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Sample records for s-nitrosoglutathione reductase gsnor

  1. S-nitrosoglutathione reductase deficiency-induced S-nitrosylation results in neuromuscular dysfunction.

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    Montagna, Costanza; Di Giacomo, Giuseppina; Rizza, Salvatore; Cardaci, Simone; Ferraro, Elisabetta; Grumati, Paolo; De Zio, Daniela; Maiani, Emiliano; Muscoli, Carolina; Lauro, Filomena; Ilari, Sara; Bernardini, Sergio; Cannata, Stefano; Gargioli, Cesare; Ciriolo, Maria R; Cecconi, Francesco; Bonaldo, Paolo; Filomeni, Giuseppe

    2014-08-01

    Nitric oxide (NO) production is implicated in muscle contraction, growth and atrophy, and in the onset of neuropathy. However, many aspects of the mechanism of action of NO are not yet clarified, mainly regarding its role in muscle wasting. Notably, whether NO production-associated neuromuscular atrophy depends on tyrosine nitration or S-nitrosothiols (SNOs) formation is still a matter of debate. Here, we aim at assessing this issue by characterizing the neuromuscular phenotype of S-nitrosoglutathione reductase-null (GSNOR-KO) mice that maintain the capability to produce NO, but are unable to reduce SNOs. We demonstrate that, without any sign of protein nitration, young GSNOR-KO mice show neuromuscular atrophy due to loss of muscle mass, reduced fiber size, and neuropathic behavior. In particular, GSNOR-KO mice show a significant decrease in nerve axon number, with the myelin sheath appearing disorganized and reduced, leading to a dramatic development of a neuropathic phenotype. Mitochondria appear fragmented and depolarized in GSNOR-KO myofibers and myotubes, conditions that are reverted by N-acetylcysteine treatment. Nevertheless, although atrogene transcription is induced, and bulk autophagy activated, no removal of damaged mitochondria is observed. These events, alongside basal increase of apoptotic markers, contribute to persistence of a neuropathic and myopathic state. Our study provides the first evidence that GSNOR deficiency, which affects exclusively SNOs reduction without altering nitrotyrosine levels, results in a clinically relevant neuromuscular phenotype. These findings provide novel insights into the involvement of GSNOR and S-nitrosylation in neuromuscular atrophy and neuropathic pain that are associated with pathological states; for example, diabetes and cancer.

  2. ROS-mediated inhibition of S-nitrosoglutathione reductase contributes to the activation of anti-oxidative mechanisms

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    Izabella Kovacs

    2016-11-01

    Full Text Available Nitric oxide (NO has emerged as a signaling molecule in plants being involved in diverse physiological processes like germination, root growth, stomata closing and response to biotic and abiotic stress. S-nitrosoglutathione (GSNO as a biological NO donor has a very important function in NO signaling since it can transfer its NO moiety to other proteins (trans-nitrosylation. Such trans-nitrosylation reactions are equilibrium reactions and depend on GSNO level. The breakdown of GSNO and thus the level of S-nitrosylated proteins are regulated by GSNO-reductase (GSNOR. In this way, this enzyme controls S-nitrosothiol levels and regulates NO signaling. Here we report that Arabidopsis thaliana GSNOR activity is reversibly inhibited by H2O2 in-vitro and by paraquat-induced oxidative stress in-vivo. Light scattering analyses of reduced and oxidized recombinant GSNOR demonstrated that GSNOR proteins form dimers under both reducing and oxidizing conditions. Moreover, mass spectrometric analyses revealed that H2O2-treatment increased the amount of oxidative modifications on Zn2+-coordinating Cys47 and Cys177. Inhibition of GSNOR results in enhanced levels of S-nitrosothiols followed by accumulation of glutathione. Moreover, transcript levels of redox-regulated genes and activities of glutathione-dependent enzymes are increased in gsnor-ko plants, which may contribute to the enhanced resistance against oxidative stress. In sum, our results demonstrate that ROS-dependent inhibition of GSNOR is playing an important role in activation of anti-oxidative mechanisms to damping oxidative damage and imply a direct crosstalk between ROS- and NO-signaling.

  3. Arsenic triggers the nitric oxide (NO) and S-nitrosoglutathione (GSNO) metabolism in Arabidopsis

    International Nuclear Information System (INIS)

    Leterrier, Marina; Airaki, Morad; Palma, José M.; Chaki, Mounira; Barroso, Juan B.; Corpas, Francisco J.

    2012-01-01

    Environmental contamination by arsenic constitutes a problem in many countries, and its accumulation in food crops may pose health complications for humans. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are involved at various levels in the mechanism of responding to environmental stress in higher plants. Using Arabidopsis seedlings exposed to different arsenate concentrations, physiological and biochemical parameters were analyzed to determine the status of ROS and RNS metabolisms. Arsenate provoked a significant reduction in growth parameters and an increase in lipid oxidation. These changes were accompanied by an alteration in antioxidative enzymes and the nitric oxide (NO) metabolism, with a significant increase in NO content, S-nitrosoglutathione reductase (GSNOR) activity and protein tyrosine nitration as well as a concomitant reduction in glutathione and S-nitrosoglutathione (GSNO) content. Our results indicate that 500 μM arsenate (AsV) causes nitro-oxidative stress in Arabidopsis, being the glutathione reductase and the GSNOR activities clearly affected. - Highlights: ► In Arabidopsis, arsenate provokes damages in the membrane integrity of root cells. ► As induces an oxidative stress according to an increase in lipid oxidation. ► NO content and protein tyrosine nitration increases under arsenate stress. ► Arsenate provokes a reduction of GSH, GSSG and GSNO content. ► Arsenate induces a nitro-oxidative stress in Arabidopsis. - Arsenic stress affects nitric oxide (NO) and glutathione (GSH) metabolism which provokes a nitro-oxidative stress.

  4. GSNOR Deficiency Enhances In Situ Skeletal Muscle Strength, Fatigue Resistance, and RyR1 S-Nitrosylation Without Impacting Mitochondrial Content and Activity

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    Moon, Younghye; Cao, Yenong; Zhu, Jingjing; Xu, Yuanyuan; Balkan, Wayne; Buys, Emmanuel S.; Diaz, Francisca; Kerrick, W. Glenn; Hare, Joshua M.

    2017-01-01

    Abstract Aim: Nitric oxide (NO) plays important, but incompletely defined roles in skeletal muscle. NO exerts its regulatory effects partly though S-nitrosylation, which is balanced by denitrosylation by enzymes such as S-nitrosoglutathione reductase (GSNOR), whose functions in skeletal muscle remain to be fully deciphered. Results: GSNOR null (GSNOR−/−) tibialis anterior (TA) muscles showed normal growth and were stronger and more fatigue resistant than controls in situ. However, GSNOR−/− lumbrical muscles showed normal contractility and Ca2+ handling in vitro, suggesting important differences in GSNOR function between muscles or between in vitro and in situ environments. GSNOR−/− TA muscles exhibited normal mitochondrial content, and capillary densities, but reduced type IIA fiber content. GSNOR inhibition did not impact mitochondrial respiratory complex I, III, or IV activities. These findings argue that enhanced GSNOR−/− TA contractility is not driven by changes in mitochondrial content or activity, fiber type, or blood vessel density. However, loss of GSNOR led to RyR1 hypernitrosylation, which is believed to increase muscle force output under physiological conditions. cGMP synthesis by soluble guanylate cyclase (sGC) was decreased in resting GSNOR−/− muscle and was more responsive to agonist (DETANO, BAY 41, and BAY 58) stimulation, suggesting that GSNOR modulates cGMP production in skeletal muscle. Innovation: GSNOR may act as a “brake” on skeletal muscle contractile performance under physiological conditions by modulating nitrosylation/denitrosylation balance. Conclusions: GSNOR may play important roles in skeletal muscle contractility, RyR1 S-nitrosylation, fiber type specification, and sGC activity. Antioxid. Redox Signal. 26, 165–181. PMID:27412893

  5. S-Nitrosoglutathione is a component of wound- and salicylic acid-induced systemic responses in Arabidopsis thaliana.

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    Espunya, M Carme; De Michele, Roberto; Gómez-Cadenas, Aurelio; Martínez, M Carmen

    2012-05-01

    S-Nitrosoglutathione (GSNO) is a bioactive, stable, and mobile reservoir of nitric oxide (NO), and an important player in defence responses to herbivory and pathogen attack in plants. It has been demonstrated previously that GSNO reductase (GSNOR) is the main enzyme responsible for the in vivo control of intracellular levels of GSNO. In this study, the role of S-nitrosothiols, in particular of GSNO, in systemic defence responses in Arabidopsis thaliana was investigated further. It was shown that GSNO levels increased rapidly and uniformly in injured Arabidopsis leaves, whereas in systemic leaves GSNO was first detected in vascular tissues and later spread over the parenchyma, suggesting that GSNO is involved in the transmission of the wound mobile signal through the vascular tissue. Moreover, GSNO accumulation was required to activate the jasmonic acid (JA)-dependent wound responses, whereas the alternative JA-independent wound-signalling pathway did not involve GSNO. Furthermore, extending previous work on the role of GSNOR in pathogenesis, it was shown that GSNO acts synergistically with salicylic acid in systemic acquired resistance activation. In conclusion, GSNOR appears to be a key regulator of systemic defence responses, in both wounding and pathogenesis.

  6. Mechanical wounding induces a nitrosative stress by down-regulation of GSNO reductase and an increase in S-nitrosothiols in sunflower (Helianthus annuus) seedlings

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    Chaki, Mounira; Valderrama, Raquel; Fernández-Ocaña, Ana M.; Carreras, Alfonso; Gómez-Rodríguez, Maria. V.; Pedrajas, José R.; Begara-Morales, Juan C.; Sánchez-Calvo, Beatriz; Luque, Francisco; Leterrier, Marina; Corpas, Francisco J.; Barroso, Juan B.

    2011-01-01

    Nitric oxide (NO) and related molecules such as peroxynitrite, S-nitrosoglutathione (GSNO), and nitrotyrosine, among others, are involved in physiological processes as well in the mechanisms of response to stress conditions. In sunflower seedlings exposed to five different adverse environmental conditions (low temperature, mechanical wounding, high light intensity, continuous light, and continuous darkness), key components of the metabolism of reactive nitrogen species (RNS) and reactive oxygen species (ROS), including the enzyme activities L-arginine-dependent nitric oxide synthase (NOS), S-nitrosogluthathione reductase (GSNOR), nitrate reductase (NR), catalase, and superoxide dismutase, the content of lipid hydroperoxide, hydrogen peroxide, S-nitrosothiols (SNOs), the cellular level of NO, GSNO, and GSNOR, and protein tyrosine nitration [nitrotyrosine (NO2-Tyr)] were analysed. Among the stress conditions studied, mechanical wounding was the only one that caused a down-regulation of NOS and GSNOR activities, which in turn provoked an accumulation of SNOs. The analyses of the cellular content of NO, GSNO, GSNOR, and NO2-Tyr by confocal laser scanning microscopy confirmed these biochemical data. Therefore, it is proposed that mechanical wounding triggers the accumulation of SNOs, specifically GSNO, due to a down-regulation of GSNOR activity, while NO2-Tyr increases. Consequently a process of nitrosative stress is induced in sunflower seedlings and SNOs constitute a new wound signal in plants. PMID:21172815

  7. Excessive Cellular S-nitrosothiol Impairs Endocytosis of Auxin Efflux Transporter PIN2

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    Min Ni

    2017-11-01

    Full Text Available S-nitrosoglutathione reductase (GSNOR1 is the key enzyme that regulates cellular levels of S-nitrosylation across kingdoms. We have previously reported that loss of GSNOR1 resulted in impaired auxin signaling and compromised auxin transport in Arabidopsis, leading to the auxin-related morphological phenotypes. However, the molecular mechanism underpinning the compromised auxin transport in gsnor1-3 mutant is still unknown. Endocytosis of plasma-membrane (PM-localized efflux PIN proteins play critical roles in auxin transport. Therefore, we investigate whether loss of GSNOR1 function has any effects on the endocytosis of PIN-FORMED (PIN proteins. It was found that the endocytosis of either the endogenous PIN2 or the transgenically expressed PIN2-GFP was compromised in the root cells of gsnor1-3 seedlings relative to Col-0. The internalization of PM-associated PIN2 or PIN2-GFP into Brefeldin A (BFA bodies was significantly reduced in gsnor1-3 upon BFA treatment in a manner independent of de novo protein synthesis. In addition, the exogenously applied GSNO not only compromised the endocytosis of PIN2-GFP but also inhibited the root elongation in a concentration-dependent manner. Taken together, our results indicate that, besides the reduced PIN2 level, one or more compromised components in the endocytosis pathway could account for the reduced endocytosis of PIN2 in gsnor1-3.

  8. Neuroprotective properties of nitric oxide and S-nitrosoglutathione

    International Nuclear Information System (INIS)

    Rauhala, Pekka; Andoh, Tsugunobu; Chiueh, C.C.

    2005-01-01

    Oxidative stress and apoptosis may play an important role in the neurodegeneration. The present paper outlines antioxidative and antiapototic mechanisms of nitric oxide and S-nitrosothiols, which could mediate neuroprotection. Nitric oxide generated by nitric oxide synthase or released from an endogenous S-nitrosothiol, S-nitrosoglutathione may up-regulate antioxidative thioredoxin system and antiapototic Bcl-2 protein through a cGMP-dependent mechanism. Moreover, nitric oxide radicals have been shown to have direct antioxidant effect through their reaction with free radicals and iron-oxygen complexes. In addition to serving as a stabilizer and carrier of nitric oxide, S-nitrosoglutathione may have protective effect through transnitrosylation reactions. Based on these new findings, a hypothesis arises that the homeostasis of nitric oxide, S-nitrosothiols, glutathione, and thioredoxin systems is important for protection against oxidative stress, apoptosis, and related neurodegenerative disorders

  9. S-Nitrosomycothiol Reductase and Mycothiol Are Required for Survival Under Aldehyde Stress and Biofilm Formation in Mycobacterium smegmatis

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    Vargas, Derek; Hageman, Samantha; Gulati, Megha; Nobile, Clarissa J.; Rawat, Mamta

    2017-01-01

    We show that Mycobacterium smegmatis mutants disrupted in mscR, coding for a dual function S-nitrosomycothiol reductase and formaldehyde dehydrogenase, and mshC, coding for a mycothiol ligase and lacking mycothiol (MSH), are more susceptible to S-nitrosoglutathione (GSNO) and aldehydes than wild type. MSH is a cofactor for MscR, and both mshC and mscR are induced by GSNO and aldehydes. We also show that a mutant disrupted in egtA, coding for a γ-glutamyl cysteine synthetase and lacking in ergothioneine, is sensitive to nitrosative stress but not to aldehydes. In addition, we find that MSH and S-nitrosomycothiol reductase are required for normal biofilm formation in M. smegmatis, suggesting potential new therapeutic pathways to target to inhibit or disrupt biofilm formation. PMID:27321674

  10. Effect of exogenous nitric oxide on antioxidative system and S-nitrosylation in leaves of Boehmeria nivea (L.) Gaud under cadmium stress.

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    Wang, Dafei; Liu, Yunguo; Tan, Xiaofei; Liu, Hongyu; Zeng, Guangming; Hu, Xinjiang; Jian, Hao; Gu, Yanling

    2015-03-01

    Cadmium (Cd)-induced growth inhibition is one of the primary factors limiting phytoremediation effect of Boehmeria nivea (L.) Gaud in contaminated soil. Sodium nitroprusside (SNP), a donor of nitric oxide (NO), has been evidenced to alleviate Cd toxicity in many plants. However, as an important mechanism of NO in orchestrating cellular functions, S-nitrosylation is still poorly understood in its relation with Cd tolerance of plants. In this study, higher exogenous NO levels were found to coincide with higher S-nitrosylation level expressed as content of S-nitrosothiols (SNO). The addition of low concentration (100 μM) SNP increased the SNO content, and it simultaneously induced an alleviating effect against Cd toxicity by enhancing the activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), and glutathione reductase (GR) and reduced the accumulation of H2O2 as compared with Cd alone. Application of S-nitrosoglutathione reductase (GSNOR) inhibitors dodecanoic acid (DA) in 100 μM SNP group brought in an extra elevation in S-nitrosylation level and further reinforced the effect of SNP. While the additions of 400 μM SNP and 400 μM SNP + 50 μM DA further elevated the S-nitrosylation level, it markedly weakened the alleviating effect against Cd toxicity as compared with the addition of 100 μM SNP. This phenomenon could be owing to excess consumption of glutathione (GSH) to form SNO under high S-nitrosylation level. Therefore, the present study indicates that S-nitrosylation is involved in the ameliorating effect of SNP against Cd toxicity. This involvement exhibited a concentration-dependent property.

  11. Interaction between neuronal nitric oxide synthase signaling and temperature influences sarcoplasmic reticulum calcium leak: role of nitroso-redox balance.

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    Dulce, Raul A; Mayo, Vera; Rangel, Erika B; Balkan, Wayne; Hare, Joshua M

    2015-01-02

    Although nitric oxide (NO) signaling modulates cardiac function and excitation-contraction coupling, opposing results because of inconsistent experimental conditions, particularly with respect to temperature, confound the ability to elucidate NO signaling pathways. Here, we show that temperature significantly modulates NO effects. To test the hypothesis that temperature profoundly affects nitroso-redox equilibrium, thereby affecting sarcoplasmic reticulum (SR) calcium (Ca(2+)) leak. We measured SR Ca(2+) leak in cardiomyocytes from wild-type (WT), NO/redox imbalance (neuronal nitric oxide synthase-deficient mice-1 [NOS1(-/-)]), and hyper S-nitrosoglutathione reductase-deficient (GSNOR(-/-)) mice. In WT cardiomyocytes, SR Ca(2+) leak increased because temperature decreased from 37°C to 23°C, whereas in NOS1(-/-) cells, the leak suddenly increased when the temperature surpassed 30°C. GSNOR(-/-) cardiomyocytes exhibited low leak throughout the temperature range. Exogenously added NO had a biphasic effect on NOS1(-/-) cardiomyocytes; reducing leak at 37°C but increasing it at subphysiological temperatures. Oxypurinol and Tempol diminished the leak in NOS1(-/-) cardiomyocytes. Cooling from 37°C to 23°C increased reactive oxygen species generation in WT but decreased it in NOS1(-/-) cardiomyocytes. Oxypurinol further reduced reactive oxygen species generation. At 23°C in WT cells, leak was decreased by tetrahydrobiopterin, an essential NOS cofactor. Cooling significantly increased SR Ca(2+) content in NOS1(-/-) cells but had no effect in WT or GSNOR(-/-). Ca(2+) leak and temperature are normally inversely proportional, whereas NOS1 deficiency reverses this effect, increasing leak and elevating reactive oxygen species production because temperature increases. Reduced denitrosylation (GSNOR deficiency) eliminates the temperature dependence of leak. Thus, temperature regulates the balance between NO and reactive oxygen species which in turn has a major effect on SR

  12. Regulation of reactive oxygen and nitrogen species by salicylic acid in rice plants under salinity stress conditions

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    Mun, Bong-Gyu; Khan, Abdul Latif; Waqas, Muhammad; Kim, Hyun-Ho; Shahzad, Raheem; Imran, Muhammad

    2018-01-01

    This study investigated the regulatory role of exogenous salicylic acid (SA) in rice and its effects on toxic reactive oxygen and nitrogen species during short-term salinity stress. SA application (0.5 and 1.0 mM) during salinity-induced stress (100 mM NaCl) resulted in significantly longer shoot length and higher chlorophyll and biomass accumulation than with salinity stress alone. NaCl-induced reactive oxygen species production led to increased levels of lipid peroxidation in rice plants, which were significantly reduced following SA application. A similar finding was observed for superoxide dismutase; however, catalase (CAT) and ascorbate peroxidase (APX) were significantly reduced in rice plants treated with SA and NaCl alone and in combination. The relative mRNA expression of OsCATA and OsAPX1 was lower in rice plants during SA stress. Regarding nitrogenous species, S-nitrosothiol (SNO) was significantly reduced initially (one day after treatment [DAT]) but then increased in plants subjected to single or combined stress conditions. Genes related to SNO biosynthesis, S-nitrosoglutathione reductase (GSNOR1), NO synthase-like activity (NOA), and nitrite reductase (NIR) were also assessed. The mRNA expression of GSNOR1 was increased relative to that of the control, whereas OsNOA was expressed at higher levels in plants treated with SA and NaCl alone relative to the control. The mRNA expression of OsNR was decreased in plants subjected to single or combination treatment, except at 2 DAT, compared to the control. In conclusion, the current findings suggest that SA can regulate the generation of NaCl-induced oxygen and nitrogen reactive species in rice plants. PMID:29558477

  13. Dual Labeling Biotin Switch Assay to Reduce Bias Derived From Different Cysteine Subpopulations: A Method to Maximize S-Nitrosylation Detection.

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    Chung, Heaseung Sophia; Murray, Christopher I; Venkatraman, Vidya; Crowgey, Erin L; Rainer, Peter P; Cole, Robert N; Bomgarden, Ryan D; Rogers, John C; Balkan, Wayne; Hare, Joshua M; Kass, David A; Van Eyk, Jennifer E

    2015-10-23

    S-nitrosylation (SNO), an oxidative post-translational modification of cysteine residues, responds to changes in the cardiac redox-environment. Classic biotin-switch assay and its derivatives are the most common methods used for detecting SNO. In this approach, the labile SNO group is selectively replaced with a single stable tag. To date, a variety of thiol-reactive tags have been introduced. However, these methods have not produced a consistent data set, which suggests an incomplete capture by a single tag and potentially the presence of different cysteine subpopulations. To investigate potential labeling bias in the existing methods with a single tag to detect SNO, explore if there are distinct cysteine subpopulations, and then, develop a strategy to maximize the coverage of SNO proteome. We obtained SNO-modified cysteine data sets for wild-type and S-nitrosoglutathione reductase knockout mouse hearts (S-nitrosoglutathione reductase is a negative regulator of S-nitrosoglutathione production) and nitric oxide-induced human embryonic kidney cell using 2 labeling reagents: the cysteine-reactive pyridyldithiol and iodoacetyl based tandem mass tags. Comparison revealed that <30% of the SNO-modified residues were detected by both tags, whereas the remaining SNO sites were only labeled by 1 reagent. Characterization of the 2 distinct subpopulations of SNO residues indicated that pyridyldithiol reagent preferentially labels cysteine residues that are more basic and hydrophobic. On the basis of this observation, we proposed a parallel dual-labeling strategy followed by an optimized proteomics workflow. This enabled the profiling of 493 SNO sites in S-nitrosoglutathione reductase knockout hearts. Using a protocol comprising 2 tags for dual-labeling maximizes overall detection of SNO by reducing the previously unrecognized labeling bias derived from different cysteine subpopulations. © 2015 American Heart Association, Inc.

  14. Synthesis of S-[13N]nitrosoglutathione (13N-GSNO) as a new potential PET imaging agent

    International Nuclear Information System (INIS)

    Llop, Jordi; Gomez-Vallejo, Vanessa; Bosque, Marta; Quincoces, Gemma; Penuelas, Ivan

    2009-01-01

    In the present paper, a fast and reproducible method for the synthesis of S-[ 13 N]nitrosoglutathione is reported for the first time. The labeling strategy is based on the production of [ 13 N]NO 3 - via the 16 O(p,α) 13 N nuclear reaction in water, as opposed to the standardized production of [ 13 N]NH 4 + in 2 mM aqueous ethanol. Following the reduction of [ 13 N]NO 3 - to [ 13 N]NO 2 - , the reaction with glutathione in the presence of hydrochloric acid led to the desired radiotracer with a good radiochemical yield (24.2±2.0% end of synthesis, n=5) in a short production time (3 min from the end of bombardment)

  15. The effect of risk elements in soil to nitric oxide metabolism in tobacco plants

    Czech Academy of Sciences Publication Activity Database

    Procházková, Dagmar; Haisel, Daniel; Pavlíková, D.; Schnablová, Renáta; Száková, J.; Vytášek, R.; Wilhelmová, Naděžda

    2012-01-01

    Roč. 58, č. 10 (2012), s. 435-440 ISSN 1214-1178 R&D Projects: GA ČR(CZ) GAP501/11/1239 Institutional research plan: CEZ:AV0Z50380511 Keywords : nitrate reductase * nitrosoglutathione reductase * nitrotyrosine Subject RIV: EF - Botanics Impact factor: 1.113, year: 2012 http://www.agriculturejournals.cz/publicFiles/75132.pdf

  16. Contribution of Fdh3 and Glr1 to Glutathione Redox State, Stress Adaptation and Virulence in Candida albicans.

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    Anna T Tillmann

    Full Text Available The major fungal pathogen of humans, Candida albicans, is exposed to reactive nitrogen and oxygen species following phagocytosis by host immune cells. In response to these toxins, this fungus activates potent anti-stress responses that include scavenging of reactive nitrosative and oxidative species via the glutathione system. Here we examine the differential roles of two glutathione recycling enzymes in redox homeostasis, stress adaptation and virulence in C. albicans: glutathione reductase (Glr1 and the S-nitrosoglutathione reductase (GSNOR, Fdh3. We show that the NADPH-dependent Glr1 recycles GSSG to GSH, is induced in response to oxidative stress and is required for resistance to macrophage killing. GLR1 deletion increases the sensitivity of C. albicans cells to H2O2, but not to formaldehyde or NO. In contrast, Fdh3 detoxifies GSNO to GSSG and NH3, and FDH3 inactivation delays NO adaptation and increases NO sensitivity. C. albicans fdh3⎔ cells are also sensitive to formaldehyde, suggesting that Fdh3 also contributes to formaldehyde detoxification. FDH3 is induced in response to nitrosative, oxidative and formaldehyde stress, and fdh3Δ cells are more sensitive to killing by macrophages. Both Glr1 and Fdh3 contribute to virulence in the Galleria mellonella and mouse models of systemic infection. We conclude that Glr1 and Fdh3 play differential roles during the adaptation of C. albicans cells to oxidative, nitrosative and formaldehyde stress, and hence during the colonisation of the host. Our findings emphasise the importance of the glutathione system and the maintenance of intracellular redox homeostasis in this major pathogen.

  17. Polymer nanocomposite particles of S-nitrosoglutathione: A suitable formulation for protection and sustained oral delivery.

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    Wu, Wen; Gaucher, Caroline; Fries, Isabelle; Hu, Xian-Ming; Maincent, Philippe; Sapin-Minet, Anne

    2015-11-10

    S-nitrosoglutathione (GSNO) is a nitric oxide (NO) donor with therapeutic potential for cardiovascular disease treatment. Chronic oral treatment with GSNO is limited by high drug sensitivity to the environment and limited oral bioavailability, requiring the development of delivery systems able to sustain NO release. The present work describes new platforms based on polymer nanocomposite particles for the delivery of GSNO. Five types of optimized nanocomposite particles have been developed (three based on chitosan, two based on alginate sodium). Those nanocomposite particles encapsulate GSNO with high efficiency from 64% to 70% and an average size of 13 to 61 μm compatible with oral delivery. Sustained release of GSNO in vitro was achieved. Indeed, chitosan nanocomposites discharged their payload within 24h; whereas alginate nanocomposites released GSNO more slowly (10% of GSNO was still remaining in the dosage form after 24h). Their cytocompatibility toward intestinal Caco-2 cells (MTT assay) was acceptable (IC50: 6.07 ± 0.07-9.46 ± 0.08 mg/mL), demonstrating their suitability as oral delivery systems for GSNO. These delivery systems presented efficient GSNO loading and sustained release as well as cytocompatibility, showing their promise as a means of improving the oral bioavailability of GSNO and as a potential new treatment. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. The nitric oxide-sensitive p21Ras-ERK pathway mediates S-nitrosoglutathione-induced apoptosis

    International Nuclear Information System (INIS)

    Tsujita, Maristela; Batista, Wagner L.; Ogata, Fernando T.; Stern, Arnold; Monteiro, Hugo P.; Arai, Roberto J.

    2008-01-01

    p21Ras protein plays a critical role in cellular signaling that induces either cell cycle progression or apoptosis. Nitric oxide (NO) has been consistently reported to activate p21Ras through the redox sensitive cysteine residue (118). In this study, we demonstrated that the p21Ras-ERK pathway regulates THP-1 monocyte/macrophage apoptosis induced by S-nitrosoglutathione (SNOG). This was apparent from studies in THP-1 cells expressing NO-insensitive p21Ras (p21Ras C118S ) where the pro-apoptotic action of SNOG was almost abrogated. Three major MAP kinase pathways (ERK, JNK, and p38) that are downstream to p21Ras were investigated. It was observed that only the activation of ERK1/2 MAP kinases by SNOG in THP-1 cells was attributable to p21Ras. The inhibition of the ERK pathway by PD98059 markedly attenuated apoptosis in SNOG-treated THP-1 cells, but had a marginal effect on SNOG-treated THP-1 cells expressing NO-insensitive p21Ras. The inhibition of the JNK and p38 pathways by selective inhibitors had no marked effects on the percentage of apoptosis. The induction of p21Waf1 expression by SNOG was observed in THP-1 cells harboring mutant and wild-type p21Ras, however in cells expressing mutant Ras, the expression of p21Waf1 was significantly attenuated. The treatment of THP-1 cells expressing wild-type p21Ras with PD98059 resulted in significant attenuation of p21Waf1 expression. These results indicate that the redox sensitive p21Ras-ERK pathway plays a critical role in sensing and delivering the pro-apoptotic signaling mediated by SNOG

  19. Polymer nanocomposites enhance S-nitrosoglutathione intestinal absorption and promote the formation of releasable nitric oxide stores in rat aorta.

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    Wu, Wen; Perrin-Sarrado, Caroline; Ming, Hui; Lartaud, Isabelle; Maincent, Philippe; Hu, Xian-Ming; Sapin-Minet, Anne; Gaucher, Caroline

    2016-10-01

    Alginate/chitosan nanocomposite particles (GSNO-acNCPs), i.e. S-nitrosoglutathione (GSNO) loaded polymeric nanoparticles incorporated into an alginate and chitosan matrix, were developed to increase the effective GSNO loading capacity, a nitric oxide (NO) donor, and to sustain its release from the intestine following oral administration. Compared with free GSNO and GSNO loaded nanoparticles, GSNO-acNCPs promoted 2.7-fold GSNO permeation through a model of intestinal barrier (Caco-2 cells). After oral administration to Wistar rats, GSNO-acNCPs promoted NO storage into the aorta during at least 17h, as highlighted by (i) a long-lasting hyporeactivity to phenylephrine (decrease in maximum vasoconstrictive effect of aortic rings) and (ii) N-acetylcysteine (a thiol which can displace NO from tissues)-induced vasodilation of aorxxtic rings preconstricted with phenylephrine. In conclusion, GSNO-acNCPs enhance GSNO intestinal absorption and promote the formation of releasable NO stores into the rat aorta. GSNO-acNCPs are promising carriers for chronic oral application devoted to the treatment of cardiovascular diseases. Copyright © 2016. Published by Elsevier Inc.

  20. Automated Online Solid-Phase Derivatization for Sensitive Quantification of Endogenous S-Nitrosoglutathione and Rapid Capture of Other Low-Molecular-Mass S-Nitrosothiols.

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    Wang, Xin; Garcia, Carlos T; Gong, Guanyu; Wishnok, John S; Tannenbaum, Steven R

    2018-02-06

    S-Nitrosothiols (RSNOs) constitute a circulating endogenous reservoir of nitric oxide and have important biological activities. In this study, an online coupling of solid-phase derivatization (SPD) with liquid chromatography-mass spectrometry (LC-MS) was developed and applied in the analysis of low-molecular-mass RSNOs. A derivatizing-reagent-modified polymer monolithic column was prepared and adapted for online SPD-LC-MS. Analytes from the LC autosampler flowed through the monolithic column for derivatization and then directly into the LC-MS for analysis. This integration of the online derivatization, LC separation, and MS detection facilitated system automation, allowing rapid, laborsaving, and sensitive detection of RSNOs. S-Nitrosoglutathione (GSNO) was quantified using this automated online method with good linearity (R 2 = 0.9994); the limit of detection was 0.015 nM. The online SPD-LC-MS method has been used to determine GSNO levels in mouse samples, 138 ± 13.2 nM of endogenous GSNO was detected in mouse plasma. Besides, the GSNO concentrations in liver (64.8 ± 11.3 pmol/mg protein), kidney (47.2 ± 6.1 pmol/mg protein), heart (8.9 ± 1.8 pmol/mg protein), muscle (1.9 ± 0.3 pmol/mg protein), hippocampus (5.3 ± 0.9 pmol/mg protein), striatum (6.7 ± 0.6 pmol/mg protein), cerebellum (31.4 ± 6.5 pmol/mg protein), and cortex (47.9 ± 4.6 pmol/mg protein) were also successfully quantified. When the derivatization was performed within 8 min, followed by LC-MS detection, samples could be rapidly analyzed compared with the offline manual method. Other low-molecular-mass RSNOs, such as S-nitrosocysteine and S-nitrosocysteinylglycine, were captured by rapid precursor-ion scanning, showing that the proposed method is a potentially powerful tool for capture, identification, and quantification of RSNOs in biological samples.

  1. S-nitrosoglutathione reduces tau hyper-phosphorylation and provides neuroprotection in rat model of chronic cerebral hypoperfusion.

    Science.gov (United States)

    Won, Je-Seong; Annamalai, Balasubramaniam; Choi, Seungho; Singh, Inderjit; Singh, Avtar K

    2015-10-22

    We have previously reported that treatment of rats subjected to permanent bilateral common carotid artery occlusion (pBCCAO), a model of chronic cerebral hypoperfusion (CCH), with S-nitrosoglutathione (GSNO), an endogenous nitric oxide carrier, improved cognitive functions and decreased amyloid-β accumulation in the brains. Since CCH has been implicated in tau hyperphosphorylation induced neurodegeneration, we investigated the role of GSNO in regulation of tau hyperphosphorylation in rat pBCCAO model. The rats subjected to pBCCAO had a significant increase in tau hyperphosphorylation with increased neuronal loss in hippocampal/cortical areas. GSNO treatment attenuated not only the tau hyperphosphorylation, but also the neurodegeneration in pBCCAO rat brains. The pBCCAO rat brains also showed increased activities of GSK-3β and Cdk5 (major tau kinases) and GSNO treatment significantly attenuated their activities. GSNO attenuated the increased calpain activities and calpain-mediated cleavage of p35 leading to production of p25 and aberrant Cdk5 activation. In in vitro studies using purified calpain protein, GSNO treatment inhibited calpain activities while 3-morpholinosydnonimine (a donor of peroxynitrite) treatment increased its activities, suggesting the opposing role of GSNO vs. peroxynitrite in regulation of calpain activities. In pBCCAO rat brains, GSNO treatment attenuated the expression of inducible nitric oxide synthase (iNOS) expression and also reduced the brain levels of nitro-tyrosine formation, thereby indicating the protective role of GSNO in iNOS/nitrosative-stress mediated calpain/tau pathologies under CCH conditions. Taken together with our previous report, these data support the therapeutic potential of GSNO, a biological NO carrier, as a neuro- and cognitive-protective agent under conditions of CCH. Published by Elsevier B.V.

  2. Characterisation of PduS, the pdu metabolosome corrin reductase, and evidence of substructural organisation within the bacterial microcompartment.

    Directory of Open Access Journals (Sweden)

    Joshua B Parsons

    2010-11-01

    Full Text Available PduS is a corrin reductase and is required for the reactivation of the cobalamin-dependent diol dehydratase. It is one component encoded within the large propanediol utilisation (pdu operon, which is responsible for the catabolism of 1,2-propanediol within a self-assembled proteinaceous bacterial microcompartment. The enzyme is responsible for the reactivation of the cobalamin coenzyme required by the diol dehydratase. The gene for the cobalamin reductase from Citrobacter freundii (pduS has been cloned to allow the protein to be overproduced recombinantly in E. coli with an N-terminal His-tag. Purified recombinant PduS is shown to be a flavoprotein with a non-covalently bound FMN that also contains two coupled [4Fe-4S] centres. It is an NADH-dependent flavin reductase that is able to mediate the one-electron reductions of cob(IIIalamin to cob(IIalamin and cob(IIalamin to cob(Ialamin. The [4Fe-4S] centres are labile to oxygen and their presence affects the midpoint redox potential of flavin. Evidence is presented that PduS is able to bind cobalamin, which is inconsistent with the view that PduS is merely a flavin reductase. PduS is also shown to interact with one of the shell proteins of the metabolosome, PduT, which is also thought to contain an [Fe-S] cluster. PduS is shown to act as a corrin reductase and its interaction with a shell protein could allow for electron passage out of the bacterial microcompartment.

  3. S-nitrosylation of the mitochondrial chaperone TRAP1 sensitizes hepatocellular carcinoma cells to inhibitors of succinate dehydrogenase

    DEFF Research Database (Denmark)

    Rizza, Salvatore; Montagna, Costanza; Cardaci, Simone

    2016-01-01

    growth and differentiation, inflammation, and metabolism. Chromosomal deletion of GSNOR results in pathologic protein S-nitrosylation that is implicated in human hepatocellular carcinoma (HCC). Here we identify a metabolic hallmark of aberrant S-nitrosylation in HCC and exploit it for therapeutic gain...

  4. Ketopantoyl-lactone reductase from Candida parapsilosis: purification and characterization as a conjugated polyketone reductase.

    Science.gov (United States)

    Hata, H; Shimizu, S; Hattori, S; Yamada, H

    1989-02-24

    Ketopantoyl-lactone reductase (2-dehydropantoyl-lactone reductase, EC 1.1.1.168) was purified and crystallized from cells of Candida parapsilosis IFO 0708. The enzyme was found to be homogeneous on ultracentrifugation, high-performance gel-permeation liquid chromatography and SDS-polyacrylamide gel electrophoresis. The relative molecular mass of the native and SDS-treated enzyme is approximately 40,000. The isoelectric point of the enzyme is 6.3. The enzyme was found to catalyze specifically the reduction of a variety of natural and unnatural polyketones and quinones other than ketopantoyl lactone in the presence of NADPH. Isatin and 5-methylisatin are rapidly reduced by the enzyme, the Km and Vmax values for isatin being 14 microM and 306 mumol/min per mg protein, respectively. Ketopantoyl lactone is also a good substrate (Km = 333 microM and Vmax = 481 mumol/min per mg protein). Reverse reaction was not detected with pantoyl lactone and NADP+. The enzyme is inhibited by quercetin, several polyketones and SH-reagents. 3,4-Dihydroxy-3-cyclobutene-1,2-dione, cyclohexenediol-1,2,3,4-tetraone and parabanic acid are uncompetitive inhibitors for the enzyme, the Ki values being 1.4, 0.2 and 3140 microM, respectively, with isatin as substrate. Comparison of the enzyme with the conjugated polyketone reductase of Mucor ambiguus (S. Shimizu, H. Hattori, H. Hata and H. Yamada (1988) Eur. J. Biochem. 174, 37-44) and ketopantoyl-lactone reductase of Saccharomyces cerevisiae suggested that ketopantoyl-lactone reductase is a kind of conjugated polyketone reductase.

  5. Ketopantoyl lactone reductase is a conjugated polyketone reductase.

    Science.gov (United States)

    Hata, H; Shimizu, S; Hattori, S; Yamada, H

    1989-03-01

    Ketopantoyl lactone reductase (EC 1.1.1.168) of Saccharomyces cerevisiae was found to catalyze the reduction of a variety of natural and unnatural conjugated polyketone compounds and quinones, such as isatin, ninhydrin, camphorquinone and beta-naphthoquinone in the presence of NADPH. 5-Bromoisatin is the best substrate for the enzyme (Km = 3.1 mM; Vmax = 650 mumol/min/mg). The enzyme is inhibited by quercetin, and several polyketones. These results suggest that ketopantoyl lactone reductase is a carbonyl reductase which specifically catalyzes the reduction of conjugated polyketones.

  6. 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.

  7. The role of extended Fe4S4 cluster ligands in mediating sulfite reductase hemoprotein activity.

    Science.gov (United States)

    Cepeda, Marisa R; McGarry, Lauren; Pennington, Joseph M; Krzystek, J; Elizabeth Stroupe, M

    2018-05-28

    The siroheme-containing subunit from the multimeric hemoflavoprotein NADPH-dependent sulfite reductase (SiR/SiRHP) catalyzes the six electron-reduction of SO 3 2- to S 2- . Siroheme is an iron-containing isobacteriochlorin that is found in sulfite and homologous siroheme-containing nitrite reductases. Siroheme does not work alone but is covalently coupled to a Fe 4 S 4 cluster through one of the cluster's ligands. One long-standing hypothesis predicted from this observation is that the environment of one iron-containing cofactor influences the properties of the other. We tested this hypothesis by identifying three amino acids (F437, M444, and T477) that interact with the Fe 4 S 4 cluster and probing the effect of altering them to alanine on the function and structure of the resulting enzymes by use of activity assays, X-ray crystallographic analysis, and EPR spectroscopy. We showed that F437 and M444 gate access for electron transfer to the siroheme-cluster assembly and the direct hydrogen bond between T477 and one of the cluster sulfides is important for determining the geometry of the siroheme active site. Copyright © 2018. Published by Elsevier B.V.

  8. Nitrite reductase activity and inhibition of H₂S biogenesis by human cystathionine ß-synthase.

    Directory of Open Access Journals (Sweden)

    Carmen Gherasim

    Full Text Available Nitrite was recognized as a potent vasodilator >130 years and has more recently emerged as an endogenous signaling molecule and modulator of gene expression. Understanding the molecular mechanisms that regulate nitrite metabolism is essential for its use as a potential diagnostic marker as well as therapeutic agent for cardiovascular diseases. In this study, we have identified human cystathionine ß-synthase (CBS as a new player in nitrite reduction with implications for the nitrite-dependent control of H₂S production. This novel activity of CBS exploits the catalytic property of its unusual heme cofactor to reduce nitrite and generate NO. Evidence for the possible physiological relevance of this reaction is provided by the formation of ferrous-nitrosyl (Fe(II-NO CBS in the presence of NADPH, the human diflavin methionine synthase reductase (MSR and nitrite. Formation of Fe(II-NO CBS via its nitrite reductase activity inhibits CBS, providing an avenue for regulating biogenesis of H₂S and cysteine, the limiting reagent for synthesis of glutathione, a major antioxidant. Our results also suggest a possible role for CBS in intracellular NO biogenesis particularly under hypoxic conditions. The participation of a regulatory heme cofactor in CBS in nitrite reduction is unexpected and expands the repertoire of proteins that can liberate NO from the intracellular nitrite pool. Our results reveal a potential molecular mechanism for cross-talk between nitrite, NO and H₂S biology.

  9. Crystallization and diffraction analysis of thioredoxin reductase from Streptomyces coelicolor

    International Nuclear Information System (INIS)

    Koháryová, Michaela; Brynda, Jiří; Řezáčová, Pavlína; Kollárová, Marta

    2011-01-01

    Thioredoxin reductase from S. coelicolor was crystallized and diffraction data were collected to 2.4 Å resolution. Thioredoxin reductases are homodimeric flavoenzymes that catalyze the transfer of electrons from NADPH to oxidized thioredoxin substrate. Bacterial thioredoxin reductases represent a promising target for the development of new antibiotics. Recombinant thioredoxin reductase TrxB from Streptomyces coelicolor was crystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected from cryocooled crystals to 2.4 Å resolution using a synchrotron-radiation source. The crystals belonged to the primitive monoclinic space group P2 1 , with unit-cell parameters a = 82.9, b = 60.6, c = 135.4 Å, α = γ = 90.0, β = 96.5°

  10. Polysulfides and products of H2S/S-nitrosoglutathione in comparison to H2S, glutathione and antioxidant Trolox are potent scavengers of superoxide anion radical and produce hydroxyl radical by decomposition of H2O2.

    Science.gov (United States)

    Misak, Anton; Grman, Marian; Bacova, Zuzana; Rezuchova, Ingeborg; Hudecova, Sona; Ondriasova, Elena; Krizanova, Olga; Brezova, Vlasta; Chovanec, Miroslav; Ondrias, Karol

    2018-06-01

    Exogenous and endogenously produced sulfide derivatives, such as H 2 S/HS - /S 2- , polysulfides and products of the H 2 S/S-nitrosoglutathione interaction (S/GSNO), affect numerous biological processes in which superoxide anion (O 2 - ) and hydroxyl (OH) radicals play an important role. Their cytoprotective-antioxidant and contrasting pro-oxidant-toxic effects have been reported. Therefore, the aim of our work was to contribute to resolving this apparent inconsistency by studying sulfide derivatives/free radical interactions and their consequent biological effects compared to the antioxidants glutathione (GSH) and Trolox. Using the electron paramagnetic resonance (EPR) spin trapping technique and O 2 - , we found that a polysulfide (Na 2 S 4 ) and S/GSNO were potent scavengers of O 2 - and cPTIO radicals compared to H 2 S (Na 2 S), GSH and Trolox, and S/GSNO scavenged the DEPMPO-OH radical. As detected by the EPR spectra of DEPMPO-OH, the formation of OH in physiological solution by S/GSNO was suggested. All the studied sulfide derivatives, but not Trolox or GSH, had a bell-shaped potency to decompose H 2 O 2 and produced OH in the following order: S/GSNO > Na 2 S 4  ≥ Na 2 S > GSH = Trolox = 0, but they scavenged OH at higher concentrations. In studies of the biological consequences of these sulfide derivatives/H 2 O 2 properties, we found the following: (i) S/GSNO alone and all sulfide derivatives in the presence of H 2 O 2 cleaved plasmid DNA; (ii) S/GSNO interfered with viral replication and consequently decreased the infectivity of viruses; (iii) the sulfide derivatives induced apoptosis in A2780 cells but inhibited apoptosis induced by H 2 O 2 ; and (iv) Na 2 S 4 modulated intracellular calcium in A87MG cells, which depended on the order of Na 2 S 4 /H 2 O 2 application. We suggest that the apparent inconsistency of the cytoprotective-antioxidant and contrasting pro-oxidant-toxic biological effects of sulfide derivatives results from their time

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

    NARCIS (Netherlands)

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

    1988-01-01

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

  12. The effect of ionic and non-ionic surfactants on the growth, nitrate reductase and nitrite reductase activities of Spirodela polyrrhiza (L. Schleiden

    Directory of Open Access Journals (Sweden)

    Józef Buczek

    2014-01-01

    Full Text Available Inclusion into the medium of 5 mg•dm-3 of non-ionic (ENF or ionic (DBST surfactant caused 50-60% inhibition of nitrite reductase MR activity in S. polyrrhiza. At the same time, increased accumulation of NO2- in the plant tissues and lowering of the total and soluble protein contents were found. DBST also lowered the nitrate reductase (NR activity and the dry mass of the plants.

  13. S-nitrosoglutathione promotes cell wall remodelling, alters the transcriptional profile and induces root hair formation in the hairless root hair defective 6 (rhd6) mutant of Arabidopsis thaliana.

    Science.gov (United States)

    Moro, Camila Fernandes; Gaspar, Marilia; da Silva, Felipe Rodrigues; Pattathil, Sivakumar; Hahn, Michael G; Salgado, Ione; Braga, Marcia Regina

    2017-03-01

    Nitric oxide (NO) exerts pleiotropic effects on plant development; however, its involvement in cell wall modification during root hair formation (RHF) has not yet been addressed. Here, mutants of Arabidopsis thaliana with altered root hair phenotypes were used to assess the involvement of S-nitrosoglutathione (GSNO), the primary NO source, in cell wall dynamics and gene expression in roots induced to form hairs. GSNO and auxin restored the root hair phenotype of the hairless root hair defective 6 (rhd6) mutant. A positive correlation was observed between increased NO production and RHF induced by auxin in rhd6 and transparent testa glabra (ttg) mutants. Deposition of an epitope within rhamnogalacturonan-I recognized by the CCRC-M2 antibody was delayed in root hair cells (trichoblasts) compared with nonhair cells (atrichoblasts). GSNO, but not auxin, restored the wild-type root glycome and transcriptome profiles in rhd6, modulating the expression of a large number of genes related to cell wall composition and metabolism, as well as those encoding ribosomal proteins, DNA and histone-modifying enzymes and proteins involved in post-translational modification. Our results demonstrate that NO plays a key role in cell wall remodelling in trichoblasts and suggest that it also participates in chromatin modification in root cells of A. thaliana. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

  14. Climate Change and the Impact of Greenhouse Gasses: CO2 and NO, Friends and Foes of Plant Oxidative Stress

    Science.gov (United States)

    Cassia, Raúl; Nocioni, Macarena; Correa-Aragunde, Natalia; Lamattina, Lorenzo

    2018-01-01

    -nitrosylation. GSNO could be decomposed by the GSNO reductase (GSNOR) to GSSG which, in turn, is reduced to GSH by glutathione reductase (GR). GSNOR may be also inhibited by S-nitrosylation and GR activated by NO. In conclusion, NO plays a central role in the tolerance of plants to climate change. PMID:29545820

  15. 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.

  16. 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.

  17. Climate Change and the Impact of Greenhouse Gasses: CO2 and NO, Friends and Foes of Plant Oxidative Stress.

    Science.gov (United States)

    Cassia, Raúl; Nocioni, Macarena; Correa-Aragunde, Natalia; Lamattina, Lorenzo

    2018-01-01

    -nitrosylation. GSNO could be decomposed by the GSNO reductase (GSNOR) to GSSG which, in turn, is reduced to GSH by glutathione reductase (GR). GSNOR may be also inhibited by S-nitrosylation and GR activated by NO. In conclusion, NO plays a central role in the tolerance of plants to climate change.

  18. 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...

  19. The structure of apo and holo forms of xylose reductase, a dimeric aldo-keto reductase from Candida tenuis.

    Science.gov (United States)

    Kavanagh, Kathryn L; Klimacek, Mario; Nidetzky, Bernd; Wilson, David K

    2002-07-16

    Xylose reductase is a homodimeric oxidoreductase dependent on NADPH or NADH and belongs to the largely monomeric aldo-keto reductase superfamily of proteins. It catalyzes the first step in the assimilation of xylose, an aldose found to be a major constituent monosaccharide of renewable plant hemicellulosic material, into yeast metabolic pathways. It does this by reducing open chain xylose to xylitol, which is reoxidized to xylulose by xylitol dehydrogenase and metabolically integrated via the pentose phosphate pathway. No structure has yet been determined for a xylose reductase, a dimeric aldo-keto reductase or a family 2 aldo-keto reductase. The structures of the Candida tenuis xylose reductase apo- and holoenzyme, which crystallize in spacegroup C2 with different unit cells, have been determined to 2.2 A resolution and an R-factor of 17.9 and 20.8%, respectively. Residues responsible for mediating the novel dimeric interface include Asp-178, Arg-181, Lys-202, Phe-206, Trp-313, and Pro-319. Alignments with other superfamily members indicate that these interactions are conserved in other dimeric xylose reductases but not throughout the remainder of the oligomeric aldo-keto reductases, predicting alternate modes of oligomerization for other families. An arrangement of side chains in a catalytic triad shows that Tyr-52 has a conserved function as a general acid. The loop that folds over the NAD(P)H cosubstrate is disordered in the apo form but becomes ordered upon cosubstrate binding. A slow conformational isomerization of this loop probably accounts for the observed rate-limiting step involving release of cosubstrate. Xylose binding (K(m) = 87 mM) is mediated by interactions with a binding pocket that is more polar than a typical aldo-keto reductase. Modeling of xylose into the active site of the holoenzyme using ordered waters as a guide for sugar hydroxyls suggests a convincing mode of substrate binding.

  20. N-terminus determines activity and specificity of styrene monooxygenase reductases.

    Science.gov (United States)

    Heine, Thomas; Scholtissek, Anika; Westphal, Adrie H; van Berkel, Willem J H; Tischler, Dirk

    2017-12-01

    Styrene monooxygenases (SMOs) are two-enzyme systems that catalyze the enantioselective epoxidation of styrene to (S)-styrene oxide. The FADH 2 co-substrate of the epoxidase component (StyA) is supplied by an NADH-dependent flavin reductase (StyB). The genome of Rhodococcus opacus 1CP encodes two SMO systems. One system, which we define as E1-type, displays homology to the SMO from Pseudomonas taiwanensis VLB120. The other system, originally reported as a fused system (RoStyA2B), is defined as E2-type. Here we found that E1-type RoStyB is inhibited by FMN, while RoStyA2B is known to be active with FMN. To rationalize the observed specificity of RoStyB for FAD, we generated an artificial reductase, designated as RoStyBart, in which the first 22 amino acid residues of RoStyB were joined to the reductase part of RoStyA2B, while the oxygenase part (A2) was removed. RoStyBart mainly purified as apo-protein and mimicked RoStyB in being inhibited by FMN. Pre-incubation with FAD yielded a turnover number at 30°C of 133.9±3.5s -1 , one of the highest rates observed for StyB reductases. RoStyBart holo-enzyme switches to a ping-pong mechanism and fluorescence analysis indicated for unproductive binding of FMN to the second (co-substrate) binding site. In summary, it is shown for the first time that optimization of the N-termini of StyB reductases allows the evolution of their activity and specificity. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. The aldo-keto reductase superfamily homepage.

    Science.gov (United States)

    Hyndman, David; Bauman, David R; Heredia, Vladi V; Penning, Trevor M

    2003-02-01

    The aldo-keto reductases (AKRs) are one of the three enzyme superfamilies that perform oxidoreduction on a wide variety of natural and foreign substrates. A systematic nomenclature for the AKR superfamily was adopted in 1996 and was updated in September 2000 (visit www.med.upenn.edu/akr). Investigators have been diligent in submitting sequences of functional proteins to the Web site. With the new additions, the superfamily contains 114 proteins expressed in prokaryotes and eukaryotes that are distributed over 14 families (AKR1-AKR14). The AKR1 family contains the aldose reductases, the aldehyde reductases, the hydroxysteroid dehydrogenases and steroid 5beta-reductases, and is the largest. Other families of interest include AKR6, which includes potassium channel beta-subunits, and AKR7 the aflatoxin aldehyde reductases. Two new families include AKR13 (yeast aldose reductase) and AKR14 (Escherichia coli aldehyde reductase). Crystal structures of many AKRs and their complexes with ligands are available in the PDB and accessible through the Web site. Each structure has the characteristic (alpha/beta)(8)-barrel motif of the superfamily, a conserved cofactor binding site and a catalytic tetrad, and variable loop structures that define substrate specificity. Although the majority of AKRs are monomeric proteins of about 320 amino acids in length, the AKR2, AKR6 and AKR7 family may form multimers. To expand the nomenclature to accommodate multimers, we recommend that the composition and stoichiometry be listed. For example, AKR7A1:AKR7A4 (1:3) would designate a tetramer of the composition indicated. The current nomenclature is recognized by the Human Genome Project (HUGO) and the Web site provides a link to genomic information including chromosomal localization, gene boundaries, human ESTs and SNPs and much more.

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

    International Nuclear Information System (INIS)

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

    1986-01-01

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

  3. 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

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

    International Nuclear Information System (INIS)

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

    2001-01-01

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

  5. Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and cholesterol biosynthesis by oxylanosterols

    Energy Technology Data Exchange (ETDEWEB)

    Panini, S.R.; Sexton, R.C.; Gupta, A.K.; Parish, E.J.; Chitrakorn, S.; Rudney, H.

    1986-11-01

    Treatment of rat intestinal epithelial cell cultures with the oxidosqualene cyclase inhibitor, 3 beta-(2-(diethylamino)-ethoxy)androst-5-en-17-one (U18666A), resulted in an accumulation of squalene 2,3:22,23-dioxide (SDO). When U18666A was withdrawn and the cells were treated with the sterol 14 alpha-demethylase inhibitor, ketoconazole, SDO was metabolized to a product identified as 24(S),25-epoxylanosterol. To test the biological effects and cellular metabolism of this compound, we prepared 24(RS),25-epoxylanosterol by chemical synthesis. The epimeric mixture of 24,25-epoxylanosterols could be resolved by high performance liquid chromatography on a wide-pore, non-endcapped, reverse phase column. Both epimers were effective suppressors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity of IEC-6 cells. The suppressive action of the natural epimer, 24(S),25-epoxylanosterol, but not that of 24(R),25-epoxylanosterol could be completely prevented by ketoconazole. IEC-6 cells could efficiently metabolize biosynthetic 24(S),25-epoxy(/sup 3/H)anosterol mainly to the known reductase-suppressor 24(S),25-epoxycholesterol. This metabolism was substantially reduced by ketoconazole. These data support the conclusion that 24(S),25-epoxylanosterol per se is not a suppressor of HMG-CoA reductase activity but is a precursor to a regulatory oxysterol(s). It has recently been reported that 25-hydroxycholesterol can occur naturally in cultured cells in amounts sufficient to effect regulation of HMG-CoA reductase. In order to investigate the biological effects of possible precursors of 25-hydroxycholesterol, we chemically synthesized 25-hydroxylanosterol and 25-hydroxylanostene-3-one. Both oxylanosterol derivatives suppressed cellular sterol synthesis at the level of HMG-CoA reductase. (Abstract Truncated)

  6. Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and cholesterol biosynthesis by oxylanosterols

    International Nuclear Information System (INIS)

    Panini, S.R.; Sexton, R.C.; Gupta, A.K.; Parish, E.J.; Chitrakorn, S.; Rudney, H.

    1986-01-01

    Treatment of rat intestinal epithelial cell cultures with the oxidosqualene cyclase inhibitor, 3 beta-[2-(diethylamino)-ethoxy]androst-5-en-17-one (U18666A), resulted in an accumulation of squalene 2,3:22,23-dioxide (SDO). When U18666A was withdrawn and the cells were treated with the sterol 14 alpha-demethylase inhibitor, ketoconazole, SDO was metabolized to a product identified as 24(S),25-epoxylanosterol. To test the biological effects and cellular metabolism of this compound, we prepared 24(RS),25-epoxylanosterol by chemical synthesis. The epimeric mixture of 24,25-epoxylanosterols could be resolved by high performance liquid chromatography on a wide-pore, non-endcapped, reverse phase column. Both epimers were effective suppressors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity of IEC-6 cells. The suppressive action of the natural epimer, 24(S),25-epoxylanosterol, but not that of 24(R),25-epoxylanosterol could be completely prevented by ketoconazole. IEC-6 cells could efficiently metabolize biosynthetic 24(S),25-epoxy[ 3 H]anosterol mainly to the known reductase-suppressor 24(S),25-epoxycholesterol. This metabolism was substantially reduced by ketoconazole. These data support the conclusion that 24(S),25-epoxylanosterol per se is not a suppressor of HMG-CoA reductase activity but is a precursor to a regulatory oxysterol(s). It has recently been reported that 25-hydroxycholesterol can occur naturally in cultured cells in amounts sufficient to effect regulation of HMG-CoA reductase. In order to investigate the biological effects of possible precursors of 25-hydroxycholesterol, we chemically synthesized 25-hydroxylanosterol and 25-hydroxylanostene-3-one. Both oxylanosterol derivatives suppressed cellular sterol synthesis at the level of HMG-CoA reductase. (Abstract Truncated)

  7. Purification and kinetic analysis of cytosolic and mitochondrial thioredoxin glutathione reductase extracted from Taenia solium cysticerci.

    Science.gov (United States)

    Plancarte, Agustin; Nava, Gabriela

    2015-02-01

    Thioredoxin glutathione reductases (TGRs) (EC 1.8.1.9) were purified to homogeneity from the cytosolic (cTsTGR) and mitochondrial (mTsTGR) fractions of Taenia solium, the agent responsible for neurocysticercosis, one of the major central nervous system parasitic diseases in humans. TsTGRs had a relative molecular weight of 132,000, while the corresponding value per subunit obtained under denaturing conditions, was of 62,000. Specific activities for thioredoxin reductase and glutathione reductase substrates for both TGRs explored were in the range or lower than values obtained for other platyhelminths and mammalian TGRs. cTsTGR and mTsTGR also showed hydroperoxide reductase activity using hydroperoxide as substrate. Km(DTNB) and Kcat(DTNB) values for cTsTGR and mTsTGR (88 µM and 1.9 s(-1); 45 µM and 12.6 s(-1), respectively) and Km(GSSG) and Kcat(GSSG) values for cTsTGR and mTsTGR (6.3 µM and 0.96 s(-1); 4 µM and 1.62 s(-1), respectively) were similar to or lower than those reported for mammalian TGRs. Mass spectrometry analysis showed that 12 peptides from cTsTGR and seven from mTsTGR were a match for gi|29825896 thioredoxin glutathione reductase [Echinococcus granulosus], confirming that both enzymes are TGRs. Both T. solium TGRs were inhibited by the gold compound auranofin, a selective inhibitor of thiol-dependent flavoreductases (I₅₀ = 3.25, 2.29 nM for DTNB and GSSG substrates, respectively for cTsTGR; I₅₀ = 5.6, 25.4 nM for mTsTGR toward the same substrates in the described order). Glutathione reductase activity of cTsTGR and mTsTGR exhibited hysteretic behavior with moderate to high concentrations of GSSG; this result was not observed either with thioredoxin, DTNB or NADPH. However, the observed hysteretic kinetics was suppressed with increasing amounts of both parasitic TGRs. These data suggest the existence of an effective substitute which may account for the lack of the detoxification enzymes glutathione reductase

  8. Directed Evolution of Carbonyl Reductase from Rhodosporidium toruloides and Its Application in Stereoselective Synthesis of tert-Butyl (3R,5S)-6-Chloro-3,5-dihydroxyhexanoate.

    Science.gov (United States)

    Liu, Zhi-Qiang; Wu, Lin; Zhang, Xiao-Jian; Xue, Ya-Ping; Zheng, Yu-Guo

    2017-05-10

    tert-Butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate ((3R,5S)-CDHH) is a key intermediate of atorvastatin and rosuvastatin synthesis. Carbonyl reductase RtSCR9 from Rhodosporidium toruloides exhibited excellent activity toward tert-butyl (S)-6-chloro-5-hydroxy-3-oxohexanoate ((S)-CHOH). For the activity of RtSCR9 to be improved, random mutagenesis and site-saturation mutagenesis were performed. Three positive mutants were obtained (mut-Gln95Asp, mut-Ile144Lys, and mut-Phe156Gln). These mutants exhibited 1.94-, 3.03-, and 1.61-fold and 1.93-, 3.15-, and 1.97-fold improvement in the specific activity and k cat /K m , respectively. Asymmetric reduction of (S)-CHOH by mut-Ile144Lys coupled with glucose dehydrogenase was conducted. The yield and enantiomeric excess of (3R,5S)-CDHH reached 98 and 99%, respectively, after 8 h bioconversion in a single batch reaction with 1 M (S)-CHOH, and the space-time yield reached 542.83 mmol L -1 h -1 g -1 wet cell weight. This study presents a new carbonyl reductase for efficient synthesis of (3R,5S)-CDHH.

  9. Nucleotide sequence of Phaseolus vulgaris L. alcohol dehydrogenase encoding cDNA and three-dimensional structure prediction of the deduced protein.

    Science.gov (United States)

    Amelia, Kassim; Khor, Chin Yin; Shah, Farida Habib; Bhore, Subhash J

    2015-01-01

    Common beans (Phaseolus vulgaris L.) are widely consumed as a source of proteins and natural products. However, its yield needs to be increased. In line with the agenda of Phaseomics (an international consortium), work of expressed sequence tags (ESTs) generation from bean pods was initiated. Altogether, 5972 ESTs have been isolated. Alcohol dehydrogenase (AD) encoding gene cDNA was a noticeable transcript among the generated ESTs. This AD is an important enzyme; therefore, to understand more about it this study was undertaken. The objective of this study was to elucidate P. vulgaris L. AD (PvAD) gene cDNA sequence and to predict the three-dimensional (3D) structure of deduced protein. positive and negative strands of the PvAD cDNA clone were sequenced using M13 forward and M13 reverse primers to elucidate the nucleotide sequence. Deduced PvAD cDNA and protein sequence was analyzed for their basic features using online bioinformatics tools. Sequence comparison was carried out using bl2seq program, and tree-view program was used to construct a phylogenetic tree. The secondary structures and 3D structure of PvAD protein were predicted by using the PHYRE automatic fold recognition server. The sequencing results analysis showed that PvAD cDNA is 1294 bp in length. It's open reading frame encodes for a protein that contains 371 amino acids. Deduced protein sequence analysis showed the presence of putative substrate binding, catalytic Zn binding, and NAD binding sites. Results indicate that the predicted 3D structure of PvAD protein is analogous to the experimentally determined crystal structure of s-nitrosoglutathione reductase from an Arabidopsis species. The 1294 bp long PvAD cDNA encodes for 371 amino acid long protein that contains conserved domains required for biological functions of AD. The predicted deduced PvAD protein's 3D structure reflects the analogy with the crystal structure of Arabidopsis thaliana s-nitrosoglutathione reductase. Further study is required

  10. Nitrate reductase activity of Staphylococcus carnosus affecting the color formation in cured raw ham.

    Science.gov (United States)

    Bosse Née Danz, Ramona; Gibis, Monika; Schmidt, Herbert; Weiss, Jochen

    2016-07-01

    The influence of the nitrate reductase activity of two Staphylococcus carnosus strains used as starter cultures on the formation of nitrate, nitrite and color pigments in cured raw ham was investigated. In this context, microbiological, chemical and multivariate image analyses were carried out on cured raw hams, which were injected with different brines containing either nitrite or nitrate, with or without the S. carnosus starter cultures. During processing and storage, the viable counts of staphylococci remained constant at 6.5logcfu/g in the hams inoculated with starter cultures, while the background microbiota of the hams processed without the starter cultures developed after 14days. Those cured hams inoculated with S. carnosus LTH 7036 (high nitrate reductase activity) showed the highest decrease in nitrate and high nitrite concentrations in the end product, but were still in the range of the legal European level. The hams cured with nitrate and without starter culture or with the other strain, S. carnosus LTH 3838 (low nitrate reductase activity) showed higher residual nitrate levels and a lower nitrite content in the end product. The multivariate image analysis identified spatial and temporal differences in the meat pigment profiles of the differently cured hams. The cured hams inoculated with S. carnosus LTH 3838 showed an uncured core due to a delay in pigment formation. Therefore, the selection of starter cultures based on their nitrate reductase activity is a key point in the formation of curing compounds and color pigments in cured raw ham manufacture. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

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

    2001-04-03

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

  12. Regulation and Turnover of Nitric Oxide by Phytoglobins in Plant Cell Responses

    DEFF Research Database (Denmark)

    Igamberdiev, Abir U; Hebelstrup, Kim; Stasolla, Claudio

    2016-01-01

    The involvement of phytoglobins in the metabolism of nitric oxide (NO) and reactive nitrogen species (RNS) produced during stress, plant growth, and development is discussed. The action of phytoglobin expression upon NO leads to the maintenance of redox status, minimization of the damage from...... to the mobility of both NO and phytohormones, plants developed strategies to regulate specific cell hormonal actions to permit differentiation during development and to respond to stress. Phytoglobins are the agents responsible for differential cellular responses to hormones that use NO as a signal transduction...... reactive oxygen and nitrogen species in the cytoplasm of the cell, and regulation of hormonal and stress responses. NO scavenging is achieved via phytoglobins, and it can also involve S-nitrosoglutathione reductase and a direct interaction of NO with superoxide anion followed by detoxification of formed...

  13. Bioinformatics approach of three partial polyprenol reductase genes in Kandelia obovata

    Science.gov (United States)

    Basyuni, M.; Wati, R.; Sagami, H.; Oku, H.; Baba, S.

    2018-03-01

    This present study describesthe bioinformatics approach to analyze three partial polyprenol reductase genes from mangrove plant, Kandeliaobovataas well aspredictedphysical and chemical properties, potential peptide, subcellular localization, and phylogenetic. The diversity was noted in the physical and chemical properties of three partial polyprenol reductase genes. The values of chloroplast were relatively high, showed that chloroplast transit peptide occurred in mangrove polyprenol reductase. The target peptide value of mitochondria varied from 0.088 to 0.198 indicated it was possible to be present. These results suggested the importance of understanding the diversity of physicochemical properties of the different amino acids in polyprenol reductase. The subcellular localization of two partial genes located in the plasma membrane. To confirm the homology among the polyprenol reductase in the database, a dendrogram was drawn. The phylogenetic tree depicts that there are three clusters, the partial genes of K. obovata joined the largest one: C23157 was close to Ricinus communis polyprenol reductase. Whereas, C23901 and C24171 were grouped with Ipomoea nil polyprenol reductase, suggested that these polyprenol reductase genes form distinct separation into tropical habitat plants.

  14. Recominant Pinoresino-Lariciresinol Reductase, Recombinant Dirigent Protein And Methods Of Use

    Science.gov (United States)

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

    2003-10-21

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

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

    DEFF Research Database (Denmark)

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

    1992-01-01

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

  16. Mechanism of Sirt1 NAD+-dependent Protein Deacetylase Inhibition by Cysteine S-Nitrosation.

    Science.gov (United States)

    Kalous, Kelsey S; Wynia-Smith, Sarah L; Olp, Michael D; Smith, Brian C

    2016-12-02

    The sirtuin family of proteins catalyze the NAD + -dependent deacylation of acyl-lysine residues. Humans encode seven sirtuins (Sirt1-7), and recent studies have suggested that post-translational modification of Sirt1 by cysteine S-nitrosation correlates with increased acetylation of Sirt1 deacetylase substrates. However, the mechanism of Sirt1 inhibition by S-nitrosation was unknown. Here, we show that Sirt1 is transnitrosated and inhibited by the physiologically relevant nitrosothiol S-nitrosoglutathione. Steady-state kinetic analyses and binding assays were consistent with Sirt1 S-nitrosation inhibiting binding of both the NAD + and acetyl-lysine substrates. Sirt1 S-nitrosation correlated with Zn 2+ release from the conserved sirtuin Zn 2+ -tetrathiolate and a loss of α-helical structure without overall thermal destabilization of the enzyme. Molecular dynamics simulations suggested that Zn 2+ loss due to Sirt1 S-nitrosation results in repositioning of the tetrathiolate subdomain away from the rest of the catalytic domain, thereby disrupting the NAD + and acetyl-lysine-binding sites. Sirt1 S-nitrosation was reversed upon exposure to the thiol-based reducing agents, including physiologically relevant concentrations of the cellular reducing agent glutathione. Reversal of S-nitrosation resulted in full restoration of Sirt1 activity only in the presence of Zn 2+ , consistent with S-nitrosation of the Zn 2+ -tetrathiolate as the primary source of Sirt1 inhibition upon S-nitrosoglutathione treatment. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Streptococcus sanguinis Class Ib Ribonucleotide Reductase

    Science.gov (United States)

    Makhlynets, Olga; Boal, Amie K.; Rhodes, DeLacy V.; Kitten, Todd; Rosenzweig, Amy C.; Stubbe, JoAnne

    2014-01-01

    Streptococcus sanguinis is a causative agent of infective endocarditis. Deletion of SsaB, a manganese transporter, drastically reduces S. sanguinis virulence. Many pathogenic organisms require class Ib ribonucleotide reductase (RNR) to catalyze the conversion of nucleotides to deoxynucleotides under aerobic conditions, and recent studies demonstrate that this enzyme uses a dimanganese-tyrosyl radical (MnIII2-Y•) cofactor in vivo. The proteins required for S. sanguinis ribonucleotide reduction (NrdE and NrdF, α and β subunits of RNR; NrdH and TrxR, a glutaredoxin-like thioredoxin and a thioredoxin reductase; and NrdI, a flavodoxin essential for assembly of the RNR metallo-cofactor) have been identified and characterized. Apo-NrdF with FeII and O2 can self-assemble a diferric-tyrosyl radical (FeIII2-Y•) cofactor (1.2 Y•/β2) and with the help of NrdI can assemble a MnIII2-Y• cofactor (0.9 Y•/β2). The activity of RNR with its endogenous reductants, NrdH and TrxR, is 5,000 and 1,500 units/mg for the Mn- and Fe-NrdFs (Fe-loaded NrdF), respectively. X-ray structures of S. sanguinis NrdIox and MnII2-NrdF are reported and provide a possible rationale for the weak affinity (2.9 μm) between them. These streptococcal proteins form a structurally distinct subclass relative to other Ib proteins with unique features likely important in cluster assembly, including a long and negatively charged loop near the NrdI flavin and a bulky residue (Thr) at a constriction in the oxidant channel to the NrdI interface. These studies set the stage for identifying the active form of S. sanguinis class Ib RNR in an animal model for infective endocarditis and establishing whether the manganese requirement for pathogenesis is associated with RNR. PMID:24381172

  18. Hydroxyurea-Mediated Cytotoxicity Without Inhibition of Ribonucleotide Reductase.

    Science.gov (United States)

    Liew, Li Phing; Lim, Zun Yi; Cohen, Matan; Kong, Ziqing; Marjavaara, Lisette; Chabes, Andrei; Bell, Stephen D

    2016-11-01

    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. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

  19. 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...

  20. 5α-reductase activity in rat adipose tissue

    International Nuclear Information System (INIS)

    Zyirek, M.; Flood, C.; Longcope, C.

    1987-01-01

    We measured the 5 α-reductase activity in isolated cell preparations of rat adipose tissue using the formation of [ 3 H] dihydrotestosterone from [ 3 H] testosterone as an endpoint. Stromal cells were prepared from the epididymal fat pad, perinephric fat, and subcutaneous fat of male rats and from perinephric fat of female rats. Adipocytes were prepared from the epididymal fat pad and perinephric fat of male rats. Stromal cells from the epididymal fat pad and perinephric fat contained greater 5α-reductase activity than did the adipocytes from these depots. Stromal cells from the epididymal fat pad contained greater activity than those from perinephric and subcutaneous depots. Perinephric stromal cells from female rats were slightly more active than those from male rats. Estradiol (10 -8 M), when added to the medium, caused a 90% decrease in 5α-reductase activity. Aromatase activity was minimal, several orders of magnitude less than 5α-reductase activity in each tissue studied

  1. Characterization of mitochondrial thioredoxin reductase from C. elegans

    International Nuclear Information System (INIS)

    Lacey, Brian M.; Hondal, Robert J.

    2006-01-01

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

  2. Dietary sources of aldose reductase inhibitors: prospects for alleviating diabetic complications.

    Science.gov (United States)

    Saraswat, Megha; Muthenna, P; Suryanarayana, P; Petrash, J Mark; Reddy, G Bhanuprakash

    2008-01-01

    Activation of polyol pathway due to increased aldose reductase activity is one of the several mechanisms that have been implicated in the development of various secondary complications of diabetes. Though numerous synthetic aldose reductase inhibitors have been tested, these have not been very successful clinically. Therefore, a number of common plant/ natural products used in Indian culinary have been evaluated for their aldose reductase inhibitory potential in the present study. The aqueous extracts of 22 plant-derived materials were prepared and evaluated for the inhibitory property against rat lens and human recombinant aldose reductase. Specificity of these extracts towards aldose reductase was established by testing their ability to inhibit a closely related enzyme viz, aldehyde reductase. The ex vivo incubation of erythrocytes in high glucose containing medium was used to underscore the significance in terms of prevention of intracellular sorbitol accumulation. Among the 22 dietary sources tested, 10 showed considerable inhibitory potential against both rat lens and human recombinant aldose reductase. Prominent inhibitory property was found in spinach, cumin, fennel, lemon, basil and black pepper with an approximate IC50 of 0.2 mg/mL with an excellent selectivity towards aldose reductase. As against this, 10 to 20 times higher concentrations were required for 50% inhibition of aldehyde reductase. Reduction in the accumulation of intracellular sorbitol by the dietary extracts further substantiated their in vivo efficacy. The findings reported here indicate the scope of adapting life-style modifications in the form of inclusion of certain common sources in the diet for the management of diabetic complications.

  3. 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.

  4. Histochemical Localization of Glutathione Dependent NBT-Reductase in Mouse Skin

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective 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. Methods The fresh frozen tissue sections (8m thickness) were prepared and incubated in medium containing NBT, reduced glutathione (GSH) and phosphate buffer. The staining for GSH was performed with mercury orange. Results  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. Conclusion The activity of the NBT-reductase seems to be dependent on the glutathione contents.

  5. Nitric oxide in plants: an assessment of the current state of knowledge

    DEFF Research Database (Denmark)

    Mur, Luis A J; Mandon, Julien; Persijn, Stefan

    2013-01-01

    Background and aims After a series of seminal works during the last decade of the 20th century nitric oxide (NO) is now firmly placed in the pantheon of plant signals. NO acts in plant-microbe interactions, responses to abiotic stress, stomatal regulation and a range of developmental processes...... of NO production from DEANO (diethylamine nitric oxide), S-nitrosoglutathione (GSNO) and sodium nitroprusside (SNP) following infiltration of tobacco leaves which could aid workers in their experiments. Further, based on current data it is difficult to define a bespoke plant NO signalling pathway, but rather....... By considering the recent advances in plant NO biology, this review will highlight certain key aspects that require further attention. Scope and conclusions The following questions will be considered. Whilst cytosolic nitrate reductase is an important source of NO, the contributions of other mechanisms...

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

    Science.gov (United States)

    Gøtterup, Jacob; Olsen, Karsten; Knøchel, Susanne; Tjener, Karsten; Stahnke, Louise H; Møller, Jens K S

    2008-04-01

    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 was followed by L(∗)a(∗)b measurements and the content of nitrosylmyoglobin (MbFe(II)NO) quantified by electron spin resonance (ESR). MbFe(II)NO was rapidly formed in sausages with added nitrite independent of the presence of nitrite reducing bacteria, whereas the rate of MbFe(II)NO formation in sausages with added 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 by autofluorescence and hexanal content, respectively. No significant direct effect of the Staphylococcus addition was observed, however, there was a clear correspondence between high initial amount of MbFe(II)NO in the different sausages and the colour stability during storage. Autofluorescence data correlated well 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 for ensuring optimal colour formation during initial fermentation stages.

  7. Regulation of schistosome egg production by HMG CoA reductase

    International Nuclear Information System (INIS)

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

    1986-01-01

    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 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

  8. Light Sensitivity of Lactococcus lactis Thioredoxin Reductase

    DEFF Research Database (Denmark)

    Skjoldager, Nicklas

    The thioredoxin system has evolved in all kingdoms of life acting as a key antioxidant system in the defense against oxidative stress. The thioredoxin system utilizes reducing equivalents from NADPH to reduce protein disulfide targets. The reducing equivalents are shuttled via a flavin and redox...... active dithiol motif in thioredoxin reductase (TrxR) to reduce the small ubiquitous thioredoxin (Trx). Trx in turn regulates the protein dithiol/disulfide balance by reduction of protein disulfide targets in e.g. ribonucleotide reductase, peroxiredoxins and methionine sulfoxide reductase. The glutathione......, thus expected to rely mainly on the Trx system for thiol-disulfide control. L. lactis is an important industrial microorganism used as starter culture in the dairy production of cheese, buttermilk etc. and known to be sensitive to oxidative stress. The L. lactis TrxR (LlTrxR) is a homodimeric...

  9. Evidence that steroid 5alpha-reductase isozyme genes are differentially methylated in human lymphocytes.

    Science.gov (United States)

    Rodríguez-Dorantes, M; Lizano-Soberón, M; Camacho-Arroyo, I; Calzada-León, R; Morimoto, S; Téllez-Ascencio, N; Cerbón, M A

    2002-03-01

    The synthesis of dihydrotestosterone (DHT) is catalyzed by steroid 5alpha-reductase isozymes 1 and 2, and this function determines the development of the male phenotype during embriogenesis and the growth of androgen sensitive tissues during puberty. The aim of this study was to determine the cytosine methylation status of 5alpha-reductase isozymes types 1 and 2 genes in normal and in 5alpha-reductase deficient men. Genomic DNA was obtained from lymphocytes of both normal subjects and patients with primary 5alpha-reductase deficiency due to point mutations in 5alpha-reductase 2 gene. Southern blot analysis of 5alpha-reductase types 1 and 2 genes from DNA samples digested with HpaII presented a different cytosine methylation pattern compared to that observed with its isoschizomer MspI, indicating that both genes are methylated in CCGG sequences. The analysis of 5alpha-reductase 1 gene from DNA samples digested with Sau3AI and its isoschizomer MboI which recognize methylation in GATC sequences showed an identical methylation pattern. In contrast, 5alpha-reductase 2 gene digested with Sau3AI presented a different methylation pattern to that of the samples digested with MboI, indicating that steroid 5alpha-reductase 2 gene possess methylated cytosines in GATC sequences. Analysis of exon 4 of 5alpha-reductase 2 gene after metabisulfite PCR showed that normal and deficient subjects present a different methylation pattern, being more methylated in patients with 5alpha-reductase 2 mutated gene. The overall results suggest that 5alpha-reductase genes 1 and 2 are differentially methylated in lymphocytes from normal and 5alpha-reductase deficient patients. Moreover, the extensive cytosine methylation pattern observed in exon 4 of 5alpha-reductase 2 gene in deficient patients, points out to an increased rate of mutations in this gene.

  10. The role of biliverdin reductase in colorectal cancer

    International Nuclear Information System (INIS)

    Bauer, M.

    2010-01-01

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

  11. Kinetics of carbonyl reductase from human brain.

    OpenAIRE

    Bohren, K M; von Wartburg, J P; Wermuth, B

    1987-01-01

    Initial-rate analysis of the carbonyl reductase-catalysed reduction of menadione by NADPH gave families of straight lines in double-reciprocal plots consistent with a sequential mechanism being obeyed. The fluorescence of NADPH was increased up to 7-fold with a concomitant shift of the emission maximum towards lower wavelength in the presence of carbonyl reductase, and both NADPH and NADP+ caused quenching of the enzyme fluorescence, indicating formation of a binary enzyme-coenzyme complex. D...

  12. Nitrate reductase activity and its relationship with applied nitrogen in soybean

    International Nuclear Information System (INIS)

    Ge Wenting; Jin Xijun; Ma Chunmei; Dong Shoukun; Gong Zhenping; Zhang Lei

    2011-01-01

    Field experiments were conducted to study the nitrate reductase activity and its relationship to nitrogen by using frame tests (pot without bottom), sand culture and 15 N-urea at transplanting in soybean variety Suinong 14. Results showed that the activity of nitrate reductase in leaf changed as a signal peak curve with the soybean growth, lower in vegetative growth phase, higher in reproductive growth period and reached the peak in blooming period, then decreased gradually. Nitrogen application showed obvious effect on the nitrate reductase activity. The activities of nitrate reductase in leaves followed the order of N 135 > N 90 > N 45 > N 0 in vegetative growth stage, no clear regularity was found during the whole reproductive growth period. The activities of nitrate reductase in leaves were accorded with the order of upper leaves > mid leaves > lower leaves, and it was very significant differences (P 15 N labeling method during beginning seed stage and full seed stage shown that 15 N abundance in various organs at different node position also followed the same order, suggesting that high level of nitrate reductase activity at upper leaves of soybean promoted the assimilation of NO 3 - . (authors)

  13. 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.

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

    Directory of Open Access Journals (Sweden)

    Jolanta Marciniak

    2014-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1988-05-17

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

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

    International Nuclear Information System (INIS)

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

    1988-01-01

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

  17. 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.

  18. Pichia stipitis xylose reductase helps detoxifying lignocellulosic hydrolysate by reducing 5-hydroxymethyl-furfural (HMF

    Directory of Open Access Journals (Sweden)

    Röder Anja

    2008-06-01

    Full Text Available Abstract Background Pichia stipitis xylose reductase (Ps-XR has been used to design Saccharomyces cerevisiae strains that are able to ferment xylose. One example is the industrial S. cerevisiae xylose-consuming strain TMB3400, which was constructed by expression of P. stipitis xylose reductase and xylitol dehydrogenase and overexpression of endogenous xylulose kinase in the industrial S. cerevisiae strain USM21. Results In this study, we demonstrate that strain TMB3400 not only converts xylose, but also displays higher tolerance to lignocellulosic hydrolysate during anaerobic batch fermentation as well as 3 times higher in vitro HMF and furfural reduction activity than the control strain USM21. Using laboratory strains producing various levels of Ps-XR, we confirm that Ps-XR is able to reduce HMF both in vitro and in vivo. Ps-XR overexpression increases the in vivo HMF conversion rate by approximately 20%, thereby improving yeast tolerance towards HMF. Further purification of Ps-XR shows that HMF is a substrate inhibitor of the enzyme. Conclusion We demonstrate for the first time that xylose reductase is also able to reduce the furaldehyde compounds that are present in undetoxified lignocellulosic hydrolysates. Possible implications of this newly characterized activity of Ps-XR on lignocellulosic hydrolysate fermentation are discussed.

  19. Lactococcus lactis Thioredoxin Reductase Is Sensitive to Light Inactivation

    DEFF Research Database (Denmark)

    Björnberg, Olof; Viennet, Thibault; Skjoldager, Nicklas

    2015-01-01

    Thioredoxin, involved in numerous redox pathways, is maintained in the dithiol state by the nicotinamide adenine dinucleotide phosphate-dependent flavoprotein thioredoxin reductase (TrxR). Here, TrxR from Lactococcus lactis is compared with the well-characterized TrxR from Escherichia coli. The two...... enzymes belong to the same class of low-molecular weight thioredoxin reductases and display similar kcat values (∼25 s-1) with their cognate thioredoxin. Remarkably, however, the L. lactis enzyme is inactivated by visible light and furthermore reduces molecular oxygen 10 times faster than E. coli Trx......-resolution mass spectrometric analysis of heat-extracted FAD from light-damaged TrxR revealed a mass increment of 13.979 Da, relative to that of unmodified FAD, corresponding to the addition of one oxygen atom and the loss of two hydrogen atoms. Tandem mass spectrometry confined the increase in mass...

  20. 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......) of bacteria and archaea were 4.59 x 10(9) and 6.68 x 10(5), respectively. Bacterial strains representing Acidithiobacillus, Leptospirillum, and Sulfobacillus were isolated from the bioreactors. To study sulfur oxidation in the reactors, pairs of new PCR primers were designed for the detection of sulfur...... oxygenase reductase (SOR) genes. Three sor-like genes, namely, sor (Fx), sor (SA), and sor (SB) were identified from metagenomic DNAs of the bioreactors. The sor (Fx) is an inactivated SOR gene and is identical to the pseudo-SOR gene of Ferroplasma acidarmanus. The sor (SA) and sor (SB) showed...

  1. 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.

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

    Science.gov (United States)

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

    2016-04-01

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

  3. In vivo photoinactivation of Escherichia coli ribonucleoside reductase by near-ultraviolet light

    International Nuclear Information System (INIS)

    Peters, J.

    1977-01-01

    Some experimental work is described showing that near-U.V. irradiation of E.coli cells selectively destroys RDP-reductase (ribonucleoside diphosphate reductase) activity in vivo are providing evidence relating the loss of RDP-reductase to loss of cellular visibility and the inactivity of irrdiated cells to support the replication of DNA phages. The data are consistent with the interpretation that the principal cause in the killing of exponentially growing E.coli cells by near-U.V., and the loss of ability of irradiated host cells to support the replication of DNA phages, is the photoinactivation of the RDP-reductase complex. (U.K.)

  4. In vivo photoinactivation of Escherichia coli ribonucleoside reductase by near-ultraviolet light

    Energy Technology Data Exchange (ETDEWEB)

    Peters, J [California Univ., Irvine (USA)

    1977-06-09

    Some experimental work is described showing that near-uv irradiation of E.coli cells selectively destroys RDP-reductase (ribonucleoside diphosphate reductase) activity in vivo are providing evidence relating the loss of RDP-reductase to loss of cellular visibility and the inactivity of irrdiated cells to support the replication of DNA phages. The data are consistent with the interpretation that the principal cause in the killing of exponentially growing E.coli cells by near-uv, and the loss of ability of irradiated host cells to support the replication of DNA phages, is the photoinactivation of the RDP-reductase complex.

  5. LC/MS/MS data analysis of the human uterine smooth muscle S-nitrosoproteome fingerprint in pregnancy, labor, and preterm labor

    Directory of Open Access Journals (Sweden)

    Craig C. Ulrich

    2015-09-01

    Full Text Available The data described in this article is the subject of an article in the American Journal of Physiology: Cell Physiology, titled “The Human Uterine Smooth Muscle S-nitrosoproteome Fingerprint in Pregnancy, Labor, and Preterm Labor” (doi:10.1152/ajpcell.00198.2013 (Ulrich et al., 2013 [1]. The data described is a large scale mass spectrometry data set that defines the human uterine smooth muscle S-nitrosoproteome differences among laboring, non-laboring, preterm laboring tissue after treatment with S-nitrosoglutathione.

  6. Sterol-induced Dislocation of 3-Hydroxy-3-methylglutaryl Coenzyme A Reductase from Endoplasmic Reticulum Membranes into the Cytosol through a Subcellular Compartment Resembling Lipid Droplets*

    Science.gov (United States)

    Hartman, Isamu Z.; Liu, Pingsheng; Zehmer, John K.; Luby-Phelps, Katherine; Jo, Youngah; Anderson, Richard G. W.; DeBose-Boyd, Russell A.

    2010-01-01

    Sterol-induced binding to Insigs in the endoplasmic reticulum (ER) allows for ubiquitination of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate-limiting enzyme in cholesterol synthesis. This ubiquitination marks reductase for recognition by the ATPase VCP/p97, which mediates extraction and delivery of reductase from ER membranes to cytosolic 26 S proteasomes for degradation. Here, we report that reductase becomes dislocated from ER membranes into the cytosol of sterol-treated cells. This dislocation exhibits an absolute requirement for the actions of Insigs and VCP/p97. Reductase also appears in a buoyant fraction of sterol-treated cells that co-purifies with lipid droplets, cytosolic organelles traditionally regarded as storage depots for neutral lipids such as triglycerides and cholesteryl esters. Genetic, biochemical, and localization studies suggest a model in which reductase is dislodged into the cytosol from an ER subdomain closely associated with lipid droplets. PMID:20406816

  7. Association between methylenetetrahydrofolate reductase (MTHFR ...

    African Journals Online (AJOL)

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

  8. Binding of Fidarestat Stereoisomers with Aldose Reductase

    Directory of Open Access Journals (Sweden)

    Dae-Sil Lee

    2006-11-01

    Full Text Available The stereospecificity in binding to aldose reductase (ALR2 of two fidarestat {6-fluoro-2',5'-dioxospiro[chroman-4,4'-imidazolidine]-2-carboxamide} stereoisomers [(2S,4Sand (2R,4S] has been investigated by means of molecular dynamics simulations using freeenergy integration techniques. The difference in the free energy of binding was found to be2.0 ± 1.7 kJ/mol in favour of the (2S,4S-form, in agreement with the experimentalinhibition data. The relative mobilities of the fidarestats complexed with ALR2 indicate alarger entropic penalty for hydrophobic binding of (2R,4S-fidarestat compared to (2S,4S-fidarestat, partially explaining its lower binding affinity. The two stereoisomers differmainly in the orientation of the carbamoyl moiety with respect to the active site and rotationof the bond joining the carbamoyl substituent to the ring. The detailed structural andenergetic insights obtained from out simulations allow for a better understanding of thefactors determining stereospecific inhibitor-ALR2 binding in the EPF charges model.

  9. Resonance Raman spectra of the copper-sulfur chromophores in Achromobacter cycloclastes nitrite reductase.

    Science.gov (United States)

    Dooley, D M; Moog, R S; Liu, M Y; Payne, W J; LeGall, J

    1988-10-15

    Resonance Raman spectroscopy at ambient temperature and 77 K has been used to probe the structures of the copper sites in Achromobacter cycloclastes nitrite reductase. This enzyme contains three copper ions per protein molecule and has two principal electronic absorption bands with lambda max values of 458 and 585 nm. Comparisons between the resonance Raman spectra of nitrite reductase and blue copper proteins establish that both the 458 and 585 nm bands are associated with Cu(II)-S(Cys) chromophores. A histidine ligand probably is also present. Different sets of vibrational frequencies are observed with 457.9 nm (ambient) or 476.1 nm (77 K) excitation as compared with 590 nm (ambient) or 593 nm (77 K) excitation. Excitation profiles indicate that the 458 and 585 nm absorption bands are associated with separate [Cu(II)-S(Cys)N(His)] sites or with inequivalent and uncoupled cysteine ligands in the same site. The former possibility is considered to be more likely.

  10. The function and properties of the iron-sulfur center in spinach ferredoxin: Thioredoxin reductase: A new biological role for iron-sulfur clusters

    Energy Technology Data Exchange (ETDEWEB)

    Staples, C.R.; Ameyibor, E.; Fu, Weiguang; Johnson, M.K. [Univ. of Georgia, Athens, GA (United States)] [and others

    1996-09-03

    Thioredoxin reduction in chloroplasts in catalyzed by a unique class of disulfide reductases which use a [2Fe-2S]{sup 2+/+} ferredoxin as the electron donor and contain an Fe-S cluster as the sole prosthetic group in addition to the active-site disulfide. The nature, properties, and function of the Fe-S cluster in spinach ferredoxin: thioredoxin reductase (FTR) have been investigated by the combination of UV/visible absorption, variable-temperature magnetic circular dichroism (MCD), EPR, and resonance Raman (RR) spectroscopies. 66 refs., 5 figs., 1 tab.

  11. A Rational Approach to Identify Inhibitors of Mycobacterium tuberculosis Enoyl Acyl Carrier Protein Reductase

    Czech Academy of Sciences Publication Activity Database

    Chhabria, M. T.; Parmar, K. B.; Brahmkshatriya, Pathik

    2013-01-01

    Roč. 19, č. 21 (2013), s. 3878-3883 ISSN 1381-6128 Institutional support: RVO:61388963 Keywords : mycobacterium tuberculosis * enoyl acyl carrier protein reductase * pharmacophore modeling * molecular docking * binding interactions Subject RIV: FR - Pharmacology ; Medidal Chemistry Impact factor: 3.288, year: 2013

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2006-12-01

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

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

    International Nuclear Information System (INIS)

    Rosenthal, Cindy; Mueller, Uwe; Panjikar, Santosh; Sun, Lianli; Ruppert, Martin; Zhao, Yu; Stöckigt, Joachim

    2006-01-01

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

  14. Identification of the 7-Hydroxymethyl Chlorophyll a Reductase of the Chlorophyll Cycle in Arabidopsis[W

    Science.gov (United States)

    Meguro, Miki; Ito, Hisashi; Takabayashi, Atsushi; Tanaka, Ryouichi; Tanaka, Ayumi

    2011-01-01

    The interconversion of chlorophyll a and chlorophyll b, referred to as the chlorophyll cycle, plays a crucial role in the processes of greening, acclimation to light intensity, and senescence. The chlorophyll cycle consists of three reactions: the conversions of chlorophyll a to chlorophyll b by chlorophyllide a oxygenase, chlorophyll b to 7-hydroxymethyl chlorophyll a by chlorophyll b reductase, and 7-hydroxymethyl chlorophyll a to chlorophyll a by 7-hydroxymethyl chlorophyll a reductase. We identified 7-hydroxymethyl chlorophyll a reductase, which is the last remaining unidentified enzyme of the chlorophyll cycle, from Arabidopsis thaliana by genetic and biochemical methods. Recombinant 7-hydroxymethyl chlorophyll a reductase converted 7-hydroxymethyl chlorophyll a to chlorophyll a using ferredoxin. Both sequence and biochemical analyses showed that 7-hydroxymethyl chlorophyll a reductase contains flavin adenine dinucleotide and an iron-sulfur center. In addition, a phylogenetic analysis elucidated the evolution of 7-hydroxymethyl chlorophyll a reductase from divinyl chlorophyllide vinyl reductase. A mutant lacking 7-hydroxymethyl chlorophyll a reductase was found to accumulate 7-hydroxymethyl chlorophyll a and pheophorbide a. Furthermore, this accumulation of pheophorbide a in the mutant was rescued by the inactivation of the chlorophyll b reductase gene. The downregulation of pheophorbide a oxygenase activity is discussed in relation to 7-hydroxymethyl chlorophyll a accumulation. PMID:21934147

  15. Genome sequence analysis of predicted polyprenol reductase gene from mangrove plant kandelia obovata

    Science.gov (United States)

    Basyuni, M.; Sagami, H.; Baba, S.; Oku, H.

    2018-03-01

    It has been previously reported that dolichols but not polyprenols were predominated in mangrove leaves and roots. Therefore, the occurrence of larger amounts of dolichol in leaves of mangrove plants implies that polyprenol reductase is responsible for the conversion of polyprenol to dolichol may be active in mangrove leaves. Here we report the early assessment of probably polyprenol reductase gene from genome sequence of mangrove plant Kandelia obovata. The functional assignment of the gene was based on a homology search of the sequences against the non-redundant (nr) peptide database of NCBI using Blastx. The degree of sequence identity between DNA sequence and known polyprenol reductase was confirmed using the Blastx probability E-value, total score, and identity. The genome sequence data resulted in three partial sequences, termed c23157 (700 bp), c23901 (960 bp), and c24171 (531 bp). The c23157 gene showed the highest similarity (61%) to predicted polyprenol reductase 2- like from Gossypium raimondii with E-value 2e-100. The second gene was c23901 to exhibit high similarity (78%) to the steroid 5-alpha-reductase Det2 from J. curcas with E-value 2e-140. Furthermore, the c24171 gene depicted highest similarity (79%) to the polyprenol reductase 2 isoform X1 from Jatropha curcas with E- value 7e-21.The present study suggested that the c23157, c23901, and c24171, genes may encode predicted polyprenol reductase. The c23157, c23901, c24171 are therefore the new type of predicted polyprenol reductase from K. obovata.

  16. 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. ...

  17. Proteomic identification of S-nitrosylated proteins in Arabidopsis

    DEFF Research Database (Denmark)

    Lindermayr, C.; Saalbach, G.; Durner, J.

    2005-01-01

    Although nitric oxide (NO) has grown into a key signaling molecule in plants during the last few years, less is known about how NO regulates different events in plants. Analyses of NO-dependent processes in animal systems have demonstrated protein S-nitrosylation of cysteine (Cys) residues...... to be one of the dominant regulation mechanisms for many animal proteins. For plants, the principle of S-nitrosylation remained to be elucidated. We generated S-nitrosothiols by treating extracts from Arabidopsis (Arabidopsis thaliana) cell suspension cultures with the NO-donor S......-nitrosoglutathione. Furthermore, Arabidopsis plants were treated with gaseous NO to analyze whether S-nitrosylation can occur in the specific redox environment of a plant cell in vivo. S-Nitrosylated proteins were detected by a biotin switch method, converting S-nitrosylated Cys to biotinylated Cys. Biotin-labeled proteins were...

  18. Bioinformatics analysis of the predicted polyprenol reductase genes in higher plants

    Science.gov (United States)

    Basyuni, M.; Wati, R.

    2018-03-01

    The present study evaluates the bioinformatics methods to analyze twenty-four predicted polyprenol reductase genes from higher plants on GenBank as well as predicted the structure, composition, similarity, subcellular localization, and phylogenetic. The physicochemical properties of plant polyprenol showed diversity among the observed genes. The percentage of the secondary structure of plant polyprenol genes followed the ratio order of α helix > random coil > extended chain structure. The values of chloroplast but not signal peptide were too low, indicated that few chloroplast transit peptide in plant polyprenol reductase genes. The possibility of the potential transit peptide showed variation among the plant polyprenol reductase, suggested the importance of understanding the variety of peptide components of plant polyprenol genes. To clarify this finding, a phylogenetic tree was drawn. The phylogenetic tree shows several branches in the tree, suggested that plant polyprenol reductase genes grouped into divergent clusters in the tree.

  19. Gene cloning and overexpression of two conjugated polyketone reductases, novel aldo-keto reductase family enzymes, of Candida parapsilosis.

    Science.gov (United States)

    Kataoka, M; Delacruz-Hidalgo, A-R G; Akond, M A; Sakuradani, E; Kita, K; Shimizu, S

    2004-04-01

    The genes encoding two conjugated polyketone reductases (CPR-C1, CPR-C2) of Candida parapsilosis IFO 0708 were cloned and sequenced. The genes encoded a total of 304 and 307 amino acid residues for CPR-C1 and CPR-C2, respectively. The deduced amino acid sequences of the two enzymes showed high similarity to each other and to several proteins of the aldo-keto reductase (AKR) superfamily. However, several amino acid residues in putative active sites of AKRs were not conserved in CPR-C1 and CPR-C2. The two CPR genes were overexpressed in Escherichia coli. The E. coli transformant bearing the CPR-C2 gene almost stoichiometrically reduced 30 mg ketopantoyl lactone/ml to D-pantoyl lactone.

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

    Science.gov (United States)

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

    2015-10-01

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

  1. Identification of the iron-sulfur center of spinach ferredoxin-nitrite reductase as a tetranuclear center, and preliminary EPR studies of mechanism.

    Science.gov (United States)

    Lancaster, J R; Vega, J M; Kamin, H; Orme-Johnson, N R; Orme-Johnson, W H; Krueger, R J; Siegel, L M

    1979-02-25

    EPR spectroscopic and chemical analyses of spinach nitrite reductase show that the enzyme contains one reducible iron-sulfur center, and one site for binding either cyanide or nitrite, per siroheme. The heme is nearly all in the high spin ferric state in the enzyme as isolated. The extinction coefficient of the enzyme has been revised to E386 = 7.6 X 10(4) cm-1 (M heme)-1. The iron-sulfur center is reduced with difficulty by agents such as reduced methyl viologen (equilibrated with 1 atm of H2 at pH 7.7 in the presence of hydrogenase) or dithionite. Complexation of the enzyme with CO (a known ligand for nitrite reductase heme) markedly increases the reducibility of the iron-sulfur center. New chemical analyses and reinterpretation of previous data show that the enzyme contains 6 mol of iron and 4 mol of acid-labile S2-/mol of siroheme. The EPR spectrum of reduced nitrite reductase in 80% dimethyl sulfoxide establishes clearly that the enzyme contains a tetranuclear iron-sulfur (Fe4S4) center. The ferriheme and Fe4S4 centers are reduced at similar rates (k = 3 to 4 s-1) by dithionite. The dithionite-reduced Fe4S4 center is rapidly (k = 100 s-1) reoxidized by nitrite. These results indicate a role for the Fe4S4 center in catalysis.

  2. A case of severe methylenetetrahydrofolate reductase deficiency presenting as neonatal encephalopathy, seizures, microcephaly and central hypoventilation

    NARCIS (Netherlands)

    Balasubramaniam, S.; Salomons, G.S.; Blom, H.J.

    2013-01-01

    Methylenetetrahydrofolate reductase (MTHFR) is a key regulatory enzyme in the remethylation of homocysteine to methionine. S-adenosylmethionine, formed from methionine and adenosine triphosphate, is the methyl donor in crucial reactions for brain development and function. MTHFR deficiency is the

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

    OpenAIRE

    Pandey, K N; Sabharwal, P S

    1982-01-01

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

  4. Identification of a Novel Epoxyqueuosine Reductase Family by Comparative Genomics.

    Science.gov (United States)

    Zallot, Rémi; Ross, Robert; Chen, Wei-Hung; Bruner, Steven D; Limbach, Patrick A; de Crécy-Lagard, Valérie

    2017-03-17

    The reduction of epoxyqueuosine (oQ) is the last step in the synthesis of the tRNA modification queuosine (Q). While the epoxyqueuosine reductase (EC 1.17.99.6) enzymatic activity was first described 30 years ago, the encoding gene queG was only identified in Escherichia coli in 2011. Interestingly, queG is absent from a large number of sequenced genomes that harbor Q synthesis or salvage genes, suggesting the existence of an alternative epoxyqueuosine reductase in these organisms. By analyzing phylogenetic distributions, physical gene clustering, and fusions, members of the Domain of Unknown Function 208 (DUF208) family were predicted to encode for an alternative epoxyqueuosine reductase. This prediction was validated with genetic methods. The Q modification is present in Lactobacillus salivarius, an organism missing queG but harboring the duf208 gene. Acinetobacter baylyi ADP1 is one of the few organisms that harbor both QueG and DUF208, and deletion of both corresponding genes was required to observe the absence of Q and the accumulation of oQ in tRNA. Finally, the conversion oQ to Q was restored in an E. coli queG mutant by complementation with plasmids harboring duf208 genes from different bacteria. Members of the DUF208 family are not homologous to QueG enzymes, and thus, duf208 is a non-orthologous replacement of queG. We propose to name DUF208 encoding genes as queH. While QueH contains conserved cysteines that could be involved in the coordination of a Fe/S center in a similar fashion to what has been identified in QueG, no cobalamin was identified associated with recombinant QueH protein.

  5. Hydroxyurea-resistant vaccinia virus: overproduction of ribonucleotide reductase

    International Nuclear Information System (INIS)

    Slabaugh, M.B.; Mathews, C.K.

    1986-01-01

    Repeated passage of vaccinia virus in increasing concentrations of hydroxyurea followed by plaque purification resulted in the isolation of variants capable of growth in 5 mM hydroxyurea, a drug concentration which inhibited the reproduction of wild-type vaccinia virus 1000-fold. Analyses of viral protein synthesis by using [ 35 S]methionine pulse-labeling at intervals throughout the infection cycle revealed that all isolates overproduced a 34,000-molecular-weight (MW) early polypeptide. Measurement of ribonucleoside-diphosphate reductase activity after infection indicated that 4- to 10-fold more activity was induced by hydroxyurea-resistant viruses than by the wild-type virus. A two-step partial purification resulted in a substantial enrichment for the 34,000-MW protein from extracts of wild-type and hydroxyurea-resistant-virus-infected, but not mock-infected, cells. In the presence of the drug, the isolates incorporated [ 3 H]thymidine into DNA earlier and a rate substantially greater than that of the wild type, although the onset of DNA synthesis was delayed in both cases. The drug resistance trait was markedly unstable in all isolates. In the absence of selective pressure, plaque-purified isolated readily segregated progeny that displayed a wide range of resistance phenotypes. The results of this study indicate that vaccinia virus encodes a subunit of ribonucleotide reductase which is 34,000-MW early protein whose overproduction confers hydroxyurea resistance on reproducing viruses

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

    Directory of Open Access Journals (Sweden)

    Yeon Bok Kim

    2014-01-01

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

  7. 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.

  8. Glutathione reductase: solvent equilibrium and kinetic isotope effects

    International Nuclear Information System (INIS)

    Wong, K.K.; Vanoni, M.A.; Blanchard, J.S.

    1988-01-01

    Glutathione reductase catalyzes the NADPH-dependent reduction of oxidized glutathione (GSSG). The kinetic mechanism is ping-pong, and we have investigated the rate-limiting nature of proton-transfer steps in the reactions catalyzed by the spinach, yeast, and human erythrocyte glutathione reductases using a combination of alternate substrate and solvent kinetic isotope effects. With NADPH or GSSG as the variable substrate, at a fixed, saturating concentration of the other substrate, solvent kinetic isotope effects were observed on V but not V/K. Plots of Vm vs mole fraction of D 2 O (proton inventories) were linear in both cases for the yeast, spinach, and human erythrocyte enzymes. When solvent kinetic isotope effect studies were performed with DTNB instead of GSSG as an alternate substrate, a solvent kinetic isotope effect of 1.0 was observed. Solvent kinetic isotope effect measurements were also performed on the asymmetric disulfides GSSNB and GSSNP by using human erythrocyte glutathione reductase. The Km values for GSSNB and GSSNP were 70 microM and 13 microM, respectively, and V values were 62 and 57% of the one calculated for GSSG, respectively. Both of these substrates yield solvent kinetic isotope effects greater than 1.0 on both V and V/K and linear proton inventories, indicating that a single proton-transfer step is still rate limiting. These data are discussed in relationship to the chemical mechanism of GSSG reduction and the identity of the proton-transfer step whose rate is sensitive to solvent isotopic composition. Finally, the solvent equilibrium isotope effect measured with yeast glutathione reductase is 4.98, which allows us to calculate a fractionation factor for the thiol moiety of GSH of 0.456

  9. Oxidation of a [Cu2S] complex by N2O and CO2: insights into a role of tetranuclearity in the CuZ site of nitrous oxide reductase.

    Science.gov (United States)

    Bagherzadeh, Sharareh; Mankad, Neal P

    2018-01-25

    Oxidation of a [Cu 2 (μ-S)] complex by N 2 O or CO 2 generated a [Cu 2 (μ-SO 4 )] product. In the presence of a sulfur trap, a [Cu 2 (μ-O)] species also formed from N 2 O. A [Cu 2 (μ-CS 3 )] species derived from CS 2 modeled initial reaction intermediates. These observations indicate that one role of tetranuclearity in the Cu Z catalytic site of nitrous oxide reductase is to protect the crucial S 2- ligand from oxidation.

  10. Nitrite-dependent vasodilation is facilitated by hypoxia and is independent of known NO-generating nitrite reductase activities

    DEFF Research Database (Denmark)

    Fago, Angela; Dalsgaard, Thomas; Fago, Angela

    2007-01-01

    is largely intrinsic to the vessel and that under hypoxia physiological nitrite concentrations are sufficient to induce NO-mediated vasodilation independently of the nitrite reductase activities investigated here. Possible reaction mechanisms for nitrite vasoactivity, including formation of S...

  11. Solution structure of an arsenate reductase-related protein, YffB, from Brucella melitensis, the etiological agent responsible for brucellosis

    International Nuclear Information System (INIS)

    Buchko, Garry W.; Hewitt, Stephen N.; Napuli, Alberto J.; Van Voorhis, Wesley C.; Myler, Peter J.

    2011-01-01

    B. melitensis is a NIAID Category B microorganism that is responsible for brucellosis and is a potential agent for biological warfare. Here, the solution structure of the 116-residue arsenate reductase-related protein Bm-YffB (BR0369) from this organism is reported. Brucella melitensis is the etiological agent responsible for brucellosis. Present in the B. melitensis genome is a 116-residue protein related to arsenate reductases (Bm-YffB; BR0369). Arsenate reductases (ArsC) convert arsenate ion (H 2 AsO 4 − ), a compound that is toxic to bacteria, to arsenite ion (AsO 2 − ), a product that may be efficiently exported out of the cell. Consequently, Bm-YffB is a potential drug target because if arsenate reduction is the protein’s major biological function then disabling the cell’s ability to reduce arsenate would make these cells more sensitive to the deleterious effects of arsenate. Size-exclusion chromatography and NMR spectroscopy indicate that Bm-YffB is a monomer in solution. The solution structure of Bm-YffB shows that the protein consists of two domains: a four-stranded mixed β-sheet flanked by two α-helices on one side and an α-helical bundle. The α/β domain is characteristic of the fold of thioredoxin-like proteins and the overall structure is generally similar to those of known arsenate reductases despite the marginal sequence similarity. Chemical shift perturbation studies with 15 N-labeled Bm-YffB show that the protein binds reduced glutathione at a site adjacent to a region similar to the HX 3 CX 3 R catalytic sequence motif that is important for arsenic detoxification activity in the classical arsenate-reductase family of proteins. The latter observation supports the hypothesis that the ArsC-YffB family of proteins may function as glutathione-dependent thiol reductases. However, comparison of the structure of Bm-YffB with the structures of proteins from the classical ArsC family suggest that the mechanism and possibly the function of Bm

  12. Comparing the xylose reductase/xylitol dehydrogenase and xylose isomerase pathways in arabinose and xylose fermenting Saccharomyces cerevisiae strains

    Directory of Open Access Journals (Sweden)

    Hahn-Hägerdal Bärbel

    2008-10-01

    Full Text Available Abstract Background Ethanolic fermentation of lignocellulosic biomass is a sustainable option for the production of bioethanol. This process would greatly benefit from recombinant Saccharomyces cerevisiae strains also able to ferment, besides the hexose sugar fraction, the pentose sugars, arabinose and xylose. Different pathways can be introduced in S. cerevisiae to provide arabinose and xylose utilisation. In this study, the bacterial arabinose isomerase pathway was combined with two different xylose utilisation pathways: the xylose reductase/xylitol dehydrogenase and xylose isomerase pathways, respectively, in genetically identical strains. The strains were compared with respect to aerobic growth in arabinose and xylose batch culture and in anaerobic batch fermentation of a mixture of glucose, arabinose and xylose. Results The specific aerobic arabinose growth rate was identical, 0.03 h-1, for the xylose reductase/xylitol dehydrogenase and xylose isomerase strain. The xylose reductase/xylitol dehydrogenase strain displayed higher aerobic growth rate on xylose, 0.14 h-1, and higher specific xylose consumption rate in anaerobic batch fermentation, 0.09 g (g cells-1 h-1 than the xylose isomerase strain, which only reached 0.03 h-1 and 0.02 g (g cells-1h-1, respectively. Whereas the xylose reductase/xylitol dehydrogenase strain produced higher ethanol yield on total sugars, 0.23 g g-1 compared with 0.18 g g-1 for the xylose isomerase strain, the xylose isomerase strain achieved higher ethanol yield on consumed sugars, 0.41 g g-1 compared with 0.32 g g-1 for the xylose reductase/xylitol dehydrogenase strain. Anaerobic fermentation of a mixture of glucose, arabinose and xylose resulted in higher final ethanol concentration, 14.7 g l-1 for the xylose reductase/xylitol dehydrogenase strain compared with 11.8 g l-1 for the xylose isomerase strain, and in higher specific ethanol productivity, 0.024 g (g cells-1 h-1 compared with 0.01 g (g cells-1 h-1

  13. Substrate and cofactor binding to nitrile reductase : A mass spectrometry based study

    NARCIS (Netherlands)

    Gjonaj, L.; Pinkse, M.W.H.; Fernandez Fueyo, E.; Hollmann, F.; Hanefeld, U.

    2016-01-01

    Nitrile reductases catalyse a two-step reduction of nitriles to amines. This requires the binding of two NADPH molecules during one catalytic cycle. For the nitrile reductase from E. coli (EcoNR) mass spectrometry studies of the catalytic mechanism were performed. EcoNR is dimeric and has no Rossman

  14. Homology modeling of dissimilatory APS reductases (AprBA of sulfur-oxidizing and sulfate-reducing prokaryotes.

    Directory of Open Access Journals (Sweden)

    Birte Meyer

    Full Text Available BACKGROUND: The dissimilatory adenosine-5'-phosphosulfate (APS reductase (cofactors flavin adenine dinucleotide, FAD, and two [4Fe-4S] centers catalyzes the transformation of APS to sulfite and AMP in sulfate-reducing prokaryotes (SRP; in sulfur-oxidizing bacteria (SOB it has been suggested to operate in the reverse direction. Recently, the three-dimensional structure of the Archaeoglobus fulgidus enzyme has been determined in different catalytically relevant states providing insights into its reaction cycle. METHODOLOGY/PRINCIPAL FINDINGS: Full-length AprBA sequences from 20 phylogenetically distinct SRP and SOB species were used for homology modeling. In general, the average accuracy of the calculated models was sufficiently good to allow a structural and functional comparison between the beta- and alpha-subunit structures (78.8-99.3% and 89.5-96.8% of the AprB and AprA main chain atoms, respectively, had root mean square deviations below 1 A with respect to the template structures. Besides their overall conformity, the SRP- and SOB-derived models revealed the existence of individual adaptations at the electron-transferring AprB protein surface presumably resulting from docking to different electron donor/acceptor proteins. These structural alterations correlated with the protein phylogeny (three major phylogenetic lineages: (1 SRP including LGT-affected Archaeoglobi and SOB of Apr lineage II, (2 crenarchaeal SRP Caldivirga and Pyrobaculum, and (3 SOB of the distinct Apr lineage I and the presence of potential APS reductase-interacting redox complexes. The almost identical protein matrices surrounding both [4Fe-4S] clusters, the FAD cofactor, the active site channel and center within the AprB/A models of SRP and SOB point to a highly similar catalytic process of APS reduction/sulfite oxidation independent of the metabolism type the APS reductase is involved in and the species it has been originated from. CONCLUSIONS: Based on the comparative

  15. Prevalence of methylenetetrahydrofolate reductase ( MTHFR ) and ...

    African Journals Online (AJOL)

    Methylenetetrahydrofolate reductase (MTHFR) and Cytosolic serine hydroxymethyltransferase (cSHMT) are enzymes involve in folate regulation in human. The C to T transition of the cSHMT and MTHFR genes at the 1420 as well as 677 nucleotides both carries TT genotype respectively. These enzymes have direct and ...

  16. 1H, 15N and 13C NMR Assignments of Mouse Methionine Sulfoxide Reductase B2

    Science.gov (United States)

    Breivik, Åshild S.; Aachmann, Finn L.; Sal, Lena S.; Kim, Hwa-Young; Del Conte, Rebecca; Gladyshev, Vadim N.; Dikiy, Alexander

    2011-01-01

    A recombinant mouse methionine-r-sulfoxide reductase 2 (MsrB2ΔS) isotopically labeled with 15N and 15N/13C was generated. We report here the 1H, 15N and 13C NMR assignments of the reduced form of this protein. PMID:19636904

  17. Methemoglobin reductase activity in intact fish red blood cells

    DEFF Research Database (Denmark)

    Jensen, Frank B; Nielsen, Karsten

    2018-01-01

    RBCs in physiological saline at normal Pco2 and pH. After initial loading of oxygenated RBCs with nitrite (partly oxidizing Hb to metHb), the nitrite is removed by three washes of the RBCs in nitrite-free physiological saline to enable the detection of RBC metHb reductase activity in the absence......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...

  18. Molecular mechanisms of drug resistance and tumor promotion involving mammalian ribonucleotide reductase

    Energy Technology Data Exchange (ETDEWEB)

    Choy, B.B.K.

    1991-01-01

    Mammalian ribonucleotide reductase is a highly regulated, rate-limiting activity responsible for converting ribonucleoside diphosphates to the deoxyribonucleotide precursors of DNA. The enzyme consists of two nonidentical proteins called M1 and M2, both of which are required for activity. Hydroxyurea is an antitumor agent which inhibits ribonucleotide reductase by interacting with the M2 component specifically at a unique tyrosyl free radical. Studies were conducted on a series of drug resistant mouse cell lines, selected by a step-wise procedure for increasing levels of resistance to the cytotoxic effects of hydroxyurea. Each successive drug selection step leading to the isolation of highly resistant cells was accompanied by stable elevations in cellular resistance and ribonucleotide reductase activity. The drug resistant cell lines exhibited gene amplification of the M2 gene, elevated M2 mRNA, and M2 protein. In addition to M2 gene amplification, posttranscriptional modulation also occurred during the drug selection. Studies of the biosynthesis rates with exogenously added iron suggest a role for iron in regulating the level of M2 protein when cells are cultured in the presence of hydroxyurea. The hydroxyurea-inactivated ribonucleotide reductase protein M2 has a destabilized iron centre, which readily releases iron. Altered expression of ferritin appears to be required for the development of hydroxyurea resistance in nammalian cells. The results show an interesting relationship between the expressions of ribonucleotide reductase and ferritin. The phorbol ester tumor promoter, TPA, is also able to alter the expression of M2. TPA was able to induce M2 mRNA levels transiently up to 18-fold within 1/2 hour. This rapid and large elevation of ribonucleotide reductase suggests that the enzyme may play a role in tumor promotion. Studies of the M2 promoter region were undertaken to better understand the mechanism of TPA induction of M2.

  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. 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.

  1. In silico docking studies of aldose reductase inhibitory activity of commercially available flavonoids

    Directory of Open Access Journals (Sweden)

    Arumugam Madeswaran

    2012-12-01

    Full Text Available The primary objective of this study was to investigate the aldose reductase inhibitory activity of flavonoids using in silico docking studies. In this perspective, flavonoids like biochanin, butein, esculatin, fisetin and herbacetin were selected. Epalrestat, a known aldose reductase inhibitor was used as the standard. In silico docking studies were carried out using AutoDock 4.2, based on the Lamarckian genetic algorithm principle. The results showed that all the selected flavonoids showed binding energy ranging between -9.33 kcal/mol to -7.23 kcal/mol when compared with that of the standard (-8.73 kcal/mol. Inhibition constant (144.13 µM to 4.98 µM and intermolecular energy (-11.42 kcal/mol to -7.83 kcal/mol of the flavonoids also coincide with the binding energy. All the selected flavonoids contributed aldose reductase inhibitory activity because of its structural properties. These molecular docking analyses could lead to the further development of potent aldose reductase inhibitors for the treatment of diabetes.

  2. 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.

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

    International Nuclear Information System (INIS)

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

    2007-01-01

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

  4. Purification of nitrate reductase from Nicotiana plumbaginifolia by affinity chromatography using 5'AMP-sepharose and monoclonal antibodies.

    Science.gov (United States)

    Moureaux, T; Leydecker, M T; Meyer, C

    1989-02-15

    Nitrate reductase was purified from leaves of Nicotiana plumbaginifolia using either 5'AMP-Sepharose chromatography or two steps of immunoaffinity chromatography involving monoclonal antibodies directed against nitrate reductase from maize and against ribulose-1,5-bisphosphate carboxylase from N. plumbaginifolia. Nitrate reductase obtained by the first method was purified 1000-fold to a specific activity of 9 units/mg protein. The second method produced an homogenous enzyme, purified 21,000-fold to a specific activity of 80 units/mg protein. SDS/PAGE of nitrate reductase always resulted in two bands of 107 and 99.5 kDa. The 107-kDa band was the nitrate reductase subunit of N. plumbaginifolia; the smaller one of 99.5 kDa is thought, as commonly reported, to result from proteolysis of the larger protein. The molecular mass of 107 kDa is close to the values calculated from the coding sequences of the two nitrate reductase genes recently cloned from tobacco (Nicotiana tabacum cv Xanthi).

  5. Constitutive non-inducible expression of the Arabidopsis thaliana Nia 2 gene in two nitrate reductase mutants of Nicotiana plumbaginifolia.

    Science.gov (United States)

    Kaye, C; Crawford, N M; Malmberg, R L

    1997-04-01

    We have isolated a haploid cell line of N. plumbaginifolia, hNP 588, that is constitutive and not inducible for nitrate reductase. Nitrate reductase mutants were isolated from hNP 588 protoplasts upon UV irradiation. Two of these nitrate reductase-deficient cell lines, nia 3 and nia 25, neither of which contained any detectable nitrate reductase activity, were selected for complementation studies. A cloned Arabidopsis thaliana nitrate reductase gene Nia 2 was introduced into each of the two mutants resulting in 56 independent kanamycin-resistant cell lines. Thirty of the 56 kanamycin-resistant cell lines were able to grow on nitrate as the sole nitrogen source. Eight of these were further analyzed for nitrate reductase enzyme activity and nitrate reductase mRNA production. All eight lines had detectable nitrate reductase activity ranging from 7% to 150% of wild-type hNP 588 callus. The enzyme activity levels were not influenced by the nitrogen source in the medium. The eight lines examined expressed a constitutive, non-inducible 3.2 kb mRNA species that was not present in untransformed controls.

  6. Overexpression of chloroplast NADPH-dependent thioredoxin reductase in Arabidopsis enhances leaf growth and elucidates in-vivo function of reductase and thioredoxin domains

    Directory of Open Access Journals (Sweden)

    Jouni eToivola

    2013-10-01

    Full Text Available Plant chloroplasts have versatile thioredoxin systems including two thioredoxin reductases and multiple types of thioredoxins. Plastid-localized NADPH-dependent thioredoxin reductase (NTRC contains both reductase (NTRd and thioredoxin (TRXd domains in a single polypeptide and forms homodimers. To study the action of NTRC and NTRC domains in vivo, we have complemented the ntrc knockout line of Arabidopsis with the wild type and full-length NTRC genes, in which 2-Cys motifs either in NTRd, or in TRXd were inactivated. The ntrc line was also transformed either with the truncated NTRd or TRXd alone. Overexpression of wild-type NTRC promoted plant growth by increasing leaf size and biomass yield of the rosettes. Complementation of the ntrc line with the full-length NTRC gene containing an active reductase but an inactive thioredoxin domain, or vice versa, recovered wild-type chloroplast phenotype and, partly, rosette biomass production, indicating that the NTRC domains are capable of interacting with other chloroplast thioredoxin systems. Overexpression of truncated NTRd or TRXd in ntrc background did not restore wild-type phenotype. Modelling of the 3-dimensional structure of the NTRC dimer indicates extensive interactions between the NTR domains and the TRX domains further stabilize the dimeric structure. The long linker region between the NTRd and TRXd, however, allows flexibility for the position of the TRXd in the dimer. Supplementation of the TRXd in the NTRC homodimer model by free chloroplast thioredoxins indicated that TRXf is the most likely partner to interact with NTRC. We propose that overexpression of NTRC promotes plant biomass yield both directly by stimulation of chloroplast biosynthetic and protected pathways controlled by NTRC and indirectly via free chloroplast thioredoxins. Our data indicate that overexpression of chloroplast thiol redox-regulator has a potential to increase biofuel yield in plant and algal species suitable for

  7. 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...

  8. Characterization and regulation of Leishmania major 3-hydroxy-3-methylglutaryl-CoA reductase

    DEFF Research Database (Denmark)

    Montalvetti, A; Pena Diaz, Javier; Hurtado, R

    2000-01-01

    In eukaryotes the enzyme 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase catalyses the synthesis of mevalonic acid, a common precursor to all isoprenoid compounds. Here we report the isolation and overexpression of the gene coding for HMG-CoA reductase from Leishmania major. The protein from L...

  9. 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 ...

  10. Characterization of human warfarin reductase

    OpenAIRE

    Sokolová, Simona

    2016-01-01

    Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Simona Sokolová Supervisor: PharmDr. Petra Malátková, Ph.D. Title of diploma thesis: Characterization of human warfarin reductase Warfarin is widely used anticoagulant drug. Considering the narrow therapeutic window of warfarin, it is important to fully understand its metabolism in human body. Oxidative, reductive and conjugation reactions are involved in warfarin metabolism. Howev...

  11. 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.

  12. Inhibitory effect of rhetsinine isolated from Evodia rutaecarpa on aldose reductase activity.

    Science.gov (United States)

    Kato, A; Yasuko, H; Goto, H; Hollinshead, J; Nash, R J; Adachi, I

    2009-03-01

    Aldose reductase inhibitors have considerable potential for the treatment of diabetic complications, without increased risk of hypoglycemia. Search for components inhibiting aldose reductase led to the discovery of active compounds contained in Evodia rutaecarpa Bentham (Rutaceae), which is the one of the component of Kampo-herbal medicine. The hot water extract from the E. rutaecarpa was subjected to distribution or gel filtration chromatography to give an active compound, N2-(2-methylaminobenzoyl)tetrahydro-1H-pyrido[3,4-b]indol-1-one (rhetsinine). It inhibited aldose reductase with IC(50) values of 24.1 microM. Furthermore, rhetsinine inhibited sorbitol accumulation by 79.3% at 100 microM. These results suggested that the E. rutaecarpa derived component, rhetsinine, would be potentially useful in the treatment of diabetic complications.

  13. 21 CFR 864.7375 - Glutathione reductase assay.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Glutathione reductase assay. 864.7375 Section 864.7375 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7375 Glutathione...

  14. Crystallization and preliminary X-ray diffraction analysis of salutaridine reductase from the opium poppy Papaver somniferum

    International Nuclear Information System (INIS)

    Higashi, Yasuhiro; Smith, Thomas J.; Jez, Joseph M.; Kutchan, Toni M.

    2010-01-01

    Recombinant P. somniferum salutaridine reductase (SalR) was purified and crystallized with NADPH using the hanging-drop vapor-diffusion method. Crystals of the SalR–NADPH complex diffracted X-rays to a resolution of 1.9 Å. The opium poppy Papaver somniferum is the source of the narcotic analgesics morphine and codeine. Salutaridine reductase (SalR; EC 1.1.1.248) reduces the C-7 keto group of salutaridine to the C-7 (S)-hydroxyl group of salutaridinol in the biosynthetic pathway that leads to morphine in the opium poppy plant. P. somniferum SalR was overproduced in Escherichia coli and purified using cobalt-affinity and size-exclusion chromatography. Hexagonal crystals belonging to space group P6 4 22 or P6 2 22 were obtained using ammonium sulfate as precipitant and diffracted to a resolution of 1.9 Å

  15. Role of a novel dual flavin reductase (NR1) and an associated histidine triad protein (DCS-1) in menadione-induced cytotoxicity

    International Nuclear Information System (INIS)

    Kwasnicka-Crawford, Dorota A.; Vincent, Steven R.

    2005-01-01

    Microsomal cytochrome P450 reductase catalyzes the one-electron transfer from NADPH via FAD and FMN to various electron acceptors, such as cytochrome P450s or to some anti-cancer quinone drugs. This results in generation of free radicals and toxic oxygen metabolites, which can contribute to the cytotoxicity of these compounds. Recently, a cytosolic NADPH-dependent flavin reductase, NR1, has been described which is highly homologous to the microsomal cytochrome P450 reductase. In this study, we show that over-expression of NR1 in human embryonic kidney cells enhances the cytotoxic action of the model quinone, menadione. Furthermore, we show that a novel human histidine triad protein DCS-1, which is expressed together with NR1 in many tissues, can significantly reduce menadione-induced cytotoxicity in these cells. We also show that DCS-1 binds NF1 and directly modulates its activity. These results suggest that NR1 may play a role in carcinogenicity and cell death associated with one-electron reductions

  16. Acidithiobacillus caldus sulfur oxidation model based on transcriptome analysis between the wild type and sulfur oxygenase reductase defective mutant.

    Directory of Open Access Journals (Sweden)

    Linxu Chen

    Full Text Available Acidithiobacillus caldus (A. caldus is widely used in bio-leaching. It gains energy and electrons from oxidation of elemental sulfur and reduced inorganic sulfur compounds (RISCs for carbon dioxide fixation and growth. Genomic analyses suggest that its sulfur oxidation system involves a truncated sulfur oxidation (Sox system (omitting SoxCD, non-Sox sulfur oxidation system similar to the sulfur oxidation in A. ferrooxidans, and sulfur oxygenase reductase (SOR. The complexity of the sulfur oxidation system of A. caldus generates a big obstacle on the research of its sulfur oxidation mechanism. However, the development of genetic manipulation method for A. caldus in recent years provides powerful tools for constructing genetic mutants to study the sulfur oxidation system.An A. caldus mutant lacking the sulfur oxygenase reductase gene (sor was created and its growth abilities were measured in media using elemental sulfur (S(0 and tetrathionate (K(2S(4O(6 as the substrates, respectively. Then, comparative transcriptome analysis (microarrays and real-time quantitative PCR of the wild type and the Δsor mutant in S(0 and K(2S(4O(6 media were employed to detect the differentially expressed genes involved in sulfur oxidation. SOR was concluded to oxidize the cytoplasmic elemental sulfur, but could not couple the sulfur oxidation with the electron transfer chain or substrate-level phosphorylation. Other elemental sulfur oxidation pathways including sulfur diooxygenase (SDO and heterodisulfide reductase (HDR, the truncated Sox pathway, and the S(4I pathway for hydrolysis of tetrathionate and oxidation of thiosulfate in A. caldus are proposed according to expression patterns of sulfur oxidation genes and growth abilities of the wild type and the mutant in different substrates media.An integrated sulfur oxidation model with various sulfur oxidation pathways of A. caldus is proposed and the features of this model are summarized.

  17. 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.

  18. Characterisation of a Desmosterol Reductase Involved in Phytosterol Dealkylation in the Silkworm, Bombyx mori

    Science.gov (United States)

    Ciufo, Leonora F.; Murray, Patricia A.; Thompson, Anu; Rigden, Daniel J.; Rees, Huw H.

    2011-01-01

    Most species of invertebrate animals cannot synthesise sterols de novo and many that feed on plants dealkylate phytosterols (mostly C29 and C28) yielding cholesterol (C27). 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 250kDa. 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. PMID:21738635

  19. Inhibition of human anthracycline reductases by emodin — A possible remedy for anthracycline resistance

    International Nuclear Information System (INIS)

    Hintzpeter, Jan; Seliger, Jan Moritz; Hofman, Jakub; Martin, Hans-Joerg; Wsol, Vladimir; Maser, Edmund

    2016-01-01

    The clinical application of anthracyclines, like daunorubicin and doxorubicin, is limited by two factors: dose-related cardiotoxicity and drug resistance. Both have been linked to reductive metabolism of the parent drug to their metabolites daunorubicinol and doxorubicinol, respectively. These metabolites show significantly less anti-neoplastic properties as their parent drugs and accumulate in cardiac tissue leading to chronic cardiotoxicity. Therefore, we aimed to identify novel and potent natural inhibitors for anthracycline reductases, which enhance the anticancer effect of anthracyclines by preventing the development of anthracycline resistance. Human enzymes responsible for the reductive metabolism of daunorubicin were tested for their sensitivity towards anthrachinones, in particular emodin and anthraflavic acid. Intense inhibition kinetic data for the most effective daunorubicin reductases, including IC 50 - and K i -values, the mode of inhibition, as well as molecular docking, were compiled. Subsequently, a cytotoxicity profile and the ability of emodin to reverse daunorubicin resistance were determined using multiresistant A549 lung cancer and HepG2 liver cancer cells. Emodin potently inhibited the four main human daunorubicin reductases in vitro. Further, we could demonstrate that emodin is able to synergistically sensitize human cancer cells towards daunorubicin at clinically relevant concentrations. Therefore, emodin may yield the potential to enhance the therapeutic effectiveness of anthracyclines by preventing anthracycline resistance via inhibition of the anthracycline reductases. In symphony with its known pharmacological properties, emodin might be a compound of particular interest in the management of anthracycline chemotherapy efficacy and their adverse effects. - Highlights: • Natural and synthetic compounds were identified as inhibitors for human daunorubicin reductases. • Emodin is a potent inhibitor for human daunorubicin reductases.

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

    African Journals Online (AJOL)

    Aldose Reductase Inhibitory and Antiglycation Activities of Four Medicinal Plant Standardized Extracts and Their Main Constituents for the Prevention of ... levels in galactosemic condition by using reverse phase high pressure liquid chromatography (RP-HPLC) and gas liquid chromatography (GLC) was determined.

  1. Xylose reductase from the thermophilic fungus Talaromyces emersonii

    Indian Academy of Sciences (India)

    Prakash

    Xylose reductase is involved in the first step of the fungal pentose catabolic pathway. The gene .... proteins with reversed coenzyme preference from NADPH to NADH ..... 399–404. Hasper A A, Visser J and de Graaff L H 2000 The Aspergillus.

  2. Biosynthesis of tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate by carbonyl reductase from Rhodosporidium toruloides in mono and biphasic media.

    Science.gov (United States)

    Liu, Zhi-Qiang; Wu, Lin; Zheng, Ling; Wang, Wen-Zhong; Zhang, Xiao-Jian; Jin, Li-Qun; Zheng, Yu-Guo

    2018-02-01

    tert-Butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate ((3R,5S)-CDHH) is the key intermediate for synthesis of atorvastatin and rosuvastatin. Carbonyl reductase exhibits excellent activity toward tert-butyl (S)-6-chloro-5-hydroxy-3-oxohexanoate ((S)-CHOH) to synthesize (3R,5S)-CDHH. In this study, a whole cell biosynthesis reaction system to produce (3R,5S)-CDHH was constructed in organic solvents. A solution of 10% (v/v) Tween-80 was introduced to the reaction system as a co-solvent, which greatly enhanced biotransformation process, giving 98.9% yield, >99% ee and 1.8-fold higher space time yield in 5 h bioconversion of 1 M (S)-CHOH, compared with 98.7% yield and >99% ee in 9 h bioconversion of a purely aqueous reaction system. Moreover, a water-octanol biphasic reaction system was built and 20% of octanol was added as reservoir of substrate resulting in 98% yield, >99% ee and 4.08 mmol L -1  h -1  g -1 (wet cell weight) space time yield. This study paved a way for the whole cell biosynthesis of (3R,5S)-CDHH in mono and biphasic media. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. 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.

  4. JS-K, a Nitric Oxide Prodrug, Has Enhanced Cytotoxicity in Colon Cancer Cells with Knockdown of Thioredoxin Reductase 1

    Science.gov (United States)

    Edes, Kornelia; Cassidy, Pamela; Shami, Paul J.; Moos, Philip J.

    2010-01-01

    Background The selenoenzyme thioredoxin reductase 1 has a complex role relating to cell growth. It is induced as a component of the cellular response to potentially mutagenic oxidants, but also appears to provide growth advantages to transformed cells by inhibiting apoptosis. In addition, selenocysteine-deficient or alkylated forms of thioredoxin reductase 1 have also demonstrated oxidative, pro-apoptotic activity. Therefore, a greater understanding of the role of thioredoxin reductase in redox initiated apoptotic processes is warranted. Methodology The role of thioredoxin reductase 1 in RKO cells was evaluated by attenuating endogenous thioredoxin reductase 1 expression with siRNA and then either inducing a selenium-deficient thioredoxin reductase or treatment with distinct redox challenges including, hydrogen peroxide, an oxidized lipid, 4-hydroxy-2-nonenol, and a nitric oxide donating prodrug. Thioredoxin redox status, cellular viability, and effector caspase activity were measured. Conclusions/Significance In cells with attenuated endogenous thioredoxin reductase 1, a stably integrated selenocysteine-deficient form of the enzyme was induced but did not alter either the thioredoxin redox status or the cellular growth kinetics. The oxidized lipid and the nitric oxide donor demonstrated enhanced cytotoxicity when thioredoxin reductase 1 was knocked-down; however, the effect was more pronounced with the nitric oxide prodrug. These results are consistent with the hypothesis that attenuation of the thioredoxin-system can promote apoptosis in a nitric oxide-dependent manner. PMID:20098717

  5. Evidence for a Ustilago maydis Steroid 5α-Reductase by Functional Expression in Arabidopsis det2-1 Mutants1

    Science.gov (United States)

    Basse, Christoph W.; Kerschbamer, Christine; Brustmann, Markus; Altmann, Thomas; Kahmann, Regine

    2002-01-01

    We have identified a gene (udh1) in the basidiomycete Ustilago maydis that is induced during the parasitic interaction with its host plant maize (Zea mays). udh1 encodes a protein with high similarity to mammalian and plant 5α-steroid reductases. Udh1 differs from those of known 5α-steroid reductases by six additional domains, partially predicted to be membrane-spanning. A fusion protein of Udh1 and the green fluorescent protein provided evidence for endoplasmic reticulum localization in U. maydis. The function of the Udh1 protein was demonstrated by complementing Arabidopsis det2-1 mutants, which display a dwarf phenotype due to a mutation in the 5α-steroid reductase encoding DET2 gene. det2-1 mutant plants expressing either the udh1 or the DET2 gene controlled by the cauliflower mosaic virus 35S promoter differed from wild-type Columbia plants by accelerated stem growth, flower and seed development and a reduction in size and number of rosette leaves. The accelerated growth phenotype of udh1 transgenic plants was stably inherited and was favored under reduced light conditions. Truncation of the N-terminal 70 amino acids of the Udh1 protein abolished the ability to restore growth in det2-1 plants. Our results demonstrate the existence of a 5α-steroid reductase encoding gene in fungi and suggest a common ancestor between fungal, plant, and mammalian proteins. PMID:12068114

  6. Synthesis of enantiomerically enriched drug precursors and an insect pheromone via reduction of ketones using commercially available carbonyl reductase screening kit "Chiralscreen® OH".

    Science.gov (United States)

    Nagai, Toshiya; Sakurai, Saki; Natori, Naoki; Hataoka, Manaka; Kinoshita, Takako; Inoue, Hiroyoshi; Hanaya, Kengo; Shoji, Mitsuru; Sugai, Takeshi

    2018-04-01

    Commercially available "Chiralscreen® OH" starter kit containing five types of carbonyl reductases (E001, E007, E031, E039, and E078) was used for the reduction of several aromatic and aliphatic ketones to obtain enantiomerically enriched drug precursors and an insect pheromone. Almost stereochemically pure secondary alcohols, used in the synthesis of drugs such as (R)-rasagiline mesylate, (S)-rivastigmine, (R)-chlorphenesin carbamate, and (R)-mexiletine, and the insect pheromone (4S,5R)-sitophilure, were conveniently obtained. The enzymes worked well with ketones containing at least one non-bulky substituent at the carbonyl group. The diverse stereochemical preference of the above five carbonyl reductases was clarified. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. Mitochondrial fumarate reductase as a target of chemotherapy: from parasites to cancer cells.

    Science.gov (United States)

    Sakai, Chika; Tomitsuka, Eriko; Esumi, Hiroyasu; Harada, Shigeharu; Kita, Kiyoshi

    2012-05-01

    Recent research on respiratory chain of the parasitic helminth, Ascaris suum has shown that the mitochondrial NADH-fumarate reductase system (fumarate respiration), which is composed of complex I (NADH-rhodoquinone reductase), rhodoquinone and complex II (rhodoquinol-fumarate reductase) plays an important role in the anaerobic energy metabolism of adult parasites inhabiting hosts. The enzymes in these parasite-specific pathways are potential target for chemotherapy. We isolated a novel compound, nafuredin, from Aspergillus niger, which inhibits NADH-fumarate reductase in helminth mitochondria at nM order. It competes for the quinone-binding site in complex I and shows high selective toxicity to the helminth enzyme. Moreover, nafuredin exerts anthelmintic activity against Haemonchus contortus in in vivo trials with sheep indicating that mitochondrial complex I is a promising target for chemotherapy. In addition to complex I, complex II is a good target because its catalytic direction is reverse of succinate-ubiquionone reductase in the host complex II. Furthermore, we found atpenin and flutolanil strongly and specifically inhibit mitochondrial complex II. Interestingly, fumarate respiration was found not only in the parasites but also in some types of human cancer cells. Analysis of the mitochondria from the cancer cells identified an anthelminthic as a specific inhibitor of the fumarate respiration. Role of isoforms of human complex II in the hypoxic condition of cancer cells and fetal tissues is a challenge. This article is part of a Special Issue entitled Biochemistry of Mitochondria, Life and Intervention 2010. Copyright © 2011 Elsevier B.V. All rights reserved.

  8. Inhibition of human anthracycline reductases by emodin — A possible remedy for anthracycline resistance

    Energy Technology Data Exchange (ETDEWEB)

    Hintzpeter, Jan, E-mail: hintzpeter@toxi.uni-kiel.de [Institute of Toxicology and Pharmacology for Natural Scientists, University Medical School Schleswig-Holstein, Campus Kiel, Brunswiker Str. 10, 24105 Kiel (Germany); Seliger, Jan Moritz [Institute of Toxicology and Pharmacology for Natural Scientists, University Medical School Schleswig-Holstein, Campus Kiel, Brunswiker Str. 10, 24105 Kiel (Germany); Hofman, Jakub [Department of Biochemical Sciences, Faculty of Pharmacy in Hradec Kralove, Charles University in Prague, Heyrovskeho 1203, 50005 Hradec Kralove (Czech Republic); Martin, Hans-Joerg [Institute of Toxicology and Pharmacology for Natural Scientists, University Medical School Schleswig-Holstein, Campus Kiel, Brunswiker Str. 10, 24105 Kiel (Germany); Wsol, Vladimir [Department of Biochemical Sciences, Faculty of Pharmacy in Hradec Kralove, Charles University in Prague, Heyrovskeho 1203, 50005 Hradec Kralove (Czech Republic); Maser, Edmund [Institute of Toxicology and Pharmacology for Natural Scientists, University Medical School Schleswig-Holstein, Campus Kiel, Brunswiker Str. 10, 24105 Kiel (Germany)

    2016-02-15

    The clinical application of anthracyclines, like daunorubicin and doxorubicin, is limited by two factors: dose-related cardiotoxicity and drug resistance. Both have been linked to reductive metabolism of the parent drug to their metabolites daunorubicinol and doxorubicinol, respectively. These metabolites show significantly less anti-neoplastic properties as their parent drugs and accumulate in cardiac tissue leading to chronic cardiotoxicity. Therefore, we aimed to identify novel and potent natural inhibitors for anthracycline reductases, which enhance the anticancer effect of anthracyclines by preventing the development of anthracycline resistance. Human enzymes responsible for the reductive metabolism of daunorubicin were tested for their sensitivity towards anthrachinones, in particular emodin and anthraflavic acid. Intense inhibition kinetic data for the most effective daunorubicin reductases, including IC{sub 50}- and K{sub i}-values, the mode of inhibition, as well as molecular docking, were compiled. Subsequently, a cytotoxicity profile and the ability of emodin to reverse daunorubicin resistance were determined using multiresistant A549 lung cancer and HepG2 liver cancer cells. Emodin potently inhibited the four main human daunorubicin reductases in vitro. Further, we could demonstrate that emodin is able to synergistically sensitize human cancer cells towards daunorubicin at clinically relevant concentrations. Therefore, emodin may yield the potential to enhance the therapeutic effectiveness of anthracyclines by preventing anthracycline resistance via inhibition of the anthracycline reductases. In symphony with its known pharmacological properties, emodin might be a compound of particular interest in the management of anthracycline chemotherapy efficacy and their adverse effects. - Highlights: • Natural and synthetic compounds were identified as inhibitors for human daunorubicin reductases. • Emodin is a potent inhibitor for human daunorubicin

  9. Cloning and sequence of the human adrenodoxin reductase gene

    International Nuclear Information System (INIS)

    Lin, Dong; Shi, Y.; Miller, W.L.

    1990-01-01

    Adrenodoxin reductase is a flavoprotein mediating electron transport to all mitochondrial forms of cytochrome P450. The authors cloned the human adrenodoxin reductase gene and characterized it by restriction endonuclease mapping and DNA sequencing. The entire gene is approximately 12 kilobases long and consists of 12 exons. The first exon encodes the first 26 of the 32 amino acids of the signal peptide, and the second exon encodes the remainder of signal peptide and the apparent FAD binding site. The remaining 10 exons are clustered in a region of only 4.3 kilobases, separated from the first two exons by a large intron of about 5.6 kilobases. Two forms of human adrenodoxin reductase mRNA, differing by the presence or absence of 18 bases in the middle of the sequence, arise from alternate splicing at the 5' end of exon 7. This alternately spliced region is directly adjacent to the NADPH binding site, which is entirely contained in exon 6. The immediate 5' flanking region lacks TATA and CAAT boxes; however, this region is rich in G+C and contains six copies of the sequence GGGCGGG, resembling promoter sequences of housekeeping genes. RNase protection experiments show that transcription is initiated from multiple sites in the 5' flanking region, located about 21-91 base pairs upstream from the AUG translational initiation codon

  10. Structural and biochemical properties of cloned and expressed human and rat steroid 5α-reductases

    International Nuclear Information System (INIS)

    Andersson, S.; Russell, D.W.

    1990-01-01

    The microsomal enzyme steroid 5α-reductase is responsible for the conversion of testosterone into the more potent androgen dihydrotestosterone. In man, this steroid acts on a variety of androgen-responsive target tissues to mediate such diverse endocrine processes as male sexual differentiation in the fetus and prostatic growth in men. Here we describe the isolation, structure, and expression of a cDNA encoding the human steroid 5α-reductase. A rat cDNA was used as a hybridization probe to screen a human prostate cDNA library. A 2.1-kilobase cDNA was identified and DNA sequence analysis indicated that the human steroid 5α-reductase was a hydrophobic protein of 259 amino acids with a predicted molecular weight of 29,462. A comparison of the human and rat protein sequences revealed a 60% identity. Transfection of expression vectors containing the human and rat cDNAs into simian COS cells resulted in the synthesis of high levels of steroid 5α-reductase enzyme activity. Both enzymes expressed in COS cells showed similar substrate specificities for naturally occurring steroid hormones. However, synthetic 4-azasteroids demonstrated marked differences in their abilities to inhibit the human and rat steroid 5α-reductases

  11. Meta-Prediction of the Effect of Methylenetetrahydrofolate Reductase Polymorphisms and Air Pollution on Alzheimer’s Disease Risk

    Directory of Open Access Journals (Sweden)

    Suh-Mian Wu

    2017-01-01

    Full Text Available Background: Alzheimer’s disease (AD is a significant public health issue. AD has been linked with methylenetetrahydrofolate reductase (MTHFR C677T polymorphism, but the findings have been inconsistent. The purpose of this meta-predictive analysis is to examine the associations between MTHFR polymorphisms and epigenetic factors, including air pollution, with AD risk using big data analytics approaches. Methods and Results: Forty-three studies (44 groups were identified by searching various databases. MTHFR C677T TT and CT genotypes had significant associations with AD risk in all racial populations (RR = 1.13, p = 0.0047; and RR = 1.12, p < 0.0001 respectively. Meta-predictive analysis showed significant increases of percentages of MTHFR C677T polymorphism with increased air pollution levels in both AD case group and control group (p = 0.0021–0.0457; with higher percentages of TT and CT genotypes in the AD case group than that in the control group with increased air pollution levels. Conclusions: The impact of MTHFR C677T polymorphism on susceptibility to AD was modified by level of air pollution. Future studies are needed to further examine the effects of gene-environment interactions including air pollution on AD risk for world populations.

  12. Overview of Catalytic Properties of Fungal Xylose Reductases and Molecular Engineering Approaches for Improved Xylose Utilisation in Yeast

    Directory of Open Access Journals (Sweden)

    Sk Amir Hossain

    2018-03-01

    Full Text Available Background and Objective: Xylose reductases belong to the aldo-keto reductase family of enzymes, which catalyse the conversion of xylose to xylitol. Yeast xylose reductases have been intensively studied in the last two decades due to their significance in biotechnological production of ethanol and xylitol from xylose. Due to its GRAS status and pronounced tolerance to harsh conditions, Saccharomyces cerevisiae is the ideal organism for industrial production of both xylitol and ethanol. However, Saccharomyces cerevisiae is unable to use xylose as the sole carbon source due to the lack of xylose specific transporters and insufficient activity of metabolic pathways for xylose utilisation. The aim of this paper is to give an overview of attempts in increasing biotechnological potential of xylose reductases and to highlight the prospective of this application. Results and Conclusion: In order to create strains with improved xylose utilization, different approaches were attempted including simultaneous overexpression of xylitol dehydrogenase, xylose reductase and pentose phosphate pathway enzymes, heterologous expression of putative xylose transporters or heterologous expression of genes coding for enzymes included in the xylose metabolism, respectively. Furthermore, number of attempts to genetically modify different xylose reductases is increasing. This review presents current knowledge about yeast xylose reductases and the different approaches applied in order to improve xylose metabolism in yeast.Conflict of interest: The authors declare no conflict of interest.

  13. 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

  14. Characterization of a cultured human T-cell line with genetically altered ribonucleotide reductase activity. Model for immunodeficiency.

    Science.gov (United States)

    Waddell, D; Ullman, B

    1983-04-10

    From human CCRF-CEM T-cells growing in continuous culture, we have selected, isolated, and characterized a clonal cell line, APHID-D2, with altered ribonucleotide reductase activity. In comparative growth rate experiments, the APHID-D2 cell line is less sensitive than the parental cell line to growth inhibition by deoxyadenosine in the presence of 10 microM erythro-9-(2-hydroxy-3-nonyl)adenine, an inhibitor of adenosine deaminase. The APHID-D2 cell line has elevated levels of all four dNTPs. The resistance of the APHID-D2 cell line to growth inhibition by deoxyadenosine and the abnormal dNTP levels can be explained by the fact that the APHID-D2 ribonucleotide reductase, unlike the parental ribonucleotide reductase, is not normally sensitive to inhibition by dATP. These results suggest that the allosteric site of ribonucleotide reductase which binds both dATP and ATP is altered in the APHID-D2 line. The isolation of a mutant clone of human T-cells which contains a ribonucleotide reductase that has lost its normal sensitivity to dATP and which is resistant to deoxyadenosine-mediated growth inhibition suggests that a primary pathogenic target of accumulated dATP in lymphocytes from patients with adenosine deaminase deficiency may be the cellular ribonucleotide reductase.

  15. YKL071W from Saccharomyces cerevisiae encodes a novel aldehyde reductase for detoxification of glycolaldehyde and furfural derived from lignocellulose.

    Science.gov (United States)

    Wang, Hanyu; Ouyang, Yidan; Zhou, Chang; Xiao, Difan; Guo, Yaping; Wu, Lan; Li, Xi; Gu, Yunfu; Xiang, Quanju; Zhao, Ke; Yu, Xiumei; Zou, Likou; Ma, Menggen

    2017-12-01

    Aldehydes generated as by-products during the pretreatment of lignocellulose are the key inhibitors to Saccharomyces cerevisiae, which is considered as the most promising microorganism for industrial production of biofuel, xylitol as well as other special chemicals from lignocellulose. S. cerevisiae has the inherent ability to in situ detoxify aldehydes to corresponding alcohols by multiple aldehyde reductases. Herein, we report that an uncharacterized open reading frame YKL071W from S. cerevisiae encodes a novel "classical" short-chain dehydrogenase/reductase (SDR) protein with NADH-dependent enzymatic activities for reduction of furfural (FF), glycolaldehyde (GA), formaldehyde (FA), and benzaldehyde (BZA). This enzyme showed much better specific activities for reduction of GA and FF than FA and BZA, and displayed much higher Km and Kcat/Km but lower Vmax and Kcat for reduction of GA than FF. For this enzyme, the optimum pH was 5.5 and 6.0 for reduction of GA and FF, and the optimum temperature was 30 °C for reduction of GA and FF. Both pH and temperature affected stability of this enzyme in a similar trend for reduction of GA and FF. Cu 2+ , Zn 2+ , Ni 2+ , and Fe 3+ had severe inhibition effects on enzyme activities of Ykl071wp for reduction of GA and FF. Transcription of YKL071W in S. cerevisiae was significantly upregulated under GA and FF stress conditions, and its transcription is most probably regulated by transcription factor genes of YAP1, CAD1, PDR3, and STB5. This research provides guidelines to identify more uncharacterized genes with reductase activities for detoxification of aldehydes derived from lignocellulose in S. cerevisiae.

  16. Purification, crystallization and preliminary X-ray diffraction analysis of enoyl-acyl carrier protein reductase (FabK) from Streptococcus mutans strain UA159

    International Nuclear Information System (INIS)

    Kim, Tae-O; Im, Dong-Won; Jung, Ha Yun; Kwon, Seong Jung; Heo, Yong-Seok

    2012-01-01

    Enoyl-acyl carrier protein reductase (FabK) from S. mutans strain UA159 was cloned, overexpressed, purified and crystallized. X-ray diffraction data were collected to a resolution of 2.40 Å. A triclosan-resistant flavoprotein termed FabK is the sole enoyl-acyl carrier protein reductase in Streptococcus pneumoniae and Streptococcus mutans. In this study, FabK from S. mutans strain UA159 was overexpressed in Escherichia coli, purified and crystallized. Diffraction data were collected to 2.40 Å resolution using a synchrotron-radiation source. The crystal belonged to space group P6 2 , with unit-cell parameters a = b = 105.79, c = 44.15 Å. The asymmetric unit contained one molecule, with a corresponding V M of 2.05 Å 3 Da −1 and a solvent content of 39.9%

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

    DEFF Research Database (Denmark)

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

    2003-01-01

    A plasmid-borne diacetyl (acetoin) reductase (butA) from Leuconostoc pseudomesenteroides CHCC2114 was sequenced and cloned. Nucleotide sequence analysis revealed an open reading frame encoding a protein of 257 amino acids which had high identity at the amino acid level to diacetyl (acetoin...

  18. X-Ray crystal structure of GarR—tartronate semialdehyde reductase from Salmonella typhimurium

    OpenAIRE

    Osipiuk, J.; Zhou, M.; Moy, S.; Collart, F.; Joachimiak, A.

    2009-01-01

    Tartronate semialdehyde reductases (TSRs), also known as 2-hydroxy-3-oxopropionate reductases, catalyze the reduction of tartronate semialdehyde using NAD as cofactor in the final stage of D-glycerate biosynthesis. These enzymes belong to family of structurally and mechanically related β-hydroxyacid dehydrogenases which differ in substrate specificity and catalyze reactions in specific metabolic pathways. Here, we present the crystal structure of GarR a TSR from Salmonella typhimurium determi...

  19. Reduced bone mass and muscle strength in male 5α-reductase type 1 inactivated mice.

    Directory of Open Access Journals (Sweden)

    Sara H Windahl

    Full Text Available Androgens are important regulators of bone mass but the relative importance of testosterone (T versus dihydrotestosterone (DHT for the activation of the androgen receptor (AR in bone is unknown. 5α-reductase is responsible for the irreversible conversion of T to the more potent AR activator DHT. There are two well established isoenzymes of 5α-reductase (type 1 and type 2, encoded by separate genes (Srd5a1 and Srd5a2. 5α-reductase type 2 is predominantly expressed in male reproductive tissues whereas 5α-reductase type 1 is highly expressed in liver and moderately expressed in several other tissues including bone. The aim of the present study was to investigate the role of 5α-reductase type 1 for bone mass using Srd5a1⁻/⁻ mice. Four-month-old male Srd5a1⁻/⁻ mice had reduced trabecular bone mineral density (-36%, p<0.05 and cortical bone mineral content (-15%, p<0.05 but unchanged serum androgen levels compared with wild type (WT mice. The cortical bone dimensions were reduced in the male Srd5a1⁻/⁻ mice as a result of a reduced cortical periosteal circumference compared with WT mice. T treatment increased the cortical periosteal circumference (p<0.05 in orchidectomized WT mice but not in orchidectomized Srd5a1⁻/⁻ mice. Male Srd5a1⁻/⁻ mice demonstrated a reduced forelimb muscle grip strength compared with WT mice (p<0.05. Female Srd5a1⁻/⁻ mice had slightly increased cortical bone mass associated with elevated circulating levels of androgens. In conclusion, 5α-reductase type 1 inactivated male mice have reduced bone mass and forelimb muscle grip strength and we propose that these effects are due to lack of 5α-reductase type 1 expression in bone and muscle. In contrast, the increased cortical bone mass in female Srd5a1⁻/⁻ mice, is an indirect effect mediated by elevated circulating androgen levels.

  20. Effect of cystamine on rat tissue GSH level and glutathione reductase activity

    International Nuclear Information System (INIS)

    Kovarova, H.; Pulpanova, J.

    1979-01-01

    Reduced glutathione (GSH) level and glutathione reductase activity were determined by means of the spectrophotometric method in various rat tissues after i.p. administration of cystamine (50 mg/kg and 20 mg/kg). GSH amount dropped in the spleen and kidney at 10 and 20 min; following this interval, an increase of GSH level was observed in the liver at 20-30 min, in the spleen and kidney at 60 min after the treatment with a radioprotective cystamine dose (50 mg/kg). The changes in GSH level induced by a non-radioprotective cystamine dose (20 mg/kg) had an opposite tendency. The activity of glutathione reductase was decreased in all tissues studied. As to the mechanism of the radioprotective action, both the inactivation of glutathione reductase activity and the changes in GSH level seem to be the factors contributing to the radioprotective effect of cystamine by strengthening the cellular radioresistance. (orig.) 891 MG/orig. 892 RKD [de

  1. Expression, purification, crystallization and preliminary X-ray diffraction analysis of carbonyl reductase from Candida parapsilosis ATCC 7330

    International Nuclear Information System (INIS)

    Aggarwal, Nidhi; Mandal, P. K.; Gautham, Namasivayam; Chadha, Anju

    2013-01-01

    The expression, purification, crystallization, preliminary X-ray diffraction and molecular-replacement studies on C. parapsilosis carbonyl reductase are reported. The NAD(P)H-dependent carbonyl reductase from Candida parapsilosis ATCC 7330 catalyses the asymmetric reduction of ethyl 4-phenyl-2-oxobutanoate to ethyl (R)-4-phenyl-2-hydroxybutanoate, a precursor of angiotensin-converting enzyme inhibitors such as Cilazapril and Benazepril. The carbonyl reductase was expressed in Escherichia coli and purified by GST-affinity and size-exclusion chromatography. Crystals were obtained by the hanging-drop vapour-diffusion method and diffracted to 1.86 Å resolution. The asymmetric unit contained two molecules of carbonyl reductase, with a solvent content of 48%. The structure was solved by molecular replacement using cinnamyl alcohol dehydrogenase from Saccharomyces cerevisiae as a search model

  2. 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.

  3. Photoaffinity labeling of steroid 5 alpha-reductase of rat liver and prostate microsomes

    International Nuclear Information System (INIS)

    Liang, T.; Cheung, A.H.; Reynolds, G.F.; Rasmusson, G.H.

    1985-01-01

    21-Diazo-4-methyl-4-aza-5 alpha-pregnane-3,20-dione (Diazo-MAPD) inhibits steroid 5 alpha-reductase in liver microsomes of female rats with a K/sub i/ value of 8.7 +/- 1.7 nM, and the inhibition is competitive with testosterone. It also inhibits the binding of a 5 alpha-reductase inhibitor, [ 3 H] 17 beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one ([ 3 H]4-MA), to the enzyme in liver microsomes. The inhibition of 5 alpha-reductase activity and of inhibitor binding activity by diazo-MAPD becomes irreversible upon UV irradiation. [1,2- 3 H]Diazo-MAPD binds to a single high affinity site in liver microsomes of female rats, and this binding requires NADPH. Without UV irradiation, this binding is reversible, and it becomes irreversible upon UV irradiation. Both the initial reversible binding and the subsequent irreversible conjugation after UV irradiation are inhibited by inhibitors (diazo-MAPD and 4-MA) and substrates (progesterone and testosterone) of 5 alpha-reductase, but they are not inhibited by 5 alpha-reduced steroids. Photoaffinity labeled liver microsomes of female rats were solubilized and fractionated by high performance gel filtration. The radioactive conjugate eluted in one major peak at Mr 50,000

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

    International Nuclear Information System (INIS)

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

    2006-01-01

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

  5. 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.

  6. Isolation and primary structural analysis of two conjugated polyketone reductases from Candida parapsilosis.

    Science.gov (United States)

    Hidalgo, A R; Akond, M A; Kita, K; Kataoka, M; Shimizu, S

    2001-12-01

    Two conjugated polyketone reductases (CPRs) were isolated from Candida parapsilosis IFO 0708. The primary structures of CPRs (C1 and C2) were analyzed by amino acid sequencing. The amino acid sequences of both enzymes had high similarity to those of several proteins of the aldo-keto-reductase (AKR) superfamily. However, several amino acid residues in the putative active sites of AKRs were not conserved in CPRs-C1 and -C2.

  7. Phylogenetic analysis of bacterial and archaeal arsC gene sequences suggests an ancient, common origin for arsenate reductase

    Directory of Open Access Journals (Sweden)

    Dugas Sandra L

    2003-07-01

    Full Text Available Abstract Background The ars gene system provides arsenic resistance for a variety of microorganisms and can be chromosomal or plasmid-borne. The arsC gene, which codes for an arsenate reductase is essential for arsenate resistance and transforms arsenate into arsenite, which is extruded from the cell. A survey of GenBank shows that arsC appears to be phylogenetically widespread both in organisms with known arsenic resistance and those organisms that have been sequenced as part of whole genome projects. Results Phylogenetic analysis of aligned arsC sequences shows broad similarities to the established 16S rRNA phylogeny, with separation of bacterial, archaeal, and subsequently eukaryotic arsC genes. However, inconsistencies between arsC and 16S rRNA are apparent for some taxa. Cyanobacteria and some of the γ-Proteobacteria appear to possess arsC genes that are similar to those of Low GC Gram-positive Bacteria, and other isolated taxa possess arsC genes that would not be expected based on known evolutionary relationships. There is no clear separation of plasmid-borne and chromosomal arsC genes, although a number of the Enterobacteriales (γ-Proteobacteria possess similar plasmid-encoded arsC sequences. Conclusion The overall phylogeny of the arsenate reductases suggests a single, early origin of the arsC gene and subsequent sequence divergence to give the distinct arsC classes that exist today. Discrepancies between 16S rRNA and arsC phylogenies support the role of horizontal gene transfer (HGT in the evolution of arsenate reductases, with a number of instances of HGT early in bacterial arsC evolution. Plasmid-borne arsC genes are not monophyletic suggesting multiple cases of chromosomal-plasmid exchange and subsequent HGT. Overall, arsC phylogeny is complex and is likely the result of a number of evolutionary mechanisms.

  8. 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 ...

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

    African Journals Online (AJOL)

    USER

    2010-06-21

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

  10. 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

  11. 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.

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

    Indian Academy of Sciences (India)

    2015-06-01

    Jun 1, 2015 ... in prostate cancer patients: a potential factor implicated ... reductase alpha polypeptides 1 and 2 in a set of 601 prostate cancer patients from four ..... tion in the key androgen-regulating genes androgen receptor, cytochrome ...

  13. Molecular and phenotypic characterization of transgenic soybean expressing the Arabidopsis ferric chelate reductase gene, FRO2.

    Science.gov (United States)

    Vasconcelos, Marta; Eckert, Helene; Arahana, Venancio; Graef, George; Grusak, Michael A; Clemente, Tom

    2006-10-01

    Soybean (Glycine max Merr.) production is reduced under iron-limiting calcareous soils throughout the upper Midwest regions of the US. Like other dicotyledonous plants, soybean responds to iron-limiting environments by induction of an active proton pump, a ferric iron reductase and an iron transporter. Here we demonstrate that heterologous expression of the Arabidopsis thaliana ferric chelate reductase gene, FRO2, in transgenic soybean significantly enhances Fe(+3) reduction in roots and leaves. Root ferric reductase activity was up to tenfold higher in transgenic plants and was not subjected to post-transcriptional regulation. In leaves, reductase activity was threefold higher in the transgenic plants when compared to control. The enhanced ferric reductase activity led to reduced chlorosis, increased chlorophyll concentration and a lessening in biomass loss in the transgenic events between Fe treatments as compared to control plants grown under hydroponics that mimicked Fe-sufficient and Fe-deficient soil environments. However, the data indicate that constitutive FRO2 expression under non-iron stress conditions may lead to a decrease in plant productivity as reflected by reduced biomass accumulation in the transgenic events under non-iron stress conditions. When grown at Fe(III)-EDDHA levels greater than 10 microM, iron concentration in the shoots of transgenic plants was significantly higher than control. The same observation was found in the roots in plants grown at iron levels higher than 32 microM Fe(III)-EDDHA. These results suggest that heterologous expression of an iron chelate reductase in soybean can provide a route to alleviate iron deficiency chlorosis.

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

    Directory of Open Access Journals (Sweden)

    Hong-Zhe Li

    2015-07-01

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

  15. Inhibition of steroid 5 alpha-reductase by specific aliphatic unsaturated fatty acids.

    Science.gov (United States)

    Liang, T; Liao, S

    1992-01-01

    Human or rat microsomal 5 alpha-reductase activity, as measured by enzymic conversion of testosterone into 5 alpha-dihydrotestosterone or by binding of a competitive inhibitor, [3H]17 beta-NN-diethulcarbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one ([3H]4-MA) to the reductase, is inhibited by low concentrations (less than 10 microM) of certain polyunsaturated fatty acids. The relative inhibitory potencies of unsaturated fatty acids are, in decreasing order: gamma-linolenic acid greater than cis-4,7,10,13,16,19-docosahexaenoic acid = cis-6,9,12,15-octatetraenoic acid = arachidonic acid = alpha-linolenic acid greater than linoleic acid greater than palmitoleic acid greater than oleic acid greater than myristoleic acid. Other unsaturated fatty acids such as undecylenic acid, erucic acid and nervonic acid, are inactive. The methyl esters and alcohol analogues of these compounds, glycerols, phospholipids, saturated fatty acids, retinoids and carotenes were inactive even at 0.2 mM. The results of the binding assay and the enzymic assay correlated well except for elaidic acid and linolelaidic acid, the trans isomers of oleic acid and linoleic acid respectively, which were much less active than their cis isomers in the binding assay but were as potent in the enzymic assay. gamma-Linolenic acid had no effect on the activities of two other rat liver microsomal enzymes: NADH:menadione reductase and glucuronosyl transferase. gamma-Linolenic acid, the most potent inhibitor tested, decreased the Vmax. and increased Km values of substrates, NADPH and testosterone, and promoted dissociation of [3H]4-MA from the microsomal reductase. gamma-Linolenic acid, but not the corresponding saturated fatty acid (stearic acid), inhibited the 5 alpha-reductase activity, but not the 17 beta-dehydrogenase activity, of human prostate cancer cells in culture. These results suggest that unsaturated fatty acids may play an important role in regulating androgen action in target cells. PMID:1637346

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

    Science.gov (United States)

    Rosenthal, Cindy; Mueller, Uwe; Panjikar, Santosh; Sun, Lianli; Ruppert, Martin; Zhao, Yu; Stöckigt, Joachim

    2006-01-01

    Perakine reductase (PR) is a novel member of the aldo-keto reductase enzyme superfamily from higher plants. PR from the plant Rauvolfia serpentina is involved in the biosynthesis of monoterpenoid indole alkaloids by performing NADPH-dependent reduction of perakine, yielding raucaffrinoline. However, PR can also reduce cinnamic aldehyde and some of its derivatives. After heterologous expression of a triple mutant of PR in Escherichia coli, crystals of the purified and methylated enzyme were obtained by the hanging-drop vapour-diffusion technique at 293 K with 100 mM sodium citrate pH 5.6 and 27% PEG 4000 as precipitant. Crystals belong to space group C2221 and diffract to 2.0 Å, with unit-cell parameters a = 58.9, b = 93.0, c = 143.4 Å. PMID:17142919

  17. Large-scale synthesis of tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate by a stereoselective carbonyl reductase with high substrate concentration and product yield.

    Science.gov (United States)

    Liu, Zhi-Qiang; Hu, Zhong-Liang; Zhang, Xiao-Jian; Tang, Xiao-Ling; Cheng, Feng; Xue, Ya-Ping; Wang, Ya-Jun; Wu, Lin; Yao, Dan-Kai; Zhou, Yi-Teng; Zheng, Yu-Guo

    2017-05-01

    To biosynthesize the (3R,5S)-CDHH in an industrial scale, a newly synthesized stereoselective short chain carbonyl reductase (SCR) was successfully cloned and expressed in Escherichia coli. The fermentation of recombinant E. coli harboring SCR was carried out in 500 L and 5000 L fermenters, with biomass and specific activity of 9.7 g DCW/L, 15749.95 U/g DCW, and 10.97 g DCW/L, 19210.12 U/g DCW, respectively. The recombinant SCR was successfully applied for efficient production of (3R,5S)-CDHH. The scale-up synthesis of (3R,5S)-CDHH was performed in 5000 L bioreactor with 400 g/L of (S)-CHOH at 30°C, resulting in a space-time yield of 13.7 mM/h/g DCW, which was the highest ever reported. After isolation and purification, the yield and d.e. of (3R,5S)-CDHH reached 97.5% and 99.5%, respectively. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:612-620, 2017. © 2017 American Institute of Chemical Engineers.

  18. The Nox/Ferric reductase/Ferric reductase-like families of Eumycetes.

    Science.gov (United States)

    Grissa, Ibtissem; Bidard, Frédérique; Grognet, Pierre; Grossetete, Sandrine; Silar, Philippe

    2010-09-01

    Reactive Oxygen Species (ROS) are involved in plant biomass degradation by fungi and development of fungal structures. While the ROS-generating NADPH oxidases from filamentous fungi are under strong scrutiny, much less is known about the related integral Membrane (or Ferric) Reductases (IMRs). Here, we present a survey of these enzymes in 29 fungal genomes covering the entire available range of fungal diversity. IMRs are present in all fungal genomes. They can be classified into at least 24 families, underscoring the high diversity of these enzymes. Some are differentially regulated during colony or fruiting body development, as well as by the nature of the carbon source of the growth medium. Importantly, functional characterization of IMRs has been made on proteins belonging to only two families, while nothing or very little is known about the proteins of the other 22 families. Copyright © 2010 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

  19. Crystallization and preliminary X-ray diffraction studies of ferredoxin reductase from Leptospira interrogans

    International Nuclear Information System (INIS)

    Nascimento, Alessandro S.; Ferrarezi, Thiago; Catalano-Dupuy, Daniela L.; Ceccarelli, Eduardo A.; Polikarpov, Igor

    2006-01-01

    Crystals adequate for X-ray diffraction analysis have been prepared from L. interrogans ferredoxin-NADP + reductase. Ferredoxin-NADP + reductase (FNR) is an FAD-containing enzyme that catalyzes electron transfer between NADP(H) and ferredoxin. Here, results are reported of the recombinant expression, purification and crystallization of FNR from Leptospira interrogans, a parasitic bacterium of animals and humans. The L. interrogans FNR crystals belong to a primitive monoclinic space group and diffract to 2.4 Å resolution at a synchrotron source

  20. 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.

  1. [Comparison of Physico-chemical Aspects between E. coli and Human Dihydrofolate Reductase: an Equilibrium Unfolding Study].

    Science.gov (United States)

    Thapliyal, Charu; Jain, Neha; Chaudhuri, Pratima

    2015-01-01

    A protein, differing in origin, may exhibit variable physicochemical behaviour, difference in sequence homology, fold and function. Thus studying structure-function relationship of proteins from altered sources is meaningful in the sense that it may give rise to comparative aspects of their sequence-structure-function relationship. Dihydrofolate reductase is an enzyme involved in cell cycle regulation. It is a significant enzyme as.a target for developing anticancer drugs. Hence, detailed understanding of structure-function relationships of wide variants of the enzyme dihydrofolate reductase would be important for developing an inhibitor or an antagonist against the enzyme involved in the cellular developmental processes. In this communication, we have reported the comparative structure-function relationship between E. coli and human dihydrofolate reductase. The differences in the unfolding behaviour of these two proteins have been investigated to understand various properties of these two proteins like relative' stability differences and variation in conformational changes under identical denaturing conditions. The equilibrium unfolding mechanism of dihydrofolate reductase proteins using guanidine hydrochloride as a denaturant in the presence of various types of osmolytes has been monitored using loss in enzymatic activity, intrinsic tryptophan fluorescence and an extrinsic fluorophore 8-anilino-1-naphthalene-sulfonic acid as probes. It has been observed that osmolytes, such as 1M sucrose, and 30% glycerol, provided enhanced stability to both variants of dihydrofolate reductase. Their level of stabilisation has been observed to be dependent on intrinsic protein stability. It was observed that 100 mM proline does not show any 'significant stabilisation to either of dihydrofolate reductases. In the present study, it has been observed that the human protein is relatively less stable than the E.coli counterpart.

  2. Crystallization of purple nitrous oxide reductase from Pseudomonas stutzeri

    International Nuclear Information System (INIS)

    Pomowski, Anja; Zumft, Walter G.; Kroneck, Peter M. H.; Einsle, Oliver

    2010-01-01

    The physiologically active form of nitrous oxide reductase was isolated and crystallized under strict exclusion of dioxygen and diffraction data were collected from crystals belonging to two different space groups. Nitrous oxide reductase (N 2 OR) from Pseudomonas stutzeri catalyzes the final step in denitrification: the two-electron reduction of nitrous oxide to molecular dinitrogen. Crystals of the enzyme were grown under strict exclusion of dioxygen by sitting-drop vapour diffusion using 2R,3R-butanediol as a cryoprotectant. N 2 OR crystallized in either space group P1 or P6 5 . Interestingly, the key determinant for the resulting space group was the crystallization temperature. Crystals belonging to space group P1 contained four 130 kDa dimers in the asymmetric unit, while crystals belonging to space group P6 5 contained a single dimer in the asymmetric unit. Diffraction data were collected to resolutions better than 2 Å

  3. Purification and characterization of Taenia crassiceps cysticerci thioredoxin: insight into thioredoxin-glutathione-reductase (TGR) substrate recognition.

    Science.gov (United States)

    Martínez-González, J J; Guevara-Flores, A; Rendón, J L; Sosa-Peinado, A; Del Arenal Mena, I P

    2015-04-01

    Thioredoxin (Trx) is an oxidoreductase central to redox homeostasis in cells and is involved in the regulation of protein activity through thiol/disulfide exchanges. Based on these facts, our goal was to purify and characterize cytosolic thioredoxin from Taenia crassiceps cysticerci, as well as to study its behavior as a substrate of thioredoxin-glutathione reductase (TGR). The enzyme was purified >133-fold with a total yield of 9.7%. A molecular mass of 11.7kDa and a pI of 4.84 were measured. Native electrophoresis was used to identify the oxidized and reduced forms of the monomer as well as the presence of a homodimer. In addition to the catalytic site cysteines, cysticerci thioredoxin contains Cys28 and Cys65 residues conserved in previously sequenced cestode thioredoxins. The following kinetic parameters were obtained for the substrate of TGR: a Km of 3.1μM, a kcat of 10s(-1) and a catalytic efficiency of 3.2×10(6)M(-1)s(-1). The negative patch around the α3-helix of Trx is involved in the interaction with TGR and suggests variable specificity and catalytic efficiency of the reductase toward thioredoxins of different origins. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  4. Role of aldose reductase C-106T polymorphism among diabetic Egyptian patients with different microvascular complications

    Directory of Open Access Journals (Sweden)

    Nermine Hossam Zakaria

    2014-04-01

    Full Text Available The aldose reductase pathway proves that elevated blood glucose promotes cellular dysfunction. The polyol pathway converts excess intracellular glucose into alcohols via activity of the aldose reductase. This enzyme catalyzes the conversion of glucose to sorbitol which triggers variety of intracellular changes in the tissues. Among diabetes, activity is drastically increased in association with three main consequences inside the cells. The aim of this study was to detect the association of the C-106 T polymorphism of the aldose reductase gene and its frequency among a sample of 150 Egyptian adults with type 2 diabetic patients having diabetic microvascular. The detection of the aldose reductase C-106 T polymorphism gene was done by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP. The genotype distribution of the C-106 T polymorphism showed that CC genotype was statistically significantly higher among patients with retinopathy compared to nephropathy. Patients with nephropathy had significant association with the TT genotype when compared with diabetic retinopathy patients. Follow up study after the genotype detection among recently diagnosed diabetic patients in order to give a prophylactic aldose reductase inhibitors; studying the microvascular complications and its relation to the genotype polymorphisms. The study may include multiple gene polymorphisms to make the relation between the gene and the occurrence of these complications more evident.

  5. Nitrate reductase and nitrous oxide production by Fusarium oxysporum 11dn1 under aerobic and anaerobic conditions.

    Science.gov (United States)

    Kurakov, A V; Nosikov, A N; Skrynnikova, E V; L'vov, N P

    2000-08-01

    The fungus Fusarium oxysporum 11dn1 was found to be able to grow and produce nitrous oxide on nitrate-containing medium in anaerobic conditions. The rate of nitrous oxide formation was three to six orders of magnitude lower than the rates of molecular nitrogen production by common denitrifying bacteria. Acetylene and ammonia did not affect the release of nitrous oxide release. It was shown that under anaerobic conditions fast increase of nitrate reductase activity occurred, caused by the synthesis of enzyme de novo and protein dephosphorylation. Reverse transfer of the mycelium to aerobic conditions led to a decline in nitrate reductase activity and stopped nitrous oxide production. The presence of two nitrate reductases was shown, which differed in molecular mass, location, temperature optima, and activity in nitrate- and ammonium-containing media. Two enzymes represent assimilatory and dissimilatory nitrate reductases, which are active in aerobic and anaerobic conditions, respectively.

  6. 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, ...

  7. DNA damage induction of ribonucleotide reductase.

    OpenAIRE

    Elledge, S J; Davis, R W

    1989-01-01

    RNR2 encodes the small subunit of ribonucleotide reductase, the enzyme that catalyzes the first step in the pathway for the production of deoxyribonucleotides needed for DNA synthesis. RNR2 is a member of a group of genes whose activities are cell cycle regulated and that are transcriptionally induced in response to the stress of DNA damage. An RNR2-lacZ fusion was used to further characterize the regulation of RNR2 and the pathway responsible for its response to DNA damage. beta-Galactosidas...

  8. Cell death by SecTRAPs: thioredoxin reductase as a prooxidant killer of cells.

    Directory of Open Access Journals (Sweden)

    Karin Anestål

    Full Text Available BACKGROUND: SecTRAPs (selenium compromised thioredoxin reductase-derived apoptotic proteins can be formed from the selenoprotein thioredoxin reductase (TrxR by targeting of its selenocysteine (Sec residue with electrophiles, or by its removal through C-terminal truncation. SecTRAPs are devoid of thioredoxin reductase activity but can induce rapid cell death in cultured cancer cell lines by a gain of function. PRINCIPAL FINDINGS: Both human and rat SecTRAPs killed human A549 and HeLa cells. The cell death displayed both apoptotic and necrotic features. It did not require novel protein synthesis nor did it show extensive nuclear fragmentation, but it was attenuated by use of caspase inhibitors. The redox active disulfide/dithiol motif in the N-terminal domain of TrxR had to be maintained for manifestation of SecTRAP cytotoxicity. Stopped-flow kinetics showed that NADPH can reduce the FAD moiety in SecTRAPs at similar rates as in native TrxR and purified SecTRAPs could maintain NADPH oxidase activity, which was accelerated by low molecular weight substrates such as juglone. In a cellular context, SecTRAPs triggered extensive formation of reactive oxygen species (ROS and consequently antioxidants could protect against the cell killing by SecTRAPs. CONCLUSIONS: We conclude that formation of SecTRAPs could contribute to the cytotoxicity seen upon exposure of cells to electrophilic agents targeting TrxR. SecTRAPs are prooxidant killers of cells, triggering mechanisms beyond those of a mere loss of thioredoxin reductase activity.

  9. Crystallization and preliminary X-ray analysis of the NADPH-dependent 3-quinuclidinone reductase from Rhodotorula rubra

    International Nuclear Information System (INIS)

    Takeshita, Daijiro; Kataoka, Michihiko; Miyakawa, Takuya; Miyazono, Ken-ichi; Uzura, Atsuko; Nagata, Koji; Shimizu, Sakayu; Tanokura, Masaru

    2009-01-01

    The NADPH-dependent 3-quinuclidinone reductase from Rhodotorula rubra was expressed, purified, and crystallized and X-ray diffraction data of this crystal were collected to 2.2 Å resolution. (R)-3-Quinuclidinol is a useful compound that is applicable to the synthesis of various pharmaceuticals. The NADPH-dependent carbonyl reductase 3-quinuclidinone reductase from Rhodotorula rubra catalyzes the stereospecific reduction of 3-quinuclidinone to (R)-3-quinuclidinol and is expected to be utilized in industrial production of this alcohol. 3-Quinuclidinone reductase from R. rubra was expressed in Escherichia coli and purified using Ni-affinity and ion-exchange column chromatography. Crystals of the protein were obtained by the sitting-drop vapour-diffusion method using PEG 8000 as the precipitant. The crystals belonged to space group P4 1 2 1 2, with unit-cell parameters a = b = 91.3, c = 265.4 Å, and diffracted X-rays to 2.2 Å resolution. The asymmetric unit contained four molecules of the protein and the solvent content was 48.4%

  10. Glutathione oxidation in response to intracellular H2O2: Key but overlapping roles for dehydroascorbate reductases.

    Science.gov (United States)

    Rahantaniaina, Marie-Sylviane; Li, Shengchun; Chatel-Innocenti, Gilles; Tuzet, Andrée; Mhamdi, Amna; Vanacker, Hélène; Noctor, Graham

    2017-08-03

    Glutathione is a pivotal molecule in oxidative stress, during which it is potentially oxidized by several pathways linked to H 2 O 2 detoxification. We have investigated the response and functional importance of 3 potential routes for glutathione oxidation pathways mediated by glutathione S-transferases (GST), glutaredoxin-dependent peroxiredoxins (PRXII), and dehydroascorbate reductases (DHAR) in Arabidopsis during oxidative stress. Loss-of-function gstU8, gstU24, gstF8, prxIIE and prxIIF mutants as well as double gstU8 gstU24, gstU8 gstF8, gstU24 gstF8, prxIIE prxIIF mutants were obtained. No mutant lines showed marked changes in their phenotype and glutathione profiles in comparison to the wild-type plants in either optimal conditions or oxidative stress triggered by catalase inhibition. By contrast, multiple loss of DHAR functions markedly decreased glutathione oxidation triggered by catalase deficiency. To assess whether this effect was mediated directly by loss of DHAR enzyme activity, or more indirectly by upregulation of other enzymes involved in glutathione and ascorbate recycling, we measured expression of glutathione reductase (GR) and expression and activity of monodehydroascorbate reductases (MDHAR). No evidence was obtained that either GRs or MDHARs were upregulated in plants lacking DHAR function. Hence, interplay between different DHARs appears to be necessary to couple ascorbate and glutathione pools and to allow glutathione-related signaling during enhanced H 2 O 2 metabolism.

  11. YNL134C from Saccharomyces cerevisiae encodes a novel protein with aldehyde reductase activity for detoxification of furfural derived from lignocellulosic biomass.

    Science.gov (United States)

    Zhao, Xianxian; Tang, Juan; Wang, Xu; Yang, Ruoheng; Zhang, Xiaoping; Gu, Yunfu; Li, Xi; Ma, Menggen

    2015-05-01

    Furfural and 5-hydroxymethylfurfural (HMF) are the two main aldehyde compounds derived from pentoses and hexoses, respectively, during lignocellulosic biomass pretreatment. These two compounds inhibit microbial growth and interfere with subsequent alcohol fermentation. Saccharomyces cerevisiae has the in situ ability to detoxify furfural and HMF to the less toxic 2-furanmethanol (FM) and furan-2,5-dimethanol (FDM), respectively. Herein, we report that an uncharacterized gene, YNL134C, was highly up-regulated under furfural or HMF stress and Yap1p and Msn2/4p transcription factors likely controlled its up-regulated expression. Enzyme activity assays showed that YNL134C is an NADH-dependent aldehyde reductase, which plays a role in detoxification of furfural to FM. However, no NADH- or NADPH-dependent enzyme activity was observed for detoxification of HMF to FDM. This enzyme did not catalyse the reverse reaction of FM to furfural or FDM to HMF. Further studies showed that YNL134C is a broad-substrate aldehyde reductase, which can reduce multiple aldehydes to their corresponding alcohols. Although YNL134C is grouped into the quinone oxidoreductase family, no quinone reductase activity was observed using 1,2-naphthoquinone or 9,10-phenanthrenequinone as a substrate, and phylogenetic analysis indicates that it is genetically distant to quinone reductases. Proteins similar to YNL134C in sequence from S. cerevisiae and other microorganisms were phylogenetically analysed. Copyright © 2015 John Wiley & Sons, Ltd.

  12. 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...

  13. Relationship between nitrate reductase and nitrate uptake in phytoplankton in the Peru upwelling region

    International Nuclear Information System (INIS)

    Blasco, D.; MacIsaac, J.J.; Packard, T.T.; Dugdale, R.C.

    1984-01-01

    Nitrate reductase (NR) activity and 15 NO 3 - uptake in phytoplankton were compared under different environmental conditions on two cruises in the upwelling region off Peru. The NR activity and NO 3 - uptake rates responded differently to light and nutrients and the differences led to variations in the uptake: reductase ratio. Analysis of these variations suggests that the re-equilibration time of the two processes in response to environmental perturbation is an important source of variability. The nitrate uptake system responds faster than the nitrate reductase system. Considering these differences in response time the basic differences in the two processes, and the differences in their measurement, the authors conclude that the Nr activity measures the current nitrate-reducing potential, which reflects NO 3 - assimilation before the sampling time, while 15 NO 3 - uptake measures NO 3 - assimilation in the 6-h period following sampling

  14. Mechanistic studies with solubilized rat liver steroid 5 alpha-reductase: Elucidation of the kinetic mechanism

    International Nuclear Information System (INIS)

    Levy, M.A.; Brandt, M.; Greway, A.T.

    1990-01-01

    A solubilized preparation of steroid 5 alpha-reductase from rat liver has been used in studies focused toward an understanding of the kinetic mechanism associated with enzyme catalysis. From the results of analyses with product and dead-end inhibitors, a preferentially ordered binding of substrates and release of products from the surface of the enzyme is proposed. The observations from these experiments were identical with those using the steroid 5 alpha-reductase activity associated with rat liver microsomes. The primary isotope effects on steady-state kinetic parameters when [4S-2H]NADPH was used also were consistent with an ordered kinetic mechanism. Normal isotope effects were observed for all three kinetic parameters (Vm/Km for both testosterone and NADPH and Vm) at all substrate concentrations used experimentally. Upon extrapolation to infinite concentration of testosterone, the isotope effect on Vm/Km for NADPH approached unity, indicating that the nicotinamide dinucleotide phosphate is the first substrate binding to and the second product released from the enzyme. The isotope effects on Vm/Km for testosterone at infinite concentration of cofactor and on Vm were 3.8 +/- 0.5 and 3.3 +/- 0.4, respectively. Data from the pH profiles of these three steady-state parameters and the inhibition constants (1/Ki) of competitive inhibitors versus both substrates indicate that the binding of nicotinamide dinucleotide phosphate involves coordination of its anionic 2'-phosphate to a protonated enzyme-associated base with an apparent pK near 8.0. From these results, relative limits have been placed on several of the internal rate constants used to describe the ordered mechanism of the rat liver steroid 5 alpha-reductase

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

    African Journals Online (AJOL)

    STORAGESEVER

    2010-03-01

    Mar 1, 2010 ... Alexander et al., 2005) and heme-type nitrite reductase gene (Smith and ... owing to a genotype-dependent response (Zhang et al.,. 1991; Sakhanokho et al., ..... Improvement of cell culture conditions for rice. Jpn. Agric. Res.

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

    Science.gov (United States)

    Poonnachit, U; Darnell, R

    2004-04-01

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

  17. Characterization of hydroxyurea (HYU) S49 T lymphoma cells

    International Nuclear Information System (INIS)

    Albert, D.A.; Gudas, L.J.

    1986-01-01

    This paper tests the hypotheses that in vivo ribonucleotide reductase activity and consequent deoxyribonucleoside triphosphate production is rate-limited by the availability of M2 activity. The authors selected and characterized cell lines with variable resistance to hydroxyurea and comparing them with wild type S49 T-lymphoma cells. Ribonucleotide reductase assay was measured and in the process C 14-CDP was added in the final volume of assay mixture. It is shown that hydroxourea reversibly binds to the tyrosine radical of the M2 subunit of ribonucleotide reductase that is required for catalytic activity

  18. Over-expression of a tobacco nitrate reductase gene in wheat (Triticum aestivum L.) increases seed protein content and weight without augmenting nitrogen supplying.

    Science.gov (United States)

    Zhao, Xiao-Qiang; Nie, Xuan-Li; Xiao, Xing-Guo

    2013-01-01

    Heavy nitrogen (N) application to gain higher yield of wheat (Triticum aestivum L.) resulted in increased production cost and environment pollution. How to diminish the N supply without losing yield and/or quality remains a challenge. To meet the challenge, we integrated and expressed a tobacco nitrate reductase gene (NR) in transgenic wheat. The 35S-NR gene was transferred into two winter cultivars, "Nongda146" and "Jimai6358", by Agrobacterium-mediation. Over-expression of the transgene remarkably enhanced T1 foliar NR activity and significantly augmented T2 seed protein content and 1000-grain weight in 63.8% and 68.1% of T1 offspring (total 67 individuals analyzed), respectively. Our results suggest that constitutive expression of foreign nitrate reductase gene(s) in wheat might improve nitrogen use efficiency and thus make it possible to increase seed protein content and weight without augmenting N supplying.

  19. Over-expression of a tobacco nitrate reductase gene in wheat (Triticum aestivum L. increases seed protein content and weight without augmenting nitrogen supplying.

    Directory of Open Access Journals (Sweden)

    Xiao-Qiang Zhao

    Full Text Available Heavy nitrogen (N application to gain higher yield of wheat (Triticum aestivum L. resulted in increased production cost and environment pollution. How to diminish the N supply without losing yield and/or quality remains a challenge. To meet the challenge, we integrated and expressed a tobacco nitrate reductase gene (NR in transgenic wheat. The 35S-NR gene was transferred into two winter cultivars, "Nongda146" and "Jimai6358", by Agrobacterium-mediation. Over-expression of the transgene remarkably enhanced T1 foliar NR activity and significantly augmented T2 seed protein content and 1000-grain weight in 63.8% and 68.1% of T1 offspring (total 67 individuals analyzed, respectively. Our results suggest that constitutive expression of foreign nitrate reductase gene(s in wheat might improve nitrogen use efficiency and thus make it possible to increase seed protein content and weight without augmenting N supplying.

  20. Expression, purification and molecular structure modeling of thioredoxin (Trx) and thioredoxin reductase (TrxR) from Acidithiobacillus ferrooxidans.

    Science.gov (United States)

    Wang, Yiping; Zhang, Xiaojian; Liu, Qing; Ai, Chenbing; Mo, Hongyu; Zeng, Jia

    2009-07-01

    The thioredoxin system consists of thioredoxin (Trx), thioredoxin reductase (TrxR) and NADPH, which plays several key roles in maintaining the redox environment of the cell. In Acidithiobacillus ferrooxidans, thioredoxin system may play important functions in the activity regulation of periplasmic proteins and energy metabolism. Here, we cloned thioredoxin (trx) and thioredoxin reductase (trxR) genes from Acidithiobacillus ferrooxidans, and expressed the genes in Escherichia coli. His-Trx and His-TrxR were purified to homogeneity with one-step Ni-NTA affinity column chromatography. Site-directed mutagenesis results confirmed that Cys33, Cys36 of thioredoxin, and Cys142, Cys145 of thioredoxin reductase were active-site residues.

  1. The NADPH thioredoxin reductase C functions as an electron donor to 2-Cys peroxiredoxin in a thermophilic cyanobacterium Thermosynechococcus elongatus BP-1

    International Nuclear Information System (INIS)

    Sueoka, Keigo; Yamazaki, Teruaki; Hiyama, Tetsuo; Nakamoto, Hitoshi

    2009-01-01

    An NADPH thioredoxin reductase C was co-purified with a 2-Cys peroxiredoxin by the combination of anion exchange chromatography and electroelution from gel slices after native PAGE from a thermophilic cyanobacterium Thermosynechococcus elongatus as an NAD(P)H oxidase complex induced by oxidative stress. The result provided a strong evidence that the NADPH thioredoxin reductase C interacts with the 2-Cys peroxiredoxin in vivo. An in vitro reconstitution assay with purified recombinant proteins revealed that both proteins were essential for an NADPH-dependent reduction of H 2 O 2 . These results suggest that the reductase transfers the reducing power from NADPH to the peroxiredoxin, which reduces peroxides in the cyanobacterium under oxidative stress. In contrast with other NADPH thioredoxin reductases, the NADPH thioredoxin reductase C contains a thioredoxin-like domain in addition to an NADPH thioredoxin reductase domain in the same polypeptide. Each domain contains a conserved CXYC motif. A point mutation at the CXYC motif in the NADPH thioredoxin reductase domain resulted in loss of the NADPH oxidation activity, while a mutation at the CXYC motif in the thioredoxin-like domain did not affect the electron transfer, indicating that this motif is not essential in the electron transport from NADPH to the 2-Cys peroxiredoxin.

  2. Alpha 1-blockers vs 5 alpha-reductase inhibitors in benign prostatic hyperplasia. A comparative review

    DEFF Research Database (Denmark)

    Andersen, J T

    1995-01-01

    During recent years, pharmacological treatment of symptomatic benign prostatic hyperplasia (BPH) has become the primary treatment choice for an increasing number of patients. The 2 principal drug classes employed are alpha 1-blockers and 5 alpha-reductase inhibitors. Current information from...... of patients who will respond well to alpha 1-blockers have yet to be identified, and data concerning the long term effects of these drugs are not yet available. 5 alpha-Reductase inhibitors have a slow onset of effect, but treatment leads to improvement in symptoms, reduction of the size of the prostate gland...... and improvement in objective parameters for bladder outflow obstruction. Approximately 30 to 50% of patients will respond to treatment with 5 alpha-reductase inhibitors. The definitive role of pharmacological treatment in symptomatic BPH remains to be established, although it seems that patients unfit...

  3. Reconstitution of FMN-free NADPH-cytochrome P-450 reductase with a phosphorothioate analog of FMN: 31P NMR studies of the reconstituted protein

    International Nuclear Information System (INIS)

    Krum, D.P.; Otvos, J.D.; Calhoun, J.P.; Miziorko, H.M.; Masters, B.S.S.

    1987-01-01

    A phosphorothioate analog of FMN (FMNS) has been synthesized and shown to be completely competent in reconstituting the FMN-free form of NADPH-cytochrome P-450 reductase as evidenced by flavin determinations and cytochrome c reductase activity assays. The FMNS-reconstituted FMN-free reductase gives rise to an air-stable semiquinone, and the fluorescence of FMNS is quenched upon addition of FMN-free reductase. 31 P NMR spectra of the FMN-free reductase reveal only two resonances (-7.3 and -11.3 ppm), which are attributable to FAD. This result confirms the assignments of Otvos et al, and demonstrates unequivocally that there are no phosphate residues other than those of FMN and FAD attached to the steapsin-solubilized reductase. The addition of FMN to the FMN-free reductase resulted in the appearance of one additional resonance at 3.9 ppm. Addition of FMNS to the FMN-free reductase caused no change, surprisingly, in the 31 P NMR spectrum until Mn(II) was added, after which a peak centered at ∼ 45 ppm was observed. This unexpected result may be explained if the T 1 for the phosphate of FMNS is significantly longer than that of FMN, and suggests that the sulfur atom of FMNS may perturb the interaction of the phosphate with its protein environment. These results demonstrate the utility of phosphorothioate analogs as mechanistic probes for proteins containing nucleotide cofactors

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

    African Journals Online (AJOL)

    STORAGESEVER

    2009-12-15

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

  5. Chemical modification of human muscle aldose reductase by pyridoxal 5'-phosphate

    International Nuclear Information System (INIS)

    Morjana, N.A.; Lyons, C.; Flynn, T.G.

    1987-01-01

    Aldose reductase (ALR2) is a monomeric oxidoreductase (Mr, 37,000). This enzyme catalyzes the reduction of a wide variety of aliphatic and aromatic aldehydes to their corresponding alcohols. The ability to reduce D-glucose and utilize NADH distinguishes ALR2 from aldehyde reductase (ALR1) which is exclusively NADPH-dependent. As part of a study to determine active site residues critical for binding and catalysis they have investigated the behavior of ALR2 with pyridoxal phosphate (PLP). In contrast to ALR1, which is inactivated by PLP, the reaction of ALR2 with PLP results in a 2-3 fold activation with the incorporation of 1 mol of PLP/mol enzyme. However, despite a 3-fold increase in k/sub cat/, there is also a 13-14 fold increase in the Km for both coenzyme and substrate and catalytic efficiency (k/sub cat//Km) is actually decreased. Reaction of ALR2 with 3 [H] PLP followed by digestion with endoproteinase Lys-C enabled the separation and purification by HPLC of a peptide containing a single pyridoxyllysine residue. The sequence of this 32 residue peptide is highly homologous with a peptide similarly obtained from pig and human ALR1 and is identical with one from pig ALR2. In all four enzymes, pig ALR1, ALR2; human ALR1, ALR2, a tetrapeptide containing the pyridoxylated lysine (I-P-K-S) shows absolute identity. Thus, despite differences in substrate and coenzyme specificity, the active site in both ALR1 and ALR2 is relatively conserved

  6. Structural, kinetic, and docking studies of artificial imine reductases based on biotin-streptavidin technology: an induced lock-and-key hypothesis.

    Science.gov (United States)

    Robles, Victor Muñoz; Dürrenberger, Marc; Heinisch, Tillmann; Lledós, Agustí; Schirmer, Tilman; Ward, Thomas R; Maréchal, Jean-Didier

    2014-11-05

    An artificial imine reductase results upon incorporation of a biotinylated Cp*Ir moiety (Cp* = C5Me5(-)) within homotetrameric streptavidin (Sav) (referred to as Cp*Ir(Biot-p-L)Cl] ⊂ Sav). Mutation of S112 reveals a marked effect of the Ir/streptavidin ratio on both the saturation kinetics as well as the enantioselectivity for the production of salsolidine. For [Cp*Ir(Biot-p-L)Cl] ⊂ S112A Sav, both the reaction rate and the selectivity (up to 96% ee (R)-salsolidine, kcat 14-4 min(-1) vs [Ir], KM 65-370 mM) decrease upon fully saturating all biotin binding sites (the ee varying between 96% ee and 45% ee R). In contrast, for [Cp*Ir(Biot-p-L)Cl] ⊂ S112K Sav, both the rate and the selectivity remain nearly constant upon varying the Ir/streptavidin ratio [up to 78% ee (S)-salsolidine, kcat 2.6 min(-1), KM 95 mM]. X-ray analysis complemented with docking studies highlight a marked preference of the S112A and S112K Sav mutants for the SIr and RIr enantiomeric forms of the cofactor, respectively. Combining both docking and saturation kinetic studies led to the formulation of an enantioselection mechanism relying on an "induced lock-and-key" hypothesis: the host protein dictates the configuration of the biotinylated Ir-cofactor which, in turn, by and large determines the enantioselectivity of the imine reductase.

  7. Structural insights into the neuroprotective-acting carbonyl reductase Sniffer of Drosophila melanogaster.

    Science.gov (United States)

    Sgraja, Tanja; Ulschmid, Julia; Becker, Katja; Schneuwly, Stephan; Klebe, Gerhard; Reuter, Klaus; Heine, Andreas

    2004-10-01

    In vivo studies with the fruit-fly Drosophila melanogaster have shown that the Sniffer protein prevents age-dependent and oxidative stress-induced neurodegenerative processes. Sniffer is a NADPH-dependent carbonyl reductase belonging to the enzyme family of short-chain dehydrogenases/reductases (SDRs). The crystal structure of the homodimeric Sniffer protein from Drosophila melanogaster in complex with NADP+ has been determined by multiple-wavelength anomalous dispersion and refined to a resolution of 1.75 A. The observed fold represents a typical dinucleotide-binding domain as detected for other SDRs. With respect to the cofactor-binding site and the region referred to as substrate-binding loop, the Sniffer protein shows a striking similarity to the porcine carbonyl reductase (PTCR). This loop, in both Sniffer and PTCR, is substantially shortened compared to other SDRs. In most enzymes of the SDR family this loop adopts a well-defined conformation only after substrate binding and remains disordered in the absence of any bound ligands or even if only the dinucleotide cofactor is bound. In the structure of the Sniffer protein, however, the conformation of this loop is well defined, although no substrate is present. Molecular modeling studies provide an idea of how binding of substrate molecules to Sniffer could possibly occur.

  8. 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 ...

  9. Ubiquinol-cytochrome c reductase (Complex III) electrochemistry at multi-walled carbon nanotubes/Nafion modified glassy carbon electrodes

    International Nuclear Information System (INIS)

    Pelster, Lindsey N.; Minteer, Shelley D.

    2012-01-01

    Highlights: ► The electron transport chain is important to the understanding of metabolism in the living cell. ► Ubiquinol-cytochrome c reductase is a membrane bound complex of the electron transport chain (Complex III). ► The paper details the first bioelectrochemical characterization of ubiquinol-cytochrome c reductase at an electrode. - Abstract: Electron transport chain complexes are critical to metabolism in living cells. Ubiquinol-cytochrome c reductase (Complex III) is responsible for carrying electrons from ubiquinol to cytochrome c, but the complex has not been evaluated electrochemically. This work details the bioelectrochemistry of ubiquinol-cytochrome c reductase of the electron transport chain of tuber mitochondria. The characterization of the electrochemistry of this enzyme is investigated in carboxylated multi-walled carbon nanotube/tetrabutyl ammonium bromide-modified Nafion ® modified glassy carbon electrodes by cyclic voltammetry. Increasing concentrations of cytochrome c result in a catalytic response from the active enzyme in the nanotube sandwich. The experiments show that the enzyme followed Michaelis–Menten kinetics with a K m for the immobilized enzyme of 2.97 (±0.11) × 10 −6 M and a V max of 6.31 (±0.82) × 10 −3 μmol min −1 at the electrode, but the K m and V max values decreased compared to the free enzyme in solution, which is expected for immobilized redox proteins. This is the first evidence of ubiquinol-cytochrome c reductase bioelectrocatalysis.

  10. Isolation and expression of the Pneumocystis carinii dihydrofolate reductase gene

    DEFF Research Database (Denmark)

    Edman, J C; Edman, U; Cao, Mi-Mi

    1989-01-01

    Pneumocystis carinii dihydrofolate reductase (DHFR; 5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) cDNA sequences have been isolated by their ability to confer trimethoprim resistance to Escherichia coli. Consistent with the recent conclusion that P. carinii is a member of the Fungi...

  11. 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 ...

  12. Overexpression of Nitrate Reductase in Tobacco Delays Drought-Induced Decreases in Nitrate Reductase Activity and mRNA1

    Science.gov (United States)

    Ferrario-Méry, Sylvie; Valadier, Marie-Hélène; Foyer, Christine H.

    1998-01-01

    Transformed (cauliflower mosaic virus 35S promoter [35S]) tobacco (Nicotiana plumbaginifolia L.) plants constitutively expressing nitrate reductase (NR) and untransformed controls were subjected to drought for 5 d. Drought-induced changes in biomass accumulation and photosynthesis were comparable in both lines of plants. After 4 d of water deprivation, a large increase in the ratio of shoot dry weight to fresh weight was observed, together with a decrease in the rate of photosynthetic CO2 assimilation. Foliar sucrose increased in both lines during water stress, but hexoses increased only in leaves from untransformed controls. Foliar NO3− decreased rapidly in both lines and was halved within 2 d of the onset of water deprivation. Total foliar amino acids decreased in leaves of both lines following water deprivation. After 4 d of water deprivation no NR activity could be detected in leaves of untransformed plants, whereas about 50% of the original activity remained in the leaves of the 35S-NR transformants. NR mRNA was much more stable than NR activity. NR mRNA abundance increased in the leaves of the 35S-NR plants and remained constant in controls for the first 3 d of drought. On the 4th d, however, NR mRNA suddenly decreased in both lines. Rehydration at d 3 caused rapid recovery (within 24 h) of 35S-NR transcripts, but no recovery was observed in the controls. The phosphorylation state of the protein was unchanged by long-term drought. There was a strong correlation between maximal extractable NR activity and ambient photosynthesis in both lines. We conclude that drought first causes increased NR protein turnover and then accelerates NR mRNA turnover. Constitutive NR expression temporarily delayed drought-induced losses in NR activity. 35S-NR expression may therefore allow more rapid recovery of N assimilation following short-term water deficit. PMID:9576799

  13. 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.

  14. Relationship between nitrate reductase and nitrate uptake in phytoplankton in the Peru upwelling region

    International Nuclear Information System (INIS)

    Blasco, D.; MacIsaac, J.J.; Packard, T.T.; Dugdale, R.C.

    1984-01-01

    Nitrate reductase (NR) activity and 15 NO 3 - uptake in phytoplankton were compared under different environmental conditions on two cruises in the upwelling region off Peru. The NR activity and NO 3 - uptake rates responded differently to light and nutrients and the differences led to variations in the uptake:reductase ratio. Analysis of these variations suggests that the re-equilibration time of the two processes in response to environmental perturbation is an important source of variability. The nitrate uptake system responds faster than the nitrate reductase system. Considering these differences in response time, the basic differences in the two processes, and the differences in their measurement, the authors conclude that the NR activity measures the current nitrate-reducing potential, which relfects NO 3 - assimilation before the sampling time, while 15 NO 3 - uptake measures NO 3 - assimilation in the 6-h period following sampling. Thus, considering the sampling time as a point of reference, the former is a measure of the past and the latter is a measure of the future

  15. Synthesis and Activity of a New Series of(Z-3-Phenyl-2-benzoylpropenoic Acid Derivatives as Aldose Reductase Inhibitors

    Directory of Open Access Journals (Sweden)

    Shao-Jie Wang

    2007-04-01

    Full Text Available During the course of studies directed towards the discovery of novel aldose reductase inhibitors for the treatment of diabetic complications, we synthesized a series of new (Z-3-phenyl-2-benzoylpropenoic acid derivatives and tested their in vitro inhibitory activities on rat lens aldose reductase. Of these compounds, (Z-3-(3,4-dihydroxyphenyl-2-(4-methylbenzoylpropenoicacid(3k was identified as the most potent inhibitor, with an IC50 of 0.49μM. The theoretical binding mode of 3k was obtained by simulation of its docking into the active site of the human aldose reductase crystal structure.

  16. Influence of rete testis fluid deprivation on the kinetic parameters of goat epididymal 5 alpha-reductase.

    Science.gov (United States)

    Kelce, W R; Lubis, A M; Braun, W F; Youngquist, R S; Ganjam, V K

    1990-01-01

    A surgical technique to cannulate the rete testis of the goat was utilized to examine the effects of rete testis fluid (RTF) deprivation on the enzymatic activity of epididymal 5 alpha-reductase. Kinetic techniques were used to determine whether the regional enzymatic effect of RTF deprivation is to decrease the apparent number of 5 alpha-reductase active sites or the catalytic activity of each active site within the epididymal epithelium. Paired comparisons of (Vmax)app and (Km)app values between control and RTF-deprived epididymides indicated that RTF deprivation affected the value of (Vmax)app with no apparent change in the values of (Km)app in caput, corpus, and cauda epididymal regions. We conclude that RTF deprivation in the goat epididymis for 7 days results in a decreased number of apparent 5 alpha-reductase active sites within the epididymal epithelium.

  17. Crystallization and preliminary X-ray analysis of the NADPH-dependent 3-quinuclidinone reductase from Rhodotorula rubra

    Science.gov (United States)

    Takeshita, Daijiro; Kataoka, Michihiko; Miyakawa, Takuya; Miyazono, Ken-ichi; Uzura, Atsuko; Nagata, Koji; Shimizu, Sakayu; Tanokura, Masaru

    2009-01-01

    (R)-3-Quinuclidinol is a useful compound that is applicable to the synthesis of various pharmaceuticals. The NADPH-dependent carbonyl reductase 3-­quinuclidinone reductase from Rhodotorula rubra catalyzes the stereospecific reduction of 3-quinuclidinone to (R)-3-quinuclidinol and is expected to be utilized in industrial production of this alcohol. 3-Quinuclidinone reductase from R. rubra was expressed in Escherichia coli and purified using Ni-affinity and ion-exchange column chromatography. Crystals of the protein were obtained by the sitting-drop vapour-diffusion method using PEG 8000 as the precipitant. The crystals belonged to space group P41212, with unit-cell parameters a = b = 91.3, c = 265.4 Å, and diffracted X-rays to 2.2 Å resolution. The asymmetric unit contained four molecules of the protein and the solvent content was 48.4%. PMID:19478454

  18. Ferric reductase genes involved in high-affinity iron uptake are differentially regulated in yeast and hyphae of Candida albicans.

    Science.gov (United States)

    Jeeves, Rose E; Mason, Robert P; Woodacre, Alexandra; Cashmore, Annette M

    2011-09-01

    The pathogenic yeast Candida albicans possesses a reductive iron uptake system which is active in iron-restricted conditions. The sequestration of iron by this mechanism initially requires the reduction of free iron to the soluble ferrous form, which is catalysed by ferric reductase proteins. Reduced iron is then taken up into the cell by a complex of a multicopper oxidase protein and an iron transport protein. Multicopper oxidase proteins require copper to function and so reductive iron and copper uptake are inextricably linked. It has previously been established that Fre10 is the major cell surface ferric reductase in C. albicans and that transcription of FRE10 is regulated in response to iron levels. We demonstrate here that Fre10 is also a cupric reductase and that Fre7 also makes a significant contribution to cell surface ferric and cupric reductase activity. It is also shown, for the first time, that transcription of FRE10 and FRE7 is lower in hyphae compared to yeast and that this leads to a corresponding decrease in cell surface ferric, but not cupric, reductase activity. This demonstrates that the regulation of two virulence determinants, the reductive iron uptake system and the morphological form of C. albicans, are linked. Copyright © 2011 John Wiley & Sons, Ltd.

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

    DEFF Research Database (Denmark)

    Farver, Ole; Brunori, Maurizio; Cutruzzolà, Francesca

    2009-01-01

    ) as the level of reduction increased in both the WT and the His mutant. Equilibrium standard enthalpy and entropy changes and activation parameters of this ET process were determined. We concluded that negative cooperativity is a common feature among the cd(1) nitrite reductases, and we discuss this control...

  20. Bisphenol A, bisphenol F and bisphenol S affect differently 5α-reductase expression and dopamine–serotonin systems in the prefrontal cortex of juvenile female rats

    International Nuclear Information System (INIS)

    Castro, Beatriz; Sánchez, Pilar; Torres, Jesús M.; Ortega, Esperanza

    2015-01-01

    Background: Early-life exposure to the endocrine disruptor bisphenol A (BPA) affects brain function and behavior, which might be attributed to its interference with hormonal steroid signaling and/or neurotransmitter systems. Alternatively, the use of structural analogs of BPA, mainly bisphenol F (BPF) and bisphenol S (BPS), has increased recently. However, limited in vivo toxicity data exist. Objectives: We investigated the effects of BPA, BPF and BPS on 5α-reductase (5α-R), a key enzyme involved in neurosteroidogenesis, as well as on dopamine (DA)- and serotonin (5-HT)-related genes, in the prefrontal cortex (PFC) of juvenile female rats. Methods: Gestating Wistar rats were treated with either vehicle or 10 μg/kg/day of BPA, BPF or BPS from gestational day 12 to parturition. Then, female pups were exposed from postnatal day 1 through day 21 (PND21), when they were euthanized and RT-PCR, western blot and quantitative PCR-array experiments were performed. Results: BPA decreased 5α-R2 and 5α-R3 mRNA and protein levels, while both BPF and BPS decreased 5α-R3 mRNA levels in PFC at PND21. Further, BPA, BPF and BPS significantly altered, respectively, the transcription of 25, 56 and 24 genes out of the 84 DA and 5-HT-related genes assayed. Of particular interest was the strong induction by all these bisphenols of Cyp2d4, implicated in corticosteroids synthesis. Conclusions: Our results demonstrate for the first time that BPA, BPF and BPS differentially affect 5α-R and genes related to DA/5-HT systems in the female PFC. In vivo evidence of the potential adverse effects of BPF and BPS in the brain of mammals is provided in this work, raising questions about the safety of these chemicals as substitutes for BPA. - Highlights: • Juvenile prefrontal cortex of female rats exposed to bisphenol A, F or S was analyzed. • We provide the first in vivo data of BPF and BPS effects in mammal brain. • BPA, BPF and BPS differently affected dopamine and serotonin

  1. Bisphenol A, bisphenol F and bisphenol S affect differently 5α-reductase expression and dopamine–serotonin systems in the prefrontal cortex of juvenile female rats

    Energy Technology Data Exchange (ETDEWEB)

    Castro, Beatriz; Sánchez, Pilar [Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Granada, Avda. de Madrid s/n, 18012 Granada (Spain); Torres, Jesús M., E-mail: torrespi@ugr.es [Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Granada, Avda. de Madrid s/n, 18012 Granada (Spain); Institute of Neurosciences, Faculty of Medicine, University of Granada, Granada (Spain); Ortega, Esperanza, E-mail: esortega@ugr.es [Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Granada, Avda. de Madrid s/n, 18012 Granada (Spain); Institute of Neurosciences, Faculty of Medicine, University of Granada, Granada (Spain)

    2015-10-15

    Background: Early-life exposure to the endocrine disruptor bisphenol A (BPA) affects brain function and behavior, which might be attributed to its interference with hormonal steroid signaling and/or neurotransmitter systems. Alternatively, the use of structural analogs of BPA, mainly bisphenol F (BPF) and bisphenol S (BPS), has increased recently. However, limited in vivo toxicity data exist. Objectives: We investigated the effects of BPA, BPF and BPS on 5α-reductase (5α-R), a key enzyme involved in neurosteroidogenesis, as well as on dopamine (DA)- and serotonin (5-HT)-related genes, in the prefrontal cortex (PFC) of juvenile female rats. Methods: Gestating Wistar rats were treated with either vehicle or 10 μg/kg/day of BPA, BPF or BPS from gestational day 12 to parturition. Then, female pups were exposed from postnatal day 1 through day 21 (PND21), when they were euthanized and RT-PCR, western blot and quantitative PCR-array experiments were performed. Results: BPA decreased 5α-R2 and 5α-R3 mRNA and protein levels, while both BPF and BPS decreased 5α-R3 mRNA levels in PFC at PND21. Further, BPA, BPF and BPS significantly altered, respectively, the transcription of 25, 56 and 24 genes out of the 84 DA and 5-HT-related genes assayed. Of particular interest was the strong induction by all these bisphenols of Cyp2d4, implicated in corticosteroids synthesis. Conclusions: Our results demonstrate for the first time that BPA, BPF and BPS differentially affect 5α-R and genes related to DA/5-HT systems in the female PFC. In vivo evidence of the potential adverse effects of BPF and BPS in the brain of mammals is provided in this work, raising questions about the safety of these chemicals as substitutes for BPA. - Highlights: • Juvenile prefrontal cortex of female rats exposed to bisphenol A, F or S was analyzed. • We provide the first in vivo data of BPF and BPS effects in mammal brain. • BPA, BPF and BPS differently affected dopamine and serotonin

  2. Location of the redox-active thiols of ribonucleotide reductase: sequences similarity between the Escherichia coli and Lactobacillus leichmannii enzymes

    International Nuclear Information System (INIS)

    Lin, A.N.I.; Ashley, G.W.; Stubbe, J.

    1987-01-01

    The redox-active thiols of Escherichia coli ribonucleoside diphosphate reductase and of Lactobacillus leichmannii ribonucleoside triphosphate reductase have been located by a procedure involving (1) prereduction of enzyme with dithiothreitol, (2) specific oxidation of the redox-active thiols by treatment with substrate in the absence of exogenous reductant, (3) alkylation of other thiols with iodoacetamide, and (4) reduction of the disulfides with dithiothreitol and alkylation with [1- 14 C]iodoacetamide. The dithiothreitol-reduce E. coli B1 subunit is able to convert 3 equiv of CDP to dCDP and is labeled with 5.4 equiv of 14 C. Sequencing of tryptic peptides shows that 2.8 equiv of 14 C is on cysteines-752 and -757 at the C-terminus of B1, while 1.0-1.5 equiv of 14 C is on cysteines-222 and -227. It thus appears that two sets of redox-active dithiols are involved in substrate reduction. The L. leichmannii reductase is able to convert 1.1 equiv of CTP to dCTP and is labeled with 2.1 equiv of 14 C. Sequencing of tryptic peptides shows that 1.4 equiv of 14 C is located on the two cysteines of C-E-G-G-A-C-P-I-K. This peptide shows remarkable and unexpected similarity to the thiol-containing region of the C-terminal peptide of E. coli B1, C-E-S-G-A-C-K-I

  3. Carbonyl Reduction of NNK by Recombinant Human Lung Enzymes. Identification of HSD17β12 as the Reductase important in (R)-NNAL formation in Human Lung.

    Science.gov (United States)

    Ashmore, Joseph H; Luo, Shaman; Watson, Christy J W; Lazarus, Philip

    2018-05-17

    4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is the most abundant and carcinogenic tobacco-specific nitrosamine in tobacco and tobacco smoke. The major metabolic pathway for NNK is carbonyl reduction to form the (R) and (S) enantiomers of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) which, like NNK, is a potent lung carcinogen. The goal of the present study was to characterize NNAL enantiomer formation in human lung and identify the enzymes responsible for this activity. While (S)-NNAL was the major enantiomer of NNAL formed in incubations with NNK in lung cytosolic fractions, (R)-NNAL comprised ~60 and ~95% of the total NNAL formed in lung whole cell lysates and microsomes, respectively. In studies examining the role of individual recombinant reductase enzymes in lung NNAL enantiomer formation, AKR1C1, AKR1C2, AKR1C3, AKR1C4 and CBR1 all exhibited (S)-NNAL formation activity. To identify the microsomal enzymes responsible for (R)-NNAL formation, 28 microsomal reductase enzymes were screened for expression by real-time PCR in normal human lung. HSD17β6, HSD17β12, KDSR, NSDHL, RDH10, RDH11 and SDR16C5 were all expressed at levels >HSD11β1, the only previously reported microsomal reductase enzyme with NNK-reducing activity, with HSD17β12 the most highly expressed. Of these lung-expressing enzymes, only HSD17β12 exhibited activity against NNK, forming primarily (>95%) (R)-NNAL, a pattern consistent with that observed in lung microsomes. siRNA knockdown of HSD17β12 resulted in significant decreases in (R)-NNAL formation activity in HEK293 cells. These data suggest that both cytosolic and microsomal enzymes are active against NNK and that HSD17β12 is the major active microsomal reductase that contributes to (R)-NNAL formation in human lung.

  4. 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

  5. Role of Lysine-54 in determining cofactor specificity and binding in human dihydrofolate reductase

    International Nuclear Information System (INIS)

    Huang, Shaoming; Tan, Xuehai; Thompson, P.D.; Freisheim, J.H.; Appleman, J.R.; Blakley, R.L.; Sheridan, R.P.; Venkataraghavan, R.

    1990-01-01

    Lysine-54 of human dihydrofolate reductase (hDHFR) appears to be involved in the interaction with the 2'-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 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 m and k 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 m (NADH)/K 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'-phosphate, makes a major contribution in terms of binding energy and differentiation of K 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

  6. Prevention of hemodynamic and vascular albumin filtration changes in diabetic rats by aldose reductase inhibitors

    International Nuclear Information System (INIS)

    Tilton, R.G.; Chang, K.; Pugliese, G.; Eades, D.M.; Province, M.A.; Sherman, W.R.; Kilo, C.; Williamson, J.R.

    1989-01-01

    This study investigated hemodynamic changes in diabetic rats and their relationship to changes in vascular albumin permeation and increased metabolism of glucose to sorbitol. The effects of 6 wk of streptozocin-induced diabetes and three structurally different inhibitors of aldose reductase were examined on (1) regional blood flow (assessed with 15-microns 85Sr-labeled microspheres) and vascular permeation by 125I-labeled bovine serum albumin (BSA) and (2) glomerular filtration rate (assessed by plasma clearance of 57Co-labeled EDTA) and urinary albumin excretion (determined by radial immunodiffusion assay). In diabetic rats, blood flow was significantly increased in ocular tissues (anterior uvea, posterior uvea, retina, and optic nerve), sciatic nerve, kidney, new granulation tissue, cecum, and brain. 125I-BSA permeation was increased in all of these tissues except brain. Glomerular filtration rate and 24-h urinary albumin excretion were increased 2- and 29-fold, respectively, in diabetic rats. All three aldose reductase inhibitors completely prevented or markedly reduced these hemodynamic and vascular filtration changes and increases in tissue sorbitol levels in the anterior uvea, posterior uvea, retina, sciatic nerve, and granulation tissue. These observations indicate that early diabetes-induced hemodynamic changes and increased vascular albumin permeation and urinary albumin excretion are aldose reductase-linked phenomena. Discordant effects of aldose reductase inhibitors on blood flow and vascular albumin permeation in some tissues suggest that increased vascular albumin permeation is not entirely attributable to hemodynamic change

  7. Genetic characterization and role in virulence of the ribonucleotide reductases of Streptococcus sanguinis.

    Science.gov (United States)

    Rhodes, DeLacy V; Crump, Katie E; Makhlynets, Olga; Snyder, Melanie; Ge, Xiuchun; Xu, Ping; Stubbe, JoAnne; Kitten, Todd

    2014-02-28

    Streptococcus sanguinis is a cause of infective endocarditis and has been shown to require a manganese transporter called SsaB for virulence and O2 tolerance. Like certain other pathogens, S. sanguinis possesses aerobic class Ib (NrdEF) and anaerobic class III (NrdDG) ribonucleotide reductases (RNRs) that perform the essential function of reducing ribonucleotides to deoxyribonucleotides. The accompanying paper (Makhlynets, O., Boal, A. K., Rhodes, D. V., Kitten, T., Rosenzweig, A. C., and Stubbe, J. (2014) J. Biol. Chem. 289, 6259-6272) indicates that in the presence of O2, the S. sanguinis class Ib RNR self-assembles an essential diferric-tyrosyl radical (Fe(III)2-Y(•)) in vitro, whereas assembly of a dimanganese-tyrosyl radical (Mn(III)2-Y(•)) cofactor requires NrdI, and Mn(III)2-Y(•) is more active than Fe(III)2-Y(•) with the endogenous reducing system of NrdH and thioredoxin reductase (TrxR1). In this study, we have shown that deletion of either nrdHEKF or nrdI completely abolishes virulence in an animal model of endocarditis, whereas nrdD mutation has no effect. The nrdHEKF, nrdI, and trxR1 mutants fail to grow aerobically, whereas anaerobic growth requires nrdD. The nrdJ gene encoding an O2-independent adenosylcobalamin-cofactored RNR was introduced into the nrdHEKF, nrdI, and trxR1 mutants. Growth of the nrdHEKF and nrdI mutants in the presence of O2 was partially restored. The combined results suggest that Mn(III)2-Y(•)-cofactored NrdF is required for growth under aerobic conditions and in animals. This could explain in part why manganese is necessary for virulence and O2 tolerance in many bacterial pathogens possessing a class Ib RNR and suggests NrdF and NrdI may serve as promising new antimicrobial targets.

  8. 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...

  9. 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...... ribonucleotide reductase, which represents the workhorse for the bioconversion of nucleotides to deoxynucleotides in many prokaryotes and eukaryotic pathogens under aerobic conditions. As the partner of the flavodoxin (NrdI), the key FdR is missing in the current model describing the class Ib system...

  10. Effect of protein S-nitrosylation on autolysis and catalytic ability of μ-calpain.

    Science.gov (United States)

    Liu, Rui; Li, Yupin; Wang, Mengqin; Zhou, Guanghong; Zhang, Wangang

    2016-12-15

    The effect of S-nitrosylation on the autolysis and catalytic ability of μ-calpain in vitro in the presence of 50μM Ca(2 +) was investigated. μ-Calpain was incubated with different concentrations of nitric oxide donor S-nitrosoglutathione (GSNO) and subsequently reacted with purified myofibrils. Results showed that the amount of 80kDa μ-calpain subunit significantly decreased as GSNO increased from 0 to 300μM, but increases of GSNO to 300, 500 and 1000μM did not result in further inhibition. The catalytic ability of nitrosylated μ-calpain to degrade titin, nebulin, troponin-T and desmin was significantly reduced when the GSNO concentration was higher than 300μM. The cysteine residues of μ-calpain at positions 49, 351, 384, and 592 in the catalytic subunit and at 142 in small subunit were S-nitrosylated, which could be responsible for decreased μ-calpain activity. Thus, S-nitrosylation can negatively regulate the activation of μ-calpain resulting in decreased proteolytic ability on myofibrils. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. Inhibitory Effect on In Vitro LDL Oxidation and HMG Co-A Reductase Activity of the Liquid-Liquid Partitioned Fractions of Hericium erinaceus (Bull. Persoon (Lion’s Mane Mushroom

    Directory of Open Access Journals (Sweden)

    Mohammad Azizur Rahman

    2014-01-01

    Full Text Available Oxidation of low-density lipoprotein (LDL has been strongly suggested as the key factor in the pathogenesis of atherosclerosis. Mushrooms have been implicated in having preventive effects against chronic diseases due especially to their antioxidant properties. In this study, in vitro inhibitory effect of Hericium erinaceus on LDL oxidation and the activity of the cholesterol biosynthetic key enzyme, 3-hydroxy-3-methyl glutaryl coenzyme A (HMG Co-A reductase, was evaluated using five liquid-liquid solvent fractions consisting of methanol : dichloromethane (M : DCM, hexane (HEX, dichloromethane (DCM, ethyl acetate (EA, and aqueous residue (AQ. The hexane fraction showed the highest inhibition of oxidation of human LDL as reflected by the increased lag time (100 mins for the formation of conjugated diene (CD at 1 µg/mL and decreased production (68.28%, IC50 0.73 mg/mL of thiobarbituric acid reactive substances (TBARS at 1 mg/mL. It also mostly inhibited (59.91% the activity of the HMG Co-A reductase at 10 mg/mL. The GC-MS profiling of the hexane fraction identified the presence of myconutrients: inter alia, ergosterol and linoleic acid. Thus, hexane fraction of Hericium erinaceus was found to be the most potent in vitro inhibitor of both LDL oxidation and HMG Co-A reductase activity having therapeutic potential for the prevention of oxidative stress-mediated vascular diseases.

  12. 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.

  13. 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.

  14. 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.

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

    Directory of Open Access Journals (Sweden)

    Giuseppe Forlani

    2017-08-01

    Full Text Available 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.

  16. Structure and Reactivity of the Glutathione Radical Cation: Radical Rearrangement from the Cysteine Sulfur to the Glutamic Acid alpha-Carbon Atom

    NARCIS (Netherlands)

    Osburn, S.; Berden, G.; Oomens, J.; Gulyuz, K.; Polfer, N.C.; O'Hair, R.A.J.; Ryzhov, V.

    2013-01-01

    A gas-phase radical rearrangement through intramolecular hydrogen-atom transfer (HAT) was studied in the glutathione radical cation, [-ECG](+.), which was generated by a homolytic cleavage of the protonated S-nitrosoglutathione. Ion-molecule reactions suggested that the radical migrates from the

  17. 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.

  18. Association study of methylenetetrahydrofolate reductase C677T mutation with cerebral venous thrombosis in an Iranian population.

    Science.gov (United States)

    Ghaznavi, Habib; Soheili, Zahra; Samiei, Shahram; Soltanpour, Mohammad S

    2015-12-01

    There are limited data on the role of methylenetetrahydrofolate reductase C677T polymorphism and hyperhomocysteinemia as risk factors for cerebral venous thrombosis in Iranian population. We examined a possible association between fasting plasma homocysteine levels, methylenetetrahydrofolate reductase C677T polymorphism, and cerebral venous thrombosis in 50 patients with a diagnosis of cerebral venous thrombosis (20-63 years old) and 75 healthy controls (18-65 years old). Genotyping of the methylenetetrahydrofolate reductase C677T gene polymorphism was performed by PCR-restriction fragment length polymorphism analysis, and homocysteine levels were measured by enzyme immunoassay. Fasting plasma homocysteine levels were significantly higher in cerebral venous thrombosis patients than in controls (P = 0.015). Moreover, plasma homocysteine levels were significantly higher in methylenetetrahydrofolate reductase 677TT genotype compared to 677CT and 677CC genotypes in both cerebral venous thrombosis patients (P = 0.01) and controls (P = 0.03). Neither 677CT heterozygote genotype [odds ratio (OR) 1.35, 95% confidence interval (CI) 0.64-2.84, P = 0.556] nor 677TT homozygote genotype (OR 1.73, 95% CI 0.32-9.21, P = 0.833) was significantly associated with cerebral venous thrombosis. Additionally, no significant differences in the frequency of 677T allele between cerebral venous thrombosis patients and controls were identified (OR 1.31, 95% CI 0.69-2.50, P = 0.512). In conclusion, our study demonstrated that elevated plasma homocysteine levels are significant risk factors for cerebral venous thrombosis. Also, methylenetetrahydrofolate reductase 677TT genotype is not linked with cerebral venous thrombosis, but is a determinant of elevated plasma homocysteine levels.

  19. The Drosophila carbonyl reductase sniffer prevents oxidative stress-induced neurodegeneration.

    Science.gov (United States)

    Botella, Jose A; Ulschmid, Julia K; Gruenewald, Christoph; Moehle, Christoph; Kretzschmar, Doris; Becker, Katja; Schneuwly, Stephan

    2004-05-04

    A growing body of evidence suggests that oxidative stress is a common underlying mechanism in the pathogenesis of neurodegenerative disorders such as Alzheimer's, Huntington's, Creutzfeld-Jakob and Parkinson's diseases. Despite the increasing number of reports finding a causal relation between oxidative stress and neurodegeneration, little is known about the genetic elements that confer protection against the deleterious effects of oxidation in neurons. We have isolated and characterized the Drosophila melanogaster gene sniffer, whose function is essential for preventing age-related neurodegeneration. In addition, we demonstrate that oxidative stress is a direct cause of neurodegeneration in the Drosophila central nervous system and that reduction of sniffer activity leads to neuronal cell death. The overexpression of the gene confers neuronal protection against oxygen-induced apoptosis, increases resistance of flies to experimental normobaric hyperoxia, and improves general locomotor fitness. Sniffer belongs to the family of short-chain dehydrogenase/reductase (SDR) enzymes and exhibits carbonyl reductase activity. This is the first in vivo evidence of the direct and important implication of this enzyme as a neuroprotective agent in the cellular defense mechanisms against oxidative stress.

  20. Cytochrome b5 reductase is the component from neuronal synaptic plasma membrane vesicles that generates superoxide anion upon stimulation by cytochrome c

    Directory of Open Access Journals (Sweden)

    Alejandro K. Samhan-Arias

    2018-05-01

    Full Text Available In this work, we measured the effect of cytochrome c on the NADH-dependent superoxide anion production by synaptic plasma membrane vesicles from rat brain. In these membranes, the cytochrome c stimulated NADH-dependent superoxide anion production was inhibited by antibodies against cytochrome b5 reductase linking the production to this enzyme. Measurement of the superoxide anion radical generated by purified recombinant soluble and membrane cytochrome b5 reductase corroborates the production of the radical by different enzyme isoforms. In the presence of cytochrome c, a burst of superoxide anion as well as the reduction of cytochrome c by cytochrome b5 reductase was measured. Complex formation between both proteins suggests that cytochrome b5 reductase is one of the major partners of cytochrome c upon its release from mitochondria to the cytosol during apoptosis. Superoxide anion production and cytochrome c reduction are the consequences of the stimulated NADH consumption by cytochrome b5 reductase upon complex formation with cytochrome c and suggest a major role of this enzyme as an anti-apoptotic protein during cell death.

  1. 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....

  2. Constituents of Musa x paradisiaca cultivar with the potential to induce the phase II enzyme, quinone reductase.

    Science.gov (United States)

    Jang, Dae Sik; Park, Eun Jung; Hawthorne, Michael E; Vigo, Jose Schunke; Graham, James G; Cabieses, Fernando; Santarsiero, Bernard D; Mesecar, Andrew D; Fong, Harry H S; Mehta, Rajendra G; Pezzuto, John M; Kinghorn, A Douglas

    2002-10-23

    A new bicyclic diarylheptanoid, rel-(3S,4aR,10bR)-8-hydroxy-3-(4-hydroxyphenyl)-9-methoxy-4a,5,6,10b-tetrahydro-3H-naphtho[2,1-b]pyran (1), as well as four known compounds, 1,2-dihydro-1,2,3-trihydroxy-9-(4-methoxyphenyl)phenalene (2), hydroxyanigorufone (3), 2-(4-hydroxyphenyl)naphthalic anhydride (4), and 1,7-bis(4-hydroxyphenyl)hepta-4(E),6(E)-dien-3-one (5), were isolated from an ethyl acetate-soluble fraction of the methanol extract of the fruits of Musa x paradisiaca cultivar, using a bioassay based on the induction of quinone reductase (QR) in cultured Hepa1c1c7 mouse hepatoma cells to monitor chromatographic fractionation. The structure and relative stereochemistry of compound 1 were elucidated unambiguously by one- and two-dimensional NMR experiments ((1)H NMR, (13)C NMR, DEPT, COSY, HMQC, HMBC, and NOESY) and single-crystal X-ray diffraction analysis. Isolates 1-5 were evaluated for their potential cancer chemopreventive properties utilizing an in vitro assay to determine quinone reductase induction and a mouse mammary organ culture assay.

  3. Expressions of the low density lipoprotein receptor and 3-hydroxy-3-methylglutaryl coenzyme A reductase genes are stimulated by recombinant platelet-derived growth factor isomers

    International Nuclear Information System (INIS)

    Roth, M.; Emmons, L.R.; Perruchoud, A.; Block, L.H.

    1991-01-01

    The plausible role that platelet-derived growth factor (PDGF) has in the localized pathophysiological changes that occur in the arterial wall during development of atherosclerotic lesions led the authors to investigate the influence of recombinant (r)PDGF isomers -AA, -AB, and -BB on the expression of low density lipoprotein receptor (LDL-R) and 3-hydroxy-3-methylglutaryl coenzyme A (HMG0CoA) reductase [(S)-mevalonate:NAD + oxidoreductase (CoA-acylating), EC 1.1.1.88] genes. In addition, they clarified the role of protein kinase C (PKC) in expression of the two genes in human skin fibroblasts and vascular smooth muscle cells. The various rPDGF isoforms are distinct in their ability to activate transcription of both genes: (i) both rPDGF-AA and -BB stimulate transcription of the LDL-R gene; in contrast, rPDGF-BB but not -AA, activates transcription of the HMG-CoA reductase gene; (ii) all recombinant isoforms of PDGF activate transcription of the c-fos gene; (iii) while rPDGF-dependent transcription of the lDL-R gene occurs independently of PKC, transcription of the HMG-CoA reductase gene appears to involve the action of that enzyme

  4. Catalase as a sulfide-sulfur oxido-reductase: An ancient (and modern?) regulator of reactive sulfur species (RSS).

    Science.gov (United States)

    Olson, Kenneth R; Gao, Yan; DeLeon, Eric R; Arif, Maaz; Arif, Faihaan; Arora, Nitin; Straub, Karl D

    2017-08-01

    Catalase is well-known as an antioxidant dismutating H 2 O 2 to O 2 and H 2 O. However, catalases evolved when metabolism was largely sulfur-based, long before O 2 and reactive oxygen species (ROS) became abundant, suggesting catalase metabolizes reactive sulfide species (RSS). Here we examine catalase metabolism of H 2 S n , the sulfur analog of H 2 O 2 , hydrogen sulfide (H 2 S) and other sulfur-bearing molecules using H 2 S-specific amperometric electrodes and fluorophores to measure polysulfides (H 2 S n ; SSP4) and ROS (dichlorofluorescein, DCF). Catalase eliminated H 2 S n , but did not anaerobically generate H 2 S, the expected product of dismutation. Instead, catalase concentration- and oxygen-dependently metabolized H 2 S and in so doing acted as a sulfide oxidase with a P 50 of 20mmHg. H 2 O 2 had little effect on catalase-mediated H 2 S metabolism but in the presence of the catalase inhibitor, sodium azide (Az), H 2 O 2 rapidly and efficiently expedited H 2 S metabolism in both normoxia and hypoxia suggesting H 2 O 2 is an effective electron acceptor in this reaction. Unexpectedly, catalase concentration-dependently generated H 2 S from dithiothreitol (DTT) in both normoxia and hypoxia, concomitantly oxidizing H 2 S in the presence of O 2 . H 2 S production from DTT was inhibited by carbon monoxide and augmented by NADPH suggesting that catalase heme-iron is the catalytic site and that NADPH provides reducing equivalents. Catalase also generated H 2 S from garlic oil, diallyltrisulfide, thioredoxin and sulfur dioxide, but not from sulfite, metabisulfite, carbonyl sulfide, cysteine, cystine, glutathione or oxidized glutathione. Oxidase activity was also present in catalase from Aspergillus niger. These results show that catalase can act as either a sulfide oxidase or sulfur reductase and they suggest that these activities likely played a prominent role in sulfur metabolism during evolution and may continue do so in modern cells as well. This also appears

  5. Crystal Structure and Catalytic Mechanism of 7-Hydroxymethyl Chlorophyll a Reductase*

    Science.gov (United States)

    Wang, Xiao; Liu, Lin

    2016-01-01

    7-Hydroxymethyl chlorophyll a reductase (HCAR) catalyzes the second half-reaction in chlorophyll b to chlorophyll a conversion. HCAR is required for the degradation of light-harvesting complexes and is necessary for efficient photosynthesis by balancing the chlorophyll a/b ratio. Reduction of the hydroxymethyl group uses redox cofactors [4Fe-4S] cluster and FAD to transfer electrons and is difficult because of the strong carbon-oxygen bond. Here, we report the crystal structure of Arabidopsis HCAR at 2.7-Å resolution and reveal that two [4Fe-4S]clusters and one FAD within a very short distance form a consecutive electron pathway to the substrate pocket. In vitro kinetic analysis confirms the ferredoxin-dependent electron transport chain, thus supporting a proton-activated electron transfer mechanism. HCAR resembles a partial reconstruction of an archaeal F420-reducing [NiFe] hydrogenase, which suggests a common mode of efficient proton-coupled electron transfer through conserved cofactor arrangements. Furthermore, the trimeric form of HCAR provides a biological clue of its interaction with light-harvesting complex II. PMID:27072131

  6. YqhD. A broad-substrate range aldehyde reductase with various applications in production of biorenewable fuels and chemicals

    Energy Technology Data Exchange (ETDEWEB)

    Jarboe, Laura R. [Iowa State Univ., Ames, IA (United States). Dept. of Chemical and Biological Engineering

    2011-01-15

    The Escherichia coli NADPH-dependent aldehyde reductase YqhD has contributed to a variety of metabolic engineering projects for production of biorenewable fuels and chemicals. As a scavenger of toxic aldehydes produced by lipid peroxidation, YqhD has reductase activity for a broad range of short-chain aldehydes, including butyraldehyde, glyceraldehyde, malondialdehyde, isobutyraldehyde, methylglyoxal, propanealdehyde, acrolein, furfural, glyoxal, 3-hydroxypropionaldehyde, glycolaldehyde, acetaldehyde, and acetol. This reductase activity has proven useful for the production of biorenewable fuels and chemicals, such as isobutanol and 1,3- and 1,2-propanediol; additional capability exists for production of 1-butanol, 1-propanol, and allyl alcohol. A drawback of this reductase activity is the diversion of valuable NADPH away from biosynthesis. This YqhD-mediated NADPH depletion provides sufficient burden to contribute to growth inhibition by furfural and 5-hydroxymethyl furfural, inhibitory contaminants of biomass hydrolysate. The structure of YqhD has been characterized, with identification of a Zn atom in the active site. Directed engineering efforts have improved utilization of 3-hydroxypropionaldehyde and NADPH. Most recently, two independent projects have demonstrated regulation of yqhD by YqhC, where YqhC appears to function as an aldehyde sensor. (orig.)

  7. Boletus edulis Nitrite Reductase Reduces Nitrite Content of Pickles and Mitigates Intoxication in Nitrite-intoxicated Mice.

    Science.gov (United States)

    Zhang, Weiwei; Tian, Guoting; Feng, Shanshan; Wong, Jack Ho; Zhao, Yongchang; Chen, Xiao; Wang, Hexiang; Ng, Tzi Bun

    2015-10-08

    Pickles are popular in China and exhibits health-promoting effects. However, nitrite produced during fermentation adversely affects health due to formation of methemoglobin and conversion to carcinogenic nitrosamine. Fruiting bodies of the mushroom Boletus edulis were capable of inhibiting nitrite production during pickle fermentation. A 90-kDa nitrite reductase (NiR), demonstrating peptide sequence homology to fungal nitrite reductase, was isolated from B. edulis fruiting bodies. The optimum temperature and pH of the enzyme was 45 °C and 6.8, respectively. B. edulis NiR was capable of prolonging the lifespan of nitrite-intoxicated mice, indicating that it had the action of an antidote. The enzyme could also eliminate nitrite from blood after intragastric administration of sodium nitrite, and after packaging into capsule, this nitrite-eliminating activity could persist for at least 120 minutes thus avoiding immediate gastric degradation. B. edulis NiR represents the first nitrite reductase purified from mushrooms and may facilitate subsequent applications.

  8. Cancer cell death induced by phosphine gold(I) compounds targeting thioredoxin reductase.

    Science.gov (United States)

    Gandin, Valentina; Fernandes, Aristi Potamitou; Rigobello, Maria Pia; Dani, Barbara; Sorrentino, Francesca; Tisato, Francesco; Björnstedt, Mikael; Bindoli, Alberto; Sturaro, Alberto; Rella, Rocco; Marzano, Cristina

    2010-01-15

    The thioredoxin system, composed of thioredoxin reductase (TrxR), thioredoxin (Trx), and NADPH (nicotinamide adenine dinucleotide phosphate), plays a central role in regulating cellular redox homeostasis and signaling pathways. TrxR, overexpressed in many tumor cells and contributing to drug resistance, has emerged as a new target for anticancer drugs. Gold complexes have been validated as potent TrxR inhibitors in vitro in the nanomolar range. In order to obtain potent and selective TrxR inhibitors, we have synthesized a series of linear, 'auranofin-like' gold(I) complexes all containing the [Au(PEt(3))](+) synthon and the ligands: Cl(-), Br(-), cyanate, thiocyanate, ethylxanthate, diethyldithiocarbamate and thiourea. Phosphine gold(I) complexes efficiently inhibited cytosolic and mitochondrial TrxR at concentrations that did not affect the two related oxidoreductases glutathione reductase (GR) and glutathione peroxidase (GPx). The inhibitory effect of the redox proteins was also observed intracellularly in cancer cells pretreated with gold(I) complexes. Gold(I) compounds were found to induce antiproliferative effects towards several human cancer cells some of which endowed with cisplatin or multidrug resistance. In addition, they were able to activate caspase-3 and induce apoptosis observed as nucleosome formation and sub-G1 cell accumulation. The complexes with thiocyanate and xanthate ligands were particularly effective in inhibiting thioredoxin reductase and inducing apoptosis. Pharmacodynamic studies in human ovarian cancer cells allowed for the correlation of intracellular drug accumulation with TrxR inhibition that leads to the induction of apoptosis via the mitochondrial pathway.

  9. X-ray crystal structure of GarR-tartronate semialdehyde reductase from Salmonella typhimurium.

    Science.gov (United States)

    Osipiuk, J; Zhou, M; Moy, S; Collart, F; Joachimiak, A

    2009-09-01

    Tartronate semialdehyde reductases (TSRs), also known as 2-hydroxy-3-oxopropionate reductases, catalyze the reduction of tartronate semialdehyde using NAD as cofactor in the final stage of D-glycerate biosynthesis. These enzymes belong to family of structurally and mechanically related beta-hydroxyacid dehydrogenases which differ in substrate specificity and catalyze reactions in specific metabolic pathways. Here, we present the crystal structure of GarR a TSR from Salmonella typhimurium determined by the single-wavelength anomalous diffraction method and refined to 1.65 A resolution. The active site of the enzyme contains L-tartrate which most likely mimics a position of a glycerate which is a product of the enzyme reaction. The analysis of the TSR structure shows also a putative NADPH binding site in the enzyme.

  10. [Aldose reductase gene polymorphism and rate of appearance of retinopathy in non insulin dependent diabetics].

    Science.gov (United States)

    Olmos, P; Acosta, A M; Schiaffino, R; Díaz, R; Alvarado, D; O'Brien, A; Muñoz, X; Arriagada, P; Claro, J C; Vega, R; Vollrath, V; Velasco, S; Emmerich, M; Maiz, A

    1999-04-01

    Recent studies suggest that polymorphisms associated to the aldose reductase gene could be related to early retinopathy in noninsulin dependent diabetics (NIDDM). There is also new interest on the genetic modulation of coagulation factors in relation to this complication. To look for a possible relationship between the rate of appearance of retinopathy and the genotype of (AC)n polymorphic marker associated to aldose reductase gene. A random sample of 27 NIDDM, aged 68.1 +/- 10.6 years, with a mean diabetes duration of 20.7 +/- 4.8 years and a mean glycosilated hemoglobin of 10.6 +/- 1.6%, was studied. The genotype of the (AC)n, polymorphic marker associated to the 5' end of the aldose reductase (ALR2) gene was determined by 32P-PCR plus sequenciation. Mutations of the factor XIII-A gene were studied by single stranded conformational polymorphism, sequenciation and restriction fragment length polymorphism. Four patients lacked the (AC)24 and had a higher rate of appearance of retinopathy than patients with the (AC)24 allele (0.0167 and 0.0907 score points per year respectively, p = 0.047). Both groups had similar glycosilated hemoglobin (11.7 +/- 0.2 and 10.5 +/- 1.6% respectively). Factor XIII gene mutations were not related to the rate of appearance of retinopathy. Our data suggest that the absence of the (AC)24 allele of the (AC)n polymorphic marker associated to the 5' end of the aldose reductase gene, is associated to a five fold reduction of retinopathy appearance rate.

  11. Regulation of Expression of Oxacillin-Inducible Methionine Sulfoxide Reductases in Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    Kyle R. Baum

    2015-01-01

    Full Text Available Cell wall-active antibiotics cause induction of a locus that leads to elevated synthesis of two methionine sulfoxide reductases (MsrA1 and MsrB in Staphylococcus aureus. To understand the regulation of this locus, reporter strains were constructed by integrating a DNA fragment consisting of the msrA1/msrB promoter in front of a promoterless lacZ gene in the chromosome of wild-type and MsrA1-, MsrB-, MsrA1/MsrB-, and SigB-deficient methicillin-sensitive S. aureus strain SH1000 and methicillin-resistant S. aureus strain COL. These reporter strains were cultured in TSB and the cellular levels of β-galactosidase activity in these cultures were assayed during different growth phases. β-galactosidase activity assays demonstrated that the lack of MsrA1, MsrB, and SigB upregulated the msrA1/msrB promoter in S. aureus strain SH1000. In S. aureus strain COL, the highest level of β-galactosidase activity was observed under the conditions when both MsrA1 and MsrB proteins were absent. The data suggest that the msrA1/msrB locus, in part, is negatively regulated by MsrA1, MsrB, and SigB in S. aureus.

  12. 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

  13. Streptococcus sanguinis class Ib ribonucleotide reductase: high activity with both iron and manganese cofactors and structural insights.

    Science.gov (United States)

    Makhlynets, Olga; Boal, Amie K; Rhodes, Delacy V; Kitten, Todd; Rosenzweig, Amy C; Stubbe, JoAnne

    2014-02-28

    Streptococcus sanguinis is a causative agent of infective endocarditis. Deletion of SsaB, a manganese transporter, drastically reduces S. sanguinis virulence. Many pathogenic organisms require class Ib ribonucleotide reductase (RNR) to catalyze the conversion of nucleotides to deoxynucleotides under aerobic conditions, and recent studies demonstrate that this enzyme uses a dimanganese-tyrosyl radical (Mn(III)2-Y(•)) cofactor in vivo. The proteins required for S. sanguinis ribonucleotide reduction (NrdE and NrdF, α and β subunits of RNR; NrdH and TrxR, a glutaredoxin-like thioredoxin and a thioredoxin reductase; and NrdI, a flavodoxin essential for assembly of the RNR metallo-cofactor) have been identified and characterized. Apo-NrdF with Fe(II) and O2 can self-assemble a diferric-tyrosyl radical (Fe(III)2-Y(•)) cofactor (1.2 Y(•)/β2) and with the help of NrdI can assemble a Mn(III)2-Y(•) cofactor (0.9 Y(•)/β2). The activity of RNR with its endogenous reductants, NrdH and TrxR, is 5,000 and 1,500 units/mg for the Mn- and Fe-NrdFs (Fe-loaded NrdF), respectively. X-ray structures of S. sanguinis NrdIox and Mn(II)2-NrdF are reported and provide a possible rationale for the weak affinity (2.9 μM) between them. These streptococcal proteins form a structurally distinct subclass relative to other Ib proteins with unique features likely important in cluster assembly, including a long and negatively charged loop near the NrdI flavin and a bulky residue (Thr) at a constriction in the oxidant channel to the NrdI interface. These studies set the stage for identifying the active form of S. sanguinis class Ib RNR in an animal model for infective endocarditis and establishing whether the manganese requirement for pathogenesis is associated with RNR.

  14. Genetic and Biochemical Analysis of Intragenic Complementation Events among Nitrate Reductase Apoenzyme-Deficient Mutants of Nicotiana Plumbaginifolia

    OpenAIRE

    Pelsy, F.; Gonneau, M.

    1991-01-01

    Intragenic complementation has been observed between apoenzyme nitrate reductase-deficient mutants (nia) of Nicotiana plumbaginifolia. In vivo as in vitro, the NADH-nitrate reductase (NR) activity in plants heterozygous for two different nia alleles was lower than in the wild type plant, but the plants were able to grow on nitrate as a sole nitrogen source. NR activity, absent in extracts of homozygous nia mutants was restored by mixing extracts from two complementing nia mutants. These obser...

  15. Expression, purification, crystallization and preliminary X-ray analysis of maleylacetate reductase from Burkholderia sp. strain SJ98

    International Nuclear Information System (INIS)

    Chauhan, Archana; Islam, Zeyaul; Jain, Rakesh Kumar; Karthikeyan, Subramanian

    2009-01-01

    Purification and preliminary X-ray crystallographic analysis of maleylacetate reductase encoded by the pnpD gene is reported. Maleylacetate reductase (EC 1.3.1.32) is an important enzyme that is involved in the degradation pathway of aromatic compounds and catalyzes the reduction of maleylacetate to 3-oxoadipate. The gene pnpD encoding maleylacetate reductase in Burkholderia sp. strain SJ98 was cloned, expressed in Escherichia coli and purified by affinity chromatography. The enzyme was crystallized in both native and SeMet-derivative forms by the sitting-drop vapour-diffusion method using PEG 3350 as a precipitant at 293 K. The crystals belonged to space group P2 1 2 1 2, with unit-cell parameters a = 72.91, b = 85.94, c = 53.07 Å. X-ray diffraction data for the native and SeMet-derivative crystal were collected to 2.7 and 2.9 Å resolution, respectively

  16. Genetic Characterization and Role in Virulence of the Ribonucleotide Reductases of Streptococcus sanguinis * ♦

    Science.gov (United States)

    Rhodes, DeLacy V.; Crump, Katie E.; Makhlynets, Olga; Snyder, Melanie; Ge, Xiuchun; Xu, Ping; Stubbe, JoAnne; Kitten, Todd

    2014-01-01

    Streptococcus sanguinis is a cause of infective endocarditis and has been shown to require a manganese transporter called SsaB for virulence and O2 tolerance. Like certain other pathogens, S. sanguinis possesses aerobic class Ib (NrdEF) and anaerobic class III (NrdDG) ribonucleotide reductases (RNRs) that perform the essential function of reducing ribonucleotides to deoxyribonucleotides. The accompanying paper (Makhlynets, O., Boal, A. K., Rhodes, D. V., Kitten, T., Rosenzweig, A. C., and Stubbe, J. (2014) J. Biol. Chem. 289, 6259–6272) indicates that in the presence of O2, the S. sanguinis class Ib RNR self-assembles an essential diferric-tyrosyl radical (FeIII2-Y•) in vitro, whereas assembly of a dimanganese-tyrosyl radical (MnIII2-Y•) cofactor requires NrdI, and MnIII2-Y• is more active than FeIII2-Y• with the endogenous reducing system of NrdH and thioredoxin reductase (TrxR1). In this study, we have shown that deletion of either nrdHEKF or nrdI completely abolishes virulence in an animal model of endocarditis, whereas nrdD mutation has no effect. The nrdHEKF, nrdI, and trxR1 mutants fail to grow aerobically, whereas anaerobic growth requires nrdD. The nrdJ gene encoding an O2-independent adenosylcobalamin-cofactored RNR was introduced into the nrdHEKF, nrdI, and trxR1 mutants. Growth of the nrdHEKF and nrdI mutants in the presence of O2 was partially restored. The combined results suggest that MnIII2-Y•-cofactored NrdF is required for growth under aerobic conditions and in animals. This could explain in part why manganese is necessary for virulence and O2 tolerance in many bacterial pathogens possessing a class Ib RNR and suggests NrdF and NrdI may serve as promising new antimicrobial targets. PMID:24381171

  17. A novel twist on molecular interactions between thioredoxin and nicotinamide adenine dinucleotide phosphate-dependent thioredoxin reductase

    DEFF Research Database (Denmark)

    Kirkensgaard, Kristine Groth; Hägglund, Per; Shahpiri, Azar

    2013-01-01

    The ubiquitous disulfide reductase thioredoxin (Trx) regulates several important biological processes such as seed germination in plants. Oxidized cytosolic Trx is regenerated by nicotinamide adenine dinucleotide phosphate (NADPH)-dependent thioredoxin reductase (NTR) in a multistep transfer...... dinucleotide (FAD)-binding domain of HvNTR2 to strongly affect the interaction with Trx. In particular, Trp42 and Met43 play key roles for recognition of the endogenous HvTrxh2. Trx from Arabidopsis thaliana is also efficiently recycled by HvNTR2 but turnover in this case appears to be less dependent...

  18. Amperometric nitrate biosensor based on Carbon nanotube/Polypyrrole/Nitrate reductase biofilm electrode

    Energy Technology Data Exchange (ETDEWEB)

    Can, Faruk; Korkut Ozoner, Seyda; Ergenekon, Pinar; Erhan, Elif, E-mail: e.erhan@gyte.edu.tr

    2012-01-01

    This study describes the construction and characterization of an amperometric nitrate biosensor based on the Polypyrrole (PPy)/Carbon nanotubes (CNTs) film. Nitrate reductase (NR) was both entrapped into the growing PPy film and chemically immobilized via the carboxyl groups of CNTs to the CNT/PPy film electrode. The optimum amperometric response for nitrate was obtained in 0.1 M phosphate buffer solution (PBS), pH 7.5 including 0.1 M lithium chloride and 7 mM potassium ferricyanide with an applied potential of 0.13 V (vs. Ag/AgCl, 3 M NaCl). Sensitivity was found to be 300 nA/mM in a linear range of 0.44-1.45 mM with a regression coefficient of 0.97. The biosensor response showed a higher linear range in comparison to standard nitrate analysis methods which were tested in this study and NADH based nitrate biosensors. A minimum detectable concentration of 0.17 mM (S/N = 3) with a relative standard deviation (RSD) of 5.4% (n = 7) was obtained for the biosensor. Phenol and glucose inhibit the electrochemical reaction strictly at a concentration of 1 {mu}g/L and 20 mg/L, respectively. The biosensor response retained 70% of its initial response over 10 day usage period when used everyday. - Highlights: Black-Right-Pointing-Pointer K{sub 3}Fe(CN){sub 6} has been used for the first time as mediator for nitrate reductase. Black-Right-Pointing-Pointer Better performance was obtained in comparison to other nitrate biosensor studies operated with various mediators. Black-Right-Pointing-Pointer Analytical parameters were better than standard nitrate analysis methods.

  19. Amperometric nitrate biosensor based on Carbon nanotube/Polypyrrole/Nitrate reductase biofilm electrode

    International Nuclear Information System (INIS)

    Can, Faruk; Korkut Ozoner, Seyda; Ergenekon, Pinar; Erhan, Elif

    2012-01-01

    This study describes the construction and characterization of an amperometric nitrate biosensor based on the Polypyrrole (PPy)/Carbon nanotubes (CNTs) film. Nitrate reductase (NR) was both entrapped into the growing PPy film and chemically immobilized via the carboxyl groups of CNTs to the CNT/PPy film electrode. The optimum amperometric response for nitrate was obtained in 0.1 M phosphate buffer solution (PBS), pH 7.5 including 0.1 M lithium chloride and 7 mM potassium ferricyanide with an applied potential of 0.13 V (vs. Ag/AgCl, 3 M NaCl). Sensitivity was found to be 300 nA/mM in a linear range of 0.44–1.45 mM with a regression coefficient of 0.97. The biosensor response showed a higher linear range in comparison to standard nitrate analysis methods which were tested in this study and NADH based nitrate biosensors. A minimum detectable concentration of 0.17 mM (S/N = 3) with a relative standard deviation (RSD) of 5.4% (n = 7) was obtained for the biosensor. Phenol and glucose inhibit the electrochemical reaction strictly at a concentration of 1 μg/L and 20 mg/L, respectively. The biosensor response retained 70% of its initial response over 10 day usage period when used everyday. - Highlights: ► K 3 Fe(CN) 6 has been used for the first time as mediator for nitrate reductase. ► Better performance was obtained in comparison to other nitrate biosensor studies operated with various mediators. ► Analytical parameters were better than standard nitrate analysis methods.

  20. Correlation of quinone reductase activity and allyl isothiocyanate formation among different genotypes and grades of horseradish roots.

    Science.gov (United States)

    Ku, Kang-Mo; Jeffery, Elizabeth H; Juvik, John A; Kushad, Mosbah M

    2015-03-25

    Horseradish (Armoracia rusticana) is a perennial crop and its ground root tissue is used in condiments because of the pungency of the glucosinolate (GS)-hydrolysis products allyl isothiocyanate (AITC) and phenethyl isothiocyanate (PEITC) derived from sinigrin and gluconasturtiin, respectively. Horseradish roots are sold in three grades: U.S. Fancy, U.S. No. 1, and U.S. No. 2 according to the USDA standards. These grading standards are primarily based on root diameter and length. There is little information on whether root grades vary in their phytochemical content or potential health promoting properties. This study measured GS, GS-hydrolysis products, potential anticancer activity (as quinone reductase inducing activity), total phenolic content, and antioxidant activities from different grades of horseradish accessions. U.S. Fancy showed significantly higher sinigrin and AITC concentrations than U.S. No. 1 ,whereas U.S. No. 1 showed significantly higher concentrations of 1-cyano 2,3-epithiopropane, the epithionitrile hydrolysis product of sinigrin, and significantly higher total phenolic concentrations than U.S. Fancy.

  1. X-Ray crystal structure of GarR—tartronate semialdehyde reductase from Salmonella typhimurium

    Science.gov (United States)

    Osipiuk, J.; Zhou, M.; Moy, S.; Collart, F.

    2009-01-01

    Tartronate semialdehyde reductases (TSRs), also known as 2-hydroxy-3-oxopropionate reductases, catalyze the reduction of tartronate semialdehyde using NAD as cofactor in the final stage of D-glycerate biosynthesis. These enzymes belong to family of structurally and mechanically related β-hydroxyacid dehydrogenases which differ in substrate specificity and catalyze reactions in specific metabolic pathways. Here, we present the crystal structure of GarR a TSR from Salmonella typhimurium determined by the single-wavelength anomalous diffraction method and refined to 1.65 Å resolution. The active site of the enzyme contains L-tartrate which most likely mimics a position of a glycerate which is a product of the enzyme reaction. The analysis of the TSR structure shows also a putative NADPH binding site in the enzyme. PMID:19184529

  2. Normal bone density in male pseudohermaphroditism due to 5a- reductase 2 deficiency

    Directory of Open Access Journals (Sweden)

    Costa Elaine Maria Frade

    2001-01-01

    Full Text Available Bone is an androgen-dependent tissue, but it is not clear whether the androgen action in bone depends on testosterone or on dihydrotestosterone. Patients with 5alpha-reductase 2 deficiency present normal levels of testosterone and low levels of dihydrotestosterone, providing an in vivo human model for the analysis of the effect of testosterone on bone. OBJECTIVE: To analyze bone mineral density in 4 adult patients with male pseudohermaphroditism due to 5alpha-reductase 2 deficiency. RESULTS: Three patients presented normal bone mineral density of the lumbar column (L1-L4 and femur neck, and the other patient presented a slight osteopenia in the lumbar column. CONCLUSION: Patients with dihydrotestosterone deficiency present normal bone mineral density, suggesting that dihydrotestosterone is not the main androgen acting in bone.

  3. The crystal structure of the bifunctional deaminase/reductase RibD of the riboflavin biosynthetic pathway in Escherichia coli: implications for the reductive mechanism.

    Science.gov (United States)

    Stenmark, Pål; Moche, Martin; Gurmu, Daniel; Nordlund, Pär

    2007-10-12

    We have determined the crystal structure of the bi-functional deaminase/reductase enzyme from Escherichia coli (EcRibD) that catalyzes two consecutive reactions during riboflavin biosynthesis. The polypeptide chain of EcRibD is folded into two domains where the 3D structure of the N-terminal domain (1-145) is similar to cytosine deaminase and the C-terminal domain (146-367) is similar to dihydrofolate reductase. We showed that EcRibD is dimeric and compared our structure to tetrameric RibG, an ortholog from Bacillus subtilis (BsRibG). We have also determined the structure of EcRibD in two binary complexes with the oxidized cofactor (NADP(+)) and with the substrate analogue ribose-5-phosphate (RP5) and superposed these two in order to mimic the ternary complex. Based on this superposition we propose that the invariant Asp200 initiates the reductive reaction by abstracting a proton from the bound substrate and that the pro-R proton from C4 of the cofactor is transferred to C1 of the substrate. A highly flexible loop is found in the reductase active site (159-173) that appears to control cofactor and substrate binding to the reductase active site and was therefore compared to the corresponding Met20 loop of E. coli dihydrofolate reductase (EcDHFR). Lys152, identified by comparing substrate analogue (RP5) coordination in the reductase active site of EcRibD with the homologous reductase from Methanocaldococcus jannaschii (MjaRED), is invariant among bacterial RibD enzymes and could contribute to the various pathways taken during riboflavin biosynthesis in bacteria and yeast.

  4. Structural basis for high substrate-binding affinity and enantioselectivity of 3-quinuclidinone reductase AtQR

    International Nuclear Information System (INIS)

    Hou, Feng; Miyakawa, Takuya; Kataoka, Michihiko; Takeshita, Daijiro; Kumashiro, Shoko; Uzura, Atsuko; Urano, Nobuyuki; Nagata, Koji; Shimizu, Sakayu; Tanokura, Masaru

    2014-01-01

    Highlights: • Crystal structure of AtQR has been determined at 1.72 Å. • NADH binding induces the formation of substrate binding site. • AtQR possesses a conserved hydrophobic wall for stereospecific binding of substrate. • Additional Glu197 residue is critical to the high binding affinity. - Abstract: (R)-3-Quinuclidinol, a useful compound for the synthesis of various pharmaceuticals, can be enantioselectively produced from 3-quinuclidinone by 3-quinuclidinone reductase. Recently, a novel NADH-dependent 3-quinuclidionone reductase (AtQR) was isolated from Agrobacterium tumefaciens, and showed much higher substrate-binding affinity (>100 fold) than the reported 3-quinuclidionone reductase (RrQR) from Rhodotorula rubra. Here, we report the crystal structure of AtQR at 1.72 Å. Three NADH-bound protomers and one NADH-free protomer form a tetrameric structure in an asymmetric unit of crystals. NADH not only acts as a proton donor, but also contributes to the stability of the α7 helix. This helix is a unique and functionally significant part of AtQR and is related to form a deep catalytic cavity. AtQR has all three catalytic residues of the short-chain dehydrogenases/reductases family and the hydrophobic wall for the enantioselective reduction of 3-quinuclidinone as well as RrQR. An additional residue on the α7 helix, Glu197, exists near the active site of AtQR. This acidic residue is considered to form a direct interaction with the amine part of 3-quinuclidinone, which contributes to substrate orientation and enhancement of substrate-binding affinity. Mutational analyses also support that Glu197 is an indispensable residue for the activity

  5. Structural basis for high substrate-binding affinity and enantioselectivity of 3-quinuclidinone reductase AtQR

    Energy Technology Data Exchange (ETDEWEB)

    Hou, Feng; Miyakawa, Takuya [Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657 (Japan); Kataoka, Michihiko [Division of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai 559-8531 (Japan); Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606-8502 (Japan); Takeshita, Daijiro [Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657 (Japan); Kumashiro, Shoko [Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606-8502 (Japan); Uzura, Atsuko [Research and Development Center, Nagase and Co., Ltd., 2-2-3 Muratani, Nishi-ku, Kobe 651-2241 (Japan); Urano, Nobuyuki [Division of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai 559-8531 (Japan); Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606-8502 (Japan); Nagata, Koji [Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657 (Japan); Shimizu, Sakayu [Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606-8502 (Japan); Faculty of Bioenvironmental Science, Kyoto Gakuen University, Sogabe-cho, Kameoka 621-8555 (Japan); Tanokura, Masaru, E-mail: amtanok@mail.ecc.u-tokyo.ac.jp [Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657 (Japan)

    2014-04-18

    Highlights: • Crystal structure of AtQR has been determined at 1.72 Å. • NADH binding induces the formation of substrate binding site. • AtQR possesses a conserved hydrophobic wall for stereospecific binding of substrate. • Additional Glu197 residue is critical to the high binding affinity. - Abstract: (R)-3-Quinuclidinol, a useful compound for the synthesis of various pharmaceuticals, can be enantioselectively produced from 3-quinuclidinone by 3-quinuclidinone reductase. Recently, a novel NADH-dependent 3-quinuclidionone reductase (AtQR) was isolated from Agrobacterium tumefaciens, and showed much higher substrate-binding affinity (>100 fold) than the reported 3-quinuclidionone reductase (RrQR) from Rhodotorula rubra. Here, we report the crystal structure of AtQR at 1.72 Å. Three NADH-bound protomers and one NADH-free protomer form a tetrameric structure in an asymmetric unit of crystals. NADH not only acts as a proton donor, but also contributes to the stability of the α7 helix. This helix is a unique and functionally significant part of AtQR and is related to form a deep catalytic cavity. AtQR has all three catalytic residues of the short-chain dehydrogenases/reductases family and the hydrophobic wall for the enantioselective reduction of 3-quinuclidinone as well as RrQR. An additional residue on the α7 helix, Glu197, exists near the active site of AtQR. This acidic residue is considered to form a direct interaction with the amine part of 3-quinuclidinone, which contributes to substrate orientation and enhancement of substrate-binding affinity. Mutational analyses also support that Glu197 is an indispensable residue for the activity.

  6. Application of photocatalytic cadmium sulfide nanoparticles to detection of enzymatic activities of glucose oxidase and glutathione reductase using oxidation of 3,3′,5,5′-tetramethylbenzidine

    Energy Technology Data Exchange (ETDEWEB)

    Grinyte, Ruta; Garai-Ibabe, Gaizka; Saa, Laura; Pavlov, Valeri, E-mail: vpavlov@cicbiomagune.es

    2015-06-30

    Highlights: • The light-powered nanosensor fabricated by enzymatic reactions was reported. • The sensor use energy of photons for oxidation of chromogenic enzymatic substrates. • Enzymatic assays for glucose oxidase and glutathione reductase were developed. - Abstract: It was found out that semiconductor CdS nanoparticles (NPs) are able to catalyze photooxidation of the well known chromogenic enzymatic substrate 3,3′,5,5′-tetramethylbenzidine (TMB) by oxygen. The photocatalytical oxidation of TMB does not require hydrogen peroxide and its rate is directly proportional to the quantity of CdS NPs produced in situ through the interaction of Cd{sup 2+} and S{sup 2−} ions in an aqueous medium. This phenomenon was applied to development of colorimetric sensitive assays for glucose oxidase and glutathione reductase based on enzymatic generation of CdS NPs acting as light-powered catalysts. Sensitivity of the developed chromogenic assays was of the same order of magnitude or even better than that of relevant fluorogenic assays. The present approach opens the possibility for the design of simple and sensitive colorimetric assays for a number of enzymes using inexpensive and available TMB as a universal chromogenic compound.

  7. ADP-ribosylation of dinitrogenase reductase in Rhodobacter capsulatus

    International Nuclear Information System (INIS)

    Jouanneau, Y.; Roby, C.; Meyer, C.M.; Vignais, P.M.

    1989-01-01

    In the photosynthetic bacterium Rhodobacter capsulatus, nitrogenase is regulated by a reversible covalent modification of Fe protein or dinitrogenase reductase (Rc2). The linkage of the modifying group to inactive Rc2 was found to be sensitive to alkali and to neutral hydroxylamine. Complete release of the modifying group was achieved by incubation of inactive Rc2 in 0.4 or 1 M hydroxylamine. After hydroxylamine treatment of the Rc2 preparation, the modifying group could be isolated and purified by affinity chromatography and ion-exchange HPLC. The modifying group comigrated with ADP-ribose on both ion-exchange HPLC and thin-layer chromatography. Analyses by 31 P NMR spectroscopy and mass spectrometry provided further evidence that the modifying group was ADP-ribose. The NMR spectrum of inactive Rc2 exhibited signals characteristic of ADP-ribose; integration of these signals allowed calculation of a molar ration ADP-ribose/Rc2 of 0.63. A hexapeptide carrying the ADP-ribose moiety was purified from a subtilisin digest of inactive Rc2. The structure of this peptide, determined by amino acid analysis and sequencing, is Gly-Arg(ADP-ribose)-Gly-Val-Ile-Thr. This structure allows identification of the binding site for ADP-ribose as Arg 101 of the polypeptide chain of Rc2. It is concluded that nitrogenase activity in R. capsulatus is regulated by reversible ADP-ribosylation of a specific arginyl residue of dinitrogenase reductase

  8. A genetic screen reveals a periplasmic copper chaperone required for nitrite reductase activity in pathogenic Neisseria.

    Science.gov (United States)

    Jen, Freda E-C; Djoko, Karrera Y; Bent, Stephen J; Day, Christopher J; McEwan, Alastair G; Jennings, Michael P

    2015-09-01

    Under conditions of low oxygen availability, Neisseria meningitidis and Neisseria gonorrhoeae are able to respire via a partial denitrification pathway in which nitrite is converted to nitrous oxide. In this process, nitrite reductase (AniA), a copper (Cu)-containing protein converts nitrite to NO, and this product is converted to nitrous oxide by nitric oxide reductase (NorB). NorB also confers protection against toxic NO, and so we devised a conditional lethal screen, using a norB mutant, to identify mutants that were resistant to nitrite-dependent killing. After random-deletion mutagenesis of N. meningitidis, this genetic screen identified a gene encoding a Cu chaperone that is essential for AniA function, AccA. Purified AccA binds one Cu (I) ion and also possesses a second binding site for Cu (II). This novel periplasmic Cu chaperone (AccA) appears to be essential for provision of Cu ions to AniA of pathogenic Neisseria to generate an active nitrite reductase. Apart from the Neisseria genus, AccA is distributed across a wide range of environmental Proteobacteria species. © FASEB.

  9. Aldose Reductase-Deficient Mice Develop Nephrogenic Diabetes Insipidus

    Science.gov (United States)

    Ho, Horace T. B.; Chung, Sookja K.; Law, Janice W. S.; Ko, Ben C. B.; Tam, Sidney C. F.; Brooks, Heddwen L.; Knepper, Mark A.; Chung, Stephen S. M.

    2000-01-01

    Aldose reductase (ALR2) is thought to be involved in the pathogenesis of various diseases associated with diabetes mellitus, such as cataract, retinopathy, neuropathy, and nephropathy. However, its physiological functions are not well understood. We developed mice deficient in this enzyme and found that they had no apparent developmental or reproductive abnormality except that they drank and urinated significantly more than their wild-type littermates. These ALR2-deficient mice exhibited a partially defective urine-concentrating ability, having a phenotype resembling that of nephrogenic diabetes insipidus. PMID:10913167

  10. In vivo assay for conversion of testosterone to dihydrotestosterone by rat prostatic steroid 5 alpha-reductase and comparison of two inhibitors

    International Nuclear Information System (INIS)

    Toomey, R.E.; Goode, R.L.; Petrow, V.; Neubauer, B.L.

    1991-01-01

    An in vivo assay for steroid 5 alpha-reductase in rat ventral prostate has been developed and used to compare the inhibitory activity of N,N-diethyl-4-methyl-3-oxo-4-aza-5 alpha-androstane-17 beta-carboxamide (4-MA) and 6-methylene-4-pregnene-3,20-dione (LY207320). Immature rats (70-80 g) received test compounds 30 min prior to s.c. injection of [3H]-T. The rats were sacrificed 30 min later and the ventral prostates were analyzed for [3H]-T metabolites. Intraprostatic [3H]-T and [3H]-DHT reached peak levels within 5 min after injection of [3H]-T and declined to about 25% of peak levels after 2 hr. 4-MA was a very potent inhibitor of [3H]-DHT formation with an estimated IC50 of 0.2 mg/kg. LY207320, an inhibitor of 5 alpha-reductase in vitro, was weakly active in vivo and did not achieve greater than 45% inhibition at high doses (greater than 200 mg/kg, s.c.). Tissue uptake of [3H]-T was also inhibited by LY207320, which may contribute to its inhibitory activity on accessory sex organ growth in the rat

  11. Molecular cloning and functional characterization of the anthocyanidin reductase gene from Vitis bellula.

    Science.gov (United States)

    Zhu, Yue; Peng, Qing-Zhong; Li, Ke-Gang; Xie, De-Yu

    2014-08-01

    Anthocyanidin reductase (ANR) is an NADPH-/NADH-dependent enzyme that transfers two hydrides to anthocyanidins to produce three types of isomeric flavan-3-ols. This reductase forms the ANR pathway toward the biosynthesis of proanthocyanidins (PAs, which are also called condensed tannins). Here, we report cloning and functional characterization of an ANR (called VbANR) homolog from the leaves of Vitis bellula, a newly developed grape crop in southern China. The open reading frame (ORF) of VbANR is 1,017 bp in length and encodes 339 amino acids. A phylogenetic analysis and an alignment using 17 sequences revealed that VbANR is approximately 99.9 % identical to the ANR homolog from Vitis vinifera. The VbANR ORF is fused to the Trx gene containing a His-tag in the pET32a(+) vector to obtain a pET32a(+)-VbANR construct for expressing the recombinant VbANR. In vitro enzyme assays show that VbANR converts cyanidin, delphinidin, and pelargonidin to their corresponding flavan-3-ols. Enzymatic products include 2S,3R-trans- and 2R,3R-cis-flavan-3-ols isomers, such as (-)-catechin and (-)-epicatechin. In addition, the third compound that is observed from the enzymatic products is most likely a 2S,3S-cis-flavan-3-ol. To analyze the kinetics and optimize pH and temperature values, a UV spectrometry method was developed to quantify the concentrations of total enzymatic products. The optimum pH and temperature values are 4.0 and 40 °C, respectively. The K m , K cat, V max, and K cat/K m values for pelargonidin and delphinidin were similar. In comparison, VbANR exhibits a slightly lower affinity to cyanidin. VbANR uses both NADPH and NADH but prefers to employ NADPH. GFP fusion and confocal microscopy analyses revealed the cytosolic localization of VbANR. The overexpression of VbANR in ban mutants reconstructed the biosynthetic pathway of PAs in the seed coat. These data demonstrate that VbANR forms the ANR pathway, leading to the formation of three types of isomeric flavan-3-ols

  12. Ebselen: A thioredoxin reductase-dependent catalyst for α-tocopherol quinone reduction

    International Nuclear Information System (INIS)

    Fang Jianguo; Zhong Liangwei; Zhao Rong; Holmgren, Arne

    2005-01-01

    The thioredoxin system, composed of thioredoxin (Trx), thioredoxin reductase (TrxR), and NADPH, is a powerful protein disulfide reductase system with a broad substrate specificity. Recently the selenazol drug ebselen was shown to be a substrate for both mammalian TrxR and Trx. We examined if α-tocopherol quinone (TQ), a product of α-tocopherol oxidation, is reduced by ebselen in the presence of TrxR, since TQ was not a substrate for the enzyme itself. Ebselen reduction of TQ in the presence of TrxR was caused by ebselen selenol, generated from fast reduction of ebselen by the enzyme. TQ has no intrinsic antioxidant activity, while the product of reduction of TQ, α-tocopherolhydroquinone (TQH 2 ), is a potent antioxidant. The thioredoxin system dependence of ebselen to catalyze reduction of other oxidized species, such as hydrogen peroxide, dehydroascorbate, and peroxynitrite, is discussed. The ability of ebselen to reduce TQ via the thioredoxin system is a novel mechanism to explain the effects of the drug as an antioxidant in vivo

  13. Protective effect of Pterocarpus marsupium bark extracts against cataract through the inhibition of aldose reductase activity in streptozotocin-induced diabetic male albino rats.

    Science.gov (United States)

    Xu, YanLi; Zhao, Yongxia; Sui, YaNan; Lei, XiaoJun

    2018-04-01

    The present study was aimed to investigate the protective effect of Pterocarpus marsupium bark extracts against cataract in streptozotocin-induced diabetic male albino rats. Aldose reductase is a key enzyme in the intracellular polyol pathway, which plays a major role in the development of diabetic cataract. Rats were divided into five groups as normal control, diabetic control, and diabetic control treated with different concentrations of Pterocarpus marsupium bark extracts. Presence of major constituents in Pterocarpus marsupium bark extract was performed by qualitative analysis. Body weight changes, blood glucose, blood insulin, and reduced glutathione (GSH) and aldose reductase mRNA and protein expression were determined. Rat body weight gain was noted following treatment with bark extracts. The blood glucose was reduced up to 36% following treatment with bark extracts. The blood insulin and tissue GSH contents were substantially increased more than 100% in diabetic rats following treatment with extracts. Aldose reductase activity was reduced up to 79.3% in diabetic rats following treatment with extracts. V max , K m , and K i of aldose reductase were reduced in the lens tissue homogenate compared to the diabetic control. Aldose reductase mRNA and protein expression were reduced more than 50% following treatment with extracts. Treatment with Pterocarpus marsupium bark was able to normalize these levels. Taking all these data together, it is concluded that the use of Pterocarpus marsupium bark extracts could be the potential therapeutic approach for the reduction of aldose reductase against diabetic cataract.

  14. Enantiocomplementary Yarrowia lipolytica Oxidoreductases: Alcohol Dehydrogenase 2 and Short Chain Dehydrogenase/Reductase

    Directory of Open Access Journals (Sweden)

    Margit Winkler

    2013-08-01

    Full Text Available Enzymes of the non-conventional yeast Yarrowia lipolytica seem to be tailor-made for the conversion of lipophilic substrates. Herein, we cloned and overexpressed the Zn-dependent alcohol dehydrogenase ADH2 from Yarrowia lipolytica in Escherichia coli. The purified enzyme was characterized in vitro. The substrate scope for YlADH2 mediated oxidation and reduction was investigated spectrophotometrically and the enzyme showed a broader substrate range than its homolog from Saccharomyces cerevisiae. A preference for secondary compared to primary alcohols in oxidation direction was observed for YlADH2. 2-Octanone was investigated in reduction mode in detail. Remarkably, YlADH2 displays perfect (S-selectivity and together with a highly (R-selective short chain dehydrogenase/ reductase from Yarrowia lipolytica it is possible to access both enantiomers of 2-octanol in >99% ee with Yarrowia lipolytica oxidoreductases.

  15. Enantiocomplementary Yarrowia lipolytica Oxidoreductases: Alcohol Dehydrogenase 2 and Short Chain Dehydrogenase/Reductase.

    Science.gov (United States)

    Napora-Wijata, Kamila; Strohmeier, Gernot A; Sonavane, Manoj N; Avi, Manuela; Robins, Karen; Winkler, Margit

    2013-08-12

    Enzymes of the non-conventional yeast Yarrowia lipolytica seem to be tailor-made for the conversion of lipophilic substrates. Herein, we cloned and overexpressed the Zn-dependent alcohol dehydrogenase ADH2 from Yarrowia lipolytica in Escherichia coli. The purified enzyme was characterized in vitro. The substrate scope for YlADH2 mediated oxidation and reduction was investigated spectrophotometrically and the enzyme showed a broader substrate range than its homolog from Saccharomyces cerevisiae. A preference for secondary compared to primary alcohols in oxidation direction was observed for YlADH2. 2-Octanone was investigated in reduction mode in detail. Remarkably, YlADH2 displays perfect (S)-selectivity and together with a highly (R)-selective short chain dehydrogenase/ reductase from Yarrowia lipolytica it is possible to access both enantiomers of 2-octanol in >99% ee with Yarrowia lipolytica oxidoreductases.

  16. Nitric oxide coordinates development of genomic instability in realization of combined effect with ionizing radiation.

    Science.gov (United States)

    Mikhailenko, V M; Diomina, E A; Muzalov, I I; Gerashchenko, B I

    2013-03-01

    The aim of this study was to investigate the ability of environmental nitrogen oxides or natural nitric oxide (NO) donors to modify free radicals ba-lance and development of genomic instability alone or in combination with ionizing radiation. Genotoxicity and cytogenetic abnormalities were assessed in vitro in peripheral blood lymphocytes (PBL) isolated from healthy humans or in vivo in rats PBL. Human PBL were treated with physiologically relevant NO donor - S-Nitrosoglutathione and X-ray irradiation. The inhalation treatment of animals with NO was carried out in chamber with purified gaseous NO mixed inside with air. Levels of S-Nitrosohemoglobin and methemoglobin in the blood were assessed with electron paramagnetic resonance. The total level of reactive oxygen and nitrogen species in PBL was determined fluorometrically, and serum levels of reactive oxygen species was determined by spectrophotometric assay. DNA damages were assessed by alkaline single-cell gel electrophoresis. The frequency of chromosomal aberrations in human PBL measured with the conventional cytogenetic assay in metaphase cells on short-term (52 h) and long-term (72 h) cultures. Environmental nitrogen oxides or release of NO from stable complexes with biomolecules (such as S-Nitrosothiols) intensified generation of free radicals, DNA damage and development of genomic instability alone or in combination with ionizing radiation. Treatment of PBL by S-Nitrosoglutathione caused prevalent induction of chromatid type but irradiation - chromosome aberrations. The dose dependence of chromatid-type aberrations observed in human PBL after combined influence of S-Nitrosoglutathione and ionizing radiation indicates a crucial role of NO in the formation of chromosomal instability. NO can deregulate free radicals balance resulted in genotoxic effect, posttranslational modification of repair enzymes and thus coordinated development of genomic instability and increase of cancer risk.

  17. 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...

  18. Influence of acute and chronic administration of methadone hydrochloride on NADPH-cytochrome c reductase and cytochrome P-450 of mouse liver microsomes.

    Science.gov (United States)

    Datta, R K; Johnson, E A; Bhattacharjee, G; Stenger, R J

    1976-03-01

    Administration of a single acute dose (20 mg/kg body weight) of methadone hydrochloride to both male and female mice increased the specific activity of NADPH-cytochrome c reductase and did not change much the content of cytochrome P-450 of their liver microsomes. Administration of multiple acute doses of methadone in male mice increased the specific activity of cytochrome c reductase and the content of cytochrome P-450 of their liver microsomes. Chronic administration of progressively increasing doses of methadone (up to 40 mg/kg body weight) to male mice increased the specific activity of c reductase. Similar chronic administration of methadone up to 28 mg/kg body weight also increased the microsomal content of P-450, but with higher doses of methadone, the content of P-450 declined and finally dropped slightly below control levels. The levels of c reductase activity and P-450 content returned to normal about two weeks after discontinuation of methadone administration.

  19. 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.

  20. Variation and inheritance of iron reductase activity in the roots of common bean (Phaseolus vulgaris L.) and association with seed iron accumulation QTL.

    Science.gov (United States)

    Blair, Matthew W; Knewtson, Sharon Jb; Astudillo, Carolina; Li, Chee-Ming; Fernandez, Andrea C; Grusak, Michael A

    2010-10-05

    Iron deficiency anemia is a global problem which often affects women and children of developing countries. Strategy I plants, such as common bean (Phaseolus vulgaris L.) take up iron through a process that involves an iron reduction mechanism in their roots; this reduction is required to convert ferric iron to ferrous iron. Root absorbed iron is critical for the iron nutrition of the plant, and for the delivery of iron to the shoot and ultimately the seeds. The objectives of this study were to determine the variability and inheritance for iron reductase activity in a range of genotypes and in a low × high seed iron cross (DOR364 x G19833), to identify quantitative trait loci (QTL) for this trait, and to assess possible associations with seed iron levels. The experiments were carried out with hydroponically grown plants provided different amounts of iron varying between 0 and 20 μM Fe(III)-EDDHA. The parents, DOR364 and G19833, plus 13 other cultivated or wild beans, were found to differ in iron reductase activity. Based on these initial experiments, two growth conditions (iron limited and iron sufficient) were selected as treatments for evaluating the DOR364 × G19833 recombinant inbred lines. A single major QTL was found for iron reductase activity under iron-limited conditions (1 μM Fe) on linkage group b02 and another major QTL was found under iron sufficient conditions (15 μM Fe) on linkage group b11. Associations between the b11 QTL were found with several QTL for seed iron. Genes conditioning iron reductase activity in iron sufficient bean plants appear to be associated with genes contributing to seed iron accumulation. Markers for bean iron reductase (FRO) homologues were found with in silico mapping based on common bean synteny with soybean and Medicago truncatula on b06 and b07; however, neither locus aligned with the QTL for iron reductase activity. In summary, the QTL for iron reductase activity under iron limited conditions may be useful in

  1. Variation and inheritance of iron reductase activity in the roots of common bean (Phaseolus vulgaris L. and association with seed iron accumulation QTL

    Directory of Open Access Journals (Sweden)

    Fernandez Andrea C

    2010-10-01

    Full Text Available Abstract Background Iron deficiency anemia is a global problem which often affects women and children of developing countries. Strategy I plants, such as common bean (Phaseolus vulgaris L. take up iron through a process that involves an iron reduction mechanism in their roots; this reduction is required to convert ferric iron to ferrous iron. Root absorbed iron is critical for the iron nutrition of the plant, and for the delivery of iron to the shoot and ultimately the seeds. The objectives of this study were to determine the variability and inheritance for iron reductase activity in a range of genotypes and in a low × high seed iron cross (DOR364 × G19833, to identify quantitative trait loci (QTL for this trait, and to assess possible associations with seed iron levels. Results The experiments were carried out with hydroponically grown plants provided different amounts of iron varying between 0 and 20 μM Fe(III-EDDHA. The parents, DOR364 and G19833, plus 13 other cultivated or wild beans, were found to differ in iron reductase activity. Based on these initial experiments, two growth conditions (iron limited and iron sufficient were selected as treatments for evaluating the DOR364 × G19833 recombinant inbred lines. A single major QTL was found for iron reductase activity under iron-limited conditions (1 μM Fe on linkage group b02 and another major QTL was found under iron sufficient conditions (15 μM Fe on linkage group b11. Associations between the b11 QTL were found with several QTL for seed iron. Conclusions Genes conditioning iron reductase activity in iron sufficient bean plants appear to be associated with genes contributing to seed iron accumulation. Markers for bean iron reductase (FRO homologues were found with in silico mapping based on common bean synteny with soybean and Medicago truncatula on b06 and b07; however, neither locus aligned with the QTL for iron reductase activity. In summary, the QTL for iron reductase activity

  2. Resolution of two native monomeric 90 kDa nitrate reductase active proteins from Shewanella gelidimarina and the sequence of two napA genes

    International Nuclear Information System (INIS)

    Simpson, Philippa J.L.; McKinzie, Audra A.; Codd, Rachel

    2010-01-01

    Research highlights: → Two monomeric 90 kDa nitrate reductase active proteins from Shewanella gelidimarina. → Sequence of napA from napEDABC-type operon and napA from NapDAGHB-type operon. → Isolation of NAP as NapA or NapAB correlated with NapA P47E amino acid substitution. -- Abstract: The reduction of nitrate to nitrite in the bacterial periplasm occurs in the 90 kDa NapA subunit of the periplasmic nitrate reductase (NAP) system. Most Shewanella genomes contain two nap operons: napEDABC and napDAGHB, which is an unusual feature of this genus. Two native, monomeric, 90 kDa nitrate reductase active proteins were resolved by hydrophobic interaction chromatography from aerobic cultures of Shewanella gelidimarina replete with reduced nitrogen compounds. The 90 kDa protein obtained in higher yield was characterized as NapA by electronic absorption and electron paramagnetic resonance spectroscopies and was identified by LC/MS/MS and MALDI-TOF/TOF MS as NapA from the napEDABC-type operon. The other 90 kDa protein, which was unstable and produced in low yields, was posited as NapA from the napDAGHB-type operon. Two napA genes have been sequenced from the napEDABC-type and napDAGHB-type operons of S. gelidimarina. Native NAP from S. putrefaciens was resolved as one NapA monomer and one NapAB heterodimer. Two amino acid substitutions in NapA correlated with the isolation of NAP as a NapA monomer or a NapAB heterodimer. The resolution of native, redox-active NapA isoforms in Shewanella provides new insight into the respiratory versatility of this genus, which has implications in bioremediation and the assembly of microbial fuel cells.

  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. 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.

  5. Fatty acyl-CoA reductases of birds

    Science.gov (United States)

    2011-01-01

    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. PMID:22151413

  6. Cold adaptation of the mononuclear molybdoenzyme periplasmic nitrate reductase from the Antarctic bacterium Shewanella gelidimarina

    Energy Technology Data Exchange (ETDEWEB)

    Simpson, Philippa J.L. [School of Chemistry, University of Sydney, New South Wales 2006 (Australia); Codd, Rachel, E-mail: rachel.codd@sydney.edu.au [School of Chemistry, University of Sydney, New South Wales 2006 (Australia); School of Medical Sciences (Pharmacology) and Bosch Institute, University of New South Wales, New South Wales 2006 (Australia)

    2011-11-04

    Highlights: Black-Right-Pointing-Pointer Cold-adapted phenotype of NapA from the Antarctic bacterium Shewanella gelidimarina. Black-Right-Pointing-Pointer Protein homology model of NapA from S. gelidimarina and mesophilic homologue. Black-Right-Pointing-Pointer Six amino acid residues identified as lead candidates governing NapA cold adaptation. Black-Right-Pointing-Pointer Molecular-level understanding of designing cool-temperature in situ oxyanion sensors. -- Abstract: The reduction of nitrate to nitrite is catalysed in bacteria by periplasmic nitrate reductase (Nap) which describes a system of variable protein subunits encoded by the nap operon. Nitrate reduction occurs in the NapA subunit, which contains a bis-molybdopterin guanine dinucleotide (Mo-MGD) cofactor and one [4Fe-4S] iron-sulfur cluster. The activity of periplasmic nitrate reductase (Nap) isolated as native protein from the cold-adapted (psychrophilic) Antarctic bacterium Shewanella gelidimarina (Nap{sub Sgel}) and middle-temperature adapted (mesophilic) Shewanella putrefaciens (Nap{sub Sput}) was examined at varied temperature. Irreversible deactivation of Nap{sub Sgel} and Nap{sub Sput} occurred at 54.5 and 65 Degree-Sign C, respectively. When Nap{sub Sgel} was preincubated at 21-70 Degree-Sign C for 30 min, the room-temperature nitrate reductase activity was maximal and invariant between 21 and 54 Degree-Sign C, which suggested that Nap{sub Sgel} was poised for optimal catalysis at modest temperatures and, unlike Nap{sub Sput}, did not benefit from thermally-induced refolding. At 20 Degree-Sign C, Nap{sub Sgel} reduced selenate at 16% of the rate of nitrate reduction. Nap{sub Sput} did not reduce selenate. Sequence alignment showed 46 amino acid residue substitutions in Nap{sub Sgel} that were conserved in NapA from mesophilic Shewanella, Rhodobacter and Escherichia species and could be associated with the Nap{sub Sgel} cold-adapted phenotype. Protein homology modeling of Nap{sub Sgel} using a

  7. Cold adaptation of the mononuclear molybdoenzyme periplasmic nitrate reductase from the Antarctic bacterium Shewanella gelidimarina

    International Nuclear Information System (INIS)

    Simpson, Philippa J.L.; Codd, Rachel

    2011-01-01

    Highlights: ► Cold-adapted phenotype of NapA from the Antarctic bacterium Shewanella gelidimarina. ► Protein homology model of NapA from S. gelidimarina and mesophilic homologue. ► Six amino acid residues identified as lead candidates governing NapA cold adaptation. ► Molecular-level understanding of designing cool-temperature in situ oxyanion sensors. -- Abstract: The reduction of nitrate to nitrite is catalysed in bacteria by periplasmic nitrate reductase (Nap) which describes a system of variable protein subunits encoded by the nap operon. Nitrate reduction occurs in the NapA subunit, which contains a bis-molybdopterin guanine dinucleotide (Mo–MGD) cofactor and one [4Fe–4S] iron–sulfur cluster. The activity of periplasmic nitrate reductase (Nap) isolated as native protein from the cold-adapted (psychrophilic) Antarctic bacterium Shewanella gelidimarina (Nap Sgel ) and middle-temperature adapted (mesophilic) Shewanella putrefaciens (Nap Sput ) was examined at varied temperature. Irreversible deactivation of Nap Sgel and Nap Sput occurred at 54.5 and 65 °C, respectively. When Nap Sgel was preincubated at 21–70 °C for 30 min, the room-temperature nitrate reductase activity was maximal and invariant between 21 and 54 °C, which suggested that Nap Sgel was poised for optimal catalysis at modest temperatures and, unlike Nap Sput , did not benefit from thermally-induced refolding. At 20 °C, Nap Sgel reduced selenate at 16% of the rate of nitrate reduction. Nap Sput did not reduce selenate. Sequence alignment showed 46 amino acid residue substitutions in Nap Sgel that were conserved in NapA from mesophilic Shewanella, Rhodobacter and Escherichia species and could be associated with the Nap Sgel cold-adapted phenotype. Protein homology modeling of Nap Sgel using a mesophilic template with 66% amino acid identity showed the majority of substitutions occurred at the protein surface distal to the Mo–MGD cofactor. Two mesophilic ↔ psychrophilic

  8. Use of 5-alpha-reductase inhibitors did not increase the risk of cardiovascular diseases in patients with benign prostate hyperplasia: a five-year follow-up study.

    Directory of Open Access Journals (Sweden)

    Teng-Fu Hsieh

    Full Text Available This nationwide population-based study investigated the risk of cardiovascular diseases after 5-alpha-reductase inhibitor therapy for benign prostate hyperplasia (BPH using the National Health Insurance Research Database (NHIRD in Taiwan.In total, 1,486 adult patients newly diagnosed with BPH and who used 5-alpha-reductase inhibitors were recruited as the study cohort, along with 9,995 subjects who did not use 5-alpha-reductase inhibitors as a comparison cohort from 2003 to 2008. Each patient was monitored for 5 years, and those who subsequently had cardiovascular diseases were identified. A Cox proportional hazards model was used to compare the risk of cardiovascular diseases between the study and comparison cohorts after adjusting for possible confounding risk factors.The patients who received 5-alpha-reductase inhibitor therapy had a lower cumulative rate of cardiovascular diseases than those who did not receive 5-alpha-reductase inhibitor therapy during the 5-year follow-up period (8.4% vs. 11.2%, P=0.003. In subgroup analysis, the 5-year cardiovascular event hazard ratio (HR was lower among the patients older than 65 years with 91 to 365 cumulative defined daily dose (cDDD 5-alpha-reductase inhibitor use (HR=0.63, 95% confidence interval (CI 0.42 to 0.92; P=0.018, however there was no difference among the patients with 28 to 90 and more than 365 cDDD 5-alpha-reductase inhibitor use (HR=1.14, 95% CI 0.77 to 1.68; P=0.518 and HR=0.83, 95% CI 0.57 to 1.20; P=0.310, respectively.5-alpha-reductase inhibitor therapy did not increase the risk of cardiovascular events in the BPH patients in 5 years of follow-up. Further mechanistic research is needed.

  9. A SAM-dependent methyltransferase cotranscribed with arsenate reductase alters resistance to peptidyl transferase center-binding antibiotics in Azospirillum brasilense Sp7.

    Science.gov (United States)

    Singh, Sudhir; Singh, Chhaya; Tripathi, Anil Kumar

    2014-05-01

    The genome of Azospirillum brasilense harbors a gene encoding S-adenosylmethionine-dependent methyltransferase, which is located downstream of an arsenate reductase gene. Both genes are cotranscribed and translationally coupled. When they were cloned and expressed individually in an arsenate-sensitive strain of Escherichia coli, arsenate reductase conferred tolerance to arsenate; however, methyltransferase failed to do so. Sequence analysis revealed that methyltransferase was more closely related to a PrmB-type N5-glutamine methyltransferase than to the arsenate detoxifying methyltransferase ArsM. Insertional inactivation of prmB gene in A. brasilense resulted in an increased sensitivity to chloramphenicol and resistance to tiamulin and clindamycin, which are known to bind at the peptidyl transferase center (PTC) in the ribosome. These observations suggested that the inability of prmB:km mutant to methylate L3 protein might alter hydrophobicity in the antibiotic-binding pocket of the PTC, which might affect the binding of chloramphenicol, clindamycin, and tiamulin differentially. This is the first report showing the role of PrmB-type N5-glutamine methyltransferases in conferring resistance to tiamulin and clindamycin in any bacterium.

  10. Crystallization and preliminary crystallographic analysis of selenomethionine-labelled progesterone 5β-reductase from Digitalis lanata Ehrh

    Energy Technology Data Exchange (ETDEWEB)

    Egerer-Sieber, Claudia [Lehrstuhl für Biotechnik, Institut für Biologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Henkestrasse 91, D-91052 Erlangen (Germany); Herl, Vanessa; Müller-Uri, Frieder; Kreis, Wolfgang [Lehrstuhl für Pharmazeutische Biologie, Institut für Biologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Staudtstrasse 5, D-91058 Erlangen (Germany); Muller, Yves A., E-mail: ymuller@biologie.uni-erlangen.de [Lehrstuhl für Biotechnik, Institut für Biologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Henkestrasse 91, D-91052 Erlangen (Germany)

    2006-03-01

    Progesterone 5β-reductase is the first stereospecific enzyme in the pathway for the synthesis of cardenolides. To elucidate the structural mechanism of this reaction, we crystallized the selenomethionine-labelled enzyme from D. lanata and report the preliminary analysis of a MAD data set collected from these crystals. Progesterone 5β-reductase (5β-POR) catalyzes the reduction of progesterone to 5β-pregnane-3,20-dione and is the first stereospecific enzyme in the putative biosynthetic pathway of Digitalis cardenolides. Selenomethionine-derivatized 5β-POR from D. lanata was successfully overproduced and crystallized. The crystals belong to space group P4{sub 3}2{sub 1}2, with unit-cell parameters a = 71.73, c = 186.64 Å. A MAD data set collected at 2.7 Å resolution allowed the identification of six out of eight possible Se-atom positions. A first inspection of the MAD-phased electron-density map shows that 5β-POR is a Rossmann-type reductase and the quality of the map is such that it is anticipated that a complete atomic model of 5β-POR will readily be built.

  11. Brevetoxin-2, is a unique inhibitor of the C-terminal redox center of mammalian thioredoxin reductase-1.

    Science.gov (United States)

    Chen, Wei; Tuladhar, Anupama; Rolle, Shantelle; Lai, Yanhao; Rodriguez Del Rey, Freddy; Zavala, Cristian E; Liu, Yuan; Rein, Kathleen S

    2017-08-15

    Karenia brevis, the Florida red tide dinoflagellate produces a suite of neurotoxins known as the brevetoxins. The most abundant of the brevetoxins PbTx-2, was found to inhibit the thioredoxin-thioredoxin reductase system, whereas the PbTx-3 has no effect on this system. On the other hand, PbTx-2 activates the reduction of small disulfides such as 5,5'-dithio-bis-(2-nitrobenzoic acid) by thioredoxin reductase. PbTx-2 has an α, β-unsaturated aldehyde moiety which functions as an efficient electrophile and selenocysteine conjugates are readily formed. PbTx-2 blocks the inhibition of TrxR by the inhibitor curcumin, whereas curcumin blocks PbTx-2 activation of TrxR. It is proposed that the mechanism of inhibition of thioredoxin reduction is via the formation of a Michael adduct between selenocysteine and the α, β-unsaturated aldehyde moiety of PbTx-2. PbTx-2 had no effect on the rates of reactions catalyzed by related enzymes such as glutathione reductase, glutathione peroxidase or glutaredoxin. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. The 5 Alpha-Reductase Isozyme Family: A Review of Basic Biology and Their Role in Human Diseases

    Directory of Open Access Journals (Sweden)

    Faris Azzouni

    2012-01-01

    Full Text Available Despite the discovery of 5 alpha-reduction as an enzymatic step in steroid metabolism in 1951, and the discovery that dihydrotestosterone is more potent than testosterone in 1968, the significance of 5 alpha-reduced steroids in human diseases was not appreciated until the discovery of 5 alpha-reductase type 2 deficiency in 1974. Affected males are born with ambiguous external genitalia, despite normal internal genitalia. The prostate is hypoplastic, nonpalpable on rectal examination and approximately 1/10th the size of age-matched normal glands. Benign prostate hyperplasia or prostate cancer does not develop in these patients. At puberty, the external genitalia virilize partially, however, secondary sexual hair remains sparse and male pattern baldness and acne develop rarely. Several compounds have been developed to inhibit the 5 alpha-reductase isozymes and they play an important role in the prevention and treatment of many common diseases. This review describes the basic biochemical properties, functions, tissue distribution, chromosomal location, and clinical significance of the 5 alpha-reductase isozyme family.

  13. Crystallization and preliminary characterization of dihydropteridine reductase from Dictyostelium discoideum

    International Nuclear Information System (INIS)

    Chen, Cong; Seo, Kyung Hye; Kim, Hye Lim; Zhuang, Ningning; Park, Young Shik; Lee, Kon Ho

    2008-01-01

    The dihydropteridine reductase from D. discoideum has been crystallized. Diffraction data were collected from a rectangular-shaped crystal to 2.16 Å resolution. Dihydropteridine reductase from Dictyostelium discoideum (dicDHPR) can produce d-threo-BH 4 [6R-(1′R,2′R)-5,6,7,8-tetrahydrobiopterin], a stereoisomer of l-erythro-BH 4 , in the last step of tetrahydrobiopterin (BH 4 ) recycling. In this reaction, DHPR uses NADH as a cofactor to reduce quinonoid dihydrobiopterin back to BH 4 . To date, the enzyme has been purified to homogeneity from many sources. In this report, the dicDHPR–NAD complex has been crystallized using the hanging-drop vapour-diffusion method with PEG 3350 as a precipitant. Rectangular-shaped crystals were obtained. Crystals grew to maximum dimensions of 0.4 × 0.6 × 0.1 mm. The crystal belonged to space group P2 1 , with unit-cell parameters a = 49.81, b = 129.90, c = 78.76 Å, β = 100.00°, and contained four molecules in the asymmetric unit, forming two closely interacting dicDHPR–NAD dimers. Diffraction data were collected to 2.16 Å resolution using synchrotron radiation. The crystal structure has been determined using the molecular-replacement method

  14. Inhibition of NADH-ubiquinone reductase activity by N,N'-dicyclohexylcarbodiimide and correlation of this inhibition with the occurrence of energy-coupling site 1 in various organisms

    International Nuclear Information System (INIS)

    Yagi, T.

    1987-01-01

    The NADH-ubiquinone reductase activity of the respiratory chains of several organisms was inhibited by the carboxyl-modifying reagent N,N'-dicyclohexylcarbodiimide (DCCD). This inhibition correlated with the presence of an energy-transducing site in this segment of the respiratory chain. Where the NADH-quinone reductase segment involved an energy-coupling site (e.g., in bovine heart and rat liver mitochondria, and in Paracoccus denitrificans, Escherichia coli, and Thermus thermophilus HB-8 membranes), DCCD acted as an inhibitor of ubiquinone reduction by NADH. By contrast, where energy-coupling site 1 was absent (e.g., in Saccharomyces cerevisiae mitochondria and BacilLus subtilis membranes), there was no inhibition of NADH-ubiquinone reductase activity by DCCD. In the bovine and P. denitrificans systems, DCCD inhibition was pseudo first order with respect to incubation time, and reaction order with respect to inhibitor concentration was close to unity, indicating that inhibition resulted from the binding of one inhibitor molecule per active unit of NADH-ubiquinone reductase. In the bovine NADH-ubiquinone reductase complex (complex I), [ 14 C]DCCD was preferentially incorporated into two subunits of molecular weight 49,000 and 29,000. The time course of labeling of the 29,000 molecular weight subunit with [ 14 C]DCCD paralleled the time course of inhibition of NADH-ubiquinone reductase activity

  15. Expression analysis of dihydroflavonol 4-reductase genes in Petunia hybrida.

    Science.gov (United States)

    Chu, Y X; Chen, H R; Wu, A Z; Cai, R; Pan, J S

    2015-05-12

    Dihydroflavonol 4-reductase (DFR) genes from Rosa chinensis (Asn type) and Calibrachoa hybrida (Asp type), driven by a CaMV 35S promoter, were integrated into the petunia (Petunia hybrida) cultivar 9702. Exogenous DFR gene expression characteristics were similar to flower-color changes, and effects on anthocyanin concentration were observed in both types of DFR gene transformants. Expression analysis showed that exogenous DFR genes were expressed in all of the tissues, but the expression levels were significantly different. However, both of them exhibited a high expression level in petals that were starting to open. The introgression of DFR genes may significantly change DFR enzyme activity. Anthocyanin ultra-performance liquid chromatography results showed that anthocyanin concentrations changed according to DFR enzyme activity. Therefore, the change in flower color was probably the result of a DFR enzyme change. Pelargonidin 3-O-glucoside was found in two different transgenic petunias, indicating that both CaDFR and RoDFR could catalyze dihydrokaempferol. Our results also suggest that transgenic petunias with DFR gene of Asp type could biosynthesize pelargonidin 3-O-glucoside.

  16. Inhibition of thioredoxin reductase but not of glutathione reductase by the major classes of alkylating and platinum-containing anticancer compounds.

    Science.gov (United States)

    Witte, Anne-Barbara; Anestål, Karin; Jerremalm, Elin; Ehrsson, Hans; Arnér, Elias S J

    2005-09-01

    Mammalian thioredoxin reductase (TrxR) is important for cell proliferation, antioxidant defense, and redox signaling. Together with glutathione reductase (GR) it is the main enzyme providing reducing equivalents to many cellular processes. GR and TrxR are flavoproteins of the same enzyme family, but only the latter is a selenoprotein. With the active site containing selenocysteine, TrxR may catalyze reduction of a wide range of substrates, but can at the same time easily be targeted by electrophilic compounds due to the extraordinarily high reactivity of a selenolate moiety. Here we addressed the inhibition of the enzyme by major anticancer alkylating agents and platinum-containing compounds and we compared it to that of GR. We confirmed prior studies suggesting that the nitrosourea carmustine can inhibit both GR and TrxR. We next found, however, that nitrogen mustards (chlorambucil and melphalan) and alkyl sulfonates (busulfan) efficiently inhibited TrxR while these compounds, surprisingly, did not inhibit GR. Inhibitions were concentration and time dependent and apparently irreversible. Anticancer anthracyclines (daunorubicin and doxorubicin) were, in contrast to the alkylating agents, not inhibitors but poor substrates of TrxR. We also found that TrxR, but not GR, was efficiently inhibited by both cisplatin, its monohydrated complex, and oxaliplatin. Carboplatin, in contrast, could not inhibit any of the two enzymes. These findings lead us to conclude that representative compounds of the major classes of clinically used anticancer alkylating agents and most platinum compounds may easily target TrxR, but not GR. The TrxR inhibition should thereby be considered as a factor that may contribute to the cytotoxicity seen upon clinical use of these drugs.

  17. Interactions of nitric oxide with α2 -adrenoceptors within the locus coeruleus underlie the facilitation of inhibitory avoidance memory by agmatine.

    Science.gov (United States)

    Shelkar, Gajanan P; Gakare, Sukanya G; Chakraborty, Suwarna; Dravid, Shashank M; Ugale, Rajesh R

    2016-09-01

    Agmatine, a putative neurotransmitter, plays a vital role in learning and memory. Although it is considered an endogenous ligand of imidazoline receptors, agmatine exhibits high affinity for α-adrenoceptors, NOS and NMDA receptors. These substrates within the locus coeruleus (LC) are critically involved in learning and memory processes. The hippocampus and LC of male Wistar rat were stereotaxically cannulated for injection. Effects of agmatine, given i.p. or intra-LC, on acquisition, consolidation and retrieval of inhibitory avoidance (IA) memory were measured. The NO donor S-nitrosoglutathione, non-specific (L-NAME) and specific NOS inhibitors (L-NIL, 7-NI, L-NIO), the α2 -adrenoceptor antagonist (yohimbine) or the corresponding agonist (clonidine) were injected intra-LC before agmatine. Intra-hippocampal injections of the NMDA antagonist, MK-801 (dizocilpine), were used to modify the memory enhancing effects of agmatine, SNG and yohimbine. Expression of tyrosine hydroxylase (TH) and eNOS in the LC was assessed immunohistochemically. Agmatine (intra-LC or i.p.) facilitated memory retrieval in the IA test. S-nitrosoglutathione potentiated, while L-NAME and L-NIO decreased, these effects of agmatine. L-NIL and 7-NI did not alter the effects of agmatine. Yohimbine potentiated, whereas clonidine attenuated, effects of agmatine within the LC. The effects of agmatine, S-nitrosoglutathione and yohimbine were blocked by intra-hippocampal MK-801. Agmatine increased the population of TH- and eNOS-immunoreactive elements in the LC. The facilitation of memory retrieval in the IA test by agmatine is probably mediated by interactions between eNOS, NO and noradrenergic pathways in the LC. © 2016 The British Pharmacological Society.

  18. The role of quinone reductase (NQO1) and quinone chemistry in quercetin cytotoxicity

    NARCIS (Netherlands)

    Gliszczynska-Swiglo, A.; Woude, van der H.; Haan, de L.H.J.; Tyrakowska, B.; Aarts, J.M.M.J.G.; Rietjens, I.M.C.M.

    2003-01-01

    The effects of quercetin on viability and proliferation of Chinese Hamster Ovary (CHO) cells and CHO cells overexpressing human quinone reductase (CHO+NQO1) were studied to investigate the involvement of the pro-oxidant quinone chemistry of quercetin. The toxicity of menadione was significantly

  19. Positive correlation between decreased cellular uptake, NADPH-glutathione reductase activity and adriamycin resistance in Ehrlich ascites tumor lines.

    Science.gov (United States)

    Scheulen, M E; Hoensch, H; Kappus, H; Seeber, S; Schmidt, C G

    1987-01-01

    From a wild type strain of Ehrlich ascites tumor (EATWT) sublines resistant to daunorubicin (EATDNM), etoposide (EATETO), and cisplatinum (EATCIS) have been developed in vivo. Increase in survival and cure rate caused by adriamycin (doxorubicin) have been determined in female NMRI mice which were inoculated i.p. with EAT cells. Adriamycin concentrations causing 50% inhibition of 3H-thymidine (ICT) and 3H-uridine incorporation (ICU) and intracellular adriamycin steady-state concentrations (SSC) were measured in vitro. Adriamycin resistance increased and SSC decreased in the following sequence: EATWT - EATCIS - EATDNM - EATETO. When ICT and ICU were corrected for intracellular adriamycin concentrations in consideration of the different SSC (ICTc, ICUc), ICTc and ICUc still varied up to the 3.2 fold in EATCIS, EATDNM and EATETO in comparison to EATWT. Thus, in addition to different SSC other factors must be responsible for adriamycin resistance. Therefore, enzymes which may play a role in the cytotoxicity related to adriamycin metabolism (NADPH-cytochrome P-450 reductase, NADPH-glutathione reductase, NADP-glucose-6-phosphate dehydrogenase, NADP-isocitrate dehydrogenase) were measured. In contrast to the other parameters determined, NADPH-glutathione reductase was significantly (p less than 0.01) increased up to the 3.2 fold parallel to adriamycin resistance as determined by increase in life span, cure rate, ICTc, and ICUc, respectively. It is concluded that high activities of NADPH-glutathione reductase may contribute to an increase in adriamycin resistance of malignant tumors.

  20. 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.

  1. Deoxynucleoside Salvage in Fission Yeast Allows Rescue of Ribonucleotide Reductase Deficiency but Not Spd1-Mediated Inhibition of Replication

    Directory of Open Access Journals (Sweden)

    Oliver Fleck

    2017-04-01

    Full Text Available 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 CRL4Cdt2 E3 ubiquitin ligase mediates degradation of Spd1 and the related protein Spd2 at S phase of the cell cycle. We have generated a conditional allele of CRL4Cdt2, by expressing the highly unstable substrate-recruiting protein Cdt2 from a repressible promoter. Unlike Spd1, Spd2 does not regulate deoxynucleotide 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 transporter human equilibrative nucleoside transporter 1 (hENT1 and the Drosophila deoxynucleoside kinase. We present evidence that this salvage pathway is functional, as 2 µM of deoxynucleosides in the culture medium is able to rescue the growth of two different temperature-sensitive alleles controlling ribonucleotide reductase. However, salvage completely failed to rescue S phase delay, checkpoint activation, and damage sensitivity, which was caused by CRL4Cdt2 inactivation, suggesting that Spd1—in addition to repressing dNTP synthesis—together with Spd2, can inhibit other replication functions. We propose that this inhibition works at the point of the replication clamp proliferating cell nuclear antigen, a co-factor for DNA replication.

  2. Community Structure of Denitrifiers, Bacteria, and Archaea along Redox Gradients in Pacific Northwest Marine Sediments by Terminal Restriction Fragment Length Polymorphism Analysis of Amplified Nitrite Reductase (nirS) and 16S rRNA Genes

    Science.gov (United States)

    Braker, Gesche; Ayala-del-Río, Héctor L.; Devol, Allan H.; Fesefeldt, Andreas; Tiedje, James M.

    2001-01-01

    Steep vertical gradients of oxidants (O2 and NO3−) in Puget Sound and Washington continental margin sediments indicate that aerobic respiration and denitrification occur within the top few millimeters to centimeters. To systematically explore the underlying communities of denitrifiers, Bacteria, and Archaea along redox gradients at distant geographic locations, nitrite reductase (nirS) genes and bacterial and archaeal 16S rRNA genes (rDNAs) were PCR amplified and analyzed by terminal restriction fragment length polymorphism (T-RFLP) analysis. The suitablility of T-RFLP analysis for investigating communities of nirS-containing denitrifiers was established by the correspondence of dominant terminal restriction fragments (T-RFs) of nirS to computer-simulated T-RFs of nirS clones. These clones belonged to clusters II, III, and IV from the same cores and were analyzed in a previous study (G. Braker, J. Zhou, L. Wu, A. H. Devol, and J. M. Tiedje, Appl. Environ. Microbiol. 66:2096–2104, 2000). T-RFLP analysis of nirS and bacterial rDNA revealed a high level of functional and phylogenetic diversity, whereas the level of diversity of Archaea was lower. A comparison of T-RFLPs based on the presence or absence of T-RFs and correspondence analysis based on the frequencies and heights of T-RFs allowed us to group sediment samples according to the sampling location and thus clearly distinguish Puget Sound and the Washington margin populations. However, changes in community structure within sediment core sections during the transition from aerobic to anaerobic conditions were minor. Thus, within the top layers of marine sediments, redox gradients seem to result from the differential metabolic activities of populations of similar communities, probably through mixing by marine invertebrates rather than from the development of distinct communities. PMID:11282647

  3. The Effects of Dose Rhizoctonia Binucleat (BNR and Phosphorus to Nitrate Reductase Activity (NRA and Chlorophyll of Vanilla Seedling (Vanilla planifolia Andrews

    Directory of Open Access Journals (Sweden)

    Haryuni Haryuni

    2016-09-01

    Full Text Available Vanilla (Vanilla planifolia Andrews is one of the important exported commodities in Indonesia. Indonesia is one of top five major vanilla exporters in the world, that produce the high quality of Indonesian vanilla with high vanillin content (2.75%. The aims of this research were to determine the effects of dose binukleat Rhizoctonia (BNR and phosphorus as well as the interaction of the nitrate reductase activity (NRA and chlorophyll of the vanilla seedling (Vanilla planifolia Andrew. Method in this research used completely randomized factorial design, by involving two factors (dose of BNR inoculation and Phosphor. The first factor is without inoculation and inoculation BNR (M0, M1, M2, M3 wich consists of (0,5, 10, 15 g/polybag, the second factor is the dose of phosphorus fertilizer (P0, P1, P2, P3 which consists of (0, 3, 6, 9 g/polibag. The results showed that the inoculation dose of BNR and doses of phosphorus not significant and lower levels of NRA and chlorophyll while the interaction dose of BNR and phosphorus significantly and increase levels of NRA and chlorophyll of vanilla seedling. Nitrate Reductase Activity and chlorophyll has important role in metabolism process as a plant growth indicator.How to CiteHaryuni, H., & Dewi, T. S. K. (2016. The Effects of Dose Rhizoctonia Binucleat (BNR and Phosphorus to Nitrate Reductase Activity (NRA and Chlorophyll of Vanilla Seedling (Vanilla planifolia Andrews. Biosaintifika: Journal of Biology & Biology Education, 8(2, 141-147.

  4. Thermophilic enzymes and their applications in biocatalysis: a robust aldo-keto reductase.

    Science.gov (United States)

    Willies, Simon; Isupov, Misha; Littlechild, Jennifer

    2010-09-01

    Extremophiles are providing a good source of novel robust enzymes for use in biocatalysis for the synthesis of new drugs. This is particularly true for the enzymes from thermophilic organisms which are more robust than their mesophilic counterparts to the conditions required for industrial bio-processes. This paper describes a new aldo-keto reductase enzyme from a thermophilic eubacteria, Thermotoga maritima which can be used for the production of primary alcohols. The enzyme has been cloned and over-expressed in Escherichia coli and has been purified and subjected to full biochemical characterization. The aldo-keto reductase can be used for production of primary alcohols using substrates including benzaldehyde, 1,2,3,6-tetrahydrobenzaldehyde and para-anisaldehyde. It is stable up to 80 degrees C, retaining over 60% activity for 5 hours at this temperature. The enzyme at pH 6.5 showed a preference for the forward, carbonyl reduction. The enzyme showed moderate stability with organic solvents, and retained 70% activity in 20% (v/v) isopropanol or DMSO. These properties are favourable for its potential industrial applications.

  5. 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.

  6. Molecular cloning and characterization of Fasciola gigantica thioredoxin-glutathione reductase.

    Science.gov (United States)

    Changklungmoa, Narin; Kueakhai, Pornanan; Sangpairoj, Kant; Chaichanasak, Pannigan; Jaikua, Wipaphorn; Riengrojpitak, Suda; Sobhon, Prasert; Chaithirayanon, Kulathida

    2015-06-01

    The Fasciola gigantica thioredoxin-glutathione reductase (FgTGR) gene is a fusion between thioredoxin reductase (TR) and a glutaredoxin (Grx) gene. FgTGR was cloned by polymerase chain reaction (PCR) from adult complementary DNA (cDNA), and its sequences showed two isoforms, i.e., the cytosolic and mitochondrial FgTGR. Cytosolic FgTGR (cytFgTGR) was composed of 2370 bp, and its peptide had no signal sequence and hence was not a secreted protein. Mitochondrial FgTGR (mitFgTGR) was composed of 2506 bp with a signal peptide of 43 amino acids; therefore, it was a secreted protein. The putative cytFgTGR and mitFgTGR peptides comprised of 598 and 641 amino acids, respectively, with a molecular weight of 65.8 kDa for cytFgTGR and mitFgTGR, with a conserved sequence (CPYC) of TR, and ACUG and CVNVGC of Grx domains. The recombinant FgTGR (rFgTGR) was expressed in Escherichia coli BL21 (DE3) and used for production for a polyclonal antibody in rabbits (anti-rFgTGR). The FgTGR protein expression, estimated by indirect ELISA using the rabbit anti-rFgTGR as probe, showed high levels of expression in eggs, and 2- and 4-week-old juveniles and adults. The rFgTGR exhibited specific activities in the 5,5'-dithiobis (2-nitro-benzoic acid) (DTNB) reductase assay for TR activity and in β-hydroxyethul disulfide (HED) for Grx activity. When analyzed by immunoblotting and immunohistochemistry, rabbit anti-rFgTGR reacted with natural FgTGR at a molecular weight of 66 kDa from eggs, whole body fraction (WB) of metacercariae, NEJ, 2- and 4-week-old juveniles and adults, and the tegumental antigen (TA) of adult. The FgTGR protein was expressed at high levels in the tegument of 2- and 4-week-old juveniles. The FgTGR may be one of the major factors acting against oxidative stresses that can damage the parasite; hence, it could be considered as a novel vaccine or a drug target.

  7. Molecular Diagnosis of 5α-Reductase Type II Deficiency in Brazilian Siblings with 46,XY Disorder of Sex Development

    Directory of Open Access Journals (Sweden)

    Maricilda Palandi de Mello

    2011-12-01

    Full Text Available The steroid 5α-reductase type II enzyme catalyzes the conversion of testosterone (T to dihydrotestosterone (DHT, and its deficiency leads to undervirilization in 46,XY individuals, due to an impairment of this conversion in genital tissues. Molecular analysis in the steroid 5α-reductase type II gene (SRD5A2 was performed in two 46,XY female siblings. SRD5A2 gene sequencing revealed that the patients were homozygous for p.Gln126Arg missense mutation, which results from the CGA > CAA nucleotide substitution. The molecular result confirmed clinical diagnosis of 46,XY disorder of sex development (DSD for the older sister and directed the investigation to other family members. Studies on SRD5A2 protein structure showed severe changes at NADPH binding region indicating that structural modeling analysis can be useful to evaluate the deleterious role of a mutation as causing 5α-reductase type II enzyme deficiency.

  8. Differential transcriptomic analysis by RNA-Seq of GSNO-responsive genes between Arabidopsis roots and leaves.

    Science.gov (United States)

    Begara-Morales, Juan C; Sánchez-Calvo, Beatriz; Luque, Francisco; Leyva-Pérez, María O; Leterrier, Marina; Corpas, Francisco J; Barroso, Juan B

    2014-06-01

    S-Nitrosoglutathione (GSNO) is a nitric oxide-derived molecule that can regulate protein function by a post-translational modification designated S-nitrosylation. GSNO has also been detected in different plant organs under physiological and stress conditions, and it can also modulate gene expression. Thirty-day-old Arabidopsis plants were grown under hydroponic conditions, and exogenous 1 mM GSNO was applied to the root systems for 3 h. Differential gene expression analyses were carried out both in roots and in leaves by RNA sequencing (RNA-seq). A total of 3,263 genes were identified as being modulated by GSNO. Most of the genes identified were associated with the mechanism of protection against stress situations, many of these having previously been identified as target genes of GSNO by array-based methods. However, new genes were identified, such as that for methionine sulfoxide reductase (MSR) in leaves or different miscellaneous RNA (miscRNA) genes in Arabidopsis roots. As a result, 1,945 GSNO-responsive genes expressed differently in leaves and roots were identified, and 114 of these corresponded exclusively to one of these organs. In summary, it is demonstrated that RNA-seq extends our knowledge of GSNO as a signaling molecule which differentially modulates gene expression in roots and leaves under non-stress conditions. © The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

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

    LENUS (Irish Health Repository)

    Crushell, Ellen

    2012-09-01

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

  10. Purification, crystallization and preliminary X-ray analysis of 3-hydroxy-3-methylglutaryl-coenzyme A reductase of Streptococcus pneumoniae

    International Nuclear Information System (INIS)

    Zhang, Liping; Feng, Lingling; Zhou, Li; Gui, Jie; Wan, Jian; Hu, Xiaopeng

    2010-01-01

    3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase of Streptococcus pneumoniae has been cloned, overexpressed and purified to homogeneity using Ni–NTA affinity chromatography. Crystals were obtained using the hanging-drop vapour-diffusion method. Class II 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductases are potential targets for novel antibiotic development. In order to obtain a precise structural model for use in virtual screening and inhibitor design, HMG-CoA reductase of Streptococcus pneumoniae was cloned, overexpressed and purified to homogeneity using Ni–NTA affinity chromatography. Crystals were obtained using the hanging-drop vapour-diffusion method. A complete data set was collected from a single frozen crystal on a home X-ray source. The crystal diffracted to 2.3 Å resolution and belonged to the orthorhombic space group C222 1 , with unit-cell parameters a = 773.4836, b = 90.3055, c = 160.5592 Å, α = β = γ = 90°. Assuming the presence of two molecules in the asymmetric unit, the solvent content was estimated to be 54.1% (V M = 2.68 Å 3 Da −1 )

  11. Biocatalysis with thermostable enzymes: structure and properties of a thermophilic 'ene'-reductase related to old yellow enzyme.

    Science.gov (United States)

    Adalbjörnsson, Björn V; Toogood, Helen S; Fryszkowska, Anna; Pudney, Christopher R; Jowitt, Thomas A; Leys, David; Scrutton, Nigel S

    2010-01-25

    We report the crystal structure of a thermophilic "ene" reductase (TOYE) isolated from Thermoanaerobacter pseudethanolicus E39. The crystal structure reveals a tetrameric enzyme and an active site that is relatively large compared to most other structurally determined and related Old Yellow Enzymes. The enzyme adopts higher order oligomeric states (octamers and dodecamers) in solution, as revealed by sedimentation velocity and multiangle laser light scattering. Bead modelling indicates that the solution structure is consistent with the basic tetrameric structure observed in crystallographic studies and electron microscopy. TOYE is stable at high temperatures (T(m)>70 degrees C) and shows increased resistance to denaturation in water-miscible organic solvents compared to the mesophilic Old Yellow Enzyme family member, pentaerythritol tetranitrate reductase. TOYE has typical ene-reductase properties of the Old Yellow Enzyme family. There is currently major interest in using Old Yellow Enzyme family members in the preparative biocatalysis of a number of activated alkenes. The increased stability of TOYE in organic solvents is advantageous for biotransformations in which water-miscible organic solvents and biphasic reaction conditions are required to both deliver novel substrates and minimize product racemisation.

  12. Functional properties and structural characterization of rice δ1-pyrroline-5-carboxylate reductase

    Directory of Open Access Journals (Sweden)

    Giuseppe eForlani

    2015-07-01

    Full Text Available The majority of plant species accumulate high intracellular levels of proline to cope with hyperosmotic stress conditions. Proline synthesis from glutamate is tightly regulated at both the transcriptional and the translational levels, yet little is known about the mechanisms for post-translational regulation of the enzymatic activities involved. The gene coding in rice (Oryza sativa L. for δ1-pyrroline-5-carboxylate (P5C reductase, the enzyme that catalyzes the second and final step in this pathway, was isolated and expressed in E. coli. The structural and functional properties of the affinity-purified protein were characterized. As for most species, rice P5C reductase was able to use in vitro either NADH or NADPH as the electron donor. However, strikingly different effects of cations and anions were found depending on the pyridine nucleotide used, namely inhibition of NADH-dependent activity and stimulation of NADPH-dependent activity. Moreover, physiological concentrations of proline and NADP+ were strongly inhibitory for the NADH-dependent reaction, whereas the NADPH-dependent activity was mildly affected. Our results suggest that only NADPH may be used in vivo and that stress-dependent variations in ion homeostasis and NADPH/NADP+ ratio could modulate enzyme activity, being functional in promoting proline accumulation and potentially also adjusting NADPH consumption during the defense against hyperosmotic stress. The apparent molecular weight of the native protein observed in size exclusion chromatography indicated a high oligomerization state. We also report the first crystal structure of a plant P5C reductase at 3.40-Å resolution, showing a decameric quaternary assembly. Based on the structure, it was possible to identify dynamic structural differences among rice, human and bacterial enzymes.

  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. Crystal structure of conjugated polyketone reductase (CPR-C1) from Candida parapsilosis IFO 0708 complexed with NADPH.

    Science.gov (United States)

    Qin, Hui-Min; Yamamura, Akihiro; Miyakawa, Takuya; Kataoka, Michihiko; Maruoka, Shintaro; Ohtsuka, Jun; Nagata, Koji; Shimizu, Sakayu; Tanokura, Masaru

    2013-11-01

    Conjugated polyketone reductase (CPR-C1) from Candida parapsilosis IFO 0708 is a member of the aldo-keto reductase (AKR) superfamily and reduces ketopantoyl lactone to d-pantoyl lactone in a NADPH-dependent and stereospecific manner. We determined the crystal structure of CPR-C1.NADPH complex at 2.20 Å resolution. CPR-C1 adopted a triose-phosphate isomerase (TIM) barrel fold at the core of the structure in which Thr25 and Lys26 of the GXGTX motif bind uniquely to the adenosine 2'-phosphate group of NADPH. This finding provides a novel structural basis for NADPH binding of the AKR superfamily. Copyright © 2013 Wiley Periodicals, Inc.

  15. Properties of latent and thiol-activated rat hepatic 3-hydroxy-3-methylglutaryl-coenzyme A reductase and regulation of enzyme activity.

    Science.gov (United States)

    Dotan, I; Shechter, I

    1983-10-15

    The effect of the thiols glutathione (GSH), dithiothreitol (DTT), and dithioerythritol (DTE) on the conversion of an inactive, latent form (El) of rat liver 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase, EC 1.1.1.34) to a catalyticaly active form (Ea) is examined. Latent hepatic microsomal HMG-CoA reductase is activated to a similar degree of activation by DTT and DTE and to a lower extent by GSH. All three thiols affect both Km and Vmax values of the enzyme toward HMG-CoA and NADPH. Studies of the effect of DTT on the affinity binding of HMG-CoA reductase to agarose-hexane-HMG-CoA (AG-HMG-CoA) resin shows that thiols are necessary for the binding of the enzyme to the resin. Removal of DTT from AG-HMG-CoA-bound soluble Ea (active enzyme) does not cause dissociation of the enzyme from the resin at low salt concentrations. Substitution of DTT by NADPH does not promote binding of soluble El (latent enzyme) to AG-HMG-CoA. The enzymatic activity of Ea in the presence of DTT and GSH indicates that these thiols compete for the same binding site on the enzyme. Diethylene glycol disulfide (ESSE) and glutathione disulfide (GSSG) inhibit the activity of Ea. ESSE is more effective for the inhibition of Ea than GSSG, causing a higher degree of maximal inhibition and affecting the enzymatic activity at lower concentrations. A method is described for the rapid conversion of soluble purified Ea to El using gel-filtration chromatography on Bio-Gel P-4 columns. These combined results point to the importance of the thiol/disulfide ratio for the modulation of hepatic HMG-CoA reductase activity.

  16. Overexpression of D-Xylose Reductase (xyl1 Gene and Antisense Inhibition of D-Xylulokinase (xyiH Gene Increase Xylitol Production in Trichoderma reesei

    Directory of Open Access Journals (Sweden)

    Yuanyuan Hong

    2014-01-01

    Full Text Available T. reesei is an efficient cellulase producer and biomass degrader. To improve xylitol production in Trichoderma reesei strains by genetic engineering, two approaches were used in this study. First, the presumptive D-xylulokinase gene in T. reesei (xyiH, which has high homology to known fungi D-xylulokinase genes, was silenced by transformation of T. reesei QM9414 strain with an antisense construct to create strain S6-2-2. The expression of the xyiH gene in the transformed strain S6-2-2 decreased at the mRNA level, and D-xylulokinase activity decreased after 48 h of incubation. This led to an increase in xylitol production from undetectable levels in wild-type T. reesei QM9414 to 8.6 mM in S6-2-2. The T. reesei Δxdh is a xylose dehydrogenase knockout strain with increased xylitol production compared to the wild-type T. reesei QM9414 (22.8 mM versus undetectable. The copy number of the xylose reductase gene (xyl1 in T. reesei Δxdh strain was increased by genetic engineering to create a new strain Δ9-5-1. The Δ9-5-1 strain showed a higher xyl1 expression and a higher yield of xylose reductase, and xylitol production was increased from 22.8 mM to 24.8 mM. Two novel strains S6-2-2 and Δ9-5-1 are capable of producing higher yields of xylitol. T. reesei has great potential in the industrial production of xylitol.

  17. Overexpression of D-Xylose Reductase (xyl1) Gene and Antisense Inhibition of D-Xylulokinase (xyiH) Gene Increase Xylitol Production in Trichoderma reesei

    Science.gov (United States)

    Hong, Yuanyuan; Dashtban, Mehdi; Kepka, Greg; Chen, Sanfeng; Qin, Wensheng

    2014-01-01

    T. reesei is an efficient cellulase producer and biomass degrader. To improve xylitol production in Trichoderma reesei strains by genetic engineering, two approaches were used in this study. First, the presumptive D-xylulokinase gene in T. reesei (xyiH), which has high homology to known fungi D-xylulokinase genes, was silenced by transformation of T. reesei QM9414 strain with an antisense construct to create strain S6-2-2. The expression of the xyiH gene in the transformed strain S6-2-2 decreased at the mRNA level, and D-xylulokinase activity decreased after 48 h of incubation. This led to an increase in xylitol production from undetectable levels in wild-type T. reesei QM9414 to 8.6 mM in S6-2-2. The T. reesei Δxdh is a xylose dehydrogenase knockout strain with increased xylitol production compared to the wild-type T. reesei QM9414 (22.8 mM versus undetectable). The copy number of the xylose reductase gene (xyl1) in T. reesei Δxdh strain was increased by genetic engineering to create a new strain Δ9-5-1. The Δ9-5-1 strain showed a higher xyl1 expression and a higher yield of xylose reductase, and xylitol production was increased from 22.8 mM to 24.8 mM. Two novel strains S6-2-2 and Δ9-5-1 are capable of producing higher yields of xylitol. T. reesei has great potential in the industrial production of xylitol. PMID:25013760

  18. S-nitrosylated proteins of a medicinal CAM plant Kalanchoe pinnata- ribulose-1,5-bisphosphate carboxylase/oxygenase activity targeted for inhibition.

    Science.gov (United States)

    Abat, Jasmeet K; Mattoo, Autar K; Deswal, Renu

    2008-06-01

    Nitric oxide (NO) is a signaling molecule that affects a myriad of processes in plants. However, the mechanistic details are limited. NO post-translationally modifies proteins by S-nitrosylation of cysteines. The soluble S-nitrosoproteome of a medicinal, crassulacean acid metabolism (CAM) plant, Kalanchoe pinnata, was purified using the biotin switch technique. Nineteen targets were identified by MALDI-TOF mass spectrometry, including proteins associated with carbon, nitrogen and sulfur metabolism, the cytoskeleton, stress and photosynthesis. Some were similar to those previously identified in Arabidopsis thaliana, but kinesin-like protein, glycolate oxidase, putative UDP glucose 4-epimerase and putative DNA topoisomerase II had not been identified as targets previously for any organism. In vitro and in vivo nitrosylation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), one of the targets, was confirmed by immunoblotting. Rubisco plays a central role in photosynthesis, and the effect of S-nitrosylation on its enzymatic activity was determined using NaH14CO3. The NO-releasing compound S-nitrosoglutathione inhibited its activity in a dose-dependent manner suggesting Rubisco inactivation by nitrosylation for the first time.

  19. A conservative region of the mercuric reductase gene (merA) as a molecular marker of bacterial mercury resistance Região conservada do gene da mercúrio redutase (merA) como marcador molecular da resistência bacteriana ao mercúrio

    OpenAIRE

    Adriana Sotero-Martins; Michele Silva de Jesus; Michele Lacerda; Josino Costa Moreira; Ana Luzia Lauria Filgueiras; Paulo Rubens Guimarães Barrocas

    2008-01-01

    The most common bacterial mercury resistance mechanism is based on the reduction of Hg(II) to Hg0, which is dependent of the mercuric reductase enzyme (MerA) activity. The use of a 431 bp fragment of a conservative region of the mercuric reductase (merA) gene was applied as a molecular marker of this mechanism, allowing the identification of mercury resistant bacterial strains.O mecanismo de resistência bacteriana ao mercúrio mais comum é baseada na redução do Hg(II) a Hg0, através da ativida...

  20. 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.

  1. Inhibition of 5α-Reductase, IL-6 Secretion, and Oxidation Process of Equisetum debile Roxb. ex Vaucher Extract as Functional Food and Nutraceuticals Ingredients

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    Wantida Chaiyana

    2017-10-01

    Full Text Available This study aims to investigate the biological activities related to hair loss of Equisetum debile extracts, including 5α-reductase inhibition, interleukin-6 (IL-6 secretion reduction, and anti-oxidation. E. debile extracts were obtained by maceration in various solvents. Crude extract (CE was obtained by maceration in 95% ethanol. Chlorophyll-free extract (CF was the CE which of the chlorophyll has been removed by electrocoagulation. Hexane extract (HE, ethyl acetate extract (EA, and ethanolic extract (ET were fraction extracts obtained from maceration in hexane, ethyl acetate, and 95% ethanol, respectively. The extracts were investigated for inhibitory activity against 5α-reductase and IL-6 secretion. Total phenolic contents (TPC were investigated and antioxidant activities were determined by means of 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS, 2,2′-diphenyl-1-picrylhydrazyl (DPPH, and ferric reducing antioxidant power (FRAP assays. The inhibition of lipid peroxidation was determined by the ferric thiocyanate method. The cytotoxicity of the extracts on dermal papilla cells and irritation test by hen's egg test chorioallantoic membrane assay were also investigated. All extracts could inhibit 5α-reductase and decrease IL-6 secretion in lipopolysaccharide-stimulated macrophage. The antioxidant activity of E. debile extracts was directly related to their TPC. ET which contained the highest TPC (68.8 ± 6.7 mg GA/g showed the highest equivalent concentration (EC1 of 289.1 ± 26.4 mM FeSO4/g, TEAC of 156.6 ± 34.6 mM Trolox/g, and 20.0 ± 6.0% DPPH inhibition. However, EA exhibited the highest inhibition against lipid peroxidation (57.2 ± 0.4%. In addition, EA showed no cytotoxicity on dermal papilla cell line and no irritation on chorioallantoic membrane of hen’s eggs. In conclusion, EA was suggested as the most attractive ingredients for functional food and nutraceuticals because of the high inhibitory activity against 5α-reductase

  2. Pharmacogenetics of aldo-keto reductase 1C (AKR1C) enzymes.

    Science.gov (United States)

    Alshogran, Osama Y

    2017-10-01

    Genetic variation in metabolizing enzymes contributes to variable drug response and disease risk. Aldo-keto reductase type 1C (AKR1C) comprises a sub-family of reductase enzymes that play critical roles in the biotransformation of various drug substrates and endogenous compounds such as steroids. Several single nucleotide polymorphisms have been reported among AKR1C encoding genes, which may affect the functional expression of the enzymes. Areas covered: This review highlights and comprehensively discusses previous pharmacogenetic reports that have examined genetic variations in AKR1C and their association with disease development, drug disposition, and therapeutic outcomes. The article also provides information about the effect of AKR1C genetic variants on enzyme function in vitro. Expert opinion: The current evidence that links the effect of AKR1C gene polymorphisms to disease progression and development is inconsistent and needs further validation, despite of the tremendous knowledge available. Information about association of AKR1C genetic variants and drug efficacy, safety, and pharmacokinetics is limited, thus, future studies that advance our understanding about these relationships and their clinical relevance are needed. It is imperative to achieve consistent findings before the potential translation and adoption of AKR1C genetic variants in clinical practice.

  3. Evaluation of constitutive iron reductase (AtFRO2 expression on mineral accumulation and distribution in soybean (Glycine max. L

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    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

  4. Purification of a NAD(P) reductase-like protein from the thermogenic appendix of the Sauromatum guttatum inflorescence.

    Science.gov (United States)

    Skubatz, Hanna; Howald, William N

    2013-03-01

    A NAD(P) reductase-like protein with a molecular mass of 34.146 ± 34 Da was purified to homogeneity from the appendix of the inflorescence of the Sauromatum guttatum. On-line liquid chromatography/electrospray ionization-mass spectrometry was used to isolate and quantify the protein. For the identification of the protein, liquid chromatography/electrospray ionization-tandem mass spectrometry analysis of tryptic digests of the protein was carried out. The acquired mass spectra were used for database searching, which led to the identification of a single tryptic peptide. The 12 amino acid tryptic peptide (FLPSEFGNDVDR) was found to be identical to amino acid residues at the positions 108-120 of isoflavone reductase in the Arabidopsis genome. A BLAST search identified this sequence region as unique and specific to a class of NAD(P)-dependent reductases involved in phenylpropanoid biosynthesis. Edman degradation revealed that the protein was N-terminally blocked. The amount of the protein (termed RL, NAD(P) reductase-like protein) increased 60-fold from D-4 (4 days before inflorescence-opening, designated as D-day) to D-Day, and declined the following day, when heat-production ceased. When salicylic acid, the endogenous trigger of heat-production in the Sauromatum appendix, was applied to premature appendices, a fivefold decrease in the amount of RL was detected in the treated section relative to the non-treated section. About 40 % of RL was found in the cytoplasm. Another 30 % was detected in Percoll-purified mitochondria and the rest, about 30 % was associated with a low speed centrifugation pellet due to nuclei and amyloplast localization. RL was also found in other thermogenic plants and detected in Arabidopsis leaves. The function of RL in thermogenic and non-thermogenic plants requires further investigation.

  5. Nitric oxide induces the alternative oxidase pathway in Arabidopsis seedlings deprived of inorganic phosphate.

    Science.gov (United States)

    Royo, Beatriz; Moran, Jose F; Ratcliffe, R George; Gupta, Kapuganti J

    2015-10-01

    Phosphate starvation compromises electron flow through the cytochrome pathway of the mitochondrial electron transport chain, and plants commonly respond to phosphate deprivation by increasing flow through the alternative oxidase (AOX). To test whether this response is linked to the increase in nitric oxide (NO) production that also increases under phosphate starvation, Arabidopsis thaliana seedlings were grown for 15 d on media containing either 0 or 1mM inorganic phosphate. The effects of the phosphate supply on growth, the production of NO, respiration, the AOX level and the production of superoxide were compared for wild-type (WT) seedlings and the nitrate reductase double mutant nia. Phosphate deprivation increased NO production in WT roots, and the AOX level and the capacity of the alternative pathway to consume electrons in WT seedlings; whereas the same treatment failed to stimulate NO production and AOX expression in the nia mutant, and the plants had an altered growth phenotype. The NO donor S-nitrosoglutathione rescued the growth phenotype of the nia mutants under phosphate deprivation to some extent, and it also increased the respiratory capacity of AOX. It is concluded that NO is required for the induction of the AOX pathway when seedlings are grown under phosphate-limiting conditions. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  6. 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

  7. 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.

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

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

    DEFF Research Database (Denmark)

    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 propo...

  10. 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

    of the genes described from eukaryotic organisms and the deduced amino acid sequence could be aligned with the C-terminal half of animal and plant reductases exhibiting pronounced similarity to other eukaryotic counterparts. Further examination of the 5' flanking region by cDNA analysis and establishment...

  11. Methylenetetrahydrofolate reductase deficiency alters levels of glutamate and γ-aminobutyric acid in brain tissue

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    N.M. Jadavji

    2015-06-01

    Full Text Available Methylenetetrahydrofolate reductase (MTHFR is an enzyme key regulator in folate metabolism. Deficiencies in MTHFR result in increased levels of homocysteine, which leads to reduced levels of S-adenosylmethionine (SAM. In the brain, SAM donates methyl groups to catechol-O-methyltransferase (COMT, which is involved in neurotransmitter analysis. Using the MTHFR-deficient mouse model the purpose of this study was to investigate levels of monoamine neurotransmitters and amino acid levels in brain tissue. MTHFR deficiency affected levels of both glutamate and γ-aminobutyric acid in within the cerebellum and hippocampus. Mthfr−/− mice had reduced levels of glutamate in the amygdala and γ-aminobutyric acid in the thalamus. The excitatory mechanisms of homocysteine through activation of the N-methyl-d-aspartate receptor in brain tissue might alter levels of glutamate and γ-aminobutyric acid.

  12. 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

  13. 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

  14. The Inhibitory Effect of Prunella vulgaris L. on Aldose Reductase and Protein Glycation

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    Hong Mei Li

    2012-01-01

    Full Text Available To evaluate the aldose reductase (AR enzyme inhibitory ability of Prunella vulgaris L. extract, six compounds were isolated and tested for their effects. The components were subjected to in vitro bioassays to investigate their inhibitory assays using rat lens aldose reductase (rAR and human recombinant AR (rhAR. Among them, caffeic acid ethylene ester showed the potent inhibition, with the IC50 values of rAR and rhAR at 3.2±0.55 μM and 12.58±0.32 μM, respectively. In the kinetic analyses using Lineweaver-Burk plots of 1/velocity and 1/concentration of substrate, this compound showed noncompetitive inhibition against rhAR. Furthermore, it inhibited galactitol formation in a rat lens incubated with a high concentration of galactose. Also it has antioxidative as well as advanced glycation end products (AGEs inhibitory effects. As a result, this compound could be offered as a leading compound for further study as a new natural products drug for diabetic complications.

  15. Structure and biocatalytic scope of thermophilic flavin-dependent halogenase and flavin reductase enzymes.

    Science.gov (United States)

    Menon, Binuraj R K; Latham, Jonathan; Dunstan, Mark S; Brandenburger, Eileen; Klemstein, Ulrike; Leys, David; Karthikeyan, Chinnan; Greaney, Michael F; Shepherd, Sarah A; Micklefield, Jason

    2016-10-04

    Flavin-dependent halogenase (Fl-Hal) enzymes have been shown to halogenate a range of synthetic as well as natural aromatic compounds. The exquisite regioselectively of Fl-Hal enzymes can provide halogenated building blocks which are inaccessible using standard halogenation chemistries. Consequently, Fl-Hal are potentially useful biocatalysts for the chemoenzymatic synthesis of pharmaceuticals and other valuable products, which are derived from haloaromatic precursors. However, the application of Fl-Hal enzymes, in vitro, has been hampered by their poor catalytic activity and lack of stability. To overcome these issues, we identified a thermophilic tryptophan halogenase (Th-Hal), which has significantly improved catalytic activity and stability, compared with other Fl-Hal characterised to date. When used in combination with a thermostable flavin reductase, Th-Hal can efficiently halogenate a number of aromatic substrates. X-ray crystal structures of Th-Hal, and the reductase partner (Th-Fre), provide insights into the factors that contribute to enzyme stability, which could guide the discovery and engineering of more robust and productive halogenase biocatalysts.

  16. The Cholesterol-Lowering Effect of Alisol Acetates Based on HMG-CoA Reductase and Its Molecular Mechanism

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    Fei Xu

    2016-01-01

    Full Text Available This study measured the impact of alisol B 23-acetate and alisol A 24-acetate, the main active ingredients of the traditional Chinese medicine Alismatis rhizoma, on total cholesterol (TC, triglyceride (TG, high density lipoprotein-cholesterol (HDL-C, and low density lipoprotein-cholesterol (LDL-C levels of hyperlipidemic mice. The binding of alisol B 23-acetate and alisol A 24-acetate to the key enzyme involved in the metabolism of TC, 3-hydroxy-3-methylglutary-coenzyme A (HMG-CoA reductase, was studied using the reagent kit method and the western blotting technique combined with a molecular simulation technique. According to the results, alisol acetates significantly lower the TC, TG, and LDL-C concentrations of hyperlipidemic mice, while raising HDL-C concentrations. Alisol acetates lower HMG-CoA reductase activity in a dose-dependent fashion, both in vivo and in vitro. Neither of these alisol acetates significantly lower the protein expression of HMG-CoA. This suggests that alisol acetates lower the TC level via inhibiting the activity of HMG-CoA reductase by its prototype drug, which may exhibit an inhibition effect via directly and competitively binding to HMG-CoA. The side chain of the alisol acetate was the steering group via molecular simulation.

  17. Low activity of superoxide dismutase and high activity of glutathione reductase in erythrocytes from centenarians

    DEFF Research Database (Denmark)

    Andersen, Helle Raun; Jeune, B; Nybo, H

    1998-01-01

    aged between 60 and 79 years. MEASUREMENTS: enzyme activities of superoxide dismutase (CuZn-SOD), glutathione peroxidase, catalase and glutathione reductase (GR) in erythrocytes. Functional capacity among the centenarians was evaluated by Katz' index of activities of daily living, the Physical...

  18. 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...

  19. Cloning, molecular characterization and expression of a cDNA encoding a functional NADH-cytochrome b5 reductase from Mucor racemosus PTCC 5305 in E. coli

    Directory of Open Access Journals (Sweden)

    NED A SETAYESH

    2009-01-01

    Full Text Available The present work aims to study a new NADH-cytochrome b5 reductase (cb5r from Mucor racemosus PTCC 5305. A cDNA coding for cb s r was isolated from a Mucor racemosus PTCC 5305 cDNA library. The nucleotide sequence of the cDNA including coding and sequences flanking regions was determined. The open reading frame starting from ATG and ending with TAG stop codon encoded 228 amino acids and displayed the closest similarity (73% with Mortierella alpina cb s r. Lack of hydrophobic residues in the N-terminal sequence was apparent, suggesting that the enzyme is a soluble isoform. The coding sequence was then cloned in the pET16b transcription vector carrying an N-terminal-linked His-Tag® sequence and expressed in Escherichia coli BL21 (DE3. The enzyme was then homogeneously purified by a metal affinity column. The recombinant Mucor enzyme was shown to have its optimal activity at pH and temperature of about 7.5 and 40 °C, respectively. The apparent Km value was calculated to be 13 μM for ferricyanide. To our knowledge, this is the first report on cloning and expression of a native fungal soluble isoform of NADH-cytochrome b5 reductase in E. coli.

  20. Induction of the Nitrate Assimilation nirA Operon and Protein-Protein Interactions in the Maturation of Nitrate and Nitrite Reductases in the Cyanobacterium Anabaena sp. Strain PCC 7120.

    Science.gov (United States)

    Frías, José E; Flores, Enrique

    2015-07-01

    Nitrate is widely used as a nitrogen source by cyanobacteria, in which the nitrate assimilation structural genes frequently constitute the so-called nirA operon. This operon contains the genes encoding nitrite reductase (nirA), a nitrate/nitrite transporter (frequently an ABC-type transporter; nrtABCD), and nitrate reductase (narB). In the model filamentous cyanobacterium Anabaena sp. strain PCC 7120, which can fix N2 in specialized cells termed heterocysts, the nirA operon is expressed at high levels only in media containing nitrate or nitrite and lacking ammonium, a preferred nitrogen source. Here we examined the genes downstream of the nirA operon in Anabaena and found that a small open reading frame of unknown function, alr0613, can be cotranscribed with the operon. The next gene in the genome, alr0614 (narM), showed an expression pattern similar to that of the nirA operon, implying correlated expression of narM and the operon. A mutant of narM with an insertion mutation failed to produce nitrate reductase activity, consistent with the idea that NarM is required for the maturation of NarB. Both narM and narB mutants were impaired in the nitrate-dependent induction of the nirA operon, suggesting that nitrite is an inducer of the operon in Anabaena. It has previously been shown that the nitrite reductase protein NirA requires NirB, a protein likely involved in protein-protein interactions, to attain maximum activity. Bacterial two-hybrid analysis confirmed possible NirA-NirB and NarB-NarM interactions, suggesting that the development of both nitrite reductase and nitrate reductase activities in cyanobacteria involves physical interaction of the corresponding enzymes with their cognate partners, NirB and NarM, respectively. Nitrate is an important source of nitrogen for many microorganisms that is utilized through the nitrate assimilation system, which includes nitrate/nitrite membrane transporters and the nitrate and nitrite reductases. Many cyanobacteria

  1. Carbohydrate restriction and dietary cholesterol modulate the expression of HMG-CoA reductase and the LDL receptor in mononuclear cells from adult men

    Directory of Open Access Journals (Sweden)

    Volek Jeff S

    2007-11-01

    Full Text Available Abstract The liver is responsible for controlling cholesterol homeostasis in the body. HMG-CoA reductase and the LDL receptor (LDL-r are involved in this regulation and are also ubiquitously expressed in all major tissues. We have previously shown in guinea pigs that there is a correlation in gene expression of HMG-CoA reductase and the LDL-r between liver and mononuclear cells. The present study evaluated human mononuclear cells as a surrogate for hepatic expression of these genes. The purpose was to evaluate the effect of dietary carbohydrate restriction with low and high cholesterol content on HMG-CoA reductase and LDL-r mRNA expression in mononuclear cells. All subjects were counseled to consume a carbohydrate restricted diet with 10–15% energy from carbohydrate, 30–35% energy from protein and 55–60% energy from fat. Subjects were randomly assigned to either EGG (640 mg/d additional dietary cholesterol or SUB groups [equivalent amount of egg substitute (0 dietary cholesterol contributions per day] for 12 weeks. At the end of the intervention, there were no changes in plasma total or LDL cholesterol (LDL-C compared to baseline (P > 0.10 or differences in plasma total or LDL-C between groups. The mRNA abundance for HMG-CoA reductase and LDL-r were measured in mononuclear cells using real time PCR. The EGG group showed a significant decrease in HMG-CoA reductase mRNA (1.98 ± 1.26 to 1.32 ± 0.92 arbitrary units P

  2. Cdna cloning and expression analyses of the isoflavone reductase-like gene of dendrobium officinale

    International Nuclear Information System (INIS)

    Qian, X.; Xu, S.Z.

    2015-01-01

    The full length of the isoflavone reductase-like gene (IRL) cDNA of Dendrobium officinale was cloned by using reverse transcription (RT) PCR combined with cDNA library, the IRL function was identified by Bioinformatics and prokaryotic expression analyses, and the IRL expression levels in the organs and tissues of D. officinale plants with different ages were determined by using real-time quantitative PCR (RT-qPCR). The results indicated that the full length of the cDNA of D. officinale IRL, DoIRL, was 1238 bp (accession no. KJ661023). Its open reading frame (ORF) was 930 bp which encoded 309 amino acids with a predicted molecular mass of 34 kDa, the 5 untranslated region (UTR) was 61 bp and the 3 UTR containing a poly (A) tail was 247 bp. The deduced amino acid sequence of DoIRL, DoIRL, was forecast to contain a NAD(P)H-binding motif (GGTGYIG) in the N-terminal region, two conserved N-glycosylation sites, a conserved nitrogen metabolite repression regulator (NmrA) domain and a phenylcoumaran benzylic ether reductase (PCBER) domain, to hold the nearest phylogenetic relationship with the PCBER of Striga asiatica, and to share both 73% identity with the isoflavone reductases-like (IRLs) of Cucumis sativus and Striga asiatica. In Escherichia coli 'BL21' cells, the DoIRL cDNA expression produced a protein band holding the predicted molecular mass of 34 kDa. DoIRL expressed in all organs and tissues of D. officinale plants with different ages at comparatively low levels, and the expression level in the leaves of the two-year-old plants was the highest. (author)

  3. Electrochemical determination of nitrate with nitrate reductase-immobilized electrodes under ambient air.

    Science.gov (United States)

    Quan, De; Shim, Jun Ho; Kim, Jong Dae; Park, Hyung Soo; Cha, Geun Sig; Nam, Hakhyun

    2005-07-15

    Nitrate monitoring biosensors were prepared by immobilizing nitrate reductase derived from yeast on a glassy carbon electrode (GCE, d = 3 mm) or screen-printed carbon paste electrode (SPCE, d = 3 mm) using a polymer (poly(vinyl alcohol)) entrapment method. The sensor could directly determine the nitrate in an unpurged aqueous solution with the aid of an appropriate oxygen scavenger: the nitrate reduction reaction driven by the enzyme and an electron-transfer mediator, methyl viologen, at -0.85 V (GCE vs Ag/AgCl) or at -0.90 V (SPCE vs Ag/AgCl) exhibited no oxygen interference in a sulfite-added solution. The electroanalytical properties of optimized biosensors were measured: the sensitivity, linear response range, and detection limit of the sensors based on GCE were 7.3 nA/microM, 15-300 microM (r2 = 0.995), and 4.1 microM (S/N = 3), respectively, and those of SPCE were 5.5 nA/microM, 15-250 microM (r2 = 0.996), and 5.5 microM (S/N = 3), respectively. The disposable SPCE-based biosensor with a built-in well- or capillary-type sample cell provided high sensor-to-sensor reproducibility (RSD sensor system was demonstrated by determining nitrate in real samples.

  4. 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....

  5. Structure and expression of human dihydropteridine reductase

    International Nuclear Information System (INIS)

    Lockyer, J.; Cook, R.G.; Milstien, S.; Kaufman, S.; Woo, S.L.C.; Ledley, F.D.

    1987-01-01

    Dihydropteridine reductase catalyzes the NADH-mediated reduction of quinonoid dihydrobiopterin and is an essential component of the pterindependent aromatic amino acid hydroxylating systems. A cDNA for human DHPR was isolated from a human liver cDNA library in the vector λgt11 using a monospecific antibody against sheep DHPR. The nucleic acid sequence and amino acid sequence of human DHPR were determined from a full-length clone. A 112 amino acid sequence of sheep DHPR was obtained by sequencing purified sheep DHPR. This sequence is highly homologous to the predicted amino acid sequence of the human protein. Gene transfer of the recombinant human DHPR into COS cells leads to expression of DHPR enzymatic activity. These results indicate that the cDNA clone identified by antibody screening is an authentic and full-length cDNA for human DHPR

  6. 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.

  7. Cyclopiazonic acid biosynthesis in Aspergillus sp.: characterization of a reductase-like R* domain in cyclopiazonate synthetase that forms and releases cyclo-acetoacetyl-L-tryptophan.

    Science.gov (United States)

    Liu, Xinyu; Walsh, Christopher T

    2009-09-15

    The fungal neurotoxin alpha-cyclopiazonic acid (CPA), a nanomolar inhibitor of Ca2+-ATPase, has a pentacyclic indole tetramic acid scaffold that arises from one molecule of tryptophan, acetyl-CoA, malonyl-CoA, and dimethylallyl pyrophosphate by consecutive action of three enzymes, CpaS, CpaD, and CpaO. CpaS is a hybrid, two module polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) that makes and releases cyclo-acetoacetyl-L-tryptophan (cAATrp), the tetramic acid that serves as substrate for subsequent prenylation and oxidative cyclization to the five ring CPA scaffold. The NRPS module in CpaS has a predicted four-domain organization of condensation, adenylation, thiolation, and reductase* (C-A-T-R*), where R* lacks the critical Ser-Tyr-Lys catalytic triad of the short chain dehydrogenase/reductase (SDR) superfamily. By heterologous overproduction in Escherichia coli of the 56 kDa Aspergillus flavus CpaS TR* didomain and the single T and R* domains, we demonstrate that CpaS catalyzes a Dieckmann-type cyclization on the N-acetoacetyl-Trp intermediate bound in thioester linkage to the phosphopantetheinyl arm of the T domain to form and release cAATrp. This occurs without any participation of NAD(P)H, so R* does not function as a canonical SDR family member. Use of the T and R* domains in in trans assays enabled multiple turnovers and evaluation of specific mutants. Mutation of the D3803 residue in the R* domain, conserved in other fungal tetramate synthetases, abolished activity both in in trans and in cis (TR*) activity assays. It is likely that cyclization of beta-ketoacylaminoacyl-S-pantetheinyl intermediates to released tetramates represents a default cyclization/release route for redox-incompetent R* domains embedded in NRPS assembly lines.

  8. 14CO2-fixation and nitrate reductase activity in vivo in relation to hybrid vigour in maize

    International Nuclear Information System (INIS)

    Balasubramanian, V.; Shanthakumari, P.; Sinha, S.K.

    1977-01-01

    Dry matter accumulation in maize shoots, leaf area, 14 CO 2 -fixation and nitrate reductase activity in vivo were measured in the field grown heterotic hybrid CM 400x CM 300 and its inbred parents CM 300 and CM 400 from seedling to maturity. Rates of dry matter accumulation and leaf area development were higher in the hybrid during the initial vegetative phase than in the inbreds. The hybrid had more absolute level of 14 CO 2 -fixation and nitrate reductase activity, although the rates of these processes on unit weight basis were not higher than those of inbreds. It is concluded that the rapid development of leaf area in hybrids during the early stages of vegetative growth is probably important for hybrid vigour. (author)

  9. S-nitrosocaptopril nanoparticles as nitric oxide-liberating and transnitrosylating anti-infective technology.

    Science.gov (United States)

    Mordorski, Breanne; Pelgrift, Robert; Adler, Brandon; Krausz, Aimee; da Costa Neto, Alexandre Batista; Liang, Hongying; Gunther, Leslie; Clendaniel, Alicea; Harper, Stacey; Friedman, Joel M; Nosanchuk, Joshua D; Nacharaju, Parimala; Friedman, Adam J

    2015-02-01

    Nitric oxide (NO), an essential agent of the innate immune system, exhibits multi-mechanistic antimicrobial activity. Previously, NO-releasing nanoparticles (NO-np) demonstrated increased antimicrobial activity when combined with glutathione (GSH) due to formation of S-nitrosoglutathione (GSNO), a transnitrosylating agent. To capitalize on this finding, we incorporated the thiol-containing ACE-inhibitor, captopril, with NO-np to form SNO-CAP-np, nanoparticles that both release NO and form S-nitrosocaptopril. In the presence of GSH, SNO-CAP-np demonstrated increased transnitrosylation activity compared to NO-np, as exhibited by increased GSNO formation. Escherichia coli and methicillin-resistant Staphylococcus aureus were highly susceptible to SNO-CAP-np in a dose-dependent fashion, with E. coli being most susceptible, and SNO-CAP-np were nontoxic in zebrafish embryos at translatable concentrations. Given SNO-CAP-np's increased transnitrosylation activity and increased E. coli susceptibility compared to NO-np, transnitrosylation rather than free NO is likely responsible for overcoming E. coli's resistance mechanisms and ultimately killing the pathogen. This team of authors incorporated the thiol-containing ACE-inhibitor, captopril, into a nitric oxide releasing nanoparticle system, generating nanoparticles that both release NO and form S-nitrosocaptopril, with pronounced toxic effects on MRSA and E. coli in the presented model system. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. Expression, purification, crystallization and preliminary X-ray analysis of ORF60, the small subunit (R2) of ribonucleotide reductase from Kaposi’s sarcoma-associated herpesvirus (KSHV)

    International Nuclear Information System (INIS)

    Gurmu, Daniel; Dahlroth, Sue-Li; Haas, Juergen; Nordlund, Pär; Erlandsen, Heidi

    2010-01-01

    Crystals of the R2 subunit from the oncovirus Kaposi’s sarcoma-associated γ-herpesvirus (KSHV) were obtained by the use of in situ proteolysis. The crystals diffracted to 2.0 Å resolution and belonged to space group P2 1 . Ribonucleotide reductase (RNR) is responsible for converting ribonucleotides to deoxyribonucleotides, which are the building blocks of DNA. The enzyme is present in all life forms as well as in some large DNA viruses such as herpesviruses. The α-herpesviruses and γ-herpesviruses encode two class Ia RNR subunits, R1 and R2, while the β-herpesvirus subfamily only encode an inactive R1 subunit. Here, the crystallization of the R2 subunit of RNR encoded by the ORF60 gene from the oncovirus Kaposi’s sarcoma-associated γ-herpesvirus (KSHV) is reported. These are the first crystals of a viral R2 subunit; the use of in situ proteolysis with chymotrypsin and the addition of hexamine cobalt(III) chloride that were necessary to obtain crystals are described. Optimization of the crystallization conditions yielded crystals that diffracted to 2.0 Å resolution. The crystals belonged to space group P2 1 , with unit-cell parameters a = 63.9, b = 71.2, c = 71.8 Å, α = 90, β = 106.7, γ = 90°. The data set collected was 95.3% complete, with an R merge of 9.6%. There are two molecules in the asymmetric unit, corresponding to a solvent content of 43.4%

  11. [Effect of UV-radiation on the level of ascorbic acid, SH-groups, and activity of glutathione reductase in the eye lens].

    Science.gov (United States)

    Byshneva, L N; Senchuk, V V

    2002-01-01

    The effect of UV radiation in vitro on the level of ascorbate, SH-groups and glutathione reductase activity in the soluble fraction of bovine eye lens was studied. UV-Irradiation increased NADPH-oxidoreductase activity, the level of ascorbate oxidation and decreased the content of SH-groups and activity of glutathione reductase. Significant activation of the NADPH-oxidoreductase activity in the presence of ascorbate and Cu2+ was observed after UV-irradiation. It is suggested that ascorbate may play an important role in the UV-induced lens pathology.

  12. P450 reductase and cytochrome b5 interactions with cytochrome P450: Effects on house fly CYP6A1 catalysis

    OpenAIRE

    Murataliev, Marat B.; Guzov, Victor M.; Walker, F. Ann; Feyereisen, René

    2008-01-01

    The interactions of protein components of the xenobiotic-metabolizing cytochrome P450 system, CYP6A1, P450 reductase, and cytochrome b5 from the house fly (Musca domestica) have been characterized. CYP6A1 activity is determined by the concentration of the CYP6A1-P450 reductase complex, regardless of which protein is present in excess. Both holo- and apo-b5 stimulated CYP6A1 heptachlor epoxidase and steroid hydroxylase activities and influenced the regioselectivity of testosterone hydroxylatio...

  13. Reduction of Hexavalent Chromium and Detection of Chromate Reductase (ChrR in Stenotrophomonas maltophilia

    Directory of Open Access Journals (Sweden)

    Rosa Baldiris

    2018-02-01

    Full Text Available An Gram negative strain of S. maltophilia, indigenous to environments contaminated by Cr(VI and identified by biochemical methods and 16S rRNA gene analysis, reduced chromate by 100%, 98–99% and 92% at concentrations in the 10–70, 80–300, and 500 mg/L range, respectively at pH 7 and temperature 37 °C. Increasing concentrations of Cr(VI in the medium lowered the growth rate but could not be directly correlated with the amount of Cr(VI reduced. The strain also exhibited multiple resistance to antibiotics and tolerance and resistance to various heavy metals (Ni, Zn and Cu, with the exception of Hg. Hexavalent chromium reduction was mainly associated with the soluble fraction of the cell evaluated with crude cell-free extracts. A protein of molecular weight around 25 kDa was detected on SDS-PAGE gel depending on the concentration of hexavalent chromium in the medium (0, 100 and 500 mg/L. In silico analysis in this contribution, revealed the presence of the chromate reductase gene ChrR in S. maltophilia, evidenced through a fragment of around 468 bp obtained experimentally. High Cr(VI concentration resistance and high Cr(VI reducing ability of the strain make it a suitable candidate for bioremediation.

  14. Identification and cloning of an NADPH-dependent hydroxycinnamoyl-CoA double bond reductase involved in dihydrochalcone formation in Malus×domestica Borkh.

    Science.gov (United States)

    Ibdah, Mwafaq; Berim, Anna; Martens, Stefan; Valderrama, Andrea Lorena Herrera; Palmieri, Luisa; Lewinsohn, Efraim; Gang, David R

    2014-11-01

    The apple tree (Malus sp.) is an agriculturally and economically important source of food and beverages. Many of the health beneficial properties of apples are due to (poly)phenolic metabolites that they contain, including various dihydrochalcones. Although many of the genes and enzymes involved in polyphenol biosynthesis are known in many plant species, the specific reactions that lead to the biosynthesis of the dihydrochalcone precursor, p-dihydrocoumaroyl-CoA (3), are unknown. To identify genes involved in the synthesis of these metabolites, existing genome databases of the Rosaceae were screened for apple genes with significant sequence similarity to Arabidopsis alkenal double bond reductases. Herein described are the isolation and characterization of a Malus hydroxycinnamoyl-CoA double bond reductase, which catalyzed the NADPH-dependent reduction of p-coumaroyl-CoA and feruloyl-CoA to p-dihydrocoumaroyl-CoA and dihydroferuloyl-CoA, respectively. Its apparent Km values for p-coumaroyl-CoA, feruloyl-CoA and NADPH were 96.6, 92.9 and 101.3μM, respectively. The Malus double bond reductase preferred feruloyl-CoA to p-coumaroyl-CoA as a substrate by a factor of 2.1 when comparing catalytic efficiencies in vitro. Expression analysis of the hydroxycinnamoyl-CoA double bond reductase gene revealed that its transcript levels showed significant variation in tissues of different developmental stages, but was expressed when expected for involvement in dihydrochalcone formation. Thus, the hydroxycinnamoyl-CoA double bond reductase appears to be responsible for the reduction of the α,β-unsaturated double bond of p-coumaroyl-CoA, the first step of dihydrochalcone biosynthesis in apple tissues, and may be involved in the production of these compounds. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

    Moon, Jaewoong; Liu, Z Lewis

    2015-04-01

    The aldehyde reductase gene ARI1 is a recently characterized member of an intermediate subfamily within the short-chain dehydrogenase/reductase (SDR) superfamily that clarified mechanisms of in situ detoxification of 2-furaldehyde and 5-hydroxymethyl-2-furaldehyde by Saccharomyces cerevisiae. Uncharacterized open reading frames (ORFs) are common among tolerant candidate genes identified for lignocellulose-to-advanced biofuels conversion. This study presents partially purified proteins of two ORFs, YDR541C and YGL039W, and direct enzyme assay evidence against aldehyde-inhibitory compounds commonly encountered during lignocellulosic biomass fermentation processes. Each of the partially purified proteins encoded by these ORFs showed a molecular mass of approximately 38 kDa, similar to Ari1p, a protein encoded by aldehyde reductase gene. Both proteins demonstrated strong aldehyde reduction activities toward 14 aldehyde substrates, with high levels of reduction activity for Ydr541cp toward both aromatic and aliphatic aldehydes. While Ydr541cp was observed to have a significantly higher specific enzyme activity at 20 U/mg using co-factor NADPH, Ygl039wp displayed a NADH preference at 25 U/mg in reduction of butylaldehyde. Amino acid sequence analysis identified a characteristic catalytic triad, Ser, Tyr and Lys; a conserved catalytic motif of Tyr-X-X-X-Lys; and a cofactor-binding sequence motif, Gly-X-X-Gly-X-X-Ala, near the N-terminus that are shared by Ydr541cp, Ygl039wp, Yol151wp/GRE2 and Ari1p. Findings of aldehyde reductase genes contribute to the yeast gene annotation and aids development of the next-generation biocatalyst for advanced biofuels production. Copyright © 2015 John Wiley & Sons, Ltd.

  16. Structure of conjugated polyketone reductase from Candida parapsilosis IFO 0708 reveals conformational changes for substrate recognition upon NADPH binding.

    Science.gov (United States)

    Qin, Hui-Min; Yamamura, Akihiro; Miyakawa, Takuya; Kataoka, Michihiko; Nagai, Takahiro; Kitamura, Nahoko; Urano, Nobuyuki; Maruoka, Shintaro; Ohtsuka, Jun; Nagata, Koji; Shimizu, Sakayu; Tanokura, Masaru

    2014-01-01

    Conjugated polyketone reductase C2 (CPR-C2) from Candida parapsilosis IFO 0708, identified as a nicotinamide adenine dinucleotide phosphate (NADPH)-dependent ketopantoyl lactone reductase, belongs to the aldo-keto reductase superfamily. This enzyme reduces ketopantoyl lactone to D-pantoyl lactone in a strictly stereospecific manner. To elucidate the structural basis of the substrate specificity, we determined the crystal structures of the apo CPR-C2 and CPR-C2/NADPH complex at 1.70 and 1.80 Å resolutions, respectively. CPR-C2 adopted a triose-phosphate isomerase barrel fold at the core of the structure. Binding with the cofactor NADPH induced conformational changes in which Thr27 and Lys28 moved 15 and 5.0 Å, respectively, in the close vicinity of the adenosine 2'-phosphate group of NADPH to form hydrogen bonds. Based on the comparison of the CPR-C2/NADPH structure with 3-α-hydroxysteroid dehydrogenase and mutation analyses, we constructed substrate binding models with ketopantoyl lactone, which provided insight into the substrate specificity by the cofactor-induced structure. The results will be useful for the rational design of CPR-C2 mutants targeted for use in the industrial manufacture of ketopantoyl lactone.

  17. Detoxification of hexavalent chromium by Leucobacter sp. uses a reductase with specificity for dihydrolipoamide.

    Science.gov (United States)

    Sarangi, Abhipsa; Krishnan, Chandraraj

    2016-02-01

    Leucobacter sp. belongs to the metal stressed community and possesses higher tolerance to metals including chromium and can detoxify toxic hexavalent chromium by reduction to less toxic trivalent chromium. But, the mechanism of reduction of hexavalent chromium by Leucobacter sp. has not been studied. Understanding the enzyme catalyzing reduction of chromium is important to improve the species for application in bioremediation. Hence, a soluble reductase catalyzing the reduction of hexavalent chromium was purified from a Leucobacter sp. and characterized. The pure chromate reductase was obtained from the cell-free extract through hydrophobic interaction and gel filtration column chromatographic methods. It was a monomeric enzyme and showed similar molecular weights in both gel filtration (∼68 KDa) and SDS-PAGE (64 KDa). It reduced Cr(VI) using both NADH and NADPH as the electron donor, but exhibited higher activity with NADH. The optimal activity was found at pH 5.5 and 30 °C. The K(m) and V(max) for Cr(VI) reduction with NADH were 46.57 μM and 0.37 μmol min(-1) (mg protein) (-1), respectively. The activity was inhibited by p-hydroxy mercury benzoate, Ag(2+) and Hg(2+) indicating the role of thiol groups in the catalysis. The spectrophotometric analysis of the purified enzyme showed the absence of bound flavin in the enzyme. The N-terminal amino acid sequence and LC/MS analysis of trypsin digested purified enzyme showed similarity to dihydrolipoyl dehydrogenase. The purified enzyme had dihydrolipoyl dehydrogenase activity with dihydrolipoamide as the substrate, which suggested that Leucobacter sp. uses reductase with multiple substrate specificity for reduction of Cr(VI) detoxification. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Expression, purification, crystallization and X-ray analysis of 3-quinuclidinone reductase from Agrobacterium tumefaciens

    International Nuclear Information System (INIS)

    Hou, Feng; Miyakawa, Takuya; Takeshita, Daijiro; Kataoka, Michihiko; Uzura, Atsuko; Nagata, Koji; Shimizu, Sakayu; Tanokura, Masaru

    2012-01-01

    The purification and crystallization of 3-quinuclidinone reductase from A. tumefaciens allowed the collection of a diffraction data set to 1.72 Å resolution. (R)-3-Quinuclidinol is a useful chiral building block for the synthesis of various pharmaceuticals and can be produced from 3-quinuclidinone by asymmetric reduction. A novel 3-quinuclidinone reductase from Agrobacterium tumefaciens (AtQR) catalyzes the stereospecific reduction of 3-quinuclidinone to (R)-3-quinuclidinol with NADH as a cofactor. Recombinant AtQR was overexpressed in Escherichia coli, purified and crystallized with NADH using the sitting-drop vapour-diffusion method at 293 K. Crystals were obtained using a reservoir solution containing PEG 3350 as a precipitant. X-ray diffraction data were collected to 1.72 Å resolution on beamline BL-5A at the Photon Factory. The crystal belonged to space group P2 1 , with unit-cell parameters a = 62.0, b = 126.4, c = 62.0 Å, β = 110.5°, and was suggested to contain four molecules in the asymmetric unit (V M = 2.08 Å 3 Da −1 )

  19. Determination of the potency of a novel saw palmetto supercritical CO2 extract (SPSE for 5α-reductase isoform II inhibition using a cell-free in vitro test system

    Directory of Open Access Journals (Sweden)

    Pais P

    2016-04-01

    Full Text Available Pilar Pais, Agustí Villar, Santiago Rull Euromed, Barcelona, Spain Background: The nicotinamide adenine dinucleotide phosphate-dependent membrane protein 5α-reductase catalyses the conversion of testosterone to the most potent androgen – 5α-dihydrotestosterone. Two 5α-reductase isoenzymes are expressed in humans: type I and type II. The latter is found primarily in prostate tissue. Saw palmetto extract (SPE has been used extensively in the treatment of lower urinary tract symptoms secondary to benign prostatic hyperplasia (BPH. The pharmacological effects of SPE include the inhibition of 5α-reductase, as well as anti-inflammatory and antiproliferative effects. Clinical studies of SPE have been inconclusive – some have shown significant results, and others have not – possibly the result of varying bioactivities of the SPEs used in the studies. Purpose: To determine the in vitro potency in a cell-free test system of a novel SP supercritical CO2 extract (SPSE, an inhibitor of the 5α-reductase isoenzyme type II. Materials and methods: The inhibitory potency of SPSE was compared to that of finasteride, an approved 5α-reductase inhibitor, on the basis of the enzymatic conversion of the substrate androstenedione to the 5α-reduced product 5α-androstanedione. Results: By concentration-dependent inhibition of 5α-reductase type II in vitro (half-maximal inhibitory concentration 3.58±0.05 µg/mL, SPSE demonstrated competitive binding toward the active site of the enzyme. Finasteride, the approved 5α-reductase inhibitor tested as positive control, led to 63%–75% inhibition of 5α-reductase type II. Conclusion: SPSE effectively inhibits the enzyme that has been linked to BPH, and the amount of extract required for activity is comparatively low. It can be confirmed from the results of this study that SPSE has bioactivity that promotes prostate health at a level that is superior to that of many other phytotherapeutic extracts. The

  20. Modulation of biosynthesis and regulatory action of 24(S),25-epoxycholesterol (S-EC) in cultured cells by progesterone (PG)

    International Nuclear Information System (INIS)

    Panini, S.R.; Gupta, A.K.; Sexton, R.C.; Parish, E.J.; Rudney, H.

    1987-01-01

    Treatment of IEC-6 cells with PG caused a strong inhibition of cholesterol biosynthesis at the level of desmosterol reductase. In addition, two new products were observed in PG-treated cells. The first compound was designated as cholesta-5,7,24-trien-3β-ol based on its HPLC chromatographic properties. The second compound was identified as S-EC based on (1) a comparison of its chromatographic properties with those of authentic EC and (2) by its conversion to 25-hydroxycholesterol (HC) upon reduction with LiAlH 4 . In spite of cellular accumulation of S-EC in the presence of PG, the activity of HMG-CoA reductase (HMGR) which is known to be sensitive to oxysterols, was elevated rather than suppressed. On the other hand, when PG-treated cells were refed fresh medium without PG, HMGR activity was suppressed. Exogenous S-EC was a potent suppressor of HMGR in untreated IEC-6 cells. Suppression of HMGR by S-EC but not by HC could be prevented by progesterone. Exogenous [ 3 H]S-EC was not metabolized by IEC-6 cells. These results support the hypothesis that S-EC plays a normal regulatory role in sterol biosynthesis and indicate that enhanced S-EC synthesis observed in the presence of PG may be due to interference with this regulatory action

  1. Laboratoire de Chimie Bactérienne C.N.R.S., Marsielle, France.

    Science.gov (United States)

    Chippaux, M; Giudici, D; Abou-Jaoudé, A; Casse, F; Pascal, M C

    1978-04-06

    Mutants of E. coli, completely devoid of nitrite reductase activity with glucose or formate as donor were studied. Biochemical analysis indicates that they are simultaneously affected in nitrate reductase, nitrite reductase, fumarate reductase and hydrogenase activities as well as in cytochrome C552 biosynthesis. The use of an antiserum specific for nitrate reductase shows that the nitrate reductase protein is probably missing. A single mutation is responsible for this phenotype: the gene affected, nir R, is located close to tyr R i.e. at 29 min on the chromosomal map.

  2. Expression of the thioredoxin-thioredoxin reductase system in the inflamed joints of patients with rheumatoid arthritis

    NARCIS (Netherlands)

    Maurice, M. M.; Nakamura, H.; Gringhuis, S.; Okamoto, T.; Yoshida, S.; Kullmann, F.; Lechner, S.; van der Voort, E. A.; Leow, A.; Versendaal, J.; Muller-Ladner, U.; Yodoi, J.; Tak, P. P.; Breedveld, F. C.; Verweij, C. L.

    1999-01-01

    OBJECTIVE: To examine the expression of the thioredoxin (TRX)-thioredoxin reductase (TR) system in patients with rheumatoid arthritis (RA) and patients with other rheumatic diseases. METHODS: Levels of TRX in plasma and synovial fluid (SF) were measured using enzyme-linked immunosorbent assay.

  3. Production and characterization of a thermostable alcohol dehydrogenase that belongs to the aldo-keto reductase superfamily

    NARCIS (Netherlands)

    Machielsen, M.P.; Uria, A.R.; Kengen, S.W.M.; Oost, van der J.

    2006-01-01

    The gene encoding a novel alcohol dehydrogenase that belongs to the aldo-keto reductase superfamily has been identified in the hyperthermophilic archaeon Pyrococcus furiosus. The gene, referred to as adhD, was functionally expressed in Escherichia coli and subsequently purified to homogeneity. The

  4. Degradation of S-nitrosocysteine in vascular tissue homogenates: role of divalent ions.

    Science.gov (United States)

    Kostka, P; Xu, B; Skiles, E H

    1999-04-01

    The objective of the study was to inquire about the mechanism(s) involved in the catabolism of S-nitrosothiols by vascular tissue under in vitro conditions. Incubations of S-nitrosocysteine (CYSNO) or S-nitrosoglutathione (GSNO) with homogenates isolated from porcine aortic smooth muscle resulted in only a marginal depletion of S-nitrosothiols from the reaction mixtures, which became statistically significant at relatively high concentrations of homogenate (> or =300 microg of protein/ml). Degradation of CYSNO (but not GSNO) was found to be potentiated several-fold by millimolar concentrations of either Mg2+ or Ca2+ ions. Under such conditions, the degradation of CYSNO was significantly suppressed by the removal of proteins by ultrafiltration (>80% inhibition) and eliminated completely by the alkylation of thiol groups with 1 mM N-ethylmaleimide. The potentiating effect of divalent ions on the degradation of CYSNO was insensitive to 0.1 mM neocuproine (selective chelator of Cu+ ions), although it was enhanced in the presence of 0.1 mM o-phenanthroline (selective chelator of Fe2+ ions). It is concluded that the degradation of CYSNO by tissue homogenate involves the interaction with protein-bound sulfhydryl groups, which is stimulated by Mg2+ or Ca2+ ions. The potentiating effect of o-phenanthroline suggests that the liberation of the nitrosonium moiety in such a process may be accompanied by its transfer to sulfur center(s) by transient formation of dinitrosyl-iron complexes.

  5. 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.

  6. Determination of the potency of a novel saw palmetto supercritical CO2 extract (SPSE) for 5α-reductase isoform II inhibition using a cell-free in vitro test system.

    Science.gov (United States)

    Pais, Pilar; Villar, Agustí; Rull, Santiago

    2016-01-01

    The nicotinamide adenine dinucleotide phosphate-dependent membrane protein 5α-reductase catalyses the conversion of testosterone to the most potent androgen - 5α-dihydrotestosterone. Two 5α-reductase isoenzymes are expressed in humans: type I and type II. The latter is found primarily in prostate tissue. Saw palmetto extract (SPE) has been used extensively in the treatment of lower urinary tract symptoms secondary to benign prostatic hyperplasia (BPH). The pharmacological effects of SPE include the inhibition of 5α-reductase, as well as anti-inflammatory and antiproliferative effects. Clinical studies of SPE have been inconclusive - some have shown significant results, and others have not - possibly the result of varying bioactivities of the SPEs used in the studies. To determine the in vitro potency in a cell-free test system of a novel SP supercritical CO2 extract (SPSE), an inhibitor of the 5α-reductase isoenzyme type II. The inhibitory potency of SPSE was compared to that of finasteride, an approved 5α-reductase inhibitor, on the basis of the enzymatic conversion of the substrate androstenedione to the 5α-reduced product 5α-androstanedione. By concentration-dependent inhibition of 5α-reductase type II in vitro (half-maximal inhibitory concentration 3.58±0.05 μg/mL), SPSE demonstrated competitive binding toward the active site of the enzyme. Finasteride, the approved 5α-reductase inhibitor tested as positive control, led to 63%-75% inhibition of 5α-reductase type II. SPSE effectively inhibits the enzyme that has been linked to BPH, and the amount of extract required for activity is comparatively low. It can be confirmed from the results of this study that SPSE has bioactivity that promotes prostate health at a level that is superior to that of many other phytotherapeutic extracts. The bioactivity of SPSE corresponds favorably to that reported for the hexane extract used in a large number of positive BPH clinical trials, as well as to finasteride

  7. Constitutive expression of nitrate reductase allows normal growth and development of Nicotiana plumbaginifolia plants.

    Science.gov (United States)

    Vincentz, M; Caboche, M

    1991-01-01

    A nitrate reductase (NR) deficient mutant of Nicotiana plumbaginifolia totally impaired in the production of NR transcript and protein was restored for NR activity by transformation with a chimaeric NR gene. This gene was composed of a full-length tobacco NR cDNA fused to the CaMV 35S promoter and to termination signals from the tobacco NR gene. The transgenic plants we obtained were viable and fertile and expressed from one-fifth to three times the wild-type NR activity in their leaves. The analysis of chimeric NR gene expression in these plants showed, by comparison with wild-type plants, that the regulation of NR gene expression by light, nitrate and circadian rhythm takes place at the transcriptional level. However, unlike nitrate, light was required for the accumulation of NR protein in transgenic plants, suggesting that NR expression is also controlled at the translational and/or post-translational level. Images PMID:2022181

  8. Glutathione-S-transferase-omega [MMA(V) reductase] knockout mice: Enzyme and arsenic species concentrations in tissues after arsenate administration

    International Nuclear Information System (INIS)

    Chowdhury, Uttam K.; Zakharyan, Robert A.; Hernandez, Alba; Avram, Mihaela D.; Kopplin, Michael J.; Aposhian, H. Vasken

    2006-01-01

    Inorganic arsenic is a human carcinogen to which millions of people are exposed via their naturally contaminated drinking water. Its molecular mechanisms of carcinogenicity have remained an enigma, perhaps because arsenate is biochemically transformed to at least five other arsenic-containing metabolites. In the biotransformation of inorganic arsenic, GSTO1 catalyzes the reduction of arsenate, MMA(V), and DMA(V) to the more toxic + 3 arsenic species. MMA(V) reductase and human (hGSTO1-1) are identical proteins. The hypothesis that GST-Omega knockout mice biotransformed inorganic arsenic differently than wild-type mice has been tested. The livers of male knockout (KO) mice, in which 222 bp of Exon 3 of the GSTO1 gene were eliminated, were analyzed by PCR for mRNA. The level of transcripts of the GSTO1 gene in KO mice was 3.3-fold less than in DBA/1lacJ wild-type (WT) mice. The GSTO2 transcripts were about two-fold less in the KO mouse. When KO and WT mice were injected intramuscularly with Na arsenate (4.16 mg As/kg body weight); tissues removed at 0.5, 1, 2, 4, 8, and 12 h after arsenate injection; and the arsenic species measured by HPLC-ICP-MS, the results indicated that the highest concentration of the recently discovered and very toxic MMA(III), a key biotransformant, was in the kidneys of both KO and WT mice. The highest concentration of DMA(III) was in the urinary bladder tissue for both the KO and WT mice. The MMA(V) reducing activity of the liver cytosol of KO mice was only 20% of that found in wild-type mice. There appears to be another enzyme(s) other than GST-O able to reduce arsenic(V) species but to a lesser extent. This and other studies suggest that each step of the biotransformation of inorganic arsenic has an alternative enzyme to biotransform the arsenic substrate

  9. A novel role of the ferric reductase Cfl1 in cell wall integrity, mitochondrial function, and invasion to host cells in Candida albicans.

    Science.gov (United States)

    Yu, Qilin; Dong, Yijie; Xu, Ning; Qian, Kefan; Chen, Yulu; Zhang, Biao; Xing, Laijun; Li, Mingchun

    2014-11-01

    Candida albicans is an important opportunistic pathogen, causing both superficial mucosal infections and life-threatening systemic diseases. Iron acquisition is an important factor for pathogen-host interaction and also a significant element for the pathogenicity of this organism. Ferric reductases, which convert ferric iron into ferrous iron, are important components of the high-affinity iron uptake system. Sequence analyses have identified at least 17 putative ferric reductase genes in C. albicans genome. CFL1 was the first ferric reductase identified in C. albicans. However, little is known about its roles in C. albicans physiology and pathogenicity. In this study, we found that disruption of CFL1 led to hypersensitivity to chemical and physical cell wall stresses, activation of the cell wall integrity (CWI) pathway, abnormal cell wall composition, and enhanced secretion, indicating a defect in CWI in this mutant. Moreover, this mutant showed abnormal mitochondrial activity and morphology, suggesting a link between ferric reductases and mitochondrial function. In addition, this mutant displayed decreased ability of adhesion to both the polystyrene microplates and buccal epithelial cells and invasion of host epithelial cells. These findings revealed a novel role of C. albicans Cfl1 in maintenance of CWI, mitochondrial function, and interaction between this pathogen and the host. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  10. Crystallization and preliminary X-ray crystallographic analysis of enoyl-ACP reductase III (FabL) from Bacillus subtilis

    International Nuclear Information System (INIS)

    Kim, Kook-Han; Park, Joon Kyu; Ha, Byung Hak; Moon, Jin Ho; Kim, Eunice EunKyeong

    2007-01-01

    Enoyl-ACP reductase III (FabL) from B. subtilis has been overexpressed, purified and crystallized. The crystal belongs to space group P622, with unit-cell parameters a = b = 139.56, c = 62.75 Å, α = β = 90, γ = 120°, and data were collected to 2.5 Å resolution using synchrotron radiation. Enoyl-[acyl-carrier protein] reductase (enoyl-ACP reductase; ENR) is a key enzyme in type II fatty-acid synthase that catalyzes the last step in each elongation cycle. It has been considered as an antibiotic target since it is an essential enzyme in bacteria. However, recent studies indicate that some pathogens have more than one ENR. Bacillus subtilis is reported to have two ENRs, namely BsFabI and BsFabL. While BsFabI is similar to other FabIs, BsFabL shows very little sequence similarity and is NADPH-dependent instead of NADH-dependent as in the case of FabI. In order to understand these differences on a structural basis, BsFabL has been cloned, expressed and and crystallized. The crystal belongs to space group P622, with unit-cell parameters a = b = 139.56, c = 62.75 Å, α = β = 90, γ = 120° and one molecule of FabL in the asymmetric unit. Data were collected using synchrotron radiation (beamline 4A at the Pohang Light Source, Korea). The crystal diffracted to 2.5 Å resolution

  11. A QM/MM–Based Computational Investigation on the Catalytic Mechanism of Saccharopine Reductase

    OpenAIRE

    Almasi, Joel N.; Bushnell, Eric A.C.; Gauld, James W.

    2011-01-01

    Saccharopine reductase from Magnaporthe grisea, an NADPH-containing enzyme in the α-aminoadipate pathway, catalyses the formation of saccharopine, a precursor to L-lysine, from the substrates glutamate and α-aminoadipate-δ-semialdehyde. Its catalytic mechanism has been investigated using quantum mechanics/molecular mechanics (QM/MM) ONIOM-based approaches. In particular, the overall catalytic pathway has been elucidated and the effects of electron correlation and the anisotropic polar protein...

  12. Loss of HMG-CoA reductase in C. elegans causes defects in protein prenylation and muscle mitochondria.

    Directory of Open Access Journals (Sweden)

    Parmida Ranji

    Full Text Available HMG-CoA reductase is the rate-limiting enzyme in the mevalonate pathway and the target of cholesterol-lowering statins. We characterized the C. elegans hmgr-1(tm4368 mutant, which lacks HMG-CoA reductase, and show that its phenotypes recapitulate that of statin treatment, though in a more severe form. Specifically, the hmgr-1(tm4368 mutant has defects in growth, reproduction and protein prenylation, is rescued by exogenous mevalonate, exhibits constitutive activation of the UPRer and requires less mevalonate to be healthy when the UPRmt is activated by a constitutively active form of ATFS-1. We also show that different amounts of mevalonate are required for different physiological processes, with reproduction requiring the highest levels. Finally, we provide evidence that the mevalonate pathway is required for the activation of the UPRmt.

  13. Analysis of dissimilatory sulfite reductase and 16S rRNA gene fragments from deep-sea hydrothermal sites of the Suiyo Seamount, Izu-Bonin Arc, Western Pacific.

    Science.gov (United States)

    Nakagawa, Tatsunori; Ishibashi, Jun-Ichiro; Maruyama, Akihiko; Yamanaka, Toshiro; Morimoto, Yusuke; Kimura, Hiroyuki; Urabe, Tetsuro; Fukui, Manabu

    2004-01-01

    This study describes the occurrence of unique dissimilatory sulfite reductase (DSR) genes at a depth of 1,380 m from the deep-sea hydrothermal vent field at the Suiyo Seamount, Izu-Bonin Arc, Western Pacific, Japan. The DSR genes were obtained from microbes that grew in a catheter-type in situ growth chamber deployed for 3 days on a vent and from the effluent water of drilled holes at 5 degrees C and natural vent fluids at 7 degrees C. DSR clones SUIYOdsr-A and SUIYOdsr-B were not closely related to cultivated species or environmental clones. Moreover, samples of microbial communities were examined by PCR-denaturing gradient gel electrophoresis (DGGE) analysis of the 16S rRNA gene. The sequence analysis of 16S rRNA gene fragments obtained from the vent catheter after a 3-day incubation revealed the occurrence of bacterial DGGE bands affiliated with the Aquificae and gamma- and epsilon-Proteobacteria as well as the occurrence of archaeal phylotypes affiliated with the Thermococcales and of a unique archaeon sequence that clustered with "Nanoarchaeota." The DGGE bands obtained from drilled holes and natural vent fluids from 7 to 300 degrees C were affiliated with the delta-Proteobacteria, genus Thiomicrospira, and Pelodictyon. The dominant DGGE bands retrieved from the effluent water of casing pipes at 3 and 4 degrees C were closely related to phylotypes obtained from the Arctic Ocean. Our results suggest the presence of microorganisms corresponding to a unique DSR lineage not detected previously from other geothermal environments.

  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 H 2 O 2 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. Cloning and sequence analysis demonstrate the chromate reduction ability of a novel chromate reductase gene from Serratia sp.

    Science.gov (United States)

    Deng, Peng; Tan, Xiaoqing; Wu, Ying; Bai, Qunhua; Jia, Yan; Xiao, Hong

    2015-03-01

    The ChrT gene encodes a chromate reductase enzyme which catalyzes the reduction of Cr(VI). The chromate reductase is also known as flavin mononucleotide (FMN) reductase (FMN_red). The aim of the present study was to clone the full-length ChrT DNA from Serratia sp. CQMUS2 and analyze the deduced amino acid sequence and three-dimensional structure. The putative ChrT gene fragment of Serratia sp. CQMUS2 was isolated by polymerase chain reaction (PCR), according to the known FMN_red gene sequence from Serratia sp. AS13. The flanking sequences of the ChrT gene were obtained by high efficiency TAIL-PCR, while the full-length gene of ChrT was cloned in Escherichia coli for subsequent sequencing. The nucleotide sequence of ChrT was submitted onto GenBank under the accession number, KF211434. Sequence analysis of the gene and amino acids was conducted using the Basic Local Alignment Search Tool, and open reading frame (ORF) analysis was performed using ORF Finder software. The ChrT gene was found to be an ORF of 567 bp that encodes a 188-amino acid enzyme with a calculated molecular weight of 20.4 kDa. In addition, the ChrT protein was hypothesized to be an NADPH-dependent FMN_red and a member of the flavodoxin-2 superfamily. The amino acid sequence of ChrT showed high sequence similarity to the FMN reductase genes of Klebsiella pneumonia and Raoultella ornithinolytica , which belong to the flavodoxin-2 superfamily. Furthermore, ChrT was shown to have a 85.6% similarity to the three-dimensional structure of Escherichia coli ChrR, sharing four common enzyme active sites for chromate reduction. Therefore, ChrT gene cloning and protein structure determination demonstrated the ability of the gene for chromate reduction. The results of the present study provide a basis for further studies on ChrT gene expression and protein function.

  16. Cloning and sequence analysis demonstrate the chromate reduction ability of a novel chromate reductase gene from Serratia sp

    Science.gov (United States)

    DENG, PENG; TAN, XIAOQING; WU, YING; BAI, QUNHUA; JIA, YAN; XIAO, HONG

    2015-01-01

    The ChrT gene encodes a chromate reductase enzyme which catalyzes the reduction of Cr(VI). The chromate reductase is also known as flavin mononucleotide (FMN) reductase (FMN_red). The aim of the present study was to clone the full-length ChrT DNA from Serratia sp. CQMUS2 and analyze the deduced amino acid sequence and three-dimensional structure. The putative ChrT gene fragment of Serratia sp. CQMUS2 was isolated by polymerase chain reaction (PCR), according to the known FMN_red gene sequence from Serratia sp. AS13. The flanking sequences of the ChrT gene were obtained by high efficiency TAIL-PCR, while the full-length gene of ChrT was cloned in Escherichia coli for subsequent sequencing. The nucleotide sequence of ChrT was submitted onto GenBank under the accession number, KF211434. Sequence analysis of the gene and amino acids was conducted using the Basic Local Alignment Search Tool, and open reading frame (ORF) analysis was performed using ORF Finder software. The ChrT gene was found to be an ORF of 567 bp that encodes a 188-amino acid enzyme with a calculated molecular weight of 20.4 kDa. In addition, the ChrT protein was hypothesized to be an NADPH-dependent FMN_red and a member of the flavodoxin-2 superfamily. The amino acid sequence of ChrT showed high sequence similarity to the FMN reductase genes of Klebsiella pneumonia and Raoultella ornithinolytica, which belong to the flavodoxin-2 superfamily. Furthermore, ChrT was shown to have a 85.6% similarity to the three-dimensional structure of Escherichia coli ChrR, sharing four common enzyme active sites for chromate reduction. Therefore, ChrT gene cloning and protein structure determination demonstrated the ability of the gene for chromate reduction. The results of the present study provide a basis for further studies on ChrT gene expression and protein function. PMID:25667630

  17. High-fat diet-induced changes in liver thioredoxin and thioredoxin reductase as a novel feature of insulin resistance

    Directory of Open Access Journals (Sweden)

    Huijun Qin

    2014-01-01

    Full Text Available High-fat diet (HFD can induce oxidative stress. Thioredoxin (Trx and thioredoxin reductase (TrxR are critical antioxidant proteins but how they are affected by HFD remains unclear. Using HFD-induced insulin-resistant mouse model, we show here that liver Trx and TrxR are significantly decreased, but, remarkably, the degree of their S-acylation is increased after consuming HFD. These HFD-induced changes in Trx/TrxR may reflect abnormalities of lipid metabolism and insulin signaling transduction. HFD-driven accumulation of 4-hydroxynonenal is another potential mechanism behind inactivation and decreased expression of Trx/TrxR. Thus, we propose HFD-induced impairment of liver Trx/TrxR as major contributor to oxidative stress and as a novel feature of insulin resistance.

  18. Expression of Xanthophyllomyces dendrorhous cytochrome-P450 hydroxylase and reductase in Mucor circinelloides.

    Science.gov (United States)

    Csernetics, Árpád; Tóth, Eszter; Farkas, Anita; Nagy, Gábor; Bencsik, Ottó; Vágvölgyi, Csaba; Papp, Tamás

    2015-02-01

    Carotenoids are natural pigments that act as powerful antioxidants and have various beneficial effects on human and animal health. Mucor circinelloides (Mucoromycotina) is a carotenoid producing zygomycetes fungus, which accumulates β-carotene as the main carotenoid but also able to produce the hydroxylated derivatives of β-carotene (i.e. zeaxanthin and β-cryptoxanthin) in low amount. These xanthophylls, together with the ketolated derivatives of β-carotene (such as canthaxanthin, echinenone and astaxanthin) have better antioxidant activity than β-carotene. In this study our aim was to modify and enhance the xanthophyll production of the M. circinelloides by expression of heterologous genes responsible for the astaxanthin biosynthesis. The crtS and crtR genes, encoding the cytochrome-P450 hydroxylase and reductase, respectively, of wild-type and astaxanthin overproducing mutant Xanthophyllomyces dendrorhous strains were amplified from cDNA and the nucleotide and the deduced amino acid sequences were compared to each other. Introduction of the crtS on autonomously replicating plasmid in the wild-type M. circinelloides resulted enhanced zeaxanthin and β-cryptoxanthin accumulation and the presence of canthaxanthin, echinenone and astaxanthin in low amount; the β-carotene hydroxylase and ketolase activity of the X. dendrorhous cytochrome-P450 hydroxylase in M. circinelloides was verified. Increased canthaxanthin and echinenone production was observed by expression of the gene in a canthaxanthin producing mutant M. circinelloides. Co-expression of the crtR and crtS genes led to increase in the total carotenoid and slight change in xanthophyll accumulation in comparison with transformants harbouring the single crtS gene.

  19. Spectroscopic and computational study of a nonheme iron nitrosyl center in a biosynthetic model of nitric oxide reductase.

    Science.gov (United States)

    Chakraborty, Saumen; Reed, Julian; Ross, Matthew; Nilges, Mark J; Petrik, Igor D; Ghosh, Soumya; Hammes-Schiffer, Sharon; Sage, J Timothy; Zhang, Yong; Schulz, Charles E; Lu, Yi

    2014-02-24

    A major barrier to understanding the mechanism of nitric oxide reductases (NORs) is the lack of a selective probe of NO binding to the nonheme FeB center. By replacing the heme in a biosynthetic model of NORs, which structurally and functionally mimics NORs, with isostructural ZnPP, the electronic structure and functional properties of the FeB nitrosyl complex was probed. This approach allowed observation of the first S=3/2 nonheme {FeNO}(7) complex in a protein-based model system of NOR. Detailed spectroscopic and computational studies show that the electronic state of the {FeNO}(7) complex is best described as a high spin ferrous iron (S=2) antiferromagnetically coupled to an NO radical (S=1/2) [Fe(2+)-NO(.)]. The radical nature of the FeB -bound NO would facilitate N-N bond formation by radical coupling with the heme-bound NO. This finding, therefore, supports the proposed trans mechanism of NO reduction by NORs. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Cloning, purification, crystallization and preliminary X-ray analysis of a chimeric NADPH-cytochrome P450 reductase

    International Nuclear Information System (INIS)

    Aigrain, Louise; Pompon, Denis; Truan, Gilles; Moréra, Solange

    2009-01-01

    A 2.5 Å resolution data set was collected from a crystal of a soluble chimeric form of NADPH-cytochrome P450 reductase (CPR) produced using a fusion gene composed of the yeast FMN and the human FAD domains. The chimeric protein was crystallized in a modified conformation compared with the previously solved structures. NADPH-cytochrome P450 reductase (CPR) is the favoured redox partner of microsomal cytochromes P450. This protein is composed of two flavin-containing domains (FMN and FAD) connected by a structured linker. An active CPR chimera consisting of the yeast FMN and human FAD domains has been produced, purified and crystallized. The crystals belonged to the monoclinic space group C2 and contained one molecule per asymmetric unit. Molecular replacement was performed using the published rat and yeast structures as search models. The initial electron-density maps revealed that the chimeric enzyme had crystallized in a conformation that differed from those of previously solved structures

  1. HMG-CoA reductase inhibitors, other lipid-lowering medication, antiplatelet therapy, and the risk of venous thrombosis

    NARCIS (Netherlands)

    Ramcharan, A.S.; van Stralen, K.J.; Snoep, J.D.; Mantel-Teeuwisse, A.K.; Doggen, Catharina Jacoba Maria

    2009-01-01

    Background: Statins [3-hydroxymethyl-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors] and antiplatelet therapy reduce the risk of atherosclerotic disease. Besides a reduction of lipid levels, statins might also have antithrombotic and anti-inflammatory properties, and anti-platelet

  2. Stereochemistry of Furfural Reduction by a Saccharomyces cerevisiae Aldehyde Reductase That Contributes to In Situ Furfural Detoxification

    Science.gov (United States)

    Ari1p from Saccharomyces cerevisiae, recently identified as an intermediate subclass short-chain dehydrogenase/reductase, contributes in situ to the detoxification of furfural. Furfural inhibits efficient ethanol production by the yeast, particularly when the carbon source is acid-treated lignocell...

  3. Glucose-6-phosphate dehydrogenase and glutathione reductase activity in methemoglobin reduction by methylene blue and cyst amine: study on glucose-6-phosphate dehydrogenase-deficient individuals, on normal subjects and on riboflavin-treated subjects

    Directory of Open Access Journals (Sweden)

    Benedito Barraviera

    1988-10-01

    Full Text Available The authors have standardized methods for evaluation of the activity of the glucose-6-phosphate dehydrogenase and of glutathione reductase. The general principle of the first method was based on methemoglobin formation by sodium nitrite followed by stimulation of the glucose-6-phosphate dehydrogenase with methylene blue. Forty six adults (23 males and 23 females were studied. Subjects were not G6PD deficient and were aged 20 to 30 years. The results showed that methemoglobin reduction by methylene blue was 154.40 and 139.90 mg/min (p<0.05 for males and females, respectively, in whole blood, and 221.10 and 207.85 mg/min (n.s., respectively, in washed red cells. These data showed that using washed red cells and 0.7g% sodium nitrite concentration produced no differences between sexes and also shortened reading time for the residual amount of methemoglobin to 90 minutes. Glutathione reductase activity was evaluated on the basis of the fact that cystamine (a thiol agent binds to the SH groups of hemoglobin, forming complexes. These complexes are reversed by the action of glutathione reductase, with methemoglobin reduction occurring simultaneously with this reaction. Thirty two adults (16 males and 16 females were studied. Subjects were not G6PD deficient and were aged 20 to 30 years. Methemoglobin reduction by cystamine was 81.27 and 91.13 mg/min (p<0.01 for males and females, respectively. These data showed that using washed red cells and 0.1 M cystamine concentration permits a reading of the residual amount of methemoglobin at 180 minutes of incubation. Glutathione reductase activity was evaluated by methemoglobin reduction by cystamine in 14 females before and after treatment with 10 mg riboflavin per day for 8 days. The results were 73.69 and 94.26 jug/min (p<0.01 before and after treatment, showing that riboflavin treatment increase glutathione reductase activity even in normal individuals. Three Black G6PD-deficient individuals (2 males and 1

  4. A QM/MM–Based Computational Investigation on the Catalytic Mechanism of Saccharopine Reductase

    Directory of Open Access Journals (Sweden)

    James W. Gauld

    2011-10-01

    Full Text Available Saccharopine reductase from Magnaporthe grisea, an NADPH-containing enzyme in the α-aminoadipate pathway, catalyses the formation of saccharopine, a precursor to L-lysine, from the substrates glutamate and α-aminoadipate-δ-semialdehyde. Its catalytic mechanism has been investigated using quantum mechanics/molecular mechanics (QM/MM ONIOM-based approaches. In particular, the overall catalytic pathway has been elucidated and the effects of electron correlation and the anisotropic polar protein environment have been examined via the use of the ONIOM(HF/6-31G(d:AMBER94 and ONIOM(MP2/6-31G(d//HF/6-31G(d:AMBER94 methods within the mechanical embedding formulism and ONIOM(MP2/6-31G(d//HF/6-31G(d:AMBER94 and ONIOM(MP2/6-311G(d,p//HF/6-31G(d:AMBER94 within the electronic embedding formulism. The results of the present study suggest that saccharopine reductase utilises a substrate-assisted catalytic pathway in which acid/base groups within the cosubstrates themselves facilitate the mechanistically required proton transfers. Thus, the enzyme appears to act most likely by binding the three required reactant molecules glutamate, α-aminoadipate-δ-semialdehyde and NADPH in a manner and polar environment conducive to reaction.

  5. Ferredoxin-thioredoxin reductase: a catalytically active dithiol group links photoreduced ferredoxin to thioredoxin functional in photosynthetic enzyme regulation

    Energy Technology Data Exchange (ETDEWEB)

    Droux, M.; Miginiac-Maslow, M.; Jacquot, J.P.; Gadal, P.; Crawford, N.A.; Kosower, N.S.; Buchanan, B.B.

    1987-07-01

    The mechanism by which the ferredoxin-thioredoxin system activates the target enzyme, NADP-malate dehydrogenase, was investigated by analyzing the sulfhydryl status of individual protein components with (/sup 14/C)iodoacetate and monobromobimane. The data indicate that ferredoxin-thioredoxin reductase (FTR)--an iron-sulfur enzyme present in oxygenic photosynthetic organisms--is the first member of a thiol chain that links light to enzyme regulation. FTR possesses a catalytically active dithiol group localized on the 13 kDa (similar) subunit, that occurs in all species investigated and accepts reducing equivalents from photoreduced ferredoxin and transfers them stoichiometrically to the disulfide form of thioredoxin m. The reduced thioredoxin m, in turn, reduces NADP-malate dehydrogenase, thereby converting it from an inactive (S-S) to an active (SH) form. The means by which FTR is able to combine electrons (from photoreduced ferredoxin) with protons (from the medium) to reduce its active disulfide group remains to be determined.

  6. Ferredoxin-thioredoxin reductase: a catalytically active dithiol group links photoreduced ferredoxin to thioredoxin functional in photosynthetic enzyme regulation

    International Nuclear Information System (INIS)

    Droux, M.; Miginiac-Maslow, M.; Jacquot, J.P.; Gadal, P.; Crawford, N.A.; Kosower, N.S.; Buchanan, B.B.

    1987-01-01

    The mechanism by which the ferredoxin-thioredoxin system activates the target enzyme, NADP-malate dehydrogenase, was investigated by analyzing the sulfhydryl status of individual protein components with [ 14 C]iodoacetate and monobromobimane. The data indicate that ferredoxin-thioredoxin reductase (FTR)--an iron-sulfur enzyme present in oxygenic photosynthetic organisms--is the first member of a thiol chain that links light to enzyme regulation. FTR possesses a catalytically active dithiol group localized on the 13 kDa (similar) subunit, that occurs in all species investigated and accepts reducing equivalents from photoreduced ferredoxin and transfers them stoichiometrically to the disulfide form of thioredoxin m. The reduced thioredoxin m, in turn, reduces NADP-malate dehydrogenase, thereby converting it from an inactive (S-S) to an active (SH) form. The means by which FTR is able to combine electrons (from photoreduced ferredoxin) with protons (from the medium) to reduce its active disulfide group remains to be determined

  7. Structure and Mechanism of Styrene Monooxygenase Reductase: New Insight into the FAD–Transfer Reaction†

    Science.gov (United States)

    Morrison, Eliot; Kantz, Auric; Gassner, George T.; Sazinsky, Matthew H.

    2013-01-01

    The two–component flavoprotein styrene monooxygenase (SMO) from Pseudomonas putida S12 catalyzes the NADH– and FAD–dependent epoxidation of styrene to styrene oxide. In this study we investigate the mechanism of flavin reduction and transfer from the reductase (SMOB) to epoxidase (NSMOA) component and report our findings in light of the 2.2–Å crystal structure of SMOB. Upon rapidly mixing with NADH, SMOB forms an NADH→FADox charge–transfer intermediate and catalyzes a hydride–transfer reaction from NADH to FAD, with a rate constant of 49.1 ± 1.4 s−1, in a step that is coupled to the rapid dissociation of NAD+. Electrochemical and equilibrium–binding studies indicate that NSMOA binds FADhq ~13–times more tightly than SMOB, which supports a vectoral transfer of FADhq from the reductase to the epoxidase. After binding to NSMOA, FADhq rapidly reacts with molecular oxygen to form a stable C(4a)–hydroperoxide intermediate. The half–life of apoSMOB generated in the FAD–transfer reaction is increased ~21–fold, supporting the model of a protein–protein interaction between apoSMOB and NSMOA with the peroxide intermediate. The mechanisms of FAD–dissociation and transport from SMOB to NSMOA were probed by monitoring the competitive reduction of cytochrome c in the presence and absence of pyridine nucleotides. Based on these studies, we propose a model in which reduced FAD binds to SMOB in equilibrium between an unreactive, sequestered state (S–state) and more reactive, transfer state (T–state). Dissociation of NAD+ after the hydride transfer–reaction transiently populates the T–state, promoting the transfer of FADhq to NSMOA. The binding of pyridine nucleotides to SMOB–FADhq shifts the FADhq–binding equilibrium from the T–state to the S–state. Additionally, the 2.2–Å crystal structure of SMOB–FADox reported in this work is discussed in light of the pyridine nucleotide–gated flavin–transfer and electron

  8. Expression, purification, crystallization and preliminary X-ray analysis of conjugated polyketone reductase C2 (CPR-C2) from Candida parapsilosis IFO 0708

    International Nuclear Information System (INIS)

    Yamamura, Akihiro; Maruoka, Shintaro; Ohtsuka, Jun; Miyakawa, Takuya; Nagata, Koji; Kataoka, Michihiko; Kitamura, Nahoko; Shimizu, Sakayu; Tanokura, Masaru

    2009-01-01

    Conjugated polyketone reductase C2 from C. parapsilosis IFO 0708 was expressed, purified and crystallized by the sitting-drop vapour-diffusion method. The crystal belonged to space group P2 1 2 1 2 1 and diffracted X-rays to 1.7 Å resolution. Conjugated polyketone reductase C2 (CPR-C2) from Candida parapsilosis IFO 0708 is a member of the NADPH-dependent aldo-keto reductase (AKR) superfamily and catalyzes the stereospecific reduction of ketopantoyl lactone to d-pantoyl lactone. A diffraction-quality crystal of recombinant CPR-C2 was obtained by the sitting-drop vapour-diffusion method using PEG 3350 as the precipitant. The crystal diffracted X-rays to 1.7 Å resolution on beamline NW12A of the Photon Factory-Advanced Ring (Tsukuba, Japan). The crystal belonged to space group P2 1 2 1 2 1 , with unit-cell parameters a = 55.02, b = 68.30, c = 68.93 Å. The Matthews coefficient (V M = 1.76 Å 3 Da −1 ) indicated that the crystal contained one CPR-C2 molecule per asymmetric unit

  9. Ferric reductase activity of low molecular weight human milk fraction is associated with enhanced iron solubility and uptake in Caco-2 cells.

    Science.gov (United States)

    Pullakhandam, Raghu; Nair, Madhavan Krishnapillai; Kasula, Sunanda; Kilari, Sreenivasulu; Thippande, Tippeswamy Gowda

    2008-09-19

    It is known that the fractional absorption of extrinsic iron from human milk is higher in infants and adults. A low molecular weight milk fraction has been proposed to increase the bioavailability of iron from human milk. Nevertheless, the mechanisms remained elusive. Here in we demonstrate ferric reductase activity (Km7.73x10(-6)M) in low molecular weight human milk fraction (10kF, filtrate derived from ultra filtration of milk whey through 10kDa cutoff membrane), which increased ferric iron solubility and iron uptake in Caco-2 cells. The 10kF fraction was as effective as ascorbic acid (1:20 iron to ascorbic acid) in increasing the ferric iron solubility and uptake in Caco-2 cells. Further, gel filtration chromatography on peptide column led to co-elution of ferric reductase and iron solubilization activities at an apparent molecular mass of iron in Caco-2 cells. Thus, it is concluded that human milk possesses ferric reductase activity and is associated with ferric iron solubilization and enhanced absorption.

  10. Thioredoxin reductase is a key factor in the oxidative stress response of Lactobacillus plantarum WCFS1

    NARCIS (Netherlands)

    Serrano, L.M.; Molenaar, D.; Wels, M.W.W.; Teusink, B.; Bron, P.A.; Vos, de W.M.; Smid, E.J.

    2007-01-01

    Background - Thioredoxin (TRX) is a powerful disulfide oxido-reductase that catalyzes a wide spectrum of redox reactions in the cell. The aim of this study is to elucidate the role of the TRX system in the oxidative stress response in Lactobacillus plantarum WCFS1. Results - We have identified the

  11. Thioredoxin reductase is a key factor in the oxidative stress response of Lactobacillus plantarum WCFS1

    NARCIS (Netherlands)

    Serrano, L.M.; Molenaar, D; Sanders, M.W.W.; Teusink, B.; Bron, P.A.; Vos, W.M. de; Smid, E.J.

    2007-01-01

    ABSTRACT: BACKGROUND: Thioredoxin (TRX) is a powerful disulfide oxido-reductase that catalyzes a wide spectrum of redox reactions in the cell. The aim of this study is to elucidate the role of the TRX system in the oxidative stress response in Lactobacillus plantarum WCFS1. RESULTS: We have

  12. 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.

  13. Kinetic mechanism of an aldehyde reductase of Saccharomyces cerevisiae that relieves toxicity of furfural and 5-hydroxymethylfurfural

    Science.gov (United States)

    An effective means of relieving the toxicity of furan aldehydes, furfural (FFA) and 5-hydroxymethylfurfural (HMF), on fermenting organisms is essential for achieving efficient fermentation of lignocellulosic biomass to ethanol and other products. Ari1p, an aldehyde reductase from Saccharomyces cerev...

  14. 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.

  15. Plant sterol metabolism. Δ7-Sterol-C5-Desaturase (STE1/DWARF7), Δ5,7-Sterol-Δ7-Reductase (DWARF5) and Δ24-Sterol-Δ24-Reductase (DIMINUTO/DWARF1) show multiple subcellular localizations in Arabidopsis thaliana (Heynh) L

    DEFF Research Database (Denmark)

    Silvestro, Daniele; Andersen, Tonni Grube; Schaller, Hubert

    2013-01-01

    in the corresponding enzymes. All fusion proteins were found to localize in the endoplasmic reticulum in functionally complemented plants. The results show that both ¿(5,7)-sterol-¿(7)-reductase and ¿(24)-sterol-¿(24)-reductase are in addition localized to the plasma membrane, whereas ¿(7)-sterol-C(5)-desaturase......Sterols are crucial lipid components that regulate membrane permeability and fluidity and are the precursors of bioactive steroids. The plant sterols exist as three major forms, free sterols, steryl glycosides and steryl esters. The storage of steryl esters in lipid droplets has been shown...... to contribute to cellular sterol homeostasis. To further document cellular aspects of sterol biosynthesis in plants, we addressed the question of the subcellular localization of the enzymes implicated in the final steps of the post-squalene biosynthetic pathway. In order to create a clear localization map...

  16. Pyranopterin Coordination Controls Molybdenum Electrochemistry in Escherichia coli Nitrate Reductase.

    Science.gov (United States)

    Wu, Sheng-Yi; Rothery, Richard A; Weiner, Joel H

    2015-10-09

    We test the hypothesis that pyranopterin (PPT) coordination plays a critical role in defining molybdenum active site redox chemistry and reactivity in the mononuclear molybdoenzymes. The molybdenum atom of Escherichia coli nitrate reductase A (NarGHI) is coordinated by two PPT-dithiolene chelates that are defined as proximal and distal based on their proximity to a [4Fe-4S] cluster known as FS0. We examined variants of two sets of residues involved in PPT coordination: (i) those interacting directly or indirectly with the pyran oxygen of the bicyclic distal PPT (NarG-Ser(719), NarG-His(1163), and NarG-His(1184)); and (ii) those involved in bridging the two PPTs and stabilizing the oxidation state of the proximal PPT (NarG-His(1092) and NarG-His(1098)). A S719A variant has essentially no effect on the overall Mo(VI/IV) reduction potential, whereas the H1163A and H1184A variants elicit large effects (ΔEm values of -88 and -36 mV, respectively). Ala variants of His(1092) and His(1098) also elicit large ΔEm values of -143 and -101 mV, respectively. An Arg variant of His(1092) elicits a small ΔEm of +18 mV on the Mo(VI/IV) reduction potential. There is a linear correlation between the molybdenum Em value and both enzyme activity and the ability to support anaerobic respiratory growth on nitrate. These data support a non-innocent role for the PPT moieties in controlling active site metal redox chemistry and catalysis. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Pyranopterin Coordination Controls Molybdenum Electrochemistry in Escherichia coli Nitrate Reductase*

    Science.gov (United States)

    Wu, Sheng-Yi; Rothery, Richard A.; Weiner, Joel H.

    2015-01-01

    We test the hypothesis that pyranopterin (PPT) coordination plays a critical role in defining molybdenum active site redox chemistry and reactivity in the mononuclear molybdoenzymes. The molybdenum atom of Escherichia coli nitrate reductase A (NarGHI) is coordinated by two PPT-dithiolene chelates that are defined as proximal and distal based on their proximity to a [4Fe-4S] cluster known as FS0. We examined variants of two sets of residues involved in PPT coordination: (i) those interacting directly or indirectly with the pyran oxygen of the bicyclic distal PPT (NarG-Ser719, NarG-His1163, and NarG-His1184); and (ii) those involved in bridging the two PPTs and stabilizing the oxidation state of the proximal PPT (NarG-His1092 and NarG-His1098). A S719A variant has essentially no effect on the overall Mo(VI/IV) reduction potential, whereas the H1163A and H1184A variants elicit large effects (ΔEm values of −88 and −36 mV, respectively). Ala variants of His1092 and His1098 also elicit large ΔEm values of −143 and −101 mV, respectively. An Arg variant of His1092 elicits a small ΔEm of +18 mV on the Mo(VI/IV) reduction potential. There is a linear correlation between the molybdenum Em value and both enzyme activity and the ability to support anaerobic respiratory growth on nitrate. These data support a non-innocent role for the PPT moieties in controlling active site metal redox chemistry and catalysis. PMID:26297003

  18. 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,...

  19. Purification and characterization of NADPH--cytochrome c reductase from the midgut of the southern armyworm (Spodoptera eridania).

    Science.gov (United States)

    Crankshaw, D L; Hetnarski, K; Wilkinson, C F

    1979-09-01

    1. NADPH-cytochrome c reductase was solubilized with bromelain and purified about 400-fold from sucrose/pyrophosphate-washed microsomal fractions from southern armyworm (Spodoptera eridania) larval midguts. 2. The enzyme has a mol.wt. of 70 035 +/- 1300 and contained 2 mol of flavin/mol of enzyme consisting of almost equimolar amounts of FMN and FAD. 3. Aerobic titration of the enzyme with NADPH caused the formation of a stable half-reduced state at 0.5 mol of NADPH/mol of flavin. 4. Kinetic analysis showed that the reduction of cytochrome c proceeded by a Bi Bi Ping Pong mechanism. 5. Apparent Km values for NADPH and cytochrome c and Ki values for NADP+ and 2'-AMP were considerably higher for the insect reductase than for the mammalian liver enzyme. 6. These are discussed in relation to possible differences in the active sites of the enzymes.

  20. A newly-detected reductase from Rauvolfia closes a gap in the biosynthesis of the antiarrhythmic alkaloid ajmaline.

    Science.gov (United States)

    Gao, Shujuan; von Schumann, Gerald; Stöckigt, Joachim

    2002-10-01

    A new enzyme, 1,2-dihydrovomilenine reductase (E.C. 1.3.1), has been detected in Rauvolfia cell suspension cultures. The enzyme specifically converts 2beta( R)-1,2-dihydrovomilenine through an NADPH-dependent reaction into 17-O-acetylnorajmaline, a close biosynthetic precursor of the antiarrhythmic alkaloid ajmaline from Rauvolfia. A five-step purification procedure using SOURCE 30Q chromatography, hydroxyapatite chromatography, 2',5'-ADP Sepharose 4B affinity chromatography and ion exchange chromatography on DEAE Sepharose and Mono Q delivered an approximately 200-fold enriched enzyme in a yield of approximately 6%. SDS-PAGE showed an M r for the enzyme of approximately 48 kDa. Optimum pH and optimum temperature of the reductase were at pH 6.0 and 37 degrees C. The enzyme shows a limited distribution in cell cultures expressing ajmaline biosynthesis, and is obviously highly specific for the ajmaline pathway.

  1. Lack of a peroxiredoxin suppresses the lethality of cells devoid of electron donors by channelling electrons to oxidized ribonucleotide reductase.

    Science.gov (United States)

    Boronat, Susanna; Domènech, Alba; Carmona, Mercè; García-Santamarina, Sarela; Bañó, M Carmen; Ayté, José; Hidalgo, Elena

    2017-06-01

    The thioredoxin and glutaredoxin pathways are responsible of recycling several enzymes which undergo intramolecular disulfide bond formation as part of their catalytic cycles such as the peroxide scavengers peroxiredoxins or the enzyme ribonucleotide reductase (RNR). RNR, the rate-limiting enzyme of deoxyribonucleotide synthesis, is an essential enzyme relying on these electron flow cascades for recycling. RNR is tightly regulated in a cell cycle-dependent manner at different levels, but little is known about the participation of electron donors in such regulation. Here, we show that cytosolic thioredoxins Trx1 and Trx3 are the primary electron donors for RNR in fission yeast. Unexpectedly, trx1 transcript and Trx1 protein levels are up-regulated in a G1-to-S phase-dependent manner, indicating that the supply of electron donors is also cell cycle-regulated. Indeed, genetic depletion of thioredoxins triggers a DNA replication checkpoint ruled by Rad3 and Cds1, with the final goal of up-regulating transcription of S phase genes and constitutive RNR synthesis. Regarding the thioredoxin and glutaredoxin cascades, one combination of gene deletions is synthetic lethal in fission yeast: cells lacking both thioredoxin reductase and cytosolic dithiol glutaredoxin. We have isolated a suppressor of this lethal phenotype: a mutation at the Tpx1-coding gene, leading to a frame shift and a loss-of-function of Tpx1, the main client of electron donors. We propose that in a mutant strain compromised in reducing equivalents, the absence of an abundant and competitive substrate such as the peroxiredoxin Tpx1 has been selected as a lethality suppressor to favor RNR function at the expense of the non-essential peroxide scavenging function, to allow DNA synthesis and cell growth.

  2. Lack of a peroxiredoxin suppresses the lethality of cells devoid of electron donors by channelling electrons to oxidized ribonucleotide reductase.

    Directory of Open Access Journals (Sweden)

    Susanna Boronat

    2017-06-01

    Full Text Available The thioredoxin and glutaredoxin pathways are responsible of recycling several enzymes which undergo intramolecular disulfide bond formation as part of their catalytic cycles such as the peroxide scavengers peroxiredoxins or the enzyme ribonucleotide reductase (RNR. RNR, the rate-limiting enzyme of deoxyribonucleotide synthesis, is an essential enzyme relying on these electron flow cascades for recycling. RNR is tightly regulated in a cell cycle-dependent manner at different levels, but little is known about the participation of electron donors in such regulation. Here, we show that cytosolic thioredoxins Trx1 and Trx3 are the primary electron donors for RNR in fission yeast. Unexpectedly, trx1 transcript and Trx1 protein levels are up-regulated in a G1-to-S phase-dependent manner, indicating that the supply of electron donors is also cell cycle-regulated. Indeed, genetic depletion of thioredoxins triggers a DNA replication checkpoint ruled by Rad3 and Cds1, with the final goal of up-regulating transcription of S phase genes and constitutive RNR synthesis. Regarding the thioredoxin and glutaredoxin cascades, one combination of gene deletions is synthetic lethal in fission yeast: cells lacking both thioredoxin reductase and cytosolic dithiol glutaredoxin. We have isolated a suppressor of this lethal phenotype: a mutation at the Tpx1-coding gene, leading to a frame shift and a loss-of-function of Tpx1, the main client of electron donors. We propose that in a mutant strain compromised in reducing equivalents, the absence of an abundant and competitive substrate such as the peroxiredoxin Tpx1 has been selected as a lethality suppressor to favor RNR function at the expense of the non-essential peroxide scavenging function, to allow DNA synthesis and cell growth.

  3. Anti-neuroinflammatory efficacy of the aldose reductase inhibitor FMHM via phospholipase C/protein kinase C-dependent NF-κB and MAPK pathways

    Energy Technology Data Exchange (ETDEWEB)

    Zeng, Ke-Wu [State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191 (China); Li, Jun [Modern Research Center for Traditional Chinese Medicine, Beijing University of Chinese Medicine, Beijing 100029 (China); Dong, Xin; Wang, Ying-Hong; Ma, Zhi-Zhong; Jiang, Yong; Jin, Hong-Wei [State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191 (China); Tu, Peng-Fei, E-mail: pengfeitu@vip.163.com [State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191 (China); Modern Research Center for Traditional Chinese Medicine, Beijing University of Chinese Medicine, Beijing 100029 (China)

    2013-11-15

    Aldose reductase (AR) has a key role in several inflammatory diseases: diabetes, cancer and cardiovascular diseases. Therefore, AR inhibition seems to be a useful strategy for anti-inflammation therapy. In the central nervous system (CNS), microglial over-activation is considered to be a central event in neuroinflammation. However, the effects of AR inhibition in CNS inflammation and its underlying mechanism of action remain unknown. In the present study, we found that FMHM (a naturally derived AR inhibitor from the roots of Polygala tricornis Gagnep.) showed potent anti-neuroinflammatory effects in vivo and in vitro by inhibiting microglial activation and expression of inflammatory mediators. Mechanistic studies showed that FMHM suppressed the activity of AR-dependent phospholipase C/protein kinase C signaling, which further resulted in downstream inactivation of the IκB kinase/IκB/nuclear factor-kappa B (NF-κB) inflammatory pathway. Therefore, AR inhibition-dependent NF-κB inactivation negatively regulated the transcription and expression of various inflammatory genes. AR inhibition by FMHM exerted neuroprotective effects in lipopolysaccharide-induced neuron–microglia co-cultures. These findings suggested that AR is a potential target for neuroinflammation inhibition and that FMHM could be an effective agent for treating or preventing neuroinflammatory diseases. - Highlights: • FMHM is a natural-derived aldose reductase (AR) inhibitor. • FMHM inhibits various neuroinflammatory mediator productions in vitro and in vivo. • FMHM inhibits neuroinflammation via aldose reductase/PLC/PKC-dependent NF-κB pathway. • FMHM inhibits neuroinflammation via aldose reductase/PLC/PKC-dependent MAPK pathway. • FMHM protects neurons against inflammatory injury in microglia-neuron co-cultures.

  4. Cell signaling mechanisms and metabolic regulation of germination and dormancy in barley seeds

    Directory of Open Access Journals (Sweden)

    Zhenguo Ma

    2017-12-01

    Full Text Available During germination of barley (Hordeum vulgare L. seeds, important morphological and physiological changes take place, including development of organs and tissues and activation of metabolic pathways. Germination and dormancy of seeds are regulated by abscisic acid, gibberellins, reactive oxygen species (ROS, reactive nitrogen species (RNS and several other factors. Activities of ascorbate–glutathione cycle enzymes, responsible for scavenging ROS, strongly increase. Catalase and superoxide dismutase activities, also scavenging ROS, decrease at the onset of seed germination and then increase. With the increase in aerobic metabolism after radicle protrusion, the activities of the fermentation enzymes lactate and alcohol dehydrogenase decline rapidly. The RNS-scavenging activity of S-nitrosoglutathione reductase decreases in the course of seed germination, in concert with elevation of nitric oxide production and protein nitrosylation. This activity supports the role of RNS in regulating seed germination. Transcription of various genes at different phases of seed germination exhibits phase-specific changes. During imbibition, genes involved in cell wall metabolism are highly expressed; in the middle phase of seed germination before radicle protrusion, genes involved in amino acid synthesis, protein synthesis, and transport and nucleic acid synthesis are upregulated significantly, and after radicle protrusion, genes involved in photosynthetic metabolism are induced. In summary, signal transduction and metabolic regulation of seed germination involve diverse reactions and complex regulation at different levels of metabolic organization. Keywords: Seed germination, Reactive oxygen species, Reactive nitrogen species, Signal transduction, Gene expression

  5. 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.

  6. Preconditioning results in S-nitrosylation of proteins involved in regulation of mitochondrial energetics and calcium transport.

    Science.gov (United States)

    Sun, Junhui; Morgan, Meghan; Shen, Rong-Fong; Steenbergen, Charles; Murphy, Elizabeth

    2007-11-26

    Nitric oxide has been shown to be an important signaling messenger in ischemic preconditioning (IPC). Accordingly, we investigated whether protein S-nitrosylation occurs in IPC hearts and whether S-nitrosoglutathione (GSNO) elicits similar effects on S-nitrosylation and cardioprotection. Preceding 20 minutes of no-flow ischemia and reperfusion, hearts from C57BL/6J mice were perfused in the Langendorff mode and subjected to the following conditions: (1) control perfusion; (2) IPC; or (3) 0.1 mmol/L GSNO treatment. Compared with control, IPC and GSNO significantly improved postischemic recovery of left ventricular developed pressure and reduced infarct size. IPC and GSNO both significantly increased S-nitrosothiol contents and S-nitrosylation levels of the L-type Ca2+ channel alpha1 subunit in heart membrane fractions. We identified several candidate S-nitrosylated proteins by proteomic analysis following the biotin switch method, including the cardiac sarcoplasmic reticulum Ca2+-ATPase, alpha-ketoglutarate dehydrogenase, and the mitochondrial F1-ATPase alpha1 subunit. The activities of these enzymes were altered in a concentration-dependent manner by GSNO treatment. We further developed a 2D DyLight fluorescence difference gel electrophoresis proteomic method that used DyLight fluors and a modified biotin switch method to identify S-nitrosylated proteins. IPC and GSNO produced a similar pattern of S-nitrosylation modification and cardiac protection against ischemia/reperfusion injury, suggesting that protein S-nitrosylation may play an important cardioprotective role in heart.

  7. Sulfite-oxido-reductase is involved in the oxidation of sulfite in Desulfocapsa sulfoexigens during disproportionation of thiosulfate and elemental sulfur.

    Science.gov (United States)

    Frederiksen, Trine-Maria; Finster, Kai

    2003-06-01

    The enzymatic pathways of elemental sulfur and thiosulfate disproportionation were investigated using cell-free extract of Desulfocapsa sulfoexigens. Sulfite was observed to be an intermediate in the metabolism of both compounds. Two distinct pathways for the oxidation of sulfite have been identified. One pathway involves APS reductase and ATP sulfurylase and can be described as the reversion of the initial steps of the dissimilatory sulfate reduction pathway. The second pathway is the direct oxidation of sulfite to sulfate by sulfite oxidoreductase. This enzyme has not been reported from sulfate reducers before. Thiosulfate reductase, which cleaves thiosulfate into sulfite and sulfide, was only present in cell-free extract from thiosulfate disproportionating cultures. We propose that this enzyme catalyzes the first step in thiosulfate disproportionation. The initial step in sulfur disproportionation was not identified. Dissimilatory sulfite reductase was present in sulfur and thiosulfate disproportionating cultures. The metabolic function of this enzyme in relation to elemental sulfur or thiosulfate disproportionation was not identified. The presence of the uncouplers HQNO and CCCP in growing cultures had negative effects on both thiosulfate and sulfur disproportionation. CCCP totally inhibited sulfur disproportionation and reduced thiosulfate disproportionation by 80% compared to an unamended control. HQNO reduced thiosulfate disproportionation by 80% and sulfur disproportionation by 90%.

  8. 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.

  9. 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

  10. Systemic and ocular pharmacokinetics of N-4-benzoylaminophenylsulfonylglycine (BAPSG), a novel aldose reductase inhibitor

    OpenAIRE

    Sunkara, Gangadhar; Ayalasomayajula, Surya P.; Rao, Cheruku S.; Vennerstrom, Jonathan L.; DeRuiter, Jack; Kompella, Uday B.

    2004-01-01

    To better develop N-[4-(benzoylamino)phenylsulfonyl]glycine (BAPSG), a potent and selective aldose reductase inhibitor capable of delaying the progression of ocular diabetic complications, the objective of this study was to assess its pharmacokinetics. The plasma pharmacokinetics of BASPG was assessed in male Sprague-Dawley rats following intravenous, intraperitoneal and oral routes of administration and its distribution to various tissues including those of the eye was studied following intr...

  11. Interaction of human biliverdin reductase with Akt/protein kinase B and phosphatidylinositol-dependent kinase 1 regulates glycogen synthase kinase 3 activity: a novel mechanism of Akt activation

    OpenAIRE

    Miralem, Tihomir; Lerner-Marmarosh, Nicole; Gibbs, Peter E. M.; Jenkins, Jermaine L.; Heimiller, Chelsea; Maines, Mahin D.

    2016-01-01

    Biliverdin reductase A (BVR) and Akt isozymes have overlapping pleiotropic functions in the insulin/PI3K/MAPK pathway. Human BVR (hBVR) also reduces the hemeoxygenase activity product biliverdin to bilirubin and is directly activated by insulin receptor kinase (IRK). Akt isoenzymes (Akt1–3) are downstream of IRK and are activated by phosphatidylinositol-dependent kinase 1 (PDK1) phosphorylating T308 before S473 autophosphorylation. Akt (RxRxxSF) and PDK1 (RFxFPxFS) binding motifs are present ...

  12. Cyclohex-1-ene carboxylic acids: synthesis and biological evaluation of novel inhibitors of human 5 alpha reductase.

    Science.gov (United States)

    Baston, Eckhard; Salem, Ola I A; Hartmann, Rolf W

    2003-03-01

    In search of novel nonsteroidal mimics of steroidal inhibitors of 5 alpha reductase, 4-(2-phenylethyl)cyclohex-1-ene carboxylic acids 1-5 were synthesized with different substituents in para position of the phenyl ring (1: N, N-diisopropylcarbamoyl, 2: phenyl, 3: phenoxy, 4: benzoyl, and 5: benzyl). The principal synthetic approach for the desired compounds consisted of a Wittig olefination between 1, 4-dioxaspiro [4.5]-decane-8-carbaldehyde (4g and the appropriate phosphonium salts. The compounds were tested for inhibition of human 5 alpha reductase isozymes 1 and 2 using DU 145 cells and preparations from prostatic tissue, respectively. They turned out to be good inhibitors of the prostatic isozyme 2 with compound 1 being the most potent one (IC(50) = 760 nM). Isozyme 1 was only slightly inhibited. It is concluded that the novel structures are appropriate for being further optimized, aiming at the development of a novel drug for the treatment of benign prostatic hyperplasia.

  13. 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...... ligase mediates degradation of Spd1 and the related protein Spd2 at S phase of the cell cycle. We have generated a conditional allele of CRL4(Cdt2), by expressing the highly unstable substrate-recruiting protein Cdt2 from a repressible promoter. Unlike Spd1, Spd2 does not regulate deoxynucleotide...... 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...

  14. 15N studies on the in-vivo assay of nitrate reductase in leaves

    International Nuclear Information System (INIS)

    Yoneyama, Tadakatsu

    1981-01-01

    The reduction of nitrate and nitrite in the leaf disks of seven di- and two mono-cotyledonous species under the in-vivo assay conditions of nitrate reductase was studied using N-15 labeled substrates. The significant reduction of both nitrate and nitrite into ammonia and amino acids was detected in the atmosphere of air. In the atmosphere of N 2 gas, anaerobic incubation enhanced the accumulation of nitrite, but the subsequent reduction to the basic nitrogen compounds was from 40 to 180 % of the aerobic rate. The present examination indicated that the in-vivo assay of nitrate reductase under aerobic condition may give greatly underestimated results due to nitrite reduction, and that the exclusion of oxygen from the in-vivo assay mixture is desirable. The addition of n- propanol may be desirable for the assay under aerobic condition. Significant difference was not observed in the reduction of nitrate supplied as sodium and potassium salts on the nitrite formation and on the incorporation of nitrate-N into basic fractions. The N-15 experiment on the dark assimilation of nitrate, nitrite and ammonia into amino acids in wheat leaves showed that these three nitrogen sources were assimilated through the same route, and that the glutamine synthetase/glutamate synthetase pathway was the main route. By anaerobic treatment, the incorporation of nitrogen into alanine and serine was relatively high. (Kako, I.)

  15. Consequence of absence of nitrate reductase activity on photosynthesis in Nicotiana plumbaginifolia plants

    International Nuclear Information System (INIS)

    Saux, C.; Lemoine, Y.; Marion-Poll, A.; Valadier, M.H.; Deng, M.; Morot-Gaudry, J.F.

    1987-01-01

    Chlorate-resistant Nicotiana plumbaginifolia (cv Viviani) mutants were found to be deficient in the nitrate reductase apoprotein (NR - nia). Because they could not grow with nitrate as sole nitrogen source, they were cultivated as graftings on wild-type Nicotiana tabacum plants. The grafts of mutant plants were chlorotic compared to the grafts of wild type. Mutant leaves did not accumulate nitrogen but contained less malate and more glutamine than wild leaves. They exhibited a slight increase of the proportion of the light-harvesting chlorophyll a/b protein complexes and a lowering of the efficiency of energy transfer between these complexes and the active centers. After a 3 second 14 CO 2 pulse, the total 14 C incorporation of the mutant leaves was approximately 20 5 of that of the control. The 14 C was essentially recovered in ribulose bisphosphate in these plants. It was consistent with a decline of ribulose bisphosphate carboxylase activity observed in the mutant. After a 3 second 14 CO 2 pulse followed by a 60 second chase with normal CO 2 , 14 C was mainly accumulated in starch which was labeled more in the mutant than in the wild type. These results confirm the observation that in the nitrate reductase deficient leaves, chloroplasts were loaded with large starch inclusions preceding disorganization of the photosynthetic apparatus

  16. Consequence of absence of nitrate reductase activity on photosynthesis in Nicotiana plumbaginifolia plants

    Energy Technology Data Exchange (ETDEWEB)

    Saux, C.; Lemoine, Y.; Marion-Poll, A.; Valadier, M.H.; Deng, M.; Morot-Gaudry, J.F.

    1987-05-01

    Chlorate-resistant Nicotiana plumbaginifolia (cv Viviani) mutants were found to be deficient in the nitrate reductase apoprotein (NR/sup -/ nia). Because they could not grow with nitrate as sole nitrogen source, they were cultivated as graftings on wild-type Nicotiana tabacum plants. The grafts of mutant plants were chlorotic compared to the grafts of wild type. Mutant leaves did not accumulate nitrogen but contained less malate and more glutamine than wild leaves. They exhibited a slight increase of the proportion of the light-harvesting chlorophyll a/b protein complexes and a lowering of the efficiency of energy transfer between these complexes and the active centers. After a 3 second /sup 14/CO/sub 2/ pulse, the total /sup 14/C incorporation of the mutant leaves was approximately 20/sup 5/ of that of the control. The /sup 14/C was essentially recovered in ribulose bisphosphate in these plants. It was consistent with a decline of ribulose bisphosphate carboxylase activity observed in the mutant. After a 3 second /sup 14/CO/sub 2/ pulse followed by a 60 second chase with normal CO/sub 2/, /sup 14/C was mainly accumulated in starch which was labeled more in the mutant than in the wild type. These results confirm the observation that in the nitrate reductase deficient leaves, chloroplasts were loaded with large starch inclusions preceding disorganization of the photosynthetic apparatus.

  17. Corynebacterium diphtheriae methionine sulfoxide reductase a exploits a unique mycothiol redox relay mechanism.

    Science.gov (United States)

    Tossounian, Maria-Armineh; Pedre, Brandán; Wahni, Khadija; Erdogan, Huriye; Vertommen, Didier; Van Molle, Inge; Messens, Joris

    2015-05-01

    Methionine sulfoxide reductases are conserved enzymes that reduce oxidized methionines in proteins and play a pivotal role in cellular redox signaling. We have unraveled the redox relay mechanisms of methionine sulfoxide reductase A of the pathogen Corynebacterium diphtheriae (Cd-MsrA) and shown that this enzyme is coupled to two independent redox relay pathways. Steady-state kinetics combined with mass spectrometry of Cd-MsrA mutants give a view of the essential cysteine residues for catalysis. Cd-MsrA combines a nucleophilic cysteine sulfenylation reaction with an intramolecular disulfide bond cascade linked to the thioredoxin pathway. Within this cascade, the oxidative equivalents are transferred to the surface of the protein while releasing the reduced substrate. Alternatively, MsrA catalyzes methionine sulfoxide reduction linked to the mycothiol/mycoredoxin-1 pathway. After the nucleophilic cysteine sulfenylation reaction, MsrA forms a mixed disulfide with mycothiol, which is transferred via a thiol disulfide relay mechanism to a second cysteine for reduction by mycoredoxin-1. With x-ray crystallography, we visualize two essential intermediates of the thioredoxin relay mechanism and a cacodylate molecule mimicking the substrate interactions in the active site. The interplay of both redox pathways in redox signaling regulation forms the basis for further research into the oxidative stress response of this pathogen. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. 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.

  19. Melatonin Regulates Oxidative Stress Initiated by Freund’s Complete Adjuvant

    Directory of Open Access Journals (Sweden)

    Miroslav Pohanka

    2015-07-01

    Full Text Available Melatonin is a hormone with strong antioxidant properties. In this experiment, Freund’s complete adjuvant was used as a stressogenic substance given to laboratory outbred mice, whereas melatonin was investigated as a protectant against the stressogenic effect. Levels of low molecular weight antioxidants, thiobarbituric acid reactive substances, and tumor necrosis factor α and activity of glutathione reductase were determined in blood from the animals. Surprisingly, melatonin was not involved in direct regulation of antioxidants, thiobarbituric acid reactive substances and tumor necrosis factor α. On the other hand, melatonin regulated glutathione reductase activity. We can conclude on regulation of metabolism caused by melatonin in the model. The effect was more important than the expected regulation of immunity and basal oxidative homeostasis.

  20. 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; 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; 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.; 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

    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

  1. 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

  2. Molecular cloning of cDNAs of human liver and placenta NADH-cytochrome b5 reductase

    International Nuclear Information System (INIS)

    Yubisui, T.; Naitoh, Y.; Zenno, S.; Tamura, M.; Takeshita, M.; Sakaki, Y.

    1987-01-01

    A cDNA coding for human liver NADH-cytochrome b 5 reductase was cloned from a human liver cDNA library constructed in phage λgt11. The library was screened by using an affinity-purified rabbit antibody against NADH-cytochrome b 5 reductase of human erythrocytes. A cDNA about 1.3 kilobase pairs long was isolated. By using the cDNA as a probe, another cDNA (pb 5 R141) of 1817 base pairs was isolated that hybridized with a synthetic oligonucleotide encoding Pro-Asp-Ile-Lys-Tyr-Pro, derived from the amino acid sequence at the amino-terminal region of the enzyme from human erythrocytes. Furthermore, by using the pb 5 R141 as a probe, cDNA clones having more 5' sequence were isolated from a human placenta cDNA library. The amino acid sequences deduced from the nucleotide sequences of these cDNA clones overlapped each other and consisted of a sequence that completely coincides with that of human erythrocytes and a sequence of 19 amino acid residues extended at the amino-terminal side. The latter sequence closely resembles that of the membrane-binding domain of steer liver microsomal enzyme

  3. Association study of sepiapterin reductase gene promoter polymorphisms with schizophrenia in a Han Chinese population

    Directory of Open Access Journals (Sweden)

    Fu JW

    2015-10-01

    Full Text Available Jiawu Fu,1,* Guoda Ma,1,* Hui Mai,1,* Xudong Luo,2 Jingwen Yin,2 Qing Chen,2 Zhixiong Lin,2 Hua Tao,1 You Li,1 Lili Cui,1 Zheng Li,3 Juda Lin,2 Bin Zhao,1 Keshen Li1 1Institute of Neurology, Affiliated Hospital of Guangdong Medical University, 2Department of Psychiatry, Affiliated Hospital of Guangdong Medical University, Zhanjiang, People’s Republic of China; 3Unit on Synapse Development and Plasticity, National Institute of Mental Health, National Institutes of Health, Bethesda, MD, USA *These authors contributed equally to this work Abstract: Sepiapterin reductase participates in the biosynthesis of tetrahydrobiopterin, which plays very important roles in the pathogenesis of schizophrenia via dysregulation of ­neurotransmitter systems. Here, two single nucleotide polymorphisms (rs1876487 and rs2421095 in the promoter region of SPR were genotyped in 941 schizophrenic patients and 944 controls in a Han Chinese population using the SNaPshot technique. No significant differences were found in the distribution of alleles or genotypes of the two single nucleotide polymorphisms (SNPs between schizophrenic patients and controls (all P>0.05. Likewise, no haplotype was found to be associated with schizophrenia. However, sex-stratified analysis revealed that the frequencies of the A allele of rs1876487 and the A–A (rs2421095–rs1876487 haplotype were all significantly different between schizophrenia and controls in females (P=0.040 and P=0.033, respectively, but not in males. Additionally, luciferase reporter gene assays revealed that the A–A haplotype had significantly higher SPR transcriptional activity compared with the A–C haplotype in SH-SY5Y cells. Our data indicate that the two SNPs do not influence the risk of schizophrenia when using the total sample, but the A allele of rs1876487 and the A–A haplotype may contribute to protective roles for schizophrenia in females. Keywords: schizophrenia, sepiapterin reductase, polymorphisms, Han

  4. Expression, purification, crystallization and preliminary X-ray analysis of NAD(P)H-dependent carbonyl reductase specifically expressed in thyroidectomized chicken fatty liver

    International Nuclear Information System (INIS)

    Yoneda, Kazunari; Fukuda, Yudai; Shibata, Takeshi; Araki, Tomohiro; Nikki, Takahiro; Sakuraba, Haruhiko; Ohshima, Toshihisa

    2012-01-01

    An NAD(P)H-dependent carbonyl reductase specifically expressed in thyroidectomized chicken fatty liver was successfully isolated and crystallized. An NAD(P)H-dependent carbonyl reductase specifically expressed in thyroidectomized chicken fatty liver was crystallized using the sitting-drop vapour-diffusion method with polyethylene glycol 300 as the precipitant. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 104.26, b = 81.32, c = 77.27 Å, β = 119.43°, and diffracted to 1.86 Å resolution on beamline NE3A at the Photon Factory. The overall R merge was 5.4% and the data completeness was 99.4%

  5. High-resolution neutron protein crystallography with radically small crystal volumes: Application of perdeuteration to human aldose reductase

    International Nuclear Information System (INIS)

    Hazemann, I.; Dauvergne, M.T.; Blakeley, M.P.; Meilleur, Flora; Haertlein, M.; Van Dorsselaer, A.; Mitschler, A.; Myles, Dean A.A.; Podjarny, A.

    2005-01-01

    Neutron diffraction data have been collected to 2.2 (angstrom) resolution from a small (0.15 mm 3 ) crystal of perdeuterated human aldose reductase (h-AR; MW = 36 kDa) in order to help to determine the protonation state of the enzyme. h-AR belongs to the aldo-keto reductase family and is implicated in diabetic complications. Its ternary complexes (h-AR-coenzyme NADPH-selected inhibitor) provide a good model to study both the enzymatic mechanism and inhibition. Here, the successful production of fully deuterated human aldose reductase (h-AR(D)), subsequent crystallization of the ternary complex h-AR(D)-NADPH-IDD594 and neutron Laue data collection at the LADI instrument at ILL using a crystal volume of just 0.15 mm 3 are reported. Neutron data were recorded to 2 (angstrom) resolution, with subsequent data analysis using data to 2.2 (angstrom). This is the first fully deuterated enzyme of this size (36 kDa) to be solved by neutron diffraction and represents a milestone in the field, as the crystal volume is at least one order of magnitude smaller than those usually required for other high-resolution neutron structures determined to date. This illustrates the significant increase in the signal-to-noise ratio of data collected from perdeuterated crystals and demonstrates that good-quality neutron data can now be collected from more typical protein crystal volumes. Indeed, the signal-to-noise ratio is then dominated by other sources of instrument background, the nature of which is under investigation. This is important for the design of future instruments, which should take maximum advantage of the reduction in the intrinsic diffraction pattern background from fully deuterated samples.

  6. Correlation of changes in rate of sterol synthesis with changes in HMG CoA reductase activity in cultured lens epithelial cells

    International Nuclear Information System (INIS)

    Cenedella, R.J.; Hitchener, W.R.

    1986-01-01

    In the present study, the authors correlated changes in HMG CoA reductase activity with changes in relative rates of sterol synthesis measured from either 3 H 2 O or 1- 14 C-acetate for bovine lens epithelial cells cultured in the presence or absence of lipoproteins. Enzyme activity and rates of incorporation of 3 H 2 O or 1- 14 C-acetate into digitonin precipitable sterols were measured in cells on the 4th day of subculture in DMEM containing 9% whole calf serum (WM) or 9% lipoprotein deficient serum (LDM). In three experiments, HMG CoA reductase activity (U/10 6 cells) averaged 2.2 +/- 0.1 times greater for cells grown in LDM than WM. Sterol synthesis averaged 3.0 +/- 0.4 times greater when measured with 3 H 2 O and 4.0 +/- 1.1 times greater when measured with 14 C-acetate. Thus, 3 H 2 O and 14 C-acetate appear to be comparable substrates for estimating changes in relative rates of sterol synthesis by cultured cells. The larger increases in rates of sterol synthesis than in reductase activity in response to decreased cholesterol could reflect stimulation at additional metabolic steps in the cholesterol pathway beyond mevalonic acid

  7. Monooxygenase system in Guerin’s carcinoma of rats under conditions of ω-3 polyunsaturated fatty acids administration

    Directory of Open Access Journals (Sweden)

    M. M. Marchenko

    2016-08-01

    Full Text Available The aim of the study was to determine the variations of function in components of monooxygenase system (MOS of rat Guerin’s carcinoma under ω-3 polyunsaturated fatty acids (PUFAs administration. The activity of Guerin’s carcinoma microsomal NADH-cytochrome b5 reductase, the content and the rate of cytochrome b5 oxidation-reduction, the content and the rate of cytochrome Р450 oxidation-reduction have been investigated in rats with tumor under conditions of ω-3 PUFAs administration. ω-3 PUFAs supplementation before and after transplantation of Guerin’s carcinoma resulted in the increase of NADH-cytochrome b5 reductase activity and decrease of cytochrome b5 level in the Guerin’s carcinoma microsomal fraction in the logarithmic phases of carcinogenesis as compared to the tumor-bearing rats. Increased activity of NADH-cytochrome b5 reductase facilitates higher electron flow in redox-chain of MOS. Under decreased cytochrome b5 levels the electrons are transferred to oxygen, which leads to heightened generation of superoxide (O2•- in comparison to control. It was shown, that the decrease of cytochrome P450 level in the Guerin’s carcinoma microsomal fraction in the logarithmic phases of oncogenesis under ω-3 PUFAs administration may be associated with its transition into an inactive form – cytochrome P420. This decrease in cytochrome P450 coincides with increased generation of superoxide by MOS oxygenase chain.

  8. Isolation and characterization of dihydrofolate reductase from trimethoprim-susceptible and trimethoprim-resistant Pseudomonas cepacia.

    OpenAIRE

    Burns, J L; Lien, D M; Hedin, L A

    1989-01-01

    Trimethoprim resistance was investigated in cystic fibrosis isolates of Pseudomonas cepacia. Determination of the MIC of trimethoprim for 111 strains revealed at least two populations of resistant organisms, suggesting the presence of more than one mechanism of resistance. Investigation of the antibiotic target, dihydrofolate reductase, was undertaken in both a susceptible strain and a strain with high-level resistance (MIC, greater than 1,000 micrograms/ml). The enzyme was purified by using ...

  9. Methylenetetrahydrofolate reductase homozygous mutation in a young boy with cerebellar infarction

    Directory of Open Access Journals (Sweden)

    Alberto Spalice

    2009-11-01

    Full Text Available Posterior circulation vascular occlusive disease in children is a rare and uncommonly reported event. Among the numerous risk factors, the methylenetetrahydrofolate reductase (MTHFR mutation is considered to be a common genetic cause of thrombosis in adults and children. Recently, a link between the MTHFR mutation and cerebrovascular disorders was reported in children. Diffusion tensor imaging (DTI is a great improvement on magnetic resonance imaging (MRI, making the in vivo anatomical and pathological study of the brain and its fibers possible. In our patient cerebellar infarction was associated with MTHFR mutation and, in a standard neurological examination, DTI revealed normal white matter tracts.

  10. 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

  11. Dimethoxycurcumin, a metabolically stable analogue of curcumin enhances the radiosensitivity of cancer cells: Possible involvement of ROS and thioredoxin reductase

    Energy Technology Data Exchange (ETDEWEB)

    Jayakumar, Sundarraj; Patwardhan, R.S.; Pal, Debojyoti [Radiation Biology & Health Sciences Division, Modular Laboratories, Bhabha Atomic Research Centre, Trombay, Mumbai 400085 (India); Sharma, Deepak [Radiation Biology & Health Sciences Division, Modular Laboratories, Bhabha Atomic Research Centre, Trombay, Mumbai 400085 (India); Homi Bhabha National Institute, Anushaktinagar, Mumbai 400094 (India); Sandur, Santosh K., E-mail: sskumar@barc.gov.in [Radiation Biology & Health Sciences Division, Modular Laboratories, Bhabha Atomic Research Centre, Trombay, Mumbai 400085 (India); Homi Bhabha National Institute, Anushaktinagar, Mumbai 400094 (India)

    2016-09-09

    Dimethoxycurcumin (DIMC), a structural analogue of curcumin, has been shown to have more stability, bioavailability, and effectiveness than its parent molecule curcumin. In this paper the radiosensitizing effect of DIMC has been investigated in A549 lung cancer cells. As compared to its parent molecule curcumin, DIMC showed a very potent radiosensitizing effect as seen by clonogenic survival assay. DIMC in combination with radiation significantly increased the apoptosis and mitotic death in A549 cells. This combinatorial treatment also lead to effective elimination of cancer stem cells. Further, there was a significant increase in cellular ROS, decrease in GSH to GSSG ratio and also significant slowdown in DNA repair when DIMC was combined with radiation. In silico docking studies and in vitro studies showed inhibition of thioredoxin reductase enzyme by DIMC. Overexpression of thioredoxin lead to the abrogation of radiosensitizing effect of DIMC underscoring the role of thioredoxin reductase in radiosensitization. Our results clearly demonstrate that DIMC can synergistically enhance the cancer cell killing when combined with radiation by targeting thioredoxin system. - Highlights: • DIMC enhances radiosensitivity of cancer cells by inducing cell death. • DIMC with radiation disrupted the cellular redox and targeted cancer stem cells. • DNA repair is hampered when cells are treated with DIMC. • DIMC inhibited thioredoxin reductase in cancer cells.

  12. Dimethoxycurcumin, a metabolically stable analogue of curcumin enhances the radiosensitivity of cancer cells: Possible involvement of ROS and thioredoxin reductase

    International Nuclear Information System (INIS)

    Jayakumar, Sundarraj; Patwardhan, R.S.; Pal, Debojyoti; Sharma, Deepak; Sandur, Santosh K.

    2016-01-01

    Dimethoxycurcumin (DIMC), a structural analogue of curcumin, has been shown to have more stability, bioavailability, and effectiveness than its parent molecule curcumin. In this paper the radiosensitizing effect of DIMC has been investigated in A549 lung cancer cells. As compared to its parent molecule curcumin, DIMC showed a very potent radiosensitizing effect as seen by clonogenic survival assay. DIMC in combination with radiation significantly increased the apoptosis and mitotic death in A549 cells. This combinatorial treatment also lead to effective elimination of cancer stem cells. Further, there was a significant increase in cellular ROS, decrease in GSH to GSSG ratio and also significant slowdown in DNA repair when DIMC was combined with radiation. In silico docking studies and in vitro studies showed inhibition of thioredoxin reductase enzyme by DIMC. Overexpression of thioredoxin lead to the abrogation of radiosensitizing effect of DIMC underscoring the role of thioredoxin reductase in radiosensitization. Our results clearly demonstrate that DIMC can synergistically enhance the cancer cell killing when combined with radiation by targeting thioredoxin system. - Highlights: • DIMC enhances radiosensitivity of cancer cells by inducing cell death. • DIMC with radiation disrupted the cellular redox and targeted cancer stem cells. • DNA repair is hampered when cells are treated with DIMC. • DIMC inhibited thioredoxin reductase in cancer cells.

  13. Clinical pattern, mutations and in vitro residual activity in 33 patients with severe 5, 10 methylenetetrahydrofolate reductase (MTHFR) deficiency

    NARCIS (Netherlands)

    Huemer, Martina; Mulder-Bleile, Regina; Burda, Patricie; Froese, D. Sean; Suormala, Terttu; Ben Zeev, Bruria; Chinnery, Patrick F.; Dionisi-Vici, Carlo; Dobbelaere, Dries; Gokcay, Gulden; Demirkol, Muebeccel; Haeberle, Johannes; Lossos, Alexander; Mengel, Eugen; Morris, Andrew A.; Niezen-Koning, Klary E.; Plecko, Barbara; Parini, Rossella; Rokicki, Dariusz; Schiff, Manuel; Schimmel, Mareike; Sewell, Adrian C.; Sperl, Wolfgang; Spiekerkoetter, Ute; Steinmann, Beat; Taddeucci, Grazia; Trejo-Gabriel-Galan, Jose M.; Trefz, Friedrich; Tsuji, Megumi; Antonia Vilaseca, Maria; von Kleist-Retzow, Juergen-Christoph; Walker, Valerie; Zeman, Jiri; Baumgartner, Matthias R.; Fowler, Brian

    Background Severe methylenetetrahydrofolate reductase (MTHFR) deficiency is a rare inborn defect disturbing the remethylation of homocysteine to methionine ( Methods Clinical, biochemical and treatment data was obtained from physicians by using a questionnaire. MTHFR activity was measured in primary

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

    DEFF Research Database (Denmark)

    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 and eu...

  15. Enhanced mutagenicity of low doses of alkylating agents and UV-light by inhibition of ribonucleotide reductase

    International Nuclear Information System (INIS)

    Jenssen, D.

    1986-01-01

    Monofunctional alkylating agents and UV-light are potent inducers of gene mutations in mammalian cells. Most data on these agent are supporting the idea that 0/sup 6/-alkylguanine is the dominating lesion responsible for the mutations induced by the alkylating agents and thymine-dimers in the case of UV-light. However, little is known about the mutagenic fate of these lesions during the replicative process. This is an essential issue to investigate not the least because of quantitative aspects. By investigating the factors affecting the mutagenic yield of these lesions, they hope to get further information on the mechanisms(s) involved. To study this, a system was applied which involves synchronized V79 Chinese hamster cells and inhibitors of the replication process. By applying hydroxyurea (HU), as inhibitor of the ribonucleotide reductase (RNR) step in DNA synthesis, the effect of nucleotide pool imbalance has been studied at the HGPRT-locus using V79 Chinese hamster cells

  16. Expression and characterization of truncated human heme oxygenase (hHO-1) and a fusion protein of hHO-1 with human cytochrome P450 reductase.

    Science.gov (United States)

    Wilks, A; Black, S M; Miller, W L; Ortiz de Montellano, P R

    1995-04-04

    A human heme oxygenase (hHO-1) gene without the sequence coding for the last 23 amino acids has been expressed in Escherichia coli behind the pho A promoter. The truncated enzyme is obtained in high yields as a soluble, catalytically-active protein, making it available for the first time for detailed mechanistic studies. The purified, truncated hHO-1/heme complex is spectroscopically indistinguishable from that of the rat enzyme and converts heme to biliverdin when reconstituted with rat liver cytochrome P450 reductase. A self-sufficient heme oxygenase system has been obtained by fusing the truncated hHO-1 gene to the gene for human cytochrome P450 reductase without the sequence coding for the 20 amino acid membrane binding domain. Expression of the fusion protein in pCWori+ yields a protein that only requires NADPH for catalytic turnover. The failure of exogenous cytochrome P450 reductase to stimulate turnover and the insensitivity of the catalytic rate toward changes in ionic strength establish that electrons are transferred intramolecularly between the reductase and heme oxygenase domains of the fusion protein. The Vmax for the fusion protein is 2.5 times higher than that for the reconstituted system. Therefore, either the covalent tether does not interfere with normal docking and electron transfer between the flavin and heme domains or alternative but equally efficient electron transfer pathways are available that do not require specific docking.

  17. 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

    . 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 (1nitrite in red musculature to more than double the value in normoxic fish......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...... concentration during anoxia in tissues with high myoglobin and mitochondria content, such as the heart. Here, we tested the hypothesis that red and white skeletal muscles develop different nitrite levels in crucian carp exposed to deep hypoxia and assessed whether this correlates with myoglobin concentration...

  18. para-Aminosalicylic acid is a prodrug targeting dihydrofolate reductase in Mycobacterium tuberculosis.

    Science.gov (United States)

    Zheng, Jun; Rubin, Eric J; Bifani, Pablo; Mathys, Vanessa; Lim, Vivian; Au, Melvin; Jang, Jichan; Nam, Jiyoun; Dick, Thomas; Walker, John R; Pethe, Kevin; Camacho, Luis R

    2013-08-09

    para-Aminosalicylic acid (PAS) is one of the antimycobacterial drugs currently used for multidrug-resistant tuberculosis. Although it has been in clinical use for over 60 years, its mechanism(s) of action remains elusive. Here we report that PAS is a prodrug targeting dihydrofolate reductase (DHFR) through an unusual and novel mechanism of action. We provide evidences that PAS is incorporated into the folate pathway by dihydropteroate synthase (DHPS) and dihydrofolate synthase (DHFS) to generate a hydroxyl dihydrofolate antimetabolite, which in turn inhibits DHFR enzymatic activity. Interestingly, PAS is recognized by DHPS as efficiently as its natural substrate para-amino benzoic acid. Chemical inhibition of DHPS or mutation in DHFS prevents the formation of the antimetabolite, thereby conferring resistance to PAS. In addition, we identified a bifunctional enzyme (riboflavin biosynthesis protein (RibD)), a putative functional analog of DHFR in a knock-out strain. This finding is further supported by the identification of PAS-resistant clinical isolates encoding a RibD overexpression mutation displaying cross-resistance to genuine DHFR inhibitors. Our findings reveal that a metabolite of PAS inhibits DHFR in the folate pathway. RibD was shown to act as a functional analog of DHFR, and as for DHFS, both were shown to be associated in PAS resistance in laboratory strains and clinical isolates.

  19. pH-tuneable binding of 2′-phospho-ADP-ribose to ketopantoate reductase: a structural and calorimetric study

    Energy Technology Data Exchange (ETDEWEB)

    Ciulli, Alessio [University Chemical Laboratory, Lensfield Road, Cambridge CB2 1EW (United Kingdom); Lobley, Carina M. C. [Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA (United Kingdom); Tuck, Kellie L. [University Chemical Laboratory, Lensfield Road, Cambridge CB2 1EW (United Kingdom); Smith, Alison G. [Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge CB2 3EA (United Kingdom); Blundell, Tom L. [Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA (United Kingdom); Abell, Chris, E-mail: ca26@cam.ac.uk [University Chemical Laboratory, Lensfield Road, Cambridge CB2 1EW (United Kingdom)

    2007-02-01

    A combined crystallographic, calorimetric and mutagenic study has been used to show how changes in pH give rise to two distinct binding modes of 2′-phospho-ADP-ribose to ketopantoate reductase. The crystal structure of Escherichia coli ketopantoate reductase in complex with 2′-monophosphoadenosine 5′-diphosphoribose, a fragment of NADP{sup +} that lacks the nicotinamide ring, is reported. The ligand is bound at the enzyme active site in the opposite orientation to that observed for NADP{sup +}, with the adenine ring occupying the lipophilic nicotinamide pocket. Isothermal titration calorimetry with R31A and N98A mutants of the enzyme is used to show that the unusual ‘reversed binding mode’ observed in the crystal is triggered by changes in the protonation of binding groups at low pH. This research has important implications for fragment-based approaches to drug design, namely that the crystallization conditions and the chemical modification of ligands can have unexpected effects on the binding modes.

  20. pH-tuneable binding of 2′-phospho-ADP-ribose to ketopantoate reductase: a structural and calorimetric study

    International Nuclear Information System (INIS)

    Ciulli, Alessio; Lobley, Carina M. C.; Tuck, Kellie L.; Smith, Alison G.; Blundell, Tom L.; Abell, Chris

    2007-01-01

    A combined crystallographic, calorimetric and mutagenic study has been used to show how changes in pH give rise to two distinct binding modes of 2′-phospho-ADP-ribose to ketopantoate reductase. The crystal structure of Escherichia coli ketopantoate reductase in complex with 2′-monophosphoadenosine 5′-diphosphoribose, a fragment of NADP + that lacks the nicotinamide ring, is reported. The ligand is bound at the enzyme active site in the opposite orientation to that observed for NADP + , with the adenine ring occupying the lipophilic nicotinamide pocket. Isothermal titration calorimetry with R31A and N98A mutants of the enzyme is used to show that the unusual ‘reversed binding mode’ observed in the crystal is triggered by changes in the protonation of binding groups at low pH. This research has important implications for fragment-based approaches to drug design, namely that the crystallization conditions and the chemical modification of ligands can have unexpected effects on the binding modes

  1. Isoniazid acetylating phenotype in patients with paracoccidioidomycosis and its relationship with serum sulfadoxin levels, glucose-6-phosphate dehydrogenase and glutathione reductase activities

    Directory of Open Access Journals (Sweden)

    Benedito Barraviera

    1991-06-01

    Full Text Available The authors evaluated the isoniazid acetylating phenotype and measured hematocrit, hemoglobin, glucose-6-phosphate dehydrogenase and glutathione reductase activities plus serum sulfadoxin levels in 39 patients with paracoccidioidomycosis (33 males and 6 females aged 17 to 58 years. Twenty one (53.84% of the patients presented a slow acetylatingphenotype and 18(46.16% a fast acetylating phenotype. Glucose-6-phosphate- dehydrogenase (G6PD acti vity was decreased in 5(23.80% slow acetylators and in 4(22.22% fast acetylators. Glutathione reductase activity was decreased in 14 (66.66% slow acetylators and in 12 (66.66% fast acetylators. Serum levels of free and total sulfadoxin Were higher in slow acetylator (p Os autores avaliaram o fenótipo acetilador da isoniazida, hematócrito, hemoglobina, atividade da glicose-6- fosfato desidrogenase, glutationa redutase e os níveis séricos de sulfadoxina de 39 doentes com paracoccidíoidomicose, senão 33 do sexo masculino e 6 do feminino, com idades compreendidas entre 17 e 58 anos. Vinte e um (53,84% doentes apresentaram fenótipo acetilador lento e 18 (46,16% rápido. A atividade da glicose-6-fosfato desidrogenase (G6PD esteve diminuída em 5 (23,80% acetiladores lentos e 4 (22,22% rápidos. A atividade da glutationa redutase esteve diminuída em 14 (66,66% acetiladores lentos e 12 (66,66% rápidos. Os níveis séricos de sulfadoxina livre e total foram maiores nos acetiladores lentos (p < 0,02. A análise dos resultados permite concluir que os níveis séricos de sulfadoxina relaciona-se com o fenótipo acetilador. Além disso, os níveis estiveram sempre acima de 50 µg/ml, níveis estes considerados terapêuticos. Por outro lado, a deficiência de glutationa redutase pode estar relacionada com a má absorção intestinal de nutrientes, entre eles riboflavina, vitamina precursora de FAD.

  2. Bee Venom Promotes Hair Growth in Association with Inhibiting 5α-Reductase Expression.

    Science.gov (United States)

    Park, Seeun; Erdogan, Sedef; Hwang, Dahyun; Hwang, Seonwook; Han, Eun Hye; Lim, Young-Hee

    2016-06-01

    Alopecia is an important issue that can occur in people of all ages. Recent studies show that bee venom can be used to treat certain diseases including rheumatoid arthritis, neuralgia, and multiple sclerosis. In this study, we investigated the preventive effect of bee venom on alopecia, which was measured by applying bee venom (0.001, 0.005, 0.01%) or minoxidil (2%) as a positive control to the dorsal skin of female C57BL/6 mice for 19 d. Growth factors responsible for hair growth were analyzed by quantitative real-time PCR and Western blot analysis using mice skins and human dermal papilla cells (hDPCs). Bee venom promoted hair growth and inhibited transition from the anagen to catagen phase. In both anagen phase mice and dexamethasone-induced catagen phase mice, hair growth was increased dose dependently compared with controls. Bee venom inhibited the expression of SRD5A2, which encodes a type II 5α-reductase that plays a major role in the conversion of testosterone into dihydrotestosterone. Moreover, bee venom stimulated proliferation of hDPCs and several growth factors (insulin-like growth factor 1 receptor (IGF-1R), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF)2 and 7) in bee venom-treated hDPCs dose dependently compared with the control group. In conclusion, bee venom is a potentially potent 5α-reductase inhibitor and hair growth promoter.

  3. Acrolein-Induced Dyslipidemia and Acute Phase Response Independenly of HMG-CoA Reductase

    Science.gov (United States)

    Conklin, Daniel J.; Prough, Russell A.; Juvan, Peter; Rezen, Tadeja; Rozman, Damjana; Haberzettl, Petra; Srivastava, Sanjay; Bhatnagar, Aruni

    2012-01-01

    Scope Aldehydes are ubiquitous natural constituents of foods, water and beverages. Dietary intake represents the greatest source of exposure to acrolein and related aldehydes. Oral acrolein induces dyslipidemia acutely and chronically increases atherosclerosis in mice, yet the mechanisms are unknown. Because lipid synthesis and trafficking are largely under hepatic control, we examined hepatic genes in murine models of acute and chronic oral acrolein exposure. Methods and results Changes in hepatic gene expression were examined using a Steroltalk microarray. Acute acrolein feeding modified plasma and hepatic proteins and increased plasma triglycerides within 15 min. By 6h, acrolein altered hepatic gene expression including Insig1, Insig2 and Hmgcr genes and stimulated an acute phase response (APR) with up-regulation of serum amyloid A genes (Saa) and systemic hypoalbuminemia. To test if decreased HMG-CoA reductase activity could modify acrolein-induced dyslipidemia or the APR, mice were pretreated with simvastatin. Statin treatment, however, did not alter acrolein-induced dyslipidemia or hypoalbuminemia associated with an APR. Few hepatic genes were dysregulated by chronic acrolein feeding in apoE-null mice. These studies confirmed that acute acrolein exposure altered expression of hepatic genes involved with lipid synthesis and trafficking and APR, and thus, indicated a hepatic locus of acrolein-induced dyslipidemia and APR that was independent of HMG CoA-reductase. Conclusion Dietary intake of acrolein could contribute to cardiovascular disease risk by disturbing hepatic function. PMID:21812109

  4. 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...

  5. Characterization of developmental and stress mediated expression of cinnamoyl-CoA reductase (CCR) in kenaf (Hibiscus cannabinus L.)

    Science.gov (United States)

    Cinnamoyl-CoA reductase (CCR) is an important enzyme for lignin biosynthesis as it catalyzes the first specific committed step in monolignol biosynthesis. We have cloned a full length coding sequence of CCR from kenaf (Hibiscus cannabinus L.), which contains a 1,020-bp open reading frame (ORF), enco...

  6. Identification of ribonucleotide reductase mutation causing temperature-sensitivity of herpes simplex virus isolates from whitlow by deep sequencing.

    Science.gov (United States)

    Daikoku, Tohru; Oyama, Yukari; Yajima, Misako; Sekizuka, Tsuyoshi; Kuroda, Makoto; Shimada, Yuka; Takehara, Kazuhiko; Miwa, Naoko; Okuda, Tomoko; Sata, Tetsutaro; Shiraki, Kimiyasu

    2015-06-01

    Herpes simplex virus 2 caused a genital ulcer, and a secondary herpetic whitlow appeared during acyclovir therapy. The secondary and recurrent whitlow isolates were acyclovir-resistant and temperature-sensitive in contrast to a genital isolate. We identified the ribonucleotide reductase mutation responsible for temperature-sensitivity by deep-sequencing analysis.

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

    DEFF Research Database (Denmark)

    Elmelund-Præstekær, 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...

  8. 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.

  9. Effect of thermal stability on protein adsorption to silica using homologous aldo-keto reductases.

    Science.gov (United States)

    Felsovalyi, Flora; Patel, Tushar; Mangiagalli, Paolo; Kumar, Sanat K; Banta, Scott

    2012-08-01

    Gaining more insight into the mechanisms governing the behavior of proteins at solid/liquid interfaces is particularly relevant in the interaction of high-value biologics with storage and delivery device surfaces, where adsorption-induced conformational changes may dramatically affect biocompatibility. The impact of structural stability on interfacial behavior has been previously investigated by engineering nonwild-type stability mutants. Potential shortcomings of such approaches include only modest changes in thermostability, and the introduction of changes in the topology of the proteins when disulfide bonds are incorporated. Here we employ two members of the aldo-keto reductase superfamily (alcohol dehydrogenase, AdhD and human aldose reductase, hAR) to gain a new perspective on the role of naturally occurring thermostability on adsorbed protein arrangement and its subsequent impact on desorption. Unexpectedly, we find that during initial adsorption events, both proteins have similar affinity to the substrate and undergo nearly identical levels of structural perturbation. Interesting differences between AdhD and hAR occur during desorption and both proteins exhibit some level of activity loss and irreversible conformational change upon desorption. Although such surface-induced denaturation is expected for the less stable hAR, it is remarkable that the extremely thermostable AdhD is similarly affected by adsorption-induced events. These results question the role of thermal stability as a predictor of protein adsorption/desorption behavior. Copyright © 2012 The Protein Society.

  10. Crystallization and preliminary X-ray crystallographic studies of the alkanesulfonate FMN reductase from Escherichia coli

    International Nuclear Information System (INIS)

    Gao, Benlian; Bertrand, Adam; Boles, William H.; Ellis, Holly R.; Mallett, T. Conn

    2005-01-01

    Crystallization of the native and SeMet FMN reductase protein of the E. coli alkanesulfonate monooxygenase two-component enzyme system is reported. The alkanesulfonate FMN reductase (SsuE) from Escherichia coli catalyzes the reduction of FMN by NADPH to provide reduced flavin for the monooxygenase (SsuD) enzyme. The vapor-diffusion technique yielded single crystals that grow as hexagonal rods and diffract to 2.9 Å resolution using synchrotron X-ray radiation. The protein crystallizes in the primitive hexagonal space group P622. The SsuE protein lacks any cysteine or methionine residues owing to the role of the SsuE enzyme in the acquisition of sulfur during sulfate starvation. Therefore, substitution of two leucine residues (Leu114 and Leu165) to methionine was performed to obtain selenomethionine-containing SsuE for MAD phasing. The selenomethionine derivative of SsuE has been expressed and purified and crystals of the protein have been obtained with and without bound FMN. These preliminary studies should lead to the structure solution of SsuE. It is anticipated that this new protein structure will provide detailed structural information on specific active-site regions of the protein and insight into the mechanism of flavin reduction and transfer of reduced flavin

  11. Synthesis of organic nitrates of luteolin as a novel class of potent aldose reductase inhibitors.

    Science.gov (United States)

    Wang, Qi-Qin; Cheng, Ning; Zheng, Xiao-Wei; Peng, Sheng-Ming; Zou, Xiao-Qing

    2013-07-15

    Aldose reductase (AR) plays an important role in the design of drugs that prevent and treat diabetic complications. Aldose reductase inhibitors (ARIs) have received significant attentions as potent therapeutic drugs. Based on combination principles, three series of luteolin derivatives were synthesised and evaluated for their AR inhibitory activity and nitric oxide (NO)-releasing capacity in vitro. Eighteen compounds were found to be potent ARIs with IC50 values ranging from (0.099±0.008) μM to (2.833±0.102) μM. O(7)-Nitrooxyethyl-O(3'),O(4')-ethylidene luteolin (La1) showed the most potent AR inhibitory activity [IC50=(0.099±0.008) μM]. All organic nitrate derivatives released low concentrations of NO in the presence of l-cysteine. Structure-activity relationship studies suggested that introduction of an NO donor, protection of the catechol structure, and the ether chain of a 2-carbon spacer as a coupling chain on the luteolin scaffold all help increase the AR inhibitory activity of the resulting compound. This class of NO-donor luteolin derivatives as efficient ARIs offer a new concept for the development and design of new drug for preventive and therapeutic drugs for diabetic complications. Copyright © 2013 Elsevier Ltd. All rights reserved.

  12. Prostate cancer cells differ in testosterone accumulation, dihydrotestosterone conversion, and androgen receptor signaling response to steroid 5α-reductase inhibitors.

    Science.gov (United States)

    Wu, Yue; Godoy, Alejandro; Azzouni, Faris; Wilton, John H; Ip, Clement; Mohler, James L

    2013-09-01

    Blocking 5α-reductase-mediated testosterone conversion to dihydrotestosterone (DHT) with finasteride or dutasteride is the driving hypothesis behind two prostate cancer prevention trials. Factors affecting intracellular androgen levels and the androgen receptor (AR) signaling axis need to be examined systematically in order to fully understand the outcome of interventions using these drugs. The expression of three 5α-reductase isozymes, as determined by immunohistochemistry and qRT-PCR, was studied in five human prostate cancer cell lines. Intracellular testosterone and DHT were analyzed using mass spectrometry. A luciferase reporter assay and AR-regulated genes were used to evaluate the modulation of AR activity. Prostate cancer cells were capable of accumulating testosterone to a level 15-50 times higher than that in the medium. The profile and expression of 5α-reductase isozymes did not predict the capacity to convert testosterone to DHT. Finasteride and dutasteride were able to depress testosterone uptake in addition to lowering intracellular DHT. The inhibition of AR activity following drug treatment often exceeded the expected response due to reduced availability of DHT. The ability to maintain high intracellular testosterone might compensate for the shortage of DHT. The biological effect of finasteride or dutasteride appears to be complex and may depend on the interplay of several factors, which include testosterone turnover, enzymology of DHT production, ability to use testosterone and DHT interchangeably, and propensity of cells for off-target AR inhibitory effect. © 2013 Wiley Periodicals, Inc.

  13. Sulfur Oxygenase Reductase (Sor) in the Moderately Thermoacidophilic Leaching Bacteria: Studies in Sulfobacillus thermosulfidooxidans and Acidithiobacillus caldus

    Science.gov (United States)

    Janosch, Claudia; Remonsellez, Francisco; Sand, Wolfgang; Vera, Mario

    2015-01-01

    The sulfur oxygenase reductase (Sor) catalyzes the oxygen dependent disproportionation of elemental sulfur, producing sulfite, thiosulfate and sulfide. Being considered an “archaeal like” enzyme, it is also encoded in the genomes of some acidophilic leaching bacteria such as Acidithiobacillus caldus, Acidithiobacillus thiooxidans, Acidithiobacillus ferrivorans and Sulfobacillus thermosulfidooxidans, among others. We measured Sor activity in crude extracts from Sb. thermosulfidooxidans DSM 9293T. The optimum temperature for its oxygenase activity was achieved at 75 °C, confirming the “thermophilic” nature of this enzyme. Additionally, a search for genes probably involved in sulfur metabolism in the genome sequence of Sb. thermosulfidooxidans DSM 9293T was done. Interestingly, no sox genes were found. Two sor genes, a complete heterodisulfidereductase (hdr) gene cluster, three tetrathionate hydrolase (tth) genes, three sulfide quinonereductase (sqr), as well as the doxD component of a thiosulfate quinonereductase (tqo) were found. Seven At. caldus strains were tested for Sor activity, which was not detected in any of them. We provide evidence that an earlier reported Sor activity from At. caldus S1 and S2 strains most likely was due to the presence of a Sulfobacillus contaminant. PMID:27682113

  14. Design of Deinococcus radiodurans thioredoxin reductase with altered thioredoxin specificity using computational alanine mutagenesis

    OpenAIRE

    Obiero, Josiah; Sanders, David AR

    2011-01-01

    In this study, the X-ray crystal structure of the complex between Escherichia coli thioredoxin reductase (EC TrxR) and its substrate thioredoxin (Trx) was used as a guide to design a Deinococcus radiodurans TrxR (DR TrxR) mutant with altered Trx specificity. Previous studies have shown that TrxRs have higher affinity for cognate Trxs (same species) than that for Trxs from different species. Computational alanine scanning mutagenesis and visual inspection of the EC TrxR–Trx interface suggested...

  15. 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...

  16. Crystal structure of isoflavone reductase from alfalfa (Medicago sativa L.).

    Science.gov (United States)

    Wang, Xiaoqiang; He, Xianzhi; Lin, Jianqiao; Shao, Hui; Chang, Zhenzhan; Dixon, Richard A

    2006-05-19

    Isoflavonoids play important roles in plant defense and exhibit a range of mammalian health-promoting activities. Isoflavone reductase (IFR) specifically recognizes isoflavones and catalyzes a stereospecific NADPH-dependent reduction to (3R)-isoflavanone. The crystal structure of Medicago sativa IFR with deletion of residues 39-47 has been determined at 1.6A resolution. Structural analysis, molecular modeling and docking, and comparison with the structures of other NADPH-dependent enzymes, defined the putative binding sites for co-factor and substrate and potential key residues for enzyme activity and substrate specificity. Further mutagenesis has confirmed the role of Lys144 as a catalytic residue. This study provides a structural basis for understanding the enzymatic mechanism and substrate specificity of IFRs as well as the functions of IFR-like proteins.

  17. 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

  18. 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...

  19. 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.  

  20. Transcriptional and metabolic regulation of denitrification in Paracoccus denitrificans allows low but significant activity of nitrous oxide reductase under oxic conditions.

    Science.gov (United States)

    Qu, Zhi; Bakken, Lars R; Molstad, Lars; Frostegård, Åsa; Bergaust, Linda L

    2016-09-01

    Oxygen is known to repress denitrification at the transcriptional and metabolic levels. It has been a common notion that nitrous oxide reductase (N2 OR) is the most sensitive enzyme among the four N-oxide reductases involved in denitrification, potentially leading to increased N2 O production under suboxic or fluctuating oxygen conditions. We present detailed gas kinetics and transcription patterns from batch culture experiments with Paracoccus denitrificans, allowing in vivo estimation of e(-) -flow to O2 and N2 O under various O2 regimes. Transcription of nosZ took place concomitantly with that of narG under suboxic conditions, whereas transcription of nirS and norB was inhibited until O2 levels approached 0 μM in the liquid. Catalytically functional N2 OR was synthesized and active in aerobically raised cells transferred to vials with 7 vol% O2 in headspace, but N2 O reduction rates were 10 times higher when anaerobic pre-cultures were subjected to the same conditions. Upon oxygen exposure, there was an incomplete and transient inactivation of N2 OR that could be ascribed to its lower ability to compete for electrons compared with terminal oxidases. The demonstrated reduction of N2 O at high O2 partial pressure and low N2 O concentrations by a bacterium not known as a typical aerobic denitrifier may provide one clue to the understanding of why some soils appear to act as sinks rather than sources for atmospheric N2 O. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

  1. A novel class of α-glucosidase and HMG-CoA reductase inhibitors from Ganoderma leucocontextum and the anti-diabetic properties of ganomycin I in KK-Ay mice.

    Science.gov (United States)

    Wang, Kai; Bao, Li; Ma, Ke; Zhang, Jinjin; Chen, Baosong; Han, Junjie; Ren, Jinwei; Luo, Huajun; Liu, Hongwei

    2017-02-15

    Three new meroterpenoids, ganoleucin A-C (1-3), together with five known meroterpenoids (4-8), were isolated from the fruiting bodies of Ganoderma leucocontextum. The structures of the new compounds were elucidated by extensive spectroscopic analysis, circular dichroism (CD) spectroscopy, and chemical transformation. The inhibitory effects of 1-8 on HMG-CoA reductase and α-glucosidase were tested in vitro. Ganomycin I (4), 5, and 8 showed stronger inhibitory activity against HMG-CoA reductase than the positive control atorvastatin. Compounds 1, and 3-8 presented potent noncompetitive inhibitory activity against α-glucosidase from both yeast and rat small intestinal mucosa. Ganomycin I (4), the most potent inhibitor against both α-glucosidase and HMG-CoA reductase, was synthesized and evaluated for its in vivo bioactivity. Pharmacological results showed that ganomycin I (4) exerted potent and efficacious hypoglycemic, hypolipidemic, and insulin-sensitizing effects in KK-A y mice. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  2. Drug-enhanced carbon monoxide production from heme by cytochrome P450 reductase

    Directory of Open Access Journals (Sweden)

    Dragic Vukomanovic

    2017-01-01

    Full Text Available Carbon monoxide (CO formed endogenously is considered to be cytoprotective, and the vast majority of CO formation is attributed to the degradation of heme by heme oxygenases-1 and -2 (HO-1, HO-2. Previously, we observed that brain microsomes containing HO-2 produced many-fold more CO in the presence of menadione and its congeners; herein we explored these observations further. We determined the effects of various drugs on CO production of rat brain microsomes and recombinant human cytochrome P450 reductase (CPR; CO was measured by gas chromatography with reductive detection. Brain microsomes of Sprague-Dawley rats or recombinant human cytochrome P450 reductase (CPR were incubated with NADPH and various drugs in closed vials in phosphate buffer at pH 7.4 and 37°C. After 15 minutes, the reaction was stopped by cooling in dry ice, and the headspace gas was analyzed for CO production using gas chromatography with reductive (mercuric oxide detection. We observed drug-enhanced CO production in the presence of both microsomes and recombinant CPR alone; the presence of HO was not required. A range of structurally diverse drugs were capable of amplifying this CO formation; these molecules had structures consistent with redox cycling capability. The addition of catalase to a reaction mixture, that contained activating drugs, inhibited the production of CO. Drug-enhanced CO formation can be catalyzed by CPR. The mechanism of CPR activation was not through classical drug-receptor mediation. Redox cycling may be involved in the drug-induced amplification of CO production by CPR through the production of reactive oxygen species.

  3. Superoxide reductase from the syphilis spirochete Treponema pallidum: crystallization and structure determination using soft X-rays

    Energy Technology Data Exchange (ETDEWEB)

    Santos-Silva, Teresa; Trincão, José; Carvalho, Ana L.; Bonifácio, Cecília; Auchère, Françoise; Moura, Isabel; Moura, José J. G.; Romão, Maria J., E-mail: mromao@dq.fct.unl.pt [REQUIMTE Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica (Portugal)

    2005-11-01

    Superoxide reductase is a non-haem iron-containing protein involved in resistance to oxidative stress. The oxidized form of the protein has been crystallized and its three-dimensional structure solved. A highly redundant X-ray diffraction data set was collected on a rotating-anode generator using Cu Kα X-ray radiation. Four Fe atoms were located in the asymmetric unit corresponding to four protein molecules arranged as a dimer of homodimers. Superoxide reductase is a 14 kDa metalloprotein containing a catalytic non-haem iron centre [Fe(His){sub 4}Cys]. It is involved in defence mechanisms against oxygen toxicity, scavenging superoxide radicals from the cell. The oxidized form of Treponema pallidum superoxide reductase was crystallized in the presence of polyethylene glycol and magnesium chloride. Two crystal forms were obtained depending on the oxidizing agents used after purification: crystals grown in the presence of K{sub 3}Fe(CN){sub 6} belonged to space group P2{sub 1} (unit-cell parameters a = 60.3, b = 59.9, c = 64.8 Å, β = 106.9°) and diffracted beyond 1.60 Å resolution, while crystals grown in the presence of Na{sub 2}IrCl{sub 6} belonged to space group C2 (a = 119.4, b = 60.1, c = 65.6 Å, β = 104.9°) and diffracted beyond 1.55 Å. A highly redundant X-ray diffraction data set from the C2 crystal form collected on a copper rotating-anode generator (λ = 1.542 Å) clearly defined the positions of the four Fe atoms present in the asymmetric unit by SAD methods. A MAD experiment at the iron absorption edge confirmed the positions of the previously determined iron sites and provided better phases for model building and refinement. Molecular replacement using the P2{sub 1} data set was successful using a preliminary trace as a search model. A similar arrangement of the four protein molecules could be observed.

  4. Superoxide reductase from the syphilis spirochete Treponema pallidum: crystallization and structure determination using soft X-rays

    International Nuclear Information System (INIS)

    Santos-Silva, Teresa; Trincão, José; Carvalho, Ana L.; Bonifácio, Cecília; Auchère, Françoise; Moura, Isabel; Moura, José J. G.; Romão, Maria J.

    2005-01-01

    Superoxide reductase is a non-haem iron-containing protein involved in resistance to oxidative stress. The oxidized form of the protein has been crystallized and its three-dimensional structure solved. A highly redundant X-ray diffraction data set was collected on a rotating-anode generator using Cu Kα X-ray radiation. Four Fe atoms were located in the asymmetric unit corresponding to four protein molecules arranged as a dimer of homodimers. Superoxide reductase is a 14 kDa metalloprotein containing a catalytic non-haem iron centre [Fe(His) 4 Cys]. It is involved in defence mechanisms against oxygen toxicity, scavenging superoxide radicals from the cell. The oxidized form of Treponema pallidum superoxide reductase was crystallized in the presence of polyethylene glycol and magnesium chloride. Two crystal forms were obtained depending on the oxidizing agents used after purification: crystals grown in the presence of K 3 Fe(CN) 6 belonged to space group P2 1 (unit-cell parameters a = 60.3, b = 59.9, c = 64.8 Å, β = 106.9°) and diffracted beyond 1.60 Å resolution, while crystals grown in the presence of Na 2 IrCl 6 belonged to space group C2 (a = 119.4, b = 60.1, c = 65.6 Å, β = 104.9°) and diffracted beyond 1.55 Å. A highly redundant X-ray diffraction data set from the C2 crystal form collected on a copper rotating-anode generator (λ = 1.542 Å) clearly defined the positions of the four Fe atoms present in the asymmetric unit by SAD methods. A MAD experiment at the iron absorption edge confirmed the positions of the previously determined iron sites and provided better phases for model building and refinement. Molecular replacement using the P2 1 data set was successful using a preliminary trace as a search model. A similar arrangement of the four protein molecules could be observed

  5. The Effect of Nitrate Levels and Harvest Times on Fe, Zn, Cu, and K, Concentrations and Nitrate Reductase Activity in Lettuce and Spinach

    Directory of Open Access Journals (Sweden)

    Z. Gheshlaghi

    2015-09-01

    Full Text Available Leafy vegetables are considered as the main sources of nitrate in the human diet. In order to investigate the effect of nitrate levels and harvest times on nitrate accumulation, nitrate reductase activity, concentrations of Fe, Zn, Cu and K in Lettuce and Spinach and their relation to nitrate accumulation in these leafy vegetables, two harvest times (29 and 46 days after transplanting, two vegetable species of lettuce and spinach and two concentrations of nitrate (10 and 20 mM were used in a hydroponics greenhouse experiment with a completely randomized design and 3 replications. Modified Hoagland and Arnon nutrient solutions were used for the experiment. The results indicated that by increasing nitrate concentration of solution, nitrate accumulation in roots and shoots of lettuce and spinach increased significantly (P ≤ 0.05, and the same trend was observed for the nitrate reductase activity in the shoots of the two species. Increasing the nitrate concentrations of solution, reduced the shoot dry weight and the concentration of Fe and Cu in both species, where as it increased the K and Zn concentrations in the shoots of the two species in each both harvest times, the nitrate accumulation increased, but the nitrate reductase activity decreased in the shoots of the two species over the course of the growth. The Concentration of Fe, Cu and K decreased in the shoots of lettuce and the spinach with the time, despite the increase in Zn concentration in the shoots. The results also indicated that increasing nitrate concentrations of solution to the levels greater than the plant capacity for reduction and net uptake of nitrate, leads to the nitrate accumulation in the plants. Nitrate accumulation in plant tissue led to decreases in fresh shoot yield and Fe and Cu concentrations and nitrate reductase activities in both lettuce and spinach.

  6. 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....

  7. 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...

  8. Effects of NADH-preferring xylose reductase expression on ethanol production from xylose in xylose-metabolizing recombinant Saccharomyces cerevisiae.

    Science.gov (United States)

    Lee, Sung-Haeng; Kodaki, Tsutomu; Park, Yong-Cheol; Seo, Jin-Ho

    2012-04-30

    Efficient conversion of xylose to ethanol is an essential factor for commercialization of lignocellulosic ethanol. To minimize production of xylitol, a major by-product in xylose metabolism and concomitantly improve ethanol production, Saccharomyces cerevisiae D452-2 was engineered to overexpress NADH-preferable xylose reductase mutant (XR(MUT)) and NAD⁺-dependent xylitol dehydrogenase (XDH) from Pichia stipitis and endogenous xylulokinase (XK). In vitro enzyme assay confirmed the functional expression of XR(MUT), XDH and XK in recombinant S. cerevisiae strains. The change of wild type XR to XR(MUT) along with XK overexpression led to reduction of xylitol accumulation in microaerobic culture. More modulation of the xylose metabolism including overexpression of XR(MUT) and transaldolase, and disruption of the chromosomal ALD6 gene encoding aldehyde dehydrogenase (SX6(MUT)) improved the performance of ethanol production from xylose remarkably. Finally, oxygen-limited fermentation of S. cerevisiae SX6(MUT) resulted in 0.64 g l⁻¹ h⁻¹ xylose consumption rate, 0.25 g l⁻¹ h⁻¹ ethanol productivity and 39% ethanol yield based on the xylose consumed, which were 1.8, 4.2 and 2.2 times higher than the corresponding values of recombinant S. cerevisiae expressing XR(MUT), XDH and XK only. Copyright © 2011 Elsevier B.V. All rights reserved.

  9. Statins: 3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitors demonstrate anti-atherosclerotic character due to their antioxidant capacity

    Digital Repository Service at National Institute of Oceanography (India)

    Puttananjaiah, M.H.; Dhale, M.A.; Gaonkar, V.; Keni, S.

    inhibitors (commonly known as statins) are widely used in cardiovascular disease prevention to lower the cholesterol. The antioxidant activity of HMG-CoA reductase inhibitors was studied by lipid peroxidation inhibition assay, DPPH, and hydroxyl radical...

  10. Fragment Discovery for the Design of Nitrogen Heterocycles as Mycobacterium tuberculosis Dihydrofolate Reductase Inhibitors.

    Science.gov (United States)

    Shelke, Rupesh U; Degani, Mariam S; Raju, Archana; Ray, Mukti Kanta; Rajan, Mysore G R

    2016-08-01

    Fragment-based drug design was used to identify Mycobacterium tuberculosis (Mtb) dihydrofolate reductase (DHFR) inhibitors. Screening of ligands against the Mtb DHFR enzyme resulted in the identification of multiple fragment hits with IC50 values in the range of 38-90 μM versus Mtb DHFR and minimum inhibitory concentration (MIC) values in the range of 31.5-125 μg/mL. These fragment scaffolds would be useful for anti-tubercular drug design. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Pharmacologically relevant receptor binding characteristics and 5alpha-reductase inhibitory activity of free Fatty acids contained in saw palmetto extract.

    Science.gov (United States)

    Abe, Masayuki; Ito, Yoshihiko; Oyunzul, Luvsandorj; Oki-Fujino, Tomomi; Yamada, Shizuo

    2009-04-01

    Saw palmetto extract (SPE), used widely for the treatment of benign prostatic hyperplasia (BPH) has been shown to bind alpha(1)-adrenergic, muscarinic and 1,4-dihydropyridine (1,4-DHP) calcium channel antagonist receptors. Major constituents of SPE are lauric acid, oleic acid, myristic acid, palmitic acid and linoleic acid. The aim of this study was to investigate binding affinities of these fatty acids for pharmacologically relevant (alpha(1)-adrenergic, muscarinic and 1,4-DHP) receptors. The fatty acids inhibited specific [(3)H]prazosin binding in rat brain in a concentration-dependent manner with IC(50) values of 23.8 to 136 microg/ml, and specific (+)-[(3)H]PN 200-110 binding with IC(50) values of 24.5 to 79.5 microg/ml. Also, lauric acid, oleic acid, myristic acid and linoleic acid inhibited specific [(3)H]N-methylscopolamine ([(3)H]NMS) binding in rat brain with IC(50) values of 56.4 to 169 microg/ml. Palmitic acid had no effect on specific [(3)H]NMS binding. The affinity of oleic acid, myristic acid and linoleic acid for each receptor was greater than the affinity of SPE. Scatchard analysis revealed that oleic acid and lauric acid caused a significant decrease in the maximal number of binding sites (B(max)) for [(3)H]prazosin, [(3)H]NMS and (+)-[(3)H]PN 200-110. The results suggest that lauric acid and oleic acid bind noncompetitively to alpha(1)-adrenergic, muscarinic and 1,4-DHP calcium channel antagonist receptors. We developed a novel and convenient method of determining 5alpha-reductase activity using LC/MS. With this method, SPE was shown to inhibit 5alpha-reductase activity in rat liver with an IC(50) of 101 microg/ml. Similarly, all the fatty acids except palmitic acid inhibited 5alpha-reductase activity, with IC(50) values of 42.1 to 67.6 microg/ml. In conclusion, lauric acid, oleic acid, myristic acid, and linoleic acid, major constituents of SPE, exerted binding activities of alpha(1)-adrenergic, muscarinic and 1,4-DHP receptors and inhibited 5

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

    Science.gov (United States)

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

    2015-12-25

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

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

    Science.gov (United States)

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

    2015-01-01

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

  14. The role of HMG-CoA reductase inhibition in endothelial dysfunction and inflammation

    Directory of Open Access Journals (Sweden)

    Paolo Gelosa

    2007-11-01

    Full Text Available Paolo Gelosa1, Mauro Cimino2, Alice Pignieri1, Elena Tremoli1,3, Uliano Guerrini1, Luigi Sironi11Department of Pharmacological Sciences, University of Milan, Italy; 2Institute of Pharmacological Sciences, Carlo Bo University of Urbino, Italy; 3Monzino Cardiologic Center IRCCS, Milan, ItalyAbstract: Statin-induced inhibition of HMG-CoA reductase reduces cholesterol production and prevents the formation of many non-steroidal isoprenoid compounds, such as farnesylpyrophosphate and geranylgeranylpyrophosphate, that act as lipid attachments for the post-translational modification of various proteins, including the G-proteins and transcription factors involved in a number of cell processes. However, the blockade of isoprenylation elicited by statin treatment also has biological effects on cell function that go beyond the decrease in cholesterol synthesis: these are the so-called “pleiotropic” effects that mainly relate to vascular function. Endothelial dysfunction is an independent predictor of cardiovascular events that correlates with inflammation markers/mediators and robust predictors of cardiovascular diseases such as increased high-sensitivity C-reactive protein levels. The results of in vivo and in vitro studies indicate that the statins have beneficial effects unrelated to cholesterol lowering, such as improving endothelial function, increasing myocardial perfusion, and enhancing the availability of nitric oxide. This review describes the pleiotropic effects of statins that may be involved in modulating/preventing endothelial dysfunction and inflammatory processes, as well as the cellular and molecular mechanisms through which they improve endothelial function.Keywords: statins; inflammation; endothelial dysfunction; nitric oxide; HMG-CoA reductase

  15. Synthesis and 5α-Reductase Inhibitory Activity of C21 Steroids Having 1,4-diene or 4,6-diene 20-ones and 4-Azasteroid 20-Oximes

    Directory of Open Access Journals (Sweden)

    Eunsook Ma

    2011-12-01

    Full Text Available The synthesis and evaluation of 5α-reductase inhibitory activity of some 4-azasteroid-20-ones and 20-oximes and 3β-hydroxy-, 3β-acetoxy-, or epoxy-substituted C21 steroidal 20-ones and 20-oximes having double bonds in the A and/or B ring are described. Inhibitory activity of synthesized compounds was assessed using 5α-reductase enzyme and [1,2,6,7-3H]testosterone as substrate. All synthesized compounds were less active than finasteride (IC50: 1.2 nM. Three 4-azasteroid-2-oximes (compounds 4, 6 and 8 showed good inhibitory activity (IC50: 26, 10 and 11 nM and were more active than corresponding 4-azasteroid 20-ones (compounds 3, 5 and 7. 3β-Hydroxy-, 3β-acetoxy- and 1α,2α-, 5α,6α- or 6α,7α-epoxysteroid-20-one and -20-oxime derivatives having double bonds in the A and/or B ring showed no inhibition of 5α-reductase enzyme.

  16. A maize gene encoding an NADPH binding enzyme highly homologous to isoflavone reductases is activated in response to sulfur starvation.

    Science.gov (United States)

    Petrucco, S; Bolchi, A; Foroni, C; Percudani, R; Rossi, G L; Ottonello, S

    1996-01-01

    we isolated a novel gene that is selectively induced both in roots and shoots in response to sulfur starvation. This gene encodes a cytosolic, monomeric protein of 33 kD that selectively binds NADPH. The predicted polypeptide is highly homologous ( > 70%) to leguminous isoflavone reductases (IFRs), but the maize protein (IRL for isoflavone reductase-like) belongs to a novel family of proteins present in a variety of plants. Anti-IRL antibodies specifically recognize IFR polypeptides, yet the maize protein is unable to use various isoflavonoids as substrates. IRL expression is correlated closely to glutathione availability: it is persistently induced in seedlings whose glutathione content is about fourfold lower than controls, and it is down-regulated rapidly when control levels of glutathione are restored. This glutathione-dependent regulation indicates that maize IRL may play a crucial role in the establishment of a thiol-independent response to oxidative stress under glutathione shortage conditions.

  17. 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...

  18. Reduced glutathione concentration and glutathione reductase activity in various rat tissues after the administration of some radioprotective agents

    International Nuclear Information System (INIS)

    Pulpanova, J.; Kovarova, H.; Ledvina, M.

    1982-01-01

    The concentrations of reduced glutathione (GSH) and activity of glutathione reductase were investigated in rat liver, kidney and spleen after intraperitoneal administration of cystamine (50 mg/kg), mexamine (10 mg/kg), or a mixture of cystamine with mexamine (20 + 10 mg/kg). The GSH concentration increased after the administration of cystamine in the liver (maximum between the 20th and 30th min), in the kidney and spleen (maximum after 60 min). The cystamine + mexamine mixture also caused a significant increase of the GSH concentration in all the organs investigated; however, the values increased at earlier intervals as after the cystamine administration. No substantial effect was shown in the case of the mexamine administration, only 30 min after the administration the values were higher. The activity of glutathione reductase was significantly lower over the entire period examined. This was found in the liver and kidney as after the administration of cystamine, as after the radioprotective mixture. There was also a less pronounced inhibition of the enzyme activity in the spleen. Mexamine as a single radioprotector had practically no influence on the activity. (author)

  19. Radiation inactivation analysis of assimilatory NADH:nitrate reductase. Apparent functional sizes of partial activities associated with intact and proteolytically modified enzyme

    International Nuclear Information System (INIS)

    Solomonson, L.P.; McCreery, M.J.; Kay, C.J.; Barber, M.J.

    1987-01-01

    Recently we demonstrated that target sizes for the partial activities of nitrate reductase were considerably smaller than the 100-kDa subunit which corresponded to the target size of the full (physiologic) activity NADH:nitrate reductase. These results suggested that the partial activities resided on functionally independent domains and that radiation inactivation may be due to localized rather than extensive damage to protein structure. The present study extends these observations and addresses several associated questions. Monophasic plots were observed over a wide range of radiation doses, suggesting a single activity component in each case. No apparent differences were observed over a 10-fold range of concentration for each substrate, suggesting that the observed slopes were not due to marked changes in Km values. Apparent target sizes estimated for partial activities associated with native enzyme and with limited proteolysis products of native enzyme suggested that the functional size obtained by radiation inactivation analysis is independent of the size of the polypeptide chain. The presence of free radical scavengers during irradiation reduced the apparent target size of both the physiologic and partial activities by an amount ranging from 24 to 43%, suggesting that a free radical mechanism is at least partially responsible for the inactivation. Immunoblot analysis of nitrate reductase irradiated in the presence of free radical scavengers revealed formation of distinct bands at 90, 75, and 40 kDa with increasing doses of irradiation rather than complete destruction of the polypeptide chain

  20. NMR evaluation of total statin content and HMG-CoA reductase inhibition in red yeast rice (Monascus spp. food supplements

    Directory of Open Access Journals (Sweden)

    Lachenmeier Dirk W

    2012-03-01

    Full Text Available Abstract Background Red yeast rice (i.e., rice fermented with Monascus spp., as a food supplement, is claimed to be blood cholesterol-lowering. The red yeast rice constituent monacolin K, also known as lovastatin, is an inhibitor of the hydroxymethylglutaryl-CoA (HMG-CoA reductase. This article aims to develop a sensitive nuclear magnetic resonance (NMR method to determine the total statin content of red yeast rice products. Methods The total statin content was determined by a 400 MHz 1H NMR spectroscopic method, based on the integration of the multiplet at δ 5.37-5.32 ppm of a hydrogen at the hexahydronaphthalene moiety in comparison to an external calibration with lovastatin. The activity of HMG-CoA reductase was measured by a commercial spectrophotometric assay kit. Results The NMR detection limit for total statins was 6 mg/L (equivalent to 0.3 mg/capsule, if two capsules are dissolved in 50 mL ethanol. The relative standard deviations were consistently lower than 11%. The total statin concentrations of five red yeast rice supplements were between 1.5 and 25.2 mg per specified daily dose. A dose-dependent inhibition of the HMG-CoA reductase enzyme activity by the red yeast rice products was demonstrated. Conclusion A simple and direct NMR assay was developed to determine the total statin content in red yeast rice. The assay can be applied for the determination of statin content for the regulatory control of red yeast rice products.

  1. Influence of the enzyme dissimilatory sulfite reductase on stable isotope fractionation during sulfate reduction

    Science.gov (United States)

    Mangalo, Muna; Einsiedl, Florian; Meckenstock, Rainer U.; Stichler, Willibald

    2008-03-01

    The stable isotopes of sulfate are often used as a tool to assess bacterial sulfate reduction on the macro scale. However, the mechanisms of stable isotope fractionation of sulfur and oxygen at the enzymatic level are not yet fully understood. In batch experiments with water enriched in 18O we investigated the effect of different nitrite concentrations on sulfur isotope fractionation by Desulfovibrio desulfuricans. With increasing nitrite concentrations, we found sulfur isotope enrichment factors ranging from -11.2 ± 1.8‰ to -22.5 ± 3.2‰. Furthermore, the δ18O values in the remaining sulfate increased from approximately 50-120‰ when 18O-enriched water was supplied. Since 18O-exchange with ambient water does not take place in sulfate, but rather in intermediates of the sulfate reduction pathway (e.g. SO32-), we suggest that nitrite affects the steady-state concentration and the extent of reoxidation of the metabolic intermediate sulfite to sulfate during sulfate reduction. Given that nitrite is known to inhibit the production of the enzyme dissimilatory sulfite reductase, our results suggest that the activity of the dissimilatory sulfite reductase regulates the kinetic isotope fractionation of sulfur and oxygen during bacterial sulfate reduction. Our novel results also imply that isotope fractionation during bacterial sulfate reduction strongly depends on the cell internal enzymatic regulation rather than on the physico-chemical features of the individual enzymes.

  2. 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.

  3. An engineered fatty acid synthase combined with a carboxylic acid reductase enables de novo production of 1-octanol in Saccharomyces cerevisiae.

    Science.gov (United States)

    Henritzi, Sandra; Fischer, Manuel; Grininger, Martin; Oreb, Mislav; Boles, Eckhard

    2018-01-01

    The ideal biofuel should not only be a regenerative fuel from renewable feedstocks, but should also be compatible with the existing fuel distribution infrastructure and with normal car engines. As the so-called drop-in biofuel, the fatty alcohol 1-octanol has been described as a valuable substitute for diesel and jet fuels and has already been produced fermentatively from sugars in small amounts with engineered bacteria via reduction of thioesterase-mediated premature release of octanoic acid from fatty acid synthase or via a reversal of the β-oxidation pathway. The previously engineered short-chain acyl-CoA producing yeast Fas1 R1834K /Fas2 fatty acid synthase variant was expressed together with carboxylic acid reductase from Mycobacterium marinum and phosphopantetheinyl transferase Sfp from Bacillus subtilis in a Saccharomyces cerevisiae Δfas1 Δfas2 Δfaa2 mutant strain. With the involvement of endogenous thioesterases, alcohol dehydrogenases, and aldehyde reductases, the synthesized octanoyl-CoA was converted to 1-octanol up to a titer of 26.0 mg L -1 in a 72-h fermentation. The additional accumulation of 90 mg L -1 octanoic acid in the medium indicated a bottleneck in 1-octanol production. When octanoic acid was supplied externally to the yeast cells, it could be efficiently converted to 1-octanol indicating that re-uptake of octanoic acid across the plasma membrane is not limiting. Additional overexpression of aldehyde reductase Ahr from Escherichia coli nearly completely prevented accumulation of octanoic acid and increased 1-octanol titers up to 49.5 mg L -1 . However, in growth tests concentrations even lower than 50.0 mg L -1 turned out to be inhibitory to yeast growth. In situ extraction in a two-phase fermentation with dodecane as second phase did not improve growth, indicating that 1-octanol acts inhibitive before secretion. Furthermore, 1-octanol production was even reduced, which results from extraction of the intermediate octanoic acid to

  4. The roles of H2S and H2O2 in regulating AsA-GSH cycle in the leaves of wheat seedlings under drought stress.

    Science.gov (United States)

    Shan, Changjuan; Zhang, Shengli; Ou, Xingqi

    2018-01-25

    This paper investigated the roles of hydrogen sulfide (H 2 S) and hydrogen peroxide (H 2 O 2 ) and the possible relationship between them in regulating the AsA-GSH cycle in wheat leaves under drought stress (DS). Results showed that DS markedly increased the production of H 2 S and H 2 O 2 , the transcript levels and activities of ascorbate peroxidase (APX), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR), and dehydroascorbate reductase (DHAR); malondialdehyde (MDA) content; and electrolyte leakage (EL). Meanwhile, DS markedly reduced plant height and biomass. Above increases induced by drought stress except MDA content and EL were all suppressed by pretreatments with H 2 S synthesis inhibitor aminooxyaceticacid (AOA) and H 2 O 2 synthesis inhibitor diphenylene iodonium (DPI). Besides, pretreatments with AOA and DPI further significantly increased MDA content and EL and significantly reduced plant height and biomass under DS. DPI reduced the production of H 2 O 2 and H 2 S induced by DS. AOA also reduced the production of H 2 S and H 2 O 2 induced by DS. Pretreatments with NaHS + AOA and H 2 O 2 + DPI reversed above effects of AOA and DPI. Our results suggested that H 2 S and H 2 O 2 all participated in the up-regulation of AsA-GSH cycle in wheat leaves by DS and possibly affected each other.

  5. Biliverdin reductase: more than a namesake - the reductase, its Peptide fragments, and biliverdin regulate activity of the three classes of protein kinase C.

    Science.gov (United States)

    Gibbs, Peter E M; Tudor, Cicerone; Maines, Mahin D

    2012-01-01

    The expanse of human biliverdin reductase (hBVR) functions in the cells is arguably unmatched 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 protein kinase C (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.

  6. Aldose reductase inhibitory compounds from Xanthium strumarium.

    Science.gov (United States)

    Yoon, Ha Na; Lee, Min Young; Kim, Jin-Kyu; Suh, Hong-Won; Lim, Soon Sung

    2013-09-01

    As part of our ongoing search for natural sources of therapeutic and preventive agents for diabetic complications, we evaluated the inhibitory effects of components of the fruit of Xanthium strumarium (X. strumarium) on aldose reductase (AR) and galactitol formation in rat lenses with high levels of glucose. To identify the bioactive components of X. strumarium, 7 caffeoylquinic acids and 3 phenolic compounds were isolated and their chemical structures were elucidated on the basis of spectroscopic evidence and comparison with published data. The abilities of 10 X. strumarium-derived components to counteract diabetic complications were investigated by means of inhibitory assays with rat lens AR (rAR) and recombinant human AR (rhAR). From the 10 isolated compounds, methyl-3,5-di-O-caffeoylquinate showed the most potent inhibition, with IC₅₀ values of 0.30 and 0.67 μM for rAR and rhAR, respectively. In the kinetic analyses using Lineweaver-Burk plots of 1/velocity and 1/substrate, methyl-3,5-di-O-caffeoylquinate showed competitive inhibition of rhAR. Furthermore, methyl-3,5-di-O-caffeoylquinate inhibited galactitol formation in the rat lens and in erythrocytes incubated with a high concentration of glucose, indicating that this compound may be effective in preventing diabetic complications.

  7. Expression and activity of sulfate transporters and APS reductase in curly kale in response to sulfate deprivation and re-supply

    NARCIS (Netherlands)

    Koralewska, Aleksandra; Buchner, Peter; Stuiver, C. Elisabeth E.; Posthumus, Freek S.; Kopriva, Stanislav; Hawkesford, Malcolm J.; De Kok, Luit J.

    2009-01-01

    Both activity and expression of sulfate transporters and APS reductase in plants are modulated by the sulfur status of the plant. To examine the regulatory mechanisms in curly kale (Brossica olerracea L.), the sulfate supply was manipulated by the transfer of seedlings to sulfate-deprived

  8. 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...

  9. Loss of nitrate reductases NIA1 and NIA2 impairs stomatal closure by altering genes of core ABA signaling components in Arabidopsis.

    Science.gov (United States)

    Zhao, Chenchen; Cai, Shengguan; Wang, Yizhou; Chen, Zhong-Hua

    2016-06-02

    Nitrate reductases NIA1 and NIA2 determine NO production in plants and are critical to abscisic acid (ABA)-induced stomatal closure. However, the role for NIA1 and NIA2 in ABA signaling has not been paid much attention in nitrate reductase loss-of-function mutant nia1nia2. Recently, we have demonstrated that ABA-inhibited K(+)in current and ABA-enhanced slow anion current were absent in nia1nia2. Exogenous NO restored regulation of these channels for stomatal closure in nia1nia2. In this study, we found that mutating NIA1 and NIA2 impaired nearly all the key components of guard cell ABA signaling pathway in Arabidopsis. We also propose a simplified model for ABA signaling in the nia1nia2 mutant.

  10. Expression, purification, crystallization and preliminary X-ray analysis of conjugated polyketone reductase C2 (CPR-C2) from Candida parapsilosis IFO 0708.

    Science.gov (United States)

    Yamamura, Akihiro; Maruoka, Shintaro; Ohtsuka, Jun; Miyakawa, Takuya; Nagata, Koji; Kataoka, Michihiko; Kitamura, Nahoko; Shimizu, Sakayu; Tanokura, Masaru

    2009-11-01

    Conjugated polyketone reductase C2 (CPR-C2) from Candida parapsilosis IFO 0708 is a member of the NADPH-dependent aldo-keto reductase (AKR) superfamily and catalyzes the stereospecific reduction of ketopantoyl lactone to d-pantoyl lactone. A diffraction-quality crystal of recombinant CPR-C2 was obtained by the sitting-drop vapour-diffusion method using PEG 3350 as the precipitant. The crystal diffracted X-rays to 1.7 angstrom resolution on beamline NW12A of the Photon Factory-Advanced Ring (Tsukuba, Japan). The crystal belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 55.02, b = 68.30, c = 68.93 angstrom. The Matthews coefficient (V(M) = 1.76 angstrom(3) Da(-1)) indicated that the crystal contained one CPR-C2 molecule per asymmetric unit.

  11. 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.

  12. 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....

  13. 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.

  14. Aldose reductase mediates retinal microglia activation

    International Nuclear Information System (INIS)

    Chang, Kun-Che; Shieh, Biehuoy; Petrash, J. Mark

    2016-01-01

    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"G"F"P mice carrying AR mutant alleles to evaluate the role of AR on RMG activation and migration in vivo. When tested on an AR"W"T 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.

  15. Selection of drug resistant mutants from random library of Plasmodium falciparum dihydrofolate reductase in Plasmodium berghei model

    OpenAIRE

    Tipsuwan, Wachiraporn; Srichairatanakool, Somdet; Kamchonwongpaisan, Sumalee; Yuthavong, Yongyuth; Uthaipibull, Chairat

    2011-01-01

    Abstract Background The prevalence of drug resistance amongst the human malaria Plasmodium species has most commonly been associated with genomic mutation within the parasites. This phenomenon necessitates evolutionary predictive studies of possible resistance mutations, which may occur when a new drug is introduced. Therefore, identification of possible new Plasmodium falciparum dihydrofolate reductase (PfDHFR) mutants that confer resistance to antifolate drugs is essential in the process of...

  16. Expression of reductases in continuous mammal cell cultures and its significance for the activation of nitroaromatics shown for the example of 1.6 dinitropyrene

    International Nuclear Information System (INIS)

    Reuter, U.

    1993-01-01

    The aim of the first part of the work was to establish the metabolism of 1,3- and 1,6-DNP in intact cells. This gave rise to the following questions. What metabolites are formed in cell lines with different enzyme outfits? What influence does the induction of P450 have on the metabolism of the two nitroaromates? Does the metabolism found in the different test cell lines permit any conclusions as to the activating mechanism of 1,6- and 1,3-DNP. In the second part these test cell lines were studied with respect to the expression of the reductases that might be involved in the metabolism of aromates. The following questions were of focal interest: Are cytochrome reductase, DT diaphorase and xanthine-oxidase expressed in the cell lines? If so, to what extent? Can these enzymes be induced in the test cell lines? In the last part the enzymes that reduce 1,6-DNP to gene-toxic products were identified. This required clarifying the following: What role do the above-mentioned reductases play in the activation of 1,6-DNP in individual cell lines? Are there other enzymes responsible for the activation of 1,6-DNP? (MG) [de

  17. Nitrous oxide reductase genes (nosZ) of denitrifying populations in soil and the earthworm gut are phylogenetically similar

    DEFF Research Database (Denmark)

    Horn, Marcus A.; Drake, Harold L.; Schramm, Andreas

    2006-01-01

    -derived sequences, or were related to N2O reductases of the genera Bradyrhizobium, Brucella, Dechloromonas, Flavobacterium, Pseudomonas, Ralstonia, and Sinorhizobium. Although the numbers of estimators for genotype richness of sequence data from the gut were higher than those of soil, only one gut-derived nos...

  18. Methylenetetrahydrofolate reductase genotypes and predisposition to atherothrombotic disease; evidence that all three MTHFR C677T genotypes confer different levels of risk.

    NARCIS (Netherlands)

    Kluijtmans, L.A.J.; Whitehead, A.S.

    2001-01-01

    AIMS: Elevated plasma homocysteine is an independent risk factor for atherothrombotic disease. Individuals homozygous for the methylenetetrahydrofolate reductase (MTHFR) 677C allele exclusively accumulate 5methyltetrahydrofolate, the methyl donor for homocysteine remethylation, in their red blood

  19. Homocysteine and the methylenetetrahydrofolate reductase 677C-->T polymorphism in relation to muscle mass and strength, physical performance and postural sway

    NARCIS (Netherlands)

    Swart, K. M. A.; Enneman, A. W.; van Wijngaarden, J. P.; van Dijk, S. C.; Brouwer-Brolsma, E. M.; Ham, A. C.; Dhonukshe-Rutten, R. A. M.; van der Velde, N.; Brug, J.; van Meurs, J. B. J.; de Groot, L. C. P. G. M.; Uitterlinden, A. G.; Lips, P.; van Schoor, N. M.

    2013-01-01

    Elevated plasma homocysteine has been linked to reduced mobility and muscle functioning in the elderly. The relation of methylenetetrahydrofolate reductase (MTHFR) 677C-->T polymorphism with these associations has not yet been studied. This study aimed to investigate (1) the association of plasma

  20. A conservative region of the mercuric reductase gene (merA as a molecular marker of bacterial mercury resistance Região conservada do gene da mercúrio redutase (merA como marcador molecular da resistência bacteriana ao mercúrio

    Directory of Open Access Journals (Sweden)

    Adriana Sotero-Martins

    2008-06-01

    Full Text Available The most common bacterial mercury resistance mechanism is based on the reduction of Hg(II to Hg0, which is dependent of the mercuric reductase enzyme (MerA activity. The use of a 431 bp fragment of a conservative region of the mercuric reductase (merA gene was applied as a molecular marker of this mechanism, allowing the identification of mercury resistant bacterial strains.O mecanismo de resistência bacteriana ao mercúrio mais comum é baseada na redução do Hg(II a Hg0, através da atividade da enzima mercúrio redutase (MerA. O uso do fragmento de 431 pb amplificado de uma região conservada do gene merA, que codifica a enzima MerA,foi utilizado como marcador molecular deste mecanismo, permitindo a identificação de bactérias resistentes ao mercúrio.