg/mL), A. mexicana (IC50 value 56.66±1.30 μg/mL), and N. nucifera (IC50 value 59.78±1.32 μg/mL). Among the alkaloidal extracts, highest inhibitory activity was shown by A. mexicana (IC50 value 25.67±1.25 μg/mL), followed by N. nucifera (IC50 value 28.82±1.85 μg/mL), P. nigrum (IC50 value 30.21±1.63 μg/mL), and M. koenigii (IC50 value 35.66±1.64 μg/mL). It may be concluded that the alkaloidal extracts of these plants possess potent aldose reductase inhibitory activity and may be therapeutically exploited in diabetes-related complications associated with increased activity of aldose reductase.

  16. Molecular study of the 5 {alpha}-reductase type 2 gene in three European families with 5 {alpha}-reductase deficiency

    Energy Technology Data Exchange (ETDEWEB)

    Boudon, C.; Lumbroso, S.; Lobaccaro, J.M. [Institut National de la Sante et de la Recherche Medicale, Montpellier (France)]|[Child Health Center, Warsaw (Poland)]|[Hopital A. de Villeneuve, Montpellier (France)] [and others

    1995-07-01

    The molecular basis of 5{alpha}-reductase (5{alpha}R) deficiency was investigated in four patients from three European families. In the French family, the first patient was raised as a female, and gonadectomy was performed before puberty. The second sibling, also raised as female, differed in that gonadal removal was performed after the onset of pubertal masculinization. The other two patients, both from Polish families, developed masculinization of external genitalia during puberty. All patients developed a female sexual identity. In all cases, no known consanguinity or family history of 5{alpha}R deficiency was reported. The genomic DNAs of the patients were sequenced after polymerase chain reaction amplification of the five exons of the 5{alpha}R type 2 gene. We found two homozygous mutations responsible for gutamine to arginine and histidine to arginine substitution in families 1 and 3, respectively. In family 2, we found a heterozygous mutation responsible for an asparagine to serine substitution at position 193. The glutamine/arginine 126 mutation in the French family was previously reported in a Creole ethnic group, and the Polish histidine/arginine 231 mutation was previously reported in a patient from Chicago, Moreover, all of the mutations created new restriction sites, which were used to determine the kindred carrier status in the three families. Because 5{alpha}R deficiency is known to be heterogenous disease in terms of clinical and biochemical expression, our data suggest that molecular biology analysis of the type 2 gene could be an essential step in diagnosing 5{alpha}R deficiency. 22 refs., 3 figs., 1 tab.

  17. A theoretical multiscale treatment of protein-protein electron transfer: The ferredoxin/ferredoxin-NADP(+) reductase and flavodoxin/ferredoxin-NADP(+) reductase systems.

    Science.gov (United States)

    Saen-Oon, Suwipa; Cabeza de Vaca, Israel; Masone, Diego; Medina, Milagros; Guallar, Victor

    2015-12-01

    In the photosynthetic electron transfer (ET) chain, two electrons transfer from photosystem I to the flavin-dependent ferredoxin-NADP(+) reductase (FNR) via two sequential independent ferredoxin (Fd) electron carriers. In some algae and cyanobacteria (as Anabaena), under low iron conditions, flavodoxin (Fld) replaces Fd as single electron carrier. Extensive mutational studies have characterized the protein-protein interaction in FNR/Fd and FNR/Fld complexes. Interestingly, even though Fd and Fld share the interaction site on FNR, individual residues on FNR do not participate to the same extent in the interaction with each of the protein partners, pointing to different electron transfer mechanisms. Despite of extensive mutational studies, only FNR/Fd X-ray structures from Anabaena and maize have been solved; structural data for FNR/Fld remains elusive. Here, we present a multiscale modelling approach including coarse-grained and all-atom protein-protein docking, the QM/MM e-Pathway analysis and electronic coupling calculations, allowing for a molecular and electronic comprehensive analysis of the ET process in both complexes. Our results, consistent with experimental mutational data, reveal the ET in FNR/Fd proceeding through a bridge-mediated mechanism in a dominant protein-protein complex, where transfer of the electron is facilitated by Fd loop-residues 40-49. In FNR/Fld, however, we observe a direct transfer between redox cofactors and less complex specificity than in Fd; more than one orientation in the encounter complex can be efficient in ET. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. A second common variant in the methylenetetrahydrofolate reductase (MTHFR) gene and its relationship to MTHFR enzyme activity, homocysteine, and cardiovascular disease risk

    NARCIS (Netherlands)

    Lievers, K.J.A.; Boers, G.H.J.; Verhoef, P.; Heijer, den M.; Kluijtmans, L.A.J.; Put, van der N.M.J.; Trijbels, F.J.M.; Blom, H.J.

    2001-01-01

    Molecular defects in genes encoding enzymes involved in homocysteine metabolism may account for mild hyperhomocysteinemia, an independent and graded risk factor for cardiovascular disease (CVD). We examined the relationship of two polymorphisms in the methylenetetrahydrofolate reductase (MTHFR)

  19. Does the interaction between maternal folate intake and the methylenetetrahydrofolate reductase polymorphisms affect the risk of cleft lip with or without cleft palate?

    NARCIS (Netherlands)

    Vermeij-Keers, C.; Kluijtmans, L.A.J.; Ocke, M.C.; Zielhuis, G.A.; Goorhuis-Brouwer, S.M.; Biezen, J.J. van der; Kuijpers-Jagtman, A.M.; Steegers-Theunissen, R.P.M.

    2003-01-01

    Periconceptional folic acid supplementation may reduce the risk of cleft lip with or without cleft palate (CL(P)). Polymorphisms in the methylenetetrahydrofolate reductase (MTHFR) gene reduce availability of 5-methyltetrahydrofolate, the predominant circulating form of folate. To determine the

  20. The periplasmic nitrate reductase in Shewanella: the resolution, distribution and functional implications of two NAP isoforms, NapEDABC and NapDAGHB

    National Research Council Canada - National Science Library

    Simpson, Philippa J. L; Richardson, David J; Codd, Rachel

    2010-01-01

    ... of Medical Sciences (Pharmacology) and Bosch Institute, University of Sydney, New South Wales 2006, Australia In the bacterial periplasm, the reduction of nitrate to nitrite is catalysed by a periplasmic nitrate reductase (NAP...

  1. The association of gastric cancer risk with plasma folate, cobalamin, and Methylenetetrahydrofolate reductase polymorphisms in the European prospective investigation into cancer and nutrition

    NARCIS (Netherlands)

    Vollset, Stein Emil; Igland, Jannicke; Jenab, Mazda; Fredriksen, Ase; Meyer, Klaus; Eussen, Simone; Gjessing, Hakon K.; Ueland, Per Magne; Pera, Guillem; Sala, Nuria; Agudo, Antonio; Capella, Gabriel; Del Giudice, Giuseppe; Palli, Domenico; Boeing, Heiner; Weikert, Cornelia; Bueno-de-Mesquita, H. Bas; Carneiro, Fatima; Pala, Valeria; Vineis, Paolo; Tumino, Rosario; Panico, Salvatore; Berglund, Goran; Manjer, Jonas; Stenling, Roger; Hallmans, Goran; Martinez, Carmen; Dorronsoro, Miren; Barricarte, Aurelio; Navarro, Carmen; Quiros, Jose R.; Allen, Naomi; Key, Timothy J.; Bingham, Sheila; Linseisen, Jakob; Kaaks, Rudolf; Overvad, Kim; Tjonneland, Anne; Buchner, Frederike L.; Peeters, Petra H. M.; Numans, Mattijs E.; Clavel-Chapelon, Francoise; Boutron-Ruault, Marie-Christine; Trichopoulou, Antonia; Lund, Eiliv; Slimani, Nadia; Ferrari, Pietro; Riboli, Elio; Gonzalez, Carlos A.

    2007-01-01

    Previous studies have shown inconsistent associations of folate intake and polymorphisms of the methylenetetrahydrofolate reductase (MTHFR) gene with gastric cancer risk. Our nested case-control study within the European Prospective Investigation into Cancer and Nutrition cohort is the first

  2. The association of gastric cancer risk with plasma folate, cobalamin, and methylenetetrahydrofolate reductase polymorphisms in the European Prospective Investigation into Cancer and Nutrition.

    NARCIS (Netherlands)

    Vollset, S.E.; Igland, J.; Jenab, M.; Fredriksen, A.; Meyer, K.; Eussen, S.; Gjessing, H.K.; Ueland, P.M.; Pera, G.; Sala, N.; Agudo, A.; Capella, G.; Giudice, G. Del; Palli, D.; Boeing, H.; Weikert, C.; Bueno-De-Mesquita, H.B.; Carneiro, F.; Pala, V.; Vineis, P.; Tumino, R.; Panico, S.; Berglund, G.; Manjer, J.; Stenling, R.; Hallmans, G.; Martinez, C.; Dorronsoro, M.; Barricarte, A.; Navarro, C.; Quiros, J.R.; Allen, N.; Key, T.J.; Bingham, S.; Linseisen, J.; Kaaks, R.; Overvad, K.; Tjonneland, A.; Buchner, F.L.; Peeters, P.H.; Numans, M.E.; Clavel-Chapelon, F.; Boutron-Ruault, M.C.; Trichopoulou, A.; Lund, E.; Slimani, N.; Ferrari, P.; Riboli, E.; Gonzalez, C.A.

    2007-01-01

    Previous studies have shown inconsistent associations of folate intake and polymorphisms of the methylenetetrahydrofolate reductase (MTHFR) gene with gastric cancer risk. Our nested case-control study within the European Prospective Investigation into Cancer and Nutrition cohort is the first

  3. Dihydrofolate reductase and dihydropteroate synthase genotypes associated with in vitro resistance of Plasmodium falciparum to pyrimethamine, trimethoprim, sulfadoxine, and sulfamethoxazole

    DEFF Research Database (Denmark)

    Khalil, Insaf; Rønn, Anita M; Alifrangis, Michael

    2003-01-01

    A total of 70 Plasmodium falciparum isolates were tested in vitro against pyrimethamine (PYR), trimethoprim (TRM), sulfadoxine (SDX), and sulfamethoxazole (SMX), and their dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genotypes were determined. dhfr genotypes correlated with ...... the cultures....

  4. Synthesis and activity of 8-substituted benzo[c]quinolizin-3-ones as dual inhibitors of human 5alpha-reductases 1 and 2.

    Science.gov (United States)

    Ferrali, Alessandro; Menchi, Gloria; Occhiato, Ernesto G; Danza, Giovanna; Mancina, Rosa; Serio, Mario; Guarna, Antonio

    2005-01-03

    Some potent dual inhibitors of 5alpha-reductases 1 and 2, based on the benzo[c]quinolizin-3-one structure and with IC(50) values ranging between 93 and 166nM for both isozymes, were found. The presence of the F atom on the ester moiety at the position 8 was crucial. This result can help in the design of other potent, dual inhibitors to be developed as drugs in the treatment of 5alpha-reductase related diseases.

  5. Age and Obesity Promote Methylation and Suppression of 5α-Reductase 2: Implications for Personalized Therapy of Benign Prostatic Hyperplasia.

    Science.gov (United States)

    Bechis, Seth K; Otsetov, Alexander G; Ge, Rongbin; Wang, Zongwei; Vangel, Mark G; Wu, Chin-Lee; Tabatabaei, Shahin; Olumi, Aria F

    2015-10-01

    In men with symptomatic benign prostatic hyperplasia 5α-reductase inhibitors are a main modality of treatment. More than 30% of men do not respond to the therapeutic effects of 5α-reductase inhibitors. We have found that a third of adult prostate samples do not express 5α-reductase type 2 secondary to epigenetic modifications. We evaluated whether 5α-reductase type 2 expression in benign prostatic hyperplasia specimens from symptomatic men was linked to methylation of the 5α-reductase type 2 gene promoter. We also identified associations with age, obesity, cardiac risk factors and prostate specific antigen. Prostate samples from men undergoing transurethral prostate resection were used. We determined 5α-reductase type 2 protein expression and gene promoter methylation status by common assays. Clinical variables included age, body mass index, hypertension, hyperlipidemia, diabetes, prostate specific antigen and prostate volume. Univariate and multivariate statistical analyses were performed followed by stepwise logistic regression modeling. Body mass index and age significantly correlated with methylation of the 5α-reductase type 2 gene promoter (p age and body mass index significantly predicted methylation status and protein expression (p age and body mass index correlate with increased 5α-reductase type 2 gene promoter methylation and decreased protein expression in men with symptomatic benign prostatic hyperplasia. These results highlight the interplay among age, obesity and gene regulation. Our findings suggest an individualized epigenetic signature for symptomatic benign prostatic hyperplasia, which may be important to choose appropriate personalized treatment options. Copyright © 2015 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

  6. Inverse relationship between age-dependent erythrocyte activity of methaemoglobin reductase and prilocaine-induced methaemoglobinaemia during infancy

    DEFF Research Database (Denmark)

    Nilsson, A; Engberg, G; Henneberg, S

    1990-01-01

    concentrations of local anaesthetics were low (maximum values: prilocaine 78 ng ml-1, lignocaine 412 ng ml-1). The activity of erythrocyte MetHb reductase (cytochrome b5 reductase) was analysed. Data from a previously studied group of infants aged 3-12 months were included also. Enzyme activity did not reach...... adult levels until after the age of 3 months. It showed a good inverse correlation with the maximum MetHb values after application of EMLA. Although the MetHb concentrations in the infants younger than 3 months were small, the enzyme capacity may be overloaded when EMLA is administered at the same time...... as other MetHb-inducing agents. It is concluded that the use of EMLA should be restricted in this age group....

  7. Association of aldose reductase gene Z+2 polymorphism with reduced susceptibility to diabetic nephropathy in Caucasian Type 1 diabetic patients

    DEFF Research Database (Denmark)

    Lajer, Mathilde; Tarnow, L; Fleckner, Jan

    2004-01-01

    AIMS: The Z-2 allele of the (AC)n polymorphism in the aldose reductase gene (ALR2) confers increased risk of microvascular diabetic complications, whereas the Z+2 allele has been proposed to be a marker of protection. However data are conflicting. Therefore, we investigated whether this polymorph......AIMS: The Z-2 allele of the (AC)n polymorphism in the aldose reductase gene (ALR2) confers increased risk of microvascular diabetic complications, whereas the Z+2 allele has been proposed to be a marker of protection. However data are conflicting. Therefore, we investigated whether...... of the ALR2 promoter polymorphism is associated with a reduced susceptibility to diabetic nephropathy in Danish Type 1 diabetic patients, suggesting a minor role for the polyol pathway in the pathogenesis of diabetic kidney disease. No association of the ALR2 polymorphism with diabetic retinopathy was found....

  8. Characterization of nineteen antimony(III) complexes as potent inhibitors of photosystem II, carbonic anhydrase, and glutathione reductase.

    Science.gov (United States)

    Karacan, Mehmet Sayım; Rodionova, Margarita V; Tunç, Turgay; Venedik, Kübra Begüm; Mamaş, Serhat; Shitov, Alexandr V; Zharmukhamedov, Sergei K; Klimov, Vyacheslav V; Karacan, Nurcan; Allakhverdiev, Suleyman I

    2016-12-01

    Nineteen antimony(III) complexes were obtained and examined as possible herbicides. Six of these were synthesized for the first time, and their structures were identified using elemental analyses, (1)H-NMR, (13)C-NMR, FTIR, LCMS, magnetic susceptibility, and conductivity measurement techniques. For the nineteen examined antimony(III) complexes their most-stable forms were determined by DFT/B3LYP/LanL2DZ calculation method. These compounds were examined for effects on photosynthetic electron transfer and carbonic anhydrase activity of photosystem II, and glutathione reductase from chloroplast as well were investigated. Our results indicated that all antimony(III) complexes inhibited glutathione reductase activity of chloroplast. A number of these also exhibited good inhibitory efficiency of the photosynthetic and carbonic anhydrase activity of Photosystem II.

  9. Late Diagnosis of 5-α-Reductase Type 2 Deficiency in an Adolescent Girl with Primary Amenorrhoea: Case report

    Directory of Open Access Journals (Sweden)

    Abdul-Rahman A. Hummadi

    2017-06-01

    Full Text Available Deficiency of the 5-α-reductase enzyme has been found to affect male sexual development. We report an 18-year-old patient who was referred to an endocrinology clinic in Jizan, Saudi Arabia, in April 2014 with primary amenorrhoea, virilisation and a lack of secondary sex characteristics. As female external genitalia were present at birth, she had been raised as a female. Magnetic resonance imaging revealed no uterine or ovarian tissue in the pelvis and the presence of a scrotal sac. She was diagnosed with 5-α-reductase type 2 deficiency, a 46,XY disorder of sexual development. Typically, affected males have pseudovaginal perineoscrotal hypospadias and ambiguous genitalia at birth. Individuals who have been raised as female manifest characteristics of virilisation at puberty, including deepening of the vocal tone, phallus enlargement, scrotal hyperpigmentation and increased muscle mass.

  10. Oxalate as a potent and selective inhibitor of spinach (Spinacia oleracea) leaf NADPH-dependent hydroxypyruvate reductase.

    Science.gov (United States)

    Kleczkowski, L A; Randall, D D; Edwards, G E

    1991-01-01

    Purified spinach (Spinacia oleracea) NADPH-preferring hydroxypyruvate reductase (HPR-2) was potently and selectively inhibited by oxalate, an end product of metabolism in plants. Both hydroxypyruvate- and glyoxylate-dependent rates of the HPR-2 enzyme were affected. Oxalate acted as an uncompetitive inhibitor of the enzyme, with Ki values of 7 and 36 microM for the NADPH/hydroxypyruvate and NADPH/glyoxylate pairs of reactants respectively. Oxalate, at millimolar levels, caused less than 10% inhibition of purified spinach NADH-preferring HPR (HPR-1) and had no effect on purified spinach NADPH-preferring glyoxylate-specific reductase (GR-1). The inhibition of spinach HPR-2 by oxalate is by far the strongest for any known inhibitor of leaf HPR and GR activities. In photosynthetic tissues, oxalate could potentially act as a primary regulator of extraperoxisomal metabolism of hydroxypyruvate and glyoxylate. PMID:2039466

  11. Association of methylenetetrahydrofolate reductase A1298C polymorphism but not of C677T with multiple sclerosis in Tunisian patients.

    Science.gov (United States)

    Fekih Mrissa, Najiba; Mrad, Meriem; Klai, Sarra; Zaouali, Jamel; Sayeh, Aycha; Mazigh, Chakib; Nsiri, Brahim; Machgoul, Salem; Gritli, Nasreddine; Mrissa, Ridha

    2013-09-01

    Multiple sclerosis (MS) is a chronic neurological disease characterized by central nervous system (CNS) inflammation and demyelination of nerve axons. The aim of this study was to investigate a possible association between the methylenetetrahydrofolate reductase (MTHFR) gene and multiple sclerosis in Tunisian patients. The genotyping of two missense variants of the methylenetetrahydrofolate reductase (MTHFR) gene, C677T and A1298C was performed in 80 multiple sclerosis patients and 200 healthy controls. No significant differences were found in the frequency of the MTHFR C677T polymorphism between MS patients and healthy controls. However, the genotype prevalence of the missense variant MTHFR A1298C was significantly different between patients and controls (A/C: 55% versus 7%, pMTHFR C677T variants and MS, there is evidence to suggest a significant association between the MTHFR A1298C polymorphisms and MS. Copyright © 2013 Elsevier B.V. All rights reserved.

  12. Thioredoxin and Its Reductase Are Present on Synaptic Vesicles, and Their Inhibition Prevents the Paralysis Induced by Botulinum Neurotoxins

    Directory of Open Access Journals (Sweden)

    Marco Pirazzini

    2014-09-01

    Full Text Available Botulinum neurotoxins consist of a metalloprotease linked via a conserved interchain disulfide bond to a heavy chain responsible for neurospecific binding and translocation of the enzymatic domain in the nerve terminal cytosol. The metalloprotease activity is enabled upon disulfide reduction and causes neuroparalysis by cleaving the SNARE proteins. Here, we show that the thioredoxin reductase-thioredoxin protein disulfide-reducing system is present on synaptic vesicles and that it is functional and responsible for the reduction of the interchain disulfide of botulinum neurotoxin serotypes A, C, and E. Specific inhibitors of thioredoxin reductase or thioredoxin prevent intoxication of cultured neurons in a dose-dependent manner and are also very effective inhibitors of the paralysis of the neuromuscular junction. We found that this group of inhibitors of botulinum neurotoxins is very effective in vivo. Most of them are nontoxic and are good candidates as preventive and therapeutic drugs for human botulism.

  13. Design, Synthesis, and Biological Evaluation of Potent Quinoline and Pyrroloquinoline Ammosamide Analogues as Inhibitors of Quinone Reductase 2

    Energy Technology Data Exchange (ETDEWEB)

    Reddy, P.V. Narasimha; Jensen, Katherine C.; Mesecar, Andrew D.; Fanwick, Phillip E.; Cushman, Mark (Purdue)

    2012-06-19

    A variety of ammosamide B analogues have been synthesized and evaluated as inhibitors of quinone reductase 2 (QR2). The potencies of the resulting series of QR2 inhibitors range from 4.1 to 25,200 nM. The data provide insight into the structural parameters necessary for QR2 inhibitory activity. The natural product ammosamide B proved to be a potent QR2 inhibitor, and the potencies of the analogues generally decreased as their structures became more distinct from that of ammosamide B. Methylation of the 8-amino group of ammosamide B was an exception, resulting in an increase in quinone reductase 2 inhibitory activity from an IC{sub 50} of 61 nM to IC{sub 50} 4.1 nM.

  14. Deoxynucleoside salvage in fission yeast allows rescue of ribonucleotide reductase deficiency but not Spd1-mediated inhibition of replication

    DEFF Research Database (Denmark)

    Fleck, Oliver; Fahnøe, Ulrik; Løvschal, Katrine Vyff

    2017-01-01

    In fission yeast, the small, intrinsically disordered protein S-phase delaying protein 1 (Spd1) blocks DNA replication and causes checkpoint activation at least in part, by inhibiting the enzyme ribonucleotide reductase, which is responsible for the synthesis of DNA. The CRL4(Cdt2) E3 ubiquitin...... triphosphate (dNTP) pools; yet we find that Spd1 and Spd2 together inhibit DNA replication upon Cdt2 depletion. To directly test whether this block of replication was solely due to insufficient dNTP levels, we established a deoxy-nucleotide salvage pathway in fission yeast by expressing the human nucleoside...... ribonucleotide reductase. However, salvage completely failed to rescue S phase delay, checkpoint activation, and damage sensitivity, which was caused by CRL4(Cdt2) inactivation, suggesting that Spd1-in addition to repressing dNTP synthesis-together with Spd2, can inhibit other replication functions. We propose...

  15. Crystallization and preliminary X-ray diffraction studies of maleylacetate reductase from Rhizobium sp. strain MTP-10005

    Energy Technology Data Exchange (ETDEWEB)

    Fujii, Tomomi; Goda, Yuko [Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011 (Japan); Yoshida, Masahiro; Oikawa, Tadao [Department of Life Science and Biotechnology, Faculty of Chemistry, Materials and Bioengineering, Kansai University, Suita, Osaka 564-8680 (Japan); Hata, Yasuo, E-mail: hata@scl.kyoto-u.ac.jp [Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011 (Japan)

    2008-08-01

    Maleylacetate reductase from Rhizobium sp. strain MTP-10005 has been crystallized using the sitting-drop vapour-diffusion method and microseeding. The crystals contained one dimeric molecule per asymmetric unit and diffracted to 1.79 Å resolution. Maleylacetate reductase (EC 1.3.1.32), which catalyzes the reduction of maleylacetate to 3-oxoadipate, plays an important role in the aerobic microbial catabolism of resorcinol. The enzyme has been crystallized at 293 K by the sitting-drop vapour-diffusion method supplemented with a microseeding technique, using ammonium sulfate as the precipitating agent. The crystal belonged to the monoclinic space group C2, with unit-cell parameters a = 56.85, b = 121.13, c = 94.09 Å, β = 101.48°, and contained one dimeric molecule in the asymmetric unit. It diffracted to 1.79 Å resolution.

  16. Evaluation of Giardia lamblia thioredoxin reductase as drug activating enzyme and as drug target.

    Science.gov (United States)

    Leitsch, David; Müller, Joachim; Müller, Norbert

    2016-12-01

    The antioxidative enzyme thioredoxin reductase (TrxR) has been suggested to be a drug target in several pathogens, including the protist parasite Giardia lamblia. TrxR is also believed to catalyse the reduction of nitro drugs, e.g. metronidazole and furazolidone, a reaction required to render these compounds toxic to G. lamblia and other microaerophiles/anaerobes. It was the objective of this study to assess the potential of TrxR as a drug target in G. lamblia and to find direct evidence for the role of this enzyme in the activation of metronidazole and other nitro drugs. TrxR was overexpressed approximately 10-fold in G. lamblia WB C6 cells by placing the trxR gene behind the arginine deiminase (ADI) promoter on a plasmid. Likewise, a mutant TrxR with a defective disulphide reductase catalytic site was strongly expressed in another G. lamblia WB C6 cell line. Susceptibilities to five antigiardial drugs, i.e. metronidazole, furazolidone, nitazoxanide, albendazole and auranofin were determined in both transfectant cell lines and compared to wildtype. Further, the impact of all five drugs on TrxR activity in vivo was measured. Overexpression of TrxR rendered G. lamblia WB C6 more susceptible to metronidazole and furazolidone but not to nitazoxanide, albendazole, and auranofin. Of all five drugs tested, only auranofin had an appreciably negative effect on TrxR activity in vivo, albeit to a much smaller extent than expected. Overexpression of TrxR and mutant TrxR had hardly any impact on growth of G. lamblia WB C6, although the enzyme also exerts a strong NADPH oxidase activity which is a source of oxidative stress. Our results constitute first direct evidence for the notion that TrxR is an activator of metronidazole and furazolidone but rather question that it is a relevant drug target of presently used antigiardial drugs. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  17. The Human Biliverdin Reductase-based Peptide Fragments and Biliverdin Regulate Protein Kinase Cδ Activity

    Science.gov (United States)

    Miralem, Tihomir; Lerner-Marmarosh, Nicole; Gibbs, Peter E. M.; Tudor, Cicerone; Hagen, Fred K.; Maines, Mahin D.

    2012-01-01

    PKCδ, a Ser/Thr kinase, promotes cell growth, tumorigenesis, and apoptosis. Human biliverdin reductase (hBVR), a Ser/Thr/Tyr kinase, inhibits apoptosis by reducing biliverdin-IX to antioxidant bilirubin. The enzymes are activated by similar stimuli. Reportedly, hBVR is a kinase-independent activator of PKCδ and is transactivated by the PKC (Gibbs, P. E., Miralem, T., Lerner-Marmarosh, N., Tudor, C., and Maines, M. D. (2012) J. Biol. Chem. 287, 1066–1079). Presently, we examined interactions between the two proteins in the context of regulation of their activities and defining targets of hBVR phosphorylation by PKCδ. LC-MS/MS analysis of PKCδ-activated intact hBVR identified phosphorylated serine positions 21, 33, 230, and 237, corresponding to the hBVR Src homology-2 domain motif (Ser230 and Ser237), flanking the ATP-binding motif (Ser21) and in PHPS sequence (Ser33) as targets of PKCδ. Ser21 and Ser230 were also phosphorylated in hBVR-based peptides. The Ser230-containing peptide was a high affinity substrate for PKCδ in vitro and in cells; the relative affinity was PKCδ > PKCβII > PKCζ. Two overlapping peptides spanning this substrate, KRNRYLSF and SFHFKSGSL, were effective inhibitors of PKCδ kinase activity and PKCδ-supported activation of transcription factors Elk1 and NF-κB. Only SFHFKSGSL, in PKCδ-transfected phorbol 12-myristate 13-acetate-stimulated cells, caused membrane blebbing and cell loss. Biliverdin noncovalently inhibited PKCδ, whereas PKCδ potentiated hBVR reductase activity and accelerated the rate of bilirubin formation. This study, together with previous findings, reveals an unexpected regulatory interplay between PKCδ and hBVR in modulating cell death/survival in response to various activating stimuli. In addition, this study has identified novel substrates for and inhibitors of PKCδ. We suggest that hBVR-based technology may have utility to modulate PKCδ-mediated functions in the cell. PMID:22584576

  18. In vivo induction of phase II detoxifying enzymes, glutathione transferase and quinone reductase by citrus triterpenoids

    Directory of Open Access Journals (Sweden)

    Ahmad Hassan

    2010-09-01

    Full Text Available Abstract Background Several cell culture and animal studies demonstrated that citrus bioactive compounds have protective effects against certain types of cancer. Among several classes of citrus bioactive compounds, limonoids were reported to prevent different types of cancer. Furthermore, the structures of citrus limonoids were reported to influence the activity of phase II detoxifying enzymes. The purpose of the study was to evaluate how variations in the structures of citrus limonoids (namely nomilin, deacetyl nomilin, and isoobacunoic acid and a mixture of limonoids would influence phase II enzyme activity in excised tissues from a mouse model. Methods In the current study, defatted sour orange seed powder was extracted with ethyl acetate and subjected to silica gel chromatography. The HPLC, NMR and mass spectra were used to elucidate the purity and structure of compounds. Female A/J mice were treated with three limonoids and a mixture in order to evaluate their effect on phase II enzymes in four different tissues. Assays for glutathione S-transferase and NAD(PH: quinone reductase (QR were used to evaluate induction of phase II enzymatic activity. Results The highest induction of GST against 1-chloro-2,4-dinitrobenzene (CDNB was observed in stomach (whole, 58% by nomilin, followed by 25% isoobacunoic acid and 19% deacetyl nomilin. Deacetyl nomilin in intestine (small as well as liver significantly reduced GST activity against CDNB. Additionally isoobacunoic acid and the limonoid mixture in liver demonstrated a significant reduction of GST activity against CDNB. Nomilin significantly induced GST activity against 4-nitroquinoline 1-oxide (4NQO, intestine (280% and stomach (75% while deacetyl nomilin showed significant induction only in intestine (73%. Induction of GST activity was also observed in intestine (93% and stomach (45% treated with the limonoid mixture. Finally, a significant induction of NAD(PH: quinone reductase (QR activity was

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

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    Josy ter Beek

    Full Text Available 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

  20. 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.