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Sample records for aldo-keto reductase 1c3

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

    SciTech Connect

    Hofman, Jakub; Malcekova, Beata; Skarka, Adam; Novotna, Eva; Wsol, Vladimir

    2014-08-01

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

  2. Aldo-keto reductase (AKR) 1C3: role in prostate disease and the development of specific inhibitors.

    PubMed

    Penning, Trevor M; Steckelbroeck, Stephan; Bauman, David R; Miller, Meredith W; Jin, Yi; Peehl, Donna M; Fung, Kar-Ming; Lin, Hseuh-Kung

    2006-03-27

    Human aldo-keto reductases (AKR) of the 1A, 1B, 1C and 1D subfamilies are involved in the pre-receptor regulation of nuclear (steroid hormone and orphan) receptors by regulating the local concentrations of their lipophilic ligands. AKR1C3 is one of the most interesting isoforms. It was cloned from human prostate and the recombinant protein was found to function as a 3-, 17- and 20-ketosteroid reductase with a preference for the conversion of Delta4-androstene-3,17-dione to testosterone implicating this enzyme in the local production of active androgens within the prostate. Using a validated isoform specific real-time RT-PCR procedure the AKR1C3 transcript was shown to be more abundant in primary cultures of epithelial cells than stromal cells, and its expression in stromal cells increased with benign and malignant disease. Using a validated isoform specific monoclonal Ab, AKR1C3 protein expression was also detected in prostate epithelial cells by immunoblot analysis. Immunohistochemical staining of prostate tissue showed that AKR1C3 was expressed in adenocarcinoma and surprisingly high expression was observed in the endothelial cells. These cells are a rich source of prostaglandin G/H synthase 2 (COX-2) and vasoactive prostaglandins (PG) and thus the ability of recombinant AKR1C enzymes to act as PGF synthases was compared. AKR1C3 had the highest catalytic efficiency (kcat/Km) for the 11-ketoreduction of PGD2 to yield 9alpha,11beta-PGF2 raising the prospect that AKR1C3 may govern ligand access to peroxisome proliferator activated receptor (PPARgamma). Activation of PPARgamma is often a pro-apoptotic signal and/or leads to terminal differentiation, while 9alpha,11beta-PGF2 is a pro-proliferative signal. AKR1C3 is potently inhibited by non-steroidal anti-inflammatory drugs suggesting that the cancer chemopreventive properties of these agents may be mediated either by inhibition of AKR1C3 or COX. To discriminate between these effects we developed potent AKR1C

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

    PubMed

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

    2015-06-05

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

  4. Roles of aldo-keto reductases 1B10 and 1C3 and ATP-binding cassette transporter in docetaxel tolerance.

    PubMed

    Matsunaga, Toshiyuki; Saito, Haruhi; Endo, Satoshi; Iguchi, Kazuhiro; Soda, Midori; El-Kabbani, Ossama; Hara, Akira; Ikari, Akira

    2016-12-01

    Docetaxel (DTX) is widely used for treatment of inveterate lung and prostate cancers, but its continuous administration elicits the hyposensitivity. Here, we established the DTX-resistant variants of human lung cancer A549 and androgen-independent prostate cancer Du145 cells and found that the resistance development provoked aberrant up-regulations of aldo-keto reductase (AKR) 1B10 and AKR1C3 in A549 and Du145 cells, respectively. In addition, the sensitivity to the DTX toxicity was significantly decreased and increased by overexpression and knockdown of the two AKR isoforms, respectively. Furthermore, the resistant cells exhibited a decreased level of reactive 4-hydroxy-2-nonenal formed during DTX treatment, and the decrease was alleviated by adding the AKR inhibitors, inferring that the two AKRs confer the chemoresistance through elevating the antioxidant properties. The development of DTX resistance was also associated with enhanced expression of an ATP-binding cassette (ABC) transporter ABCB1 among the ABC transporter isoforms. The combined treatment with inhibitors of the two AKRs and ABCB1 additively sensitized the resistant cells to DTX. Intriguingly, the AKR1B10 inhibitor also suppressed the lung cancer cross-resistance against cisplatin. The results suggest that combined treatment with AKRs (1B10 and 1C3) and ABCB1 inhibitors exerts overcoming effect against the cancer resistance to DTX and cisplatin, and can be used as the adjuvant therapy.

  5. Aldo-keto reductases AKR1C1, AKR1C2 and AKR1C3 may enhance progesterone metabolism in ovarian endometriosis.

    PubMed

    Hevir, N; Vouk, K; Sinkovec, J; Ribič-Pucelj, M; Rižner, T Lanišnik

    2011-05-30

    Endometriosis is a very common disease that is characterized by increased formation of estradiol and disturbed progesterone action. This latter is usually explained by a lack of progesterone receptor B (PR-B) expression, while the role of pre-receptor metabolism of progesterone is not yet fully understood. In normal endometrium, progesterone is metabolized by reductive 20α-hydroxysteroid dehydrogenases (20α-HSDs), 3α/β-HSDs and 5α/β-reductases. The aldo-keto reductases 1C1 and 1C3 (AKR1C1 and AKR1C3) are the major reductive 20α-HSDs, while the oxidative reaction is catalyzed by 17β-HSD type 2 (HSD17B2). Also, 3α-HSD and 3β-HSD activities have been associated with the AKR1C isozymes. Additionally, 5α-reductase types 1 and 2 (SRD5A1, SRD5A2) and 5β-reductase (AKR1D1) are responsible for the formation of 5α- and 5β-reduced pregnanes. In this study, we examined the expression of PR-AB and the progesterone metabolizing enzymes in 31 specimens of ovarian endometriosis and 28 specimens of normal endometrium. Real-time PCR analysis revealed significantly decreased mRNA levels of PR-AB, HSD17B2 and SRD5A2, significantly increased mRNA levels of AKR1C1, AKR1C2, AKR1C3 and SRD5A1, and negligible mRNA levels of AKR1D1. Immunohistochemistry staining of endometriotic tissue compared to control endometrium showed significantly lower PR-B levels in epithelial cells and no significant differences in stromal cells, there were no significant differences in the expression of AKR1C3 and significantly higher AKR1C2 levels were seen only in stromal cells. Our expression analysis data at the mRNA level and partially at the cellular level thus suggest enhanced metabolism of progesterone by SRD5A1 and the 20α-HSD and 3α/β-HSD activities of AKR1C1, AKR1C2 and AKR1C3.

  6. The aldo-keto reductase superfamily homepage.

    PubMed

    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.

  7. Development of Potent and Selective Inhibitors of Aldo-Keto Reductase 1C3 (type 5 17β-Hydroxysteroid Dehydrogenase) Based on N-Phenyl-Aminobenzoates and Their Structure Activity Relationships

    PubMed Central

    Adeniji, Adegoke O.; Twenter, Barry M.; Byrns, Michael C.; Jin, Yi; Chen, Mo; Winkler, Jeffrey D.; Penning, Trevor M.

    2012-01-01

    Aldo-keto reductase 1C3 (AKR1C3; type 5 17β-hydroxysteroid dehydrogenase) is overexpressed in castrate resistant prostate cancer (CRPC) and is implicated in the intratumoral biosynthesis of testosterone and 5α-dihydrotestosterone. Selective AKR1C3 inhibitors are required since compounds should not inhibit the highly related AKR1C1 and AKR1C2 isoforms which are involved in the inactivation of 5α-dihydrotestosterone. NSAIDs, N-phenylanthranilates in particular are potent but non-selective AKR1C3 inhibitors. Using flufenamic acid, 2-{[3-(trifluoromethyl)phenyl]amino}benzoic acid as lead compound, five classes of structural analogs were synthesized and evaluated for AKR1C3 inhibitory potency and selectivity. Structure activity relationship (SAR) studies revealed that a meta-carboxylic acid group relative to the amine conferred pronounced AKR1C3 selectivity without loss of potency, while electron withdrawing groups on the phenylamino B-ring were optimal for AKR1C3 inhibition. Lead compounds did not inhibit COX-1 or COX-2 but blocked the AKR1C3 mediated production of testosterone in LNCaP-AKR1C3 cells. These compounds offer promising leads towards new therapeutics for CRPC. PMID:22263837

  8. Comparative anatomy of the aldo-keto reductase superfamily.

    PubMed

    Jez, J M; Bennett, M J; Schlegel, B P; Lewis, M; Penning, T M

    1997-09-15

    The aldo-keto reductases metabolize a wide range of substrates and are potential drug targets. This protein superfamily includes aldose reductases, aldehyde reductases, hydroxysteroid dehydrogenases and dihydrodiol dehydrogenases. By combining multiple sequence alignments with known three-dimensional structures and the results of site-directed mutagenesis studies, we have developed a structure/function analysis of this superfamily. Our studies suggest that the (alpha/beta)8-barrel fold provides a common scaffold for an NAD(P)(H)-dependent catalytic activity, with substrate specificity determined by variation of loops on the C-terminal side of the barrel. All the aldo-keto reductases are dependent on nicotinamide cofactors for catalysis and retain a similar cofactor binding site, even among proteins with less than 30% amino acid sequence identity. Likewise, the aldo-keto reductase active site is highly conserved. However, our alignments indicate that variation ofa single residue in the active site may alter the reaction mechanism from carbonyl oxidoreduction to carbon-carbon double-bond reduction, as in the 3-oxo-5beta-steroid 4-dehydrogenases (Delta4-3-ketosteroid 5beta-reductases) of the superfamily. Comparison of the proposed substrate binding pocket suggests residues 54 and 118, near the active site, as possible discriminators between sugar and steroid substrates. In addition, sequence alignment and subsequent homology modelling of mouse liver 17beta-hydroxysteroid dehydrogenase and rat ovary 20alpha-hydroxysteroid dehydrogenase indicate that three loops on the C-terminal side of the barrel play potential roles in determining the positional and stereo-specificity of the hydroxysteroid dehydrogenases. Finally, we propose that the aldo-keto reductase superfamily may represent an example of divergent evolution from an ancestral multifunctional oxidoreductase and an example of convergent evolution to the same active-site constellation as the short

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

    PubMed

    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.

  10. The Aldo-Keto Reductase Superfamily and its Role in Drug Metabolism and Detoxification

    PubMed Central

    Barski, Oleg A.; Tipparaju, Srinivas M.; Bhatnagar, Aruni

    2008-01-01

    The Aldo-Keto Reductase (AKR) superfamily comprises of several enzymes that catalyze redox transformations involved in biosynthesis, intermediary metabolism and detoxification. Substrates of the family include glucose, steroids, glycosylation end products, lipid peroxidation products, and environmental pollutants. These proteins adopt a (β/α)8 barrel structural motif interrupted by a number of extraneous loops and helixes that vary between proteins and bring structural identity to individual families. The human AKR family differs from the rodent families. Due to their broad substrate specificity, AKRs play an important role in the Phase II detoxification of a large number of pharmaceuticals, drugs, and xenobiotics. PMID:18949601

  11. Pyrithione-based ruthenium complexes as inhibitors of aldo-keto reductase 1C enzymes and anticancer agents.

    PubMed

    Kljun, Jakob; Anko, Maja; Traven, Katja; Sinreih, Maša; Pavlič, Renata; Peršič, Špela; Ude, Žiga; Codina, Elisa Esteve; Stojan, Jure; Lanišnik Rižner, Tea; Turel, Iztok

    2016-08-07

    Four ruthenium complexes of clinically used zinc ionophore pyrithione and its oxygen analog 2-hydroxypyridine N-oxide were prepared and evaluated as inhibitors of enzymes of the aldo-keto reductase subfamily 1C (AKR1C). A kinetic study assisted with docking simulations showed a mixed type of inhibition consisting of a fast reversible and a slow irreversible step in the case of both organometallic compounds 1A and 1B. Both compounds also showed a remarkable selectivity towards AKR1C1 and AKR1C3 which are targets for breast cancer drug design. The organoruthenium complex of ligand pyrithione as well as pyrithione itself also displayed toxicity on the hormone-dependent MCF-7 breast cancer cell line with EC50 values in the low micromolar range.

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

    SciTech Connect

    Scharf, Michael; Sethi, Amit

    2016-09-13

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

  13. Enzymatic detection of γ-hydroxybutyrate using aldo-keto reductase 7A2.

    PubMed

    Bendinskas, Kestutis; Sattelberg, Patricia; Crossett, Daniel; Banyikwa, Andrew; Dempsey, Daniel; MacKenzie, James A

    2011-05-01

    Gamma-hydroxybutyrate (GHB) is a prescribed medication as well as a drug of abuse. Its detection in various matrices for in-field forensic scientists remains a challenge. We have developed an assay that uses aldo-keto reductase 7A2 (AKR7A2) for the specific determination of GHB in various drinks. AKR7A2 was purified using Ni-affinity chromatography. The Michaelis-Menten constant for the GHB oxidation reaction was 10 mM, and the minimum detection limit was 4 mM. Ethanol was not a substrate for AKR7A2. In a coupled reaction with NADP(+), phenazine methosulfate (PMS), and 2,6-dichlorophenolindophenol, various beverages (orange juice, milk, soda, and numerous alcoholic drinks) containing GHB turned from blue to light yellow. In a second coupled reaction where diaphorase replaced PMS, the presence of GHB also caused the expected change of color in various beers.

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

    PubMed

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

    2016-11-01

    A human member of the aldo-keto reductase (AKR) superfamily, AKR1B10, is a cytosolic NADPH-dependent reductase toward various carbonyl compounds including reactive aldehydes, and is normally expressed in intestines. The enzyme is overexpressed in several extraintestinal cancers, and suggested as a potential target for cancer treatment. We found that saturated and cis-unsaturated fatty acids inhibit AKR1B10. Among the saturated fatty acids, myristic acid was the most potent, showing the IC50 value of 4.2 μM cis-Unsaturated fatty acids inhibited AKR1B10 more potently, and linoleic, arachidonic, and docosahexaenoic acids showed the lowest IC50 values of 1.1 μM. The inhibition by these fatty acids was reversible and kinetically competitive with respect to the substrate, showing the Ki values of 0.24-1.1 μM. These fatty acids, except for α-linoleic acid, were much less inhibitory to structurally similar aldose reductase. Site-directed mutagenesis study suggested that the fatty acids interact with several active site residues of AKR1B10, of which Gln114, Val301 and Gln303 are responsible for the inhibitory selectivity. Linoleic and arachidonic acids also effectively inhibited AKR1B10-mediated 4-oxo-2-nonenal metabolism in HCT-15 cells. Thus, the cis-unsaturated fatty acids may be used as an adjuvant therapy for treatment of cancers that up-regulate AKR1B10.

  15. Putative Role of the Aldo-Keto Reductase from Trypanosoma cruzi in Benznidazole Metabolism

    PubMed Central

    Garavaglia, Patricia Andrea; Laverrière, Marc; Cannata, Joaquín J. B.

    2016-01-01

    Benznidazole (Bz), the drug used for treatment of Chagas' disease (caused by the protozoan Trypanosoma cruzi), is activated by a parasitic NADH-dependent type I nitroreductase (NTR I). However, several studies have shown that other enzymes are involved. The aim of this study was to evaluate whether the aldo-keto reductase from T. cruzi (TcAKR), a NADPH-dependent oxido-reductase previously described by our group, uses Bz as the substrate. We demonstrated that both recombinant and native TcAKR enzymes reduce Bz by using NADPH, but not NADH, as a cofactor. TcAKR-overexpressing epimastigotes showed higher NADPH-dependent Bz reductase activity and a 50% inhibitory concentration (IC50) value for Bz 1.8-fold higher than that of the controls, suggesting that TcAKR is involved in Bz detoxification instead of activation. To understand the role of TcAKR in Bz metabolism, we studied TcAKR expression and NADPH/NADH-dependent Bz reductase activities in two T. cruzi strains with differential susceptibility to Bz: CL Brener and Nicaragua. Taking into account the results obtained with TcAKR-overexpressing epimastigotes, we expected the more resistant strain, Nicaragua, to have higher TcAKR levels than CL Brener. However, the results were the opposite. CL Brener showed 2-fold higher TcAKR expression and 5.7-fold higher NADPH-Bz reduction than the Nicaragua strain. In addition, NADH-dependent Bz reductase activity, characteristic of NTR I, was also higher in CL Brener than in Nicaragua. We conclude that although TcAKR uses Bz as the substrate, TcAKR activity is not a determinant of Bz resistance in wild-type strains and may be overcome by other enzymes involved in Bz activation, such as NADPH- and NADH-dependent reductases. PMID:26856844

  16. Putative Role of the Aldo-Keto Reductase from Trypanosoma cruzi in Benznidazole Metabolism.

    PubMed

    Garavaglia, Patricia Andrea; Laverrière, Marc; Cannata, Joaquín J B; García, Gabriela Andrea

    2016-05-01

    Benznidazole (Bz), the drug used for treatment of Chagas' disease (caused by the protozoan Trypanosoma cruzi), is activated by a parasitic NADH-dependent type I nitroreductase (NTR I). However, several studies have shown that other enzymes are involved. The aim of this study was to evaluate whether the aldo-keto reductase from T. cruzi (TcAKR), a NADPH-dependent oxido-reductase previously described by our group, uses Bz as the substrate. We demonstrated that both recombinant and native TcAKR enzymes reduce Bz by using NADPH, but not NADH, as a cofactor. TcAKR-overexpressing epimastigotes showed higher NADPH-dependent Bz reductase activity and a 50% inhibitory concentration (IC50) value for Bz 1.8-fold higher than that of the controls, suggesting that TcAKR is involved in Bz detoxification instead of activation. To understand the role of TcAKR in Bz metabolism, we studied TcAKR expression and NADPH/NADH-dependent Bz reductase activities in two T. cruzi strains with differential susceptibility to Bz: CL Brener and Nicaragua. Taking into account the results obtained with TcAKR-overexpressing epimastigotes, we expected the more resistant strain, Nicaragua, to have higher TcAKR levels than CL Brener. However, the results were the opposite. CL Brener showed 2-fold higher TcAKR expression and 5.7-fold higher NADPH-Bz reduction than the Nicaragua strain. In addition, NADH-dependent Bz reductase activity, characteristic of NTR I, was also higher in CL Brener than in Nicaragua. We conclude that although TcAKR uses Bz as the substrate, TcAKR activity is not a determinant of Bz resistance in wild-type strains and may be overcome by other enzymes involved in Bz activation, such as NADPH- and NADH-dependent reductases.

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

    PubMed

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

    2016-09-09

    In recent years, concerns about the use of glyphosate-resistant (GR) crops have increased because of glyphosate residual levels in plants and development of herbicide-resistant weeds. In spite of identifying glyphosate detoxifying genes from microorganisms, the plant mechanism to detoxify glyphosate has not been studied. We characterized an Aldo-keto reductase gene from Pseudomonas (PsAKR1) and rice (OsAKR1) and showed, by docking studies, both PsAKR1 and OsAKR1 can efficiently bind to glyphosate. Silencing AKR1 homologs in rice and Nicotiana benthamiana or mutation of AKR1 in yeast and Arabidopsis showed increased sensitivity to glyphosate. External application of AKR proteins rescued glyphosate-mediated cucumber seedlings growth inhibition. Regeneration of tobacco transgenic lines expressing PsAKR1 or OsAKRI on glyphosate suggests that AKR can be used as selectable marker to develop transgenic crops. PsAKR1 or OsAKRI expressing tobacco and rice transgenic plants showed improved tolerance to glyphosate with reduced accumulation of shikimic acid without affecting the normal photosynthetic rates. These results suggested that AKR1 when overexpressed detoxifies glyphosate in planta. This article is protected by copyright. All rights reserved.

  18. Structure and Promoter Characterization of Aldo-Keto Reductase Family 1 B10 Gene

    PubMed Central

    Liu, Ziwen; Zhong, Linlin; Krishack, Paulette A; Robbins, Sarah; Cao, Julia X; Zhao, Yupei; Chung, Stephen; Cao, Deliang

    2009-01-01

    Aldo-keto reductase family 1 member B10 (AKR1B10) is overexpressed in human hepatocellular carcinoma, lung squamous carcinoma, and lung adenocarcinoma in smokers. Our recent studies have showed that AKR1B10 plays a critical role in the growth and proliferation of cancer cells by detoxifying reactive carbonyls and regulating fatty acid biosynthesis. However, little is known about the regulatory mechanisms of AKR1B10 expression. In this study, we determined the structure of AKR1B10 gene and characterized its promoter. The results demonstrated that AKR1B10 consists of 10 exons and 9 introns, stretching approximately 13.8 kb. A 5′-RACE study determined the transcriptional start site of AKR1B10 at 320 bp upstream of the ATG translational start codon. A TATA-like (TAATAA) and a CAAT box are present from −145 to −140 bp and −193 to −190 bp upstream of the transcriptional start site, respectively. Motif analysis recognized multiple putative oncogenic and tumor suppressor protein binding sites in the AKR1B10 promoter, including c-Ets-1, C/EBP, AP-1, and p53, but osmolytic response elements were not found. A -4,091 bp of the 5′-flanking fragment of the AKR1B10 gene was capable of driving GFP and luciferase reporter gene expression in HepG2 cells derived from human hepatocellular carcinoma; progressive 5′-deletions revealed that a −255 bp fragment possesses full promoter activity. PMID:19236911

  19. Human Aldo-Keto Reductases and the Metabolic Activation of Polycyclic Aromatic Hydrocarbons

    PubMed Central

    2015-01-01

    Aldo-keto reductases (AKRs) are promiscuous NAD(P)(H) dependent oxidoreductases implicated in the metabolic activation of polycyclic aromatic hydrocarbons (PAH). These enzymes catalyze the oxidation of non-K-region trans-dihydrodiols to the corresponding o-quinones with the concomitant production of reactive oxygen species (ROS). The PAH o-quinones are Michael acceptors and can form adducts but are also redox-active and enter into futile redox cycles to amplify ROS formation. Evidence exists to support this metabolic pathway in humans. The human recombinant AKR1A1 and AKR1C1–AKR1C4 enzymes all catalyze the oxidation of PAH trans-dihydrodiols to PAH o-quinones. Many human AKRs also catalyze the NADPH-dependent reduction of the o-quinone products to air-sensitive catechols, exacerbating ROS formation. Moreover, this pathway of PAH activation occurs in a panel of human lung cell lines, resulting in the production of ROS and oxidative DNA damage in the form of 8-oxo-2′-deoxyguanosine. Using stable-isotope dilution liquid chromatography tandem mass spectrometry, this pathway of benzo[a]pyrene (B[a]P) metabolism was found to contribute equally with the diol-epoxide pathway to the activation of this human carcinogen in human lung cells. Evaluation of the mutagenicity of anti-B[a]P-diol epoxide with B[a]P-7,8-dione on p53 showed that the o-quinone produced by AKRs was the more potent mutagen, provided that it was permitted to redox cycle, and that the mutations observed were G to T transversions, reminiscent of those observed in human lung cancer. It is concluded that there is sufficient evidence to support the role of human AKRs in the metabolic activation of PAH in human lung cell lines and that they may contribute to the causation of human lung cancer. PMID:25279998

  20. Aldo-keto Reductase 1B15 (AKR1B15)

    PubMed Central

    Weber, Susanne; Salabei, Joshua K.; Möller, Gabriele; Kremmer, Elisabeth; Bhatnagar, Aruni; Adamski, Jerzy; Barski, Oleg A.

    2015-01-01

    Aldo-keto reductases (AKRs) comprise a superfamily of proteins involved in the reduction and oxidation of biogenic and xenobiotic carbonyls. In humans, at least 15 AKR superfamily members have been identified so far. One of these is a newly identified gene locus, AKR1B15, which clusters on chromosome 7 with the other human AKR1B subfamily members (i.e. AKR1B1 and AKR1B10). We show that alternative splicing of the AKR1B15 gene transcript gives rise to two protein isoforms with different N termini: AKR1B15.1 is a 316-amino acid protein with 91% amino acid identity to AKR1B10; AKR1B15.2 has a prolonged N terminus and consists of 344 amino acid residues. The two gene products differ in their expression level, subcellular localization, and activity. In contrast with other AKR enzymes, which are mostly cytosolic, AKR1B15.1 co-localizes with the mitochondria. Kinetic studies show that AKR1B15.1 is predominantly a reductive enzyme that catalyzes the reduction of androgens and estrogens with high positional selectivity (17β-hydroxysteroid dehydrogenase activity) as well as 3-keto-acyl-CoA conjugates and exhibits strong cofactor selectivity toward NADP(H). In accordance with its substrate spectrum, the enzyme is expressed at the highest levels in steroid-sensitive tissues, namely placenta, testis, and adipose tissue. Placental and adipose expression could be reproduced in the BeWo and SGBS cell lines, respectively. In contrast, AKR1B15.2 localizes to the cytosol and displays no enzymatic activity with the substrates tested. Collectively, these results demonstrate the existence of a novel catalytically active AKR, which is associated with mitochondria and expressed mainly in steroid-sensitive tissues. PMID:25577493

  1. Genomic Rearrangements Leading to Overexpression of Aldo-Keto Reductase YafB of Escherichia coli Confer Resistance to Glyoxal

    PubMed Central

    Kwon, Minsuk; Lee, Junghoon; Lee, Changhan

    2012-01-01

    Glyoxal is toxic and mutagenic α-oxoaldehyde generated in vivo as an oxidation by-product of sugar metabolism. We selected glyoxal-resistant mutants from an Escherichia coli strain lacking major glyoxal-detoxifying genes, gloA and yqhD, by growing cells in medium containing a lethal concentration of glyoxal. The mutants carried diverse genomic rearrangements, such as multibase deletions and recombination, in the upstream region of the yafB gene, encoding an aldo-keto reductase. Since these genomic lesions create transcriptional fusions of the yafB gene to the upstream rrn regulon or eliminate a negative regulatory site, the mutants generally enhanced an expression of the yafB gene. Glyoxal resistances of the mutants are correlated with the levels of yafB transcripts as well as the activities of aldo-keto reductase. An overproduction of YafB in the glyoxal-resistant mutant lacking the putative NsrR-binding site provides evidence that the yafB gene is negatively regulated by this protein. We also observed that the expression of yafB is enhanced with an increased concentration of glyoxal as well as a mutation in the fnr gene, encoding a putative regulator. The bindings of NsrR and Fnr to the yafB promoter were also demonstrated by gel mobility shift assays. PMID:22328670

  2. The aldo-keto reductase AKR1B7 coexpresses with renin without influencing renin production and secretion.

    PubMed

    Machura, Katharina; Iankilevitch, Elina; Neubauer, Björn; Theuring, Franz; Kurtz, Armin

    2013-03-01

    On the basis of evidence that within the adult kidney, the aldo-keto reductase AKR1B7 (aldo-keto reductase family 1, member 7, also known as mouse vas deferens protein, MVDP) is selectively expressed in renin-producing cells, we aimed to define a possible role of AKR1B7 for the regulation and function of renin cells in the kidney. We could confirm colocalization and corecruitment of renin and of AKR1B7 in wild-type kidneys. Renin cells in AKR1B7-deficient kidneys showed normal morphology, numbers, and intrarenal distribution. Plasma renin concentration (PRC) and renin mRNA levels of AKR1B7-deficient mice were normal at standard chow and were lowered by a high-salt diet directly comparable to wild-type mice. Treatment with a low-salt diet in combination with an angiotensin-converting enzyme inhibitor strongly increased PRC and renin mRNA in a similar fashion both in AKR1B7-deficient and wild-type mice. Under this condition, we also observed a strong retrograde recruitment of renin-expressing cell along the preglomerular vessels, however, without a difference between AKR1B7-deficient and wild-type mice. The isolated perfused mouse kidney model was used to study the acute regulation of renin secretion by ANG II and by perfusion pressure. Regarding these parameters, no differences were observed between AKR1B7-deficient and wild-type kidneys. In summary, our data suggest that AKR1B7 is not of major relevance for the regulation of renin production and secretion in spite of its striking coregulation with renin expression.

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

    PubMed

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

    2012-05-01

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

  4. Chicken muscle aldose reductase: purification, properties and relationship to other chicken aldo/keto reductases.

    PubMed

    Murphy, D G; Davidson, W S

    1986-01-01

    An enzyme that catalyzes the NADPH-dependent reduction of a wide range of aromatic and hydroxy-aliphatic aldehydes was purified from chicken breast muscle. This enzyme shares many properties with mammalian aldose reductases including molecular weight, relative substrate specificity, Michaelis constants, an inhibitor specificity. Therefore, it seems appropriate to call this enzyme an aldose reductase (EC 1.1.1.21). Chicken muscle aldose reductase appears to be kinetically identical to an aldose reductase that has been purified from chicken kidney (Hara et al., Eur. J. Biochem. 133, 207-214) and to hen muscle L-glycol dehydrogenase (Bernado et al., Biochim. biophys. Acta 659, 189-198). The association of this aldose reductase with muscular dystrophy in the chick is discussed.

  5. Catalytic mechanism and substrate selectivity of aldo-keto reductases: insights from structure-function studies of Candida tenuis xylose reductase.

    PubMed

    Kratzer, Regina; Wilson, David K; Nidetzky, Bernd

    2006-09-01

    Aldo-keto reductases (AKRs) constitute a large protein superfamily of mainly NAD(P)-dependent oxidoreductases involved in carbonyl metabolism. Catalysis is promoted by a conserved tetrad of active site residues (Tyr, Lys, Asp and His). Recent results of structure-function relationship studies for xylose reductase (AKR2B5) require an update of the proposed catalytic mechanism. Electrostatic stabilization by the epsilon-NH3+ group of Lys is a key source of catalytic power of xylose reductase. A molecular-level analysis of the substrate binding pocket of xylose reductase provides a case of how a very broadly specific AKR achieves the requisite selectivity for its physiological substrate and could serve as the basis for the design of novel reductases with improved specificities for biocatalytic applications.

  6. Aldo-keto reductase 1b7, a novel marker for renin cells, is regulated by cyclic AMP signaling

    PubMed Central

    Lin, Eugene E.; Pentz, Ellen S.; Sequeira-Lopez, Maria Luisa S.

    2015-01-01

    We previously identified aldo-keto reductase 1b7 (AKR1B7) as a marker for juxtaglomerular renin cells in the adult mouse kidney. However, the distribution of renin cells varies dynamically, and it was unknown whether AKR1B7 maintains coexpression with renin in response to different developmental, physiological, and pathological situations, and furthermore, whether similar factor(s) simultaneously regulate both proteins. We show here that throughout kidney development, AKR1B7 expression—together with renin—is progressively restricted in the kidney arteries toward the glomerulus. Subsequently, when formerly renin-expressing cells reacquire renin expression, AKR1B7 is reexpressed as well. This pattern of coexpression persists in extreme pathological situations, such as deletion of the genes for aldosterone synthase or Dicer. However, the two proteins do not colocalize within the same organelles: renin is found in the secretory granules, whereas AKR1B7 localizes to the endoplasmic reticulum. Interestingly, upon deletion of the renin gene, AKR1B7 expression is maintained in a pattern mimicking the embryonic expression of renin, while ablation of renin cells resulted in complete abolition of AKR1B7 expression. Finally, we demonstrate that AKR1B7 transcription is controlled by cAMP. Cultured cells of the renin lineage reacquire the ability to express both renin and AKR1B7 upon elevation of intracellular cAMP. In vivo, deleting elements of the cAMP-response pathway (CBP/P300) results in a stark decrease in AKR1B7- and renin-positive cells. In summary, AKR1B7 is expressed within the renin cell throughout development and perturbations to homeostasis, and AKR1B7 is regulated by cAMP levels within the renin cell. PMID:26180185

  7. Oxidation of PAH trans-Dihydrodiols by Human Aldo-Keto Reductase AKR1B10

    PubMed Central

    Quinn, Amy M.; Harvey, Ronald G.; Penning, Trevor M.

    2009-01-01

    AKR1B10 has been identified as a potential biomarker for human non-small cell lung carcinoma and as a tobacco exposure and response gene. AKR1B10 functions as an efficient retinal reductase in vitro, and may regulate retinoic acid homeostasis. However, the possibility that this enzyme is able to activate polycyclic aromatic hydrocarbon (PAH) trans-dihydrodiols to form reactive and redox-active o-quinones has not been investigated to date. AKR1B10 was found to oxidize a wide range of PAH trans-dihydrodiol substrates in vitro to yield PAH o-quinones. Reactions of AKR1B10 proceeded with improper stereochemistry, since it was specific for the minor (+)-benzo[a]pyrene-7S,8S-dihydrodiol diastereomer formed in vivo. However, AKR1B10 displayed reasonable activity in the oxidation of both the (−)-R,R and (+)-S,S stereoisomers of benzo[g]chrysene-11,12-dihydrodiol and oxidized the potentially relevant, albeit minor, (+)-benz[a]anthracene-3S,4S-dihydrodiol metabolite. We find that AKR1B10 is therefore likely to play a contributing role in the activation of PAH trans-dihydrodiols in human lung. AKR1B10 retinal reductase activity was confirmed in vitro and found to be 5- to 150-fold greater than the oxidation of PAH trans-dihydrodiols examined. AKR1B10 was highly expressed at the mRNA and protein levels in human lung adenocarcinoma A549 cells, and robust retinal reductase activity was measured in lysates of these cells. The much greater catalytic efficiency of retinal reduction compared to PAH trans-dihydrodiol metabolism suggests AKR1B10 may play a greater role in lung carcinogenesis through dysregulation of retinoic acid homeostasis than through oxidation of PAH trans-dihydrodiols. PMID:18788756

  8. Heightened aggressive behavior in mice deficient in aldo-keto reductase 1a (Akr1a).

    PubMed

    Homma, Takujiro; Akihara, Ryusuke; Okano, Satoshi; Shichiri, Mototada; Yoshida, Yasukazu; Yamada, Ken-Ichi; Miyata, Satoshi; Nakajima, Osamu; Fujii, Junichi

    2017-02-15

    Aldehyde reductase (Akr1a) is involved in the synthesis of ascorbic acid (AsA) which may play a role in social behavior. In the current study, we performed analyses on Akr1a-deficient (Akr1a(-/-)) mice that synthesize about 10% as much AsA as wild-type mice from the viewpoint of intermale aggression. The use of the resident-intruder test revealed that the Akr1a(-/-) mice exhibited more aggressive phenotypes than wild-type control mice. Unexpectedly, however, the oral administration of additional AsA failed to reduce the aggressive behavior of Akr1a(-/-) mice, suggesting that the heightened aggression was independent of AsA biosynthesis. The findings also show that the plasma levels of corticosterone, but not serotonin and testosterone, were increased in the absence of Akr1a in mice, suggesting that the mice were highly stressed. These results suggest that Akr1a might be involved in the metabolism of steroids and other carbonyl-containing compounds and, hence, the absence of Akr1a results in heightened aggression via a malfunction in a metabolic pathway.

  9. Bioequivalence studies of tibolone in premenopausal women and effects on expression of the tibolone-metabolizing enzyme AKR1C (aldo-keto reductase) family caused by estradiol.

    PubMed

    Kang, Keon W; Kim, Yoon G

    2008-12-01

    This study aimed to investigate the bioequivalence of a test formulation of tibolone with the marketed reference formulation in 24 young healthy female volunteers. Tibolone is a synthetic steroid hormone for menopausal women. Volunteers were treated with the 2 formulations of tibolone (total dose of active ingredient 2.5 mg) according to a 2 x 2 crossover design with a 1-week washout period. Plasma concentrations of 3alpha- and 3beta-hydroxytibolone, which are major metabolites of tibolone, were assayed in timed samples over a 24-hour period with a validated gas chromatography/mass spectrometry (GC/MS) method that had a lower limit of quantification of 0.5 ng/mL. The reference and test formulations gave a mean 3alpha-hydroxytibolone C(max) of 5.0 and 5.2 ng/mL, respectively, and a mean 3beta-hydroxytibolone C(max) of 16.4 and 16.5 ng/mL, respectively. The mean AUC(t) of 3alpha-hydroxytibolone was 24.7 and 24.3 ng h/mL, whereas the mean AUC(t) of 3beta-hydroxytibolone was 57.6 and 54.8 ng h/mL for the test and reference formulations, respectively. The authors did not find significant differences in pharmacokinetic parameters between the 2 formulations, but metabolite formation was different from reports in postmenopausal women. The authors therefore measured the effects of estradiol on the expression of the tibolone-metabolizing enzymes, from the aldo-keto reductase (AKR1C) family, using HepG2 cell (human hepatoma cells) and MCF-7 cell (human breast cancer cells). Estradiol increased mRNA levels of AKR1C1, AKR1C2, and AKR1C3 and protein levels of total AKR1C in HepG2 cells. Estradiol selectively enhanced levels of AKR1C2 mRNA in MCF-7 cells. Thus, changes in the major metabolites of tibolone might result from changes in AKR1C family expression by patient estrogen status.

  10. The Role of Human Aldo-Keto Reductases in the Metabolic Activation and Detoxication of Polycyclic Aromatic Hydrocarbons: Interconversion of PAH Catechols and PAH o-Quinones.

    PubMed

    Zhang, Li; Jin, Yi; Huang, Meng; Penning, Trevor M

    2012-01-01

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

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

    PubMed

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

    2016-05-20

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

  12. Aldo-keto reductase 1C1 induced by interleukin-1β mediates the invasive potential and drug resistance of metastatic bladder cancer cells

    PubMed Central

    Matsumoto, Ryuji; Tsuda, Masumi; Yoshida, Kazuhiko; Tanino, Mishie; Kimura, Taichi; Nishihara, Hiroshi; Abe, Takashige; Shinohara, Nobuo; Nonomura, Katsuya; Tanaka, Shinya

    2016-01-01

    In treating bladder cancer, determining the molecular mechanisms of tumor invasion, metastasis, and drug resistance are urgent to improving long-term patient survival. One of the metabolic enzymes, aldo-keto reductase 1C1 (AKR1C1), plays an essential role in cancer invasion/metastasis and chemoresistance. In orthotopic xenograft models of a human bladder cancer cell line, UM-UC-3, metastatic sublines were established from tumors in the liver, lung, and bone. These cells possessed elevated levels of EMT-associated markers, such as Snail, Slug, or CD44, and exhibited enhanced invasion. By microarray analysis, AKR1C1 was found to be up-regulated in metastatic lesions, which was verified in metastatic human bladder cancer specimens. Decreased invasion caused by AKR1C1 knockdown suggests a novel role of AKR1C1 in cancer invasion, which is probably due to the regulation of Rac1, Src, or Akt. An inflammatory cytokine, interleukin-1β, was found to increase AKR1C1 in bladder cancer cell lines. One particular non-steroidal anti-inflammatory drug, flufenamic acid, antagonized AKR1C1 and decreased the cisplatin-resistance and invasion potential of metastatic sublines. These data uncover the crucial role of AKR1C1 in regulating both metastasis and drug resistance; as a result, AKR1C1 should be a potent molecular target in invasive bladder cancer treatment. PMID:27698389

  13. Aldo-keto reductases are biomarkers of NRF2 activity and are co-ordinately overexpressed in non-small cell lung cancer

    PubMed Central

    MacLeod, A Kenneth; Acosta-Jimenez, Lourdes; Coates, Philip J; McMahon, Michael; Carey, Frank A; Honda, Tadashi; Henderson, Colin J; Wolf, C Roland

    2016-01-01

    Background: Although the nuclear factor-erythroid 2-related factor 2 (NRF2) pathway is one of the most frequently dysregulated in cancer, it is not clear whether mutational status is a good predictor of NRF2 activity. Here we utilise four members of the aldo-keto reductase (AKR) superfamily as biomarkers to address this question. Methods: Twenty-three cell lines of diverse origin and NRF2-pathway mutational status were used to determine the relationship between AKR expression and NRF2 activity. AKR expression was evaluated in lung cancer biopsies and Cancer Genome Atlas (TCGA) and Oncomine data sets. Results: AKRs were expressed at a high basal level in cell lines carrying mutations in the NRF2 pathway. In non-mutant cell lines, co-ordinate induction of AKRs was consistently observed following activation of NRF2. Immunohistochemical analysis of lung tumour biopsies and interrogation of TCGA data revealed that AKRs are enriched in both squamous cell carcinomas (SCCs) and adenocarcinomas that contain somatic alterations in the NRF2 pathway but, in the case of SCC, AKRs were also enriched in most other tumours. Conclusions: An AKR biomarker panel can be used to determine NRF2 status in tumours. Hyperactivation of the NRF2 pathway is far more prevalent in lung SCC than previously predicted by genomic analyses. PMID:27824809

  14. Cloning of a novel aldo-keto reductase gene from Klebsiella sp. strain F51-1-2 and its functional expression in Escherichia coli.

    PubMed

    Jiang, Hong; Yang, Chao; Qu, Hong; Liu, Zheng; Fu, Q S; Qiao, Chuanling

    2007-08-01

    A soil bacterium capable of metabolizing organophosphorus compounds by reducing the P S group in the molecules was taxonomically identified as Klebsiella sp. strain F51-1-2. The gene involved in the reduction of organophosphorus compounds was cloned from this strain by the shotgun technique, and the deduced protein (named AKR5F1) showed homology to members of the aldo-keto reductase (AKR) superfamily. The intact coding region for AKR5F1 was subcloned into vector pET28a and overexpressed in Escherichia coli BL21(DE3). Recombinant His(6)-tagged AKR5F1 was purified in one step using Ni-nitrilotriacetic acid affinity chromatography. Assays for cofactor specificity indicated that reductive transformation of organophosphorus compounds by the recombinant AKR5F1 specifically required NADH. The kinetic constants of the purified recombinant AKR5F1 toward six thion organophosphorus compounds were determined. For example, the K(m) and k(cat) values of reductive transformation of malathion by the purified recombinant AKR5F1 are 269.5 +/- 47.0 microM and 25.7 +/- 1.7 min(-1), respectively. Furthermore, the reductive transformation of organophosphorus compounds can be largely explained by structural modeling.

  15. Three-dimensional structure of rat liver 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase: a member of the aldo-keto reductase superfamily.

    PubMed Central

    Hoog, S S; Pawlowski, J E; Alzari, P M; Penning, T M; Lewis, M

    1994-01-01

    The 3.0-A-resolution x-ray structure of rat liver 3 alpha-hydroxysteroid dehydrogenase/dihydrodiol dehydrogenase (3 alpha-HSD, EC 1.1.1.50) was determined by molecular replacement using human placental aldose reductase as the search model. The protein folds into an alpha/beta or triose-phosphate isomerase barrel and lacks a canonical Rossmann fold for binding pyridine nucleotide. The structure contains a concentration of hydrophobic amino acids that lie in a cavity near the top of the barrel and that are presumed to be involved in binding hydrophobic substrates (steroids, prostaglandins, and polycyclic aromatic hydrocarbons) and inhibitors (nonsteroidal antiinflammatory drugs). At the distal end of this cavity lie three residues in close proximity that have been implicated in catalysis by site-directed mutagenesis--Tyr-55, Asp-50, and Lys-84. Tyr-55 is postulated to act as the general acid. 3 alpha-HSD shares significant sequence identity with other HSDs that belong to the aldo-keto reductase superfamily and these may show similar architecture. Other members of this family include prostaglandin F synthase and rho-crystallin. By contrast, 3 alpha-HSD shares no sequence identity with HSDs that are members of the short-chain alcohol dehydrogenase family but does contain the Tyr-Xaa-Xaa-Xaa-Lys consensus sequence implicated in catalysis in this family. In the 3 alpha-HSD structure these residues are on the periphery of the barrel and are unlikely to participate in catalysis. Images PMID:8146147

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

    PubMed Central

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

    2014-01-01

    Background and Purpose Fluoxetine, a selective serotonin reuptake inhibitor, elevates brain concentrations of the neuroactive progesterone metabolite allopregnanolone, an effect suggested to underlie its use in the treatment of premenstrual dysphoria. One report showed fluoxetine to activate the aldo-keto reductase (AKR) component of 3α-hydroxysteroid dehydrogenase (3α-HSD), which catalyses production of allopregnanolone from 5α-dihydroprogesterone. However, this action was not observed by others. The present study sought to clarify the site of action for fluoxetine in elevating brain allopregnanolone. Experimental Approach Adult male rats and female rats in dioestrus were treated with fluoxetine and their brains assayed for allopregnanolone and its precursors, progesterone and 5α-dihydroprogesterone. Subcellular fractions of rat brain were also used to investigate the actions of fluoxetine on 3α-HSD activity in both the reductive direction, producing allopregnanolone from 5α-dihydroprogesterone, and the reverse oxidative direction. Fluoxetine was also tested on these recombinant enzyme activities expressed in HEK cells. Key Results Short-term treatment with fluoxetine increased brain allopregnanolone concentrations in female, but not male, rats. Enzyme assays on native rat brain fractions and on activities expressed in HEK cells showed fluoxetine did not affect the AKR producing allopregnanolone from 5α-dihydroprogesterone but did inhibit the microsomal dehydrogenase oxidizing allopregnanolone to 5α-dihydroprogesterone. Conclusions and Implications Fluoxetine elevated allopregnanolone in female rat brain by inhibiting its oxidation to 5α-dihydroprogesterone by a microsomal dehydrogenase. This is a novel site of action for fluoxetine, with implications for the development of new agents and/or dosing regimens to raise brain allopregnanolone. PMID:25161074

  17. The responses of mitochondrial proteome in rat liver to the consumption of moderate ethanol: the possible roles of aldo-keto reductases.

    PubMed

    Shi, Liang; Wang, Yuan; Tu, Shuyang; Li, Xiaolei; Sun, Maomao; Srivastava, Sanjay; Xu, Ningzhi; Bhatnagar, Aruni; Liu, Siqi

    2008-08-01

    A large body of evidence supports the view that mitochondria are a primary target of alcohol stress. Changes in mitochondrial proteins due to moderate ethanol intake, however, have not been broadly and accurately estimated. For this study, rats were fed low doses of ethanol and the mitochondria were isolated from heart, kidney, and liver, using ultracentrifugation with Nycodenz density gradient. The mitochondrial proteins were well resolved upon two-dimensional electrophoresis (2DE), and the alcohol-responsive 2DE spots were identified by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF/TOF MS). Compared with the control group, the proteins extracted from liver mitochondria of ethanol-fed rats exhibited the significant changes on 2DE images, whereas the 2DE images obtained from the kidney and the heart mitochondria remained almost unchanged by ethanol feeding. Significantly, over 50% of the alcohol-responsive proteins in liver mitochondria were members of aldo-keto reductase family (AKR), which were usually present in cytoplasm. The organelle distributions of AKR proteins in liver mitochondria were further confirmed by Western blot analysis as well as by confocal microscopy. In addition, translocations of AKR were examined in the CHANG cell line, which was cultured with and without ethanol. The results of Western blot strongly suggested that the abundances of AKR proteins in the mitochondria were greatly reduced by the presence of ethanol in culture medium. The results of this study show that, even with moderate ethanol feeding, the mitochondrial proteome in rat liver was more sensitive to alcohol stress than that of either the kidney or the heart. The translocation of AKR proteins may be involved in the detoxification of liver cells.

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

    SciTech Connect

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

    2014-07-15

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

  19. Aldo-keto reductase-1 (AKR1) protect cellular enzymes from salt stress by detoxifying reactive cytotoxic compounds.

    PubMed

    Vemanna, Ramu S; Babitha, K C; Solanki, Jayant K; Amarnatha Reddy, V; Sarangi, S K; Udayakumar, M

    2017-04-01

    Cytotoxic compounds like reactive carbonyl compounds such as methylglyoxal (MG), melandialdehyde (MDA), besides the ROS accumulate significantly at higher levels under salinity stress conditions and affect lipids and proteins that inhibit plant growth and productivity. The detoxification of these cytotoxic compounds by overexpression of NADPH-dependent Aldo-ketoreductase (AKR1) enzyme enhances the salinity stress tolerance in tobacco. The PsAKR1 overexpression plants showed higher survival and chlorophyll content and reduced MDA, H2O2, and MG levels under NaCl stress. The transgenic plants showed reduced levels of Na(+) levels in both root and shoot due to reduced reactive carbonyl compounds (RCCs) and showed enhanced membrane stability resulted in higher root growth and biomass. The increased levels of antioxidant glutathione and enhanced activity of superoxide dismutase (SOD), ascorbate peroxidase (APX) and glutathione reductase (GR) suggest AKR1 could protect these enzymes from the RCC induced protein carbonylation by detoxification process. The transgenics also showed higher activity of delta 1-pyrroline-5- carboxylate synthase (P5CS) enzyme resulted in increasedproline levels to maintain osmotic homeostasis. The results demonstrates that the AKR1 protects proteins or enzymes that are involved in scavenging of cytotoxic compounds by detoxifying RCCs generated under salinity stress.

  20. Knockdown or inhibition of aldo-keto reductase 1B10 inhibits pancreatic carcinoma growth via modulating Kras-E-cadherin pathway.

    PubMed

    Zhang, Wanying; Li, Haonan; Yang, Yihe; Liao, Jie; Yang, Guang-Yu

    2014-12-28

    Aldo-keto reductase 1B10 (AKR1B10) has relatively specific lipid substrates including carbonyls, retinal and farnesal/geranylgeranial. Metabolizing these lipid substrates appears crucial to carcinogenesis, particularly for farnesal/geranylgeranial that involves protein prenylation. Mutant Kras is a most common active oncogene in pancreatic cancer, and its activation requires protein prenylation. To directly determine the role of AKR1B10 in pancreatic carcinogenesis, we knocked down AKR1B10 in CD18 human pancreatic carcinoma cells using shRNA approach. Silencing AKR1B10 resulted in a significant inhibition of anchor-dependent growth (knockdown cells vs. vector-control cells: 67 ± 9.5 colonies/HPF vs. 170 ± 3.7 colonies/HPF, p < 0.01), invasion index (0.27 vs. 1.00, p < 0.05), and cell migration (at 16 hours 9.2 ± 1.2% vs. 14.0 ± 1.8%, at 24 hours 21.0 ± 1.1% vs. 30.5 ± 3.5%, and at 48 hours 51.9 ± 5.7% vs. 88.9 ± 3.0%, p < 0.01). Inhibition of AKR1B10 by oleanolic acid (OA) showed a dose-dependent inhibition of cell growth with IC50 at 30 µM. Kras pull-down and Western blot analysis revealed a significant down-regulation of active form Kras and phosphorylated C-Raf, and Erk, as well as an up-regulation of E-cadherin. A significant reduction of in vivo tumor growth was observed in nude mice implanted with the CD18 pancreatic carcinoma cells with AKR1B10 knockdown (tumor weight: 0.25 ± 0.06 g vs. 0.52 ± 0.07 g, p = 0.01), and with OA treatment (tumor weight: 0.35 ± 0.05 g vs. 0.52 ± 0.07 g, p = 0.05). Our findings indicate AKR1B10 is a unique enzyme involved in pancreatic carcinogenesis via modulation of the Kras-E-cadherin pathway.

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

    DTIC Science & Technology

    2013-10-01

    distribution. Mol Endocrinol 1997, 11, 1971-1984. 17. Stanbrough, M.; Bubley, G. J.; Ross , K.; Golub, T. R.; Rubin, M. A.; Penning, T. M.; Febbo, P...21. Cherian, M. T.; Wilson , E. M.; Shapiro, D. J. A competitive inhibitor that reduces recruitment of androgen receptor to androgen-responsive

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

    PubMed Central

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

    2015-01-01

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

  3. A Novel Aldo-Keto Reductase (AKR17A1) of Anabaena sp. PCC 7120 Degrades the Rice Field Herbicide Butachlor and Confers Tolerance to Abiotic Stresses in E. coli

    PubMed Central

    Agrawal, Chhavi; Sen, Sonia; Yadav, Shivam; Rai, Shweta; Rai, Lal Chand

    2015-01-01

    Present study deals with the identification of a novel aldo/keto reductase, AKR17A1 from Anabaena sp. PCC7120 and adds on as 17th family of AKR superfamily drawn from a wide variety of organisms. AKR17A1 shares many characteristics of a typical AKR such as— (i) conferring tolerance to multiple stresses like heat, UV-B, and cadmium, (ii) excellent activity towards known AKR substrates (isatin and 2-nitrobenzaldehyde), and (iii) obligate dependence on NADPH as a cofactor for enzyme activity. The most novel attribute of AKR17A1, first reported in this study, is its capability to metabolize butachlor, a persistent rice field herbicide that adversely affects agro-ecosystem and non-target organisms. The AKR17A1 catalyzed- degradation of butachlor resulted into formation of 1,2-benzene dicarboxylic acid and 2,6 bis (1,1, dimethylethyl) 4,-methyl phenol as the major products confirmed by GC-MS analysis. PMID:26372161

  4. Reduction of lipid peroxidation products and advanced glycation end-product precursors by cyanobacterial aldo-keto reductase AKR3G1—a founding member of the AKR3G subfamily.

    PubMed

    Hintzpeter, Jan; Martin, Hans-Joerg; Maser, Edmund

    2015-01-01

    The purpose of this study was to investigate the origin and function of the aldo-keto reductase (AKR) superfamily as enzymes involved in the detoxification of xenobiotics. We used the cyanobacterium Synechocystis sp. PCC 6803 as a model organism and sequence alignments to find bacterial AKRs with highest identity to human enzymes. Disappearance of NADPH was monitored spectrophotometrically to calculate enzymatic activity. The molecular weight of the native protein was determined by size exclusion chromatography. Substrate docking was performed by SwissDock. Sequence alignments identified the NADPH-dependent AKR3G1 having 41.5 and 40% identity with the human enzymes AKR1B1 and AKR1B10, respectively. Highest enzymatic efficiency was observed with 4-oxonon-2-enal (4-ONE; k(cat)/K(m), 561 s(-1) mM(-1)) and 4-hydroxynonenal (k(cat)/K(m), 26.5 s(-1) mM(-1)), respectively. P74308 is the most efficient enzyme for 4-ONE discovered until now. Cooperativity of this monomeric enzyme was observed with some substrates. Enzyme inactivation or oligomerization as possible explanations for nonhyperbolic enzyme kinetics were ruled out by Selwyn's test and gel filtration. The role of the little investigated carbonyl-reducing enzymes in detoxification seems to be in fact a very old process with rarely observed nonhyperbolic enzyme kinetics as an adaptation mechanism to higher concentrations of reactive oxygen species.

  5. Aldo-keto reductase 1B10 promotes development of cisplatin resistance in gastrointestinal cancer cells through down-regulating peroxisome proliferator-activated receptor-γ-dependent mechanism.

    PubMed

    Matsunaga, Toshiyuki; Suzuki, Ayaka; Kezuka, Chihiro; Okumura, Naoko; Iguchi, Kazuhiro; Inoue, Ikuo; Soda, Midori; Endo, Satoshi; El-Kabbani, Ossama; Hara, Akira; Ikari, Akira

    2016-08-25

    Cisplatin (cis-diamminedichloroplatinum, CDDP) is one of the most effective chemotherapeutic drugs that are used for treatment of patients with gastrointestinal cancer cells, but its continuous administration often evokes the development of chemoresistance. In this study, we investigated alterations in antioxidant molecules and functions using a newly established CDDP-resistant variant of gastric cancer MKN45 cells, and found that aldo-keto reductase 1B10 (AKR1B10) is significantly up-regulated with acquisition of the CDDP resistance. In the nonresistant MKN45 cells, the sensitivity to cytotoxic effect of CDDP was decreased and increased by overexpression and silencing of AKR1B10, respectively. In addition, the AKR1B10 overexpression markedly suppressed accumulation and cytotoxicity of 4-hydroxy-2-nonenal that is produced during lipid peroxidation by CDDP treatment, suggesting that the enzyme acts as a crucial factor for facilitation of the CDDP resistance through inhibiting induction of oxidative stress by the drug. Transient exposure to CDDP and induction of the CDDP resistance decreased expression of peroxisome proliferator-activated receptor-γ (PPARγ) in MKN45 and colon cancer LoVo cells. Additionally, overexpression of PPARγ in the cells elevated the sensitivity to the CDDP toxicity, which was further augmented by concomitant treatment with a PPARγ ligand rosiglitazone. Intriguingly, overexpression of AKR1B10 in the cells resulted in a decrease in PPARγ expression, which was recovered by addition of an AKR1B10 inhibitor oleanolic acid, inferring that PPARγ is a downstream target of AKR1B10-dependent mechanism underlying the CDDP resistance. Combined treatment with the AKR1B10 inhibitor and PPARγ ligand elevated the CDDP sensitivity, which was almost the same level as that in the parental cells. These results suggest that combined treatment with the AKR1B10 inhibitor and PPARγ ligand is an effective adjuvant therapy for overcoming CDDP resistance of

  6. Identification of a determinant for strict NADP(H)-specificity and high sensitivity to mixed-type steroid inhibitor of rabbit aldo-keto reductase 1C33 by site-directed mutagenesis.

    PubMed

    Endo, Satoshi; Matsunaga, Toshiyuki; Ikari, Akira; El-Kabbani, Ossama; Hara, Akira; Kitade, Yukio

    2015-03-01

    In rabbit tissues, hydroxysteroid dehydrogenase belonging to the aldo-keto reductase (AKR) superfamily exists in six isoforms (AKRs: 1C5 and 1C29-1C33), sharing >73% amino acid sequence identity. AKR1C33 is strictly NADPH-specific, in contrast to dual NADPH/NADH specificity of the other isoforms. All coenzyme-binding residues of the structurally elucidated AKR1C5 are conserved in other isoforms, except that S217 (interacting with the pyrophosphate moiety) and T273 (interacting with the 2'-phosphate moiety) are replaced with F217 and N272, respectively, in AKR1C33. To explore the determinants for the NADPH specificity of AKR1C33, we prepared its F217S and N272T mutant enzymes. The mutation of F217S, but not N272T, converted AKR1C33 into a dually coenzyme-specific form that showed similar kcat values for NAD(P)H to those of AKR1C32. The reverse mutation (S217F) in dually coenzyme-specific AKR1C32 produced a strictly NADPH-specific form. The F217S mutation also abolished the activity towards 3-keto-5β-cholestanes that are substrates specific to AKR1C33, and markedly decreased the sensitivity to 4-pregnenes (such as deoxycorticosterone and medroxyprogesterone acetate) that were found to be potent mixed-type inhibitors of the wild-type enzyme. The results indicate the important role of F217 in the strict NADPH-dependency, as well as its involvement in the unique catalytic properties of AKR1C33.

  7. Sulindac inhibits pancreatic carcinogenesis in LSL-KrasG12D-LSL-Trp53R172H-Pdx-1-Cre mice via suppressing aldo-keto reductase family 1B10 (AKR1B10)

    PubMed Central

    Li, Haonan; Yang, Allison L.; Yang, Guang-Yu

    2013-01-01

    Sulindac has been identified as a competitive inhibitor of aldo-keto reductase 1B10 (AKR1B10), an enzyme that plays a key role in carcinogenesis. AKR1B10 is overexpressed in pancreatic ductal adenocarcinoma (PDAC) and exhibits lipid substrate specificity, especially for farnesyl and geranylgeranyl. There have been no studies though showing that the inhibition of PDAC by sulindac is via inhibition of AKR1B10, particularly the metabolism of farnesyl/geranylgeranyl and Kras protein prenylation. To determine the chemopreventive effects of sulindac on pancreatic carcinogenesis, 5-week-old LSL-KrasG12D-LSL-Trp53R172H-Pdx-1-Cre mice (Pankras/p53 mice) were fed an AIN93M diet with or without 200 p.p.m. sulindac (n = 20/group). Kaplan–Meier survival analysis showed that average animal survival in Pankras/p53 mice was 143.7±8.8 days, and average survival with sulindac was increased to 168.0±8.8 days (P < 0.005). Histopathological analyses revealed that 90% of mice developed PDAC, 10% with metastasis to the liver and lymph nodes. With sulindac, the incidence of PDAC was reduced to 56% (P < 0.01) and only one mouse had lymph node metastasis. Immunochemical analysis showed that sulindac significantly decreased Ki-67-labeled cell proliferation and markedly reduced the expression of phosphorylated extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Raf and mitogen-activated protein kinase kinase 1 and 2. In in vitro experiments with PDAC cells from Pankras/p53 mice, sulindac exhibited dose-dependent inhibition of AKR1B10 activity. By silencing AKR1B10 expression through small interfering RNA or by sulindac treatment, these in vitro models showed a reduction in Kras and human DNA-J homolog 2 protein prenylation, and downregulation of phosphorylated C-raf, ERK1/2 and MEK1/2 expression. Our results demonstrate that sulindac inhibits pancreatic carcinogenesis by the inhibition of Kras protein prenylation by targeting AKR1B10. PMID:23689354

  8. Fine tuning of coenzyme specificity in family 2 aldo-keto reductases revealed by crystal structures of the Lys-274 → Arg mutant of Candida tenuis xylose reductase (AKR2B5) bound to NAD + and NADP +

    SciTech Connect

    Leitgeb, Stefan; Petschacher, Barbara; Wilson, David K.; Nidetzky, Bernd

    2005-01-11

    Aldo-keto reductases of family 2 employ single site replacement Lys → Arg to switch their cosubstrate preference from NADPH to NADH. X-ray crystal structures of Lys-274 → Arg mutant of Candida tenuis xylose reductase (AKR2B5) bound to NAD+ and NADP+ were determined at a resolution of 2.4 and 2.3 Å, respectively. Due to steric conflicts in the NADP+-bound form, the arginine side chain must rotate away from the position of the original lysine side chain, thereby disrupting a network of direct and water-mediated interactions between Glu-227, Lys-274 and the cofactor 2'-phosphate and 3'-hydroxy groups. Because anchoring contacts of its Glu-227 are lost, the coenzyme-enfolding loop that becomes ordered upon binding of NAD(P)+ in the wild-type remains partly disordered in the NADP+-bound mutant. The results delineate a catalytic reaction profile for the mutant in comparison to wild-type.

  9. AKR1C3 as a target in castrate resistant prostate cancer.

    PubMed

    Adeniji, Adegoke O; Chen, Mo; Penning, Trevor M

    2013-09-01

    Aberrant androgen receptor (AR) activation is the major driver of castrate resistant prostate cancer (CRPC). CRPC is ultimately fatal and more therapeutic agents are needed to treat this disease. Compounds that target the androgen axis by inhibiting androgen biosynthesis and or AR signaling are potential candidates for use in CRPC treatment and are currently being pursued aggressively. Aldo-keto reductase 1C3 (AKR1C3) plays a pivotal role in androgen biosynthesis within the prostate. It catalyzes the 17-ketoreduction of weak androgen precursors to give testosterone and 5α-dihydrotestosterone. AKR1C3 expression and activity has been implicated in the development of CRPC, making it a rational target. Selective inhibition of AKR1C3 will be important, however, due to the presence of closely related isoforms, AKR1C1 and AKR1C2 that are also involved in androgen inactivation. We examine the evidence that supports the vital role of AKR1C3 in CRPC and recent developments in the discovery of potent and selective AKR1C3 inhibitors. This article is part of a Special Issue entitled 'CSR 2013'.

  10. Knockdown of AKR1C3 exposes a potential epigenetic susceptibility in prostate cancer cells

    PubMed Central

    Doig, Craig L.; Battaglia, Sebastiano; Khanim, Farhat L.; Bunce, Christopher M.; Campbell, Moray J.

    2017-01-01

    Background The aldo-keto reductase 1C3 (AKR1C3) has been heavily implicated in the propagation of prostate malignancy. AKR1C3 protein is elevated within prostate cancer tissue, it contributes to the formation of androgens and downstream stimulation of the androgen receptor (AR). Elevated expression of AKR1C3 is also reported in acute myeloid leukemia but the target nuclear receptors have been identified as members of the peroxisome-proliferator activated receptor (PPARs) subfamily. Thus, AKR1C3 cancer biology is likely to be tissue dependent and hormonally linked to the availability of ligands for both the steroidogenic and non-steroidogenic nuclear receptors. Methods In the current study we investigated the potential for AKR1C3 to regulate the availability of prostaglandin-derived ligands for PPARg mainly, prostaglandin J2 (PGJ2). Using prostate cancer cell lines with stably reduced AKR1C3 levels we examined the impact of AKR1C3 upon proliferation mediated by PPAR ligands. Results These studies revealed knockdown of AKR1C3 had no effect upon the sensitivity of androgen receptor independent prostate cancer cells towards PPAR ligands. However, the reduction of levels of AKR1C3 was accompanied by a significantly reduced mRNA expression of a range of HDACs, transcriptional co-regulators, and increased sensitivity towards SAHA, a clinically approved histone deacetylase inhibitor. Conclusions These results suggest a hitherto unidentified link between AKR1C3 levels and the epigenetic status in prostate cancer cells. This raises an interesting possibility of a novel rational to target AKR1C3, the utilization of AKRIC3 selective inhibitors in combination with HDAC inhibition as part of novel epigenetic therapies in androgen deprivation therapy recurrent prostate cancer. PMID:26429394

  11. Overexpression of AKR1C3 significantly enhances human prostate cancer cells resistance to radiation

    PubMed Central

    Gao, Xian-Shu; Li, Yi; Yu, Hongliang; Xiong, Wei; Yu, Hao; Wang, Wen; Li, Yingbo; Teng, Yingqi; Zhou, Demin

    2016-01-01

    Aldo-keto reductase 1C3(AKR1C3) is an enzyme involved in prostaglandins metabolism. Studies suggest that AKR1C3 has a pivotal role in the radioresistance of esophageal cancer and non-small-cell lung cancer, yet the role of AKR1C3 in prostate cancer cells radiation resistance has not yet been clarified. In our study, we established a stable overexpressing AKR1C3 cell line (AKR1C3-over) derived from the prostate cell line DU145 and its control cell line (Control). We conducted colony formation assay to determine the role of AKR1C3 in radioresistance and we used its chemical inhibitor to detect whether it can restored the sensitivity of the acquired tumor cells. Flow cytometry assay was carried out to detect IR-induced ROS accumulation. Elisa was adopted to dedect the concentration of PGF2α in the suspension of the cells after 6GY radiation. Western blotting was used to dedect the MAPK and PPAR γ. The results demonstrated that overexpression of AKR1C3 in prostate cancer can result in radioresistance and suppression of AKR1C3 via its chemical inhibitor indocin restored the sensitivity of the acquired tumor cells. According to the flow cytometry assay, ROS was decreased by 80% in DU145-over cells. Also overexpression of AKR1C3 could result in the accumulation of prostaglandin F2α (PGF2α), which can not only promote prostate cancer cell 's proliferation but also could enhance prostate cancer cells resistance to radiation and activated the MAPK pathway and inhibited the expression of PPARγ. In conclusion, we found that overexpression of AKR1C3 significantly enhanced human prostate cancer cells resistance to radiation through activation of MAPK pathway. PMID:27385003

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

    DTIC Science & Technology

    2010-08-01

    1Department of Medical Microbiology , Immunology, & Cell Biology, SimmonsCooper Cancer Institute and 2Division of Statistics and Research Consulting, Southern...University, Beijing 100084,People’s Republic of China. ¥To whom requests reprints: Deliang Cao, Department of Medical Microbiology , Immunology, & Cell

  13. Screening Baccharin Analogs as Selective Inhibitors Against Type 5 17β-Hydroxysteroid Dehydrogenase (AKR1C3)

    PubMed Central

    Zang, Tianzhu; Verma, Kshitij; Chen, Mo; Jin, Yi; Trippier, Paul C.; Penning, Trevor M.

    2015-01-01

    Aldo-keto reductase 1C3 (AKR1C3), also known as type 5 17β-hydroxysteroid dehydrogenase, is a downstream steroidogenic enzyme and converts androgen precursors to the potent androgen receptor ligands: testosterone and 5α-dihydrotestosterone. Studies have shown that AKR1C3 is involved in the development of castration resistant prostate cancer (CRPC) and that it is a rational drug target for the treatment of CRPC. Baccharin, a component of Brazilian propolis, has been observed to exhibit a high inhibitory potency and selectivity for AKR1C3 over other AKR1C isoforms and is a promising lead compound for developing more potent and selective inhibitors. Here, we report the screening of fifteen baccharin analogs as selective inhibitors against AKR1C3 versus AKR1C2 (type 3 3α-hydroxysteroid dehydrogenase). Among these analogs, the inhibitory activity and selectivity of thirteen compounds were evaluated for the first time. The substitution of the 4-dihydrocinnamoyloxy group of baccharin by an acetate group displayed nanomolar inhibitory potency (IC50: 440 nM) and a 102-fold selectivity over AKR1C2. By contrast, when the cinnamic acid group of baccharin was esterified, there was a dramatic decrease in potency and selectivity for AKR1C3 in comparison to baccharin. Low or sub- micromolar inhibition was observed when the 3-prenyl group of baccharin was removed, and the selectivity over AKR1C2 was low. Although unsubstituted baccharin was still the most potent (IC50: 100 nM) and selective inhibitor for AKR1C3, these data provide structure-activity relationships required for the optimization of new baccharin analogs. They suggest that the carboxylate group on cinnamic acid, the prenyl group, and either retention of 4′-dihydrocinnamoyloxy group or acetate substituent on cinnamic acid are important to maintain the high potency and selectivity for AKR1C3. PMID:25555457

  14. Morpholylureas are a new class of potent and selective inhibitors of the type 5 17-β-hydroxysteroid dehydrogenase (AKR1C3).

    PubMed

    Flanagan, Jack U; Atwell, Graham J; Heinrich, Daniel M; Brooke, Darby G; Silva, Shevan; Rigoreau, Laurent J M; Trivier, Elisabeth; Turnbull, Andrew P; Raynham, Tony; Jamieson, Stephen M F; Denny, William A

    2014-02-01

    Inhibitors of the aldo-keto reductase enzyme AKR1C3 are of interest as potential drugs for leukemia and hormone-related cancers. A series of non-carboxylate morpholino(phenylpiperazin-1-yl)methanones were prepared by palladium-catalysed coupling of substituted phenyl or pyridyl bromides with the known morpholino(piperazin-1-yl)methanone, and shown to be potent (IC50∼100nM) and very isoform-selective inhibitors of AKR1C3. Lipophilic electron-withdrawing substituents on the phenyl ring were positive for activity, as was an H-bond acceptor on the other terminal ring, and the ketone moiety (as a urea) was essential. These structure-activity relationships are consistent with an X-ray structure of a representative compound bound in the AKR1C3 active site, which showed H-bonding between the carbonyl oxygen of the drug and Tyr55 and His117 in the 'oxyanion hole' of the enzyme, with the piperazine bridging unit providing the correct twist to allow the terminal benzene ring to occupy the lipophilic pocket and align with Phe311.

  15. Human carbonyl reductase (CBR) localized to band 21q22. 1 by high-resolution fluorescence in situ hybridization displays gene dosage effects in trisomy 21 cells

    SciTech Connect

    Lemieux, N. ); Malfoy, B. ); Forrest, G.L. )

    1993-01-01

    Human carbonyl reductase (CBR) belongs to a group of NADPH-dependent enzymes called aldo-keto reductases. The enzyme can function as an aldo-keto reductase or as a quinone reductase with potential for modulating quinone-mediated oxygen free radicals. The CBR gene was mapped by high-resolution fluorescence in situ hybridization to band 21q22.12, very close to the SOD1 locus at position 2lq22.11. CBR displayed gene dosage effects in trisomy 21 human lymphoblasts at the DNA and mRNA levels. Lymphoblasts with increasing chromosome 21 ploidy also showed increased aldo-keto reductase activity and increased quinone reductase activity. Both aldo-keto reductase activity and quinone reductase activity have been shown to be associated with carbonyl reductase. The location of CBR near SOD1 and the increased enzyme activity and potential for free radical modulation in trisomy 21 cells implicate CBR as a candidate for contributing to the pathology of certain diseases such as Down syndrome and Alzheimer disease. 28 refs., 1 fig., 1 tab.

  16. Oxidative damage-related genes AKR1C3 and OGG1 modulate risks for lung cancer due to exposure to PAH-rich coal combustion emissions.

    PubMed

    Lan, Qing; Mumford, Judy L; Shen, Min; Demarini, David M; Bonner, Matthew R; He, Xingzhou; Yeager, Meredith; Welch, Robert; Chanock, Stephen; Tian, Linwei; Chapman, Robert S; Zheng, Tongzhang; Keohavong, Phouthone; Caporaso, Neil; Rothman, Nathaniel

    2004-11-01

    Lung cancer rates among men and particularly among women, almost all of whom are non-smokers, in Xuan Wei County, China are among the highest in China and have been causally associated with exposure to indoor smoky coal emissions that contain very high levels of polycyclic aromatic hydrocarbons (PAHs). As such, this population provides a unique opportunity to study the pathogenesis of PAH-induced lung cancer that is not substantially influenced by the large number of other carcinogenic constituents of tobacco smoke. Aldo-keto reductases (AKRs) activate PAH dihydrodiols to yield their corresponding reactive and redox-active o-quinones, which can then generate reactive oxygen species that cause oxidative DNA damage. We therefore examined the association between single nucleotide polymorphisms (SNPs) in four genes (AKR1C3-Gln5His, NQO1-Pro187Ser, MnSOD-Val16Ala and OGG1-Ser326Cys) that play a role in the generation, prevention or repair of oxidative damage and lung cancer risk in a population-based, case-control study of 119 cases and 113 controls in Xuan Wei, China. The AKR1C3-Gln/Gln genotype was associated with a 1.84-fold [95% confidence interval (CI) = 0.98-3.45] increased risk and the combined OGG1-Cys/Cys and Ser/Cys genotypes were associated with a 1.93-fold (95% CI = 1.12-3.34) increased risk of lung cancer. Subgroup analysis revealed that the effects were particularly elevated among women who had relatively high cumulative exposure to smoky coal. SNPs in MnSOD and NQO1 were not associated with lung cancer risk. These results suggest that SNPs in the oxidative stress related-genes AKR1C3 and OGG1 may play a role in the pathogenesis of lung cancer in this population, particularly among heavily exposed women. However, due to the small sample size, additional studies are needed to evaluate these associations within Xuan Wei and other populations with substantial exposure to PAHs.

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

    PubMed Central

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

    2010-01-01

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

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

    SciTech Connect

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

    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.

  19. Catalytic reaction profile for NADH-dependent reduction of aromatic aldehydes by xylose reductase from Candida tenuis.

    PubMed Central

    Mayr, Peter; Nidetzky, Bernd

    2002-01-01

    Kinetic substituent effects have been used to examine the catalytic reaction profile of xylose reductase from the yeast Candida tenuis, a representative aldo/keto reductase of primary carbohydrate metabolism. Michaelis-Menten parameters (k(cat) and K(m)) for NADH-dependent enzymic aldehyde reductions have been determined using a homologous series of benzaldehyde derivatives in which substituents in meta and para positions were employed to systematically perturb the properties of the reactive carbonyl group. Kinetic isotope effects (KIEs) on k(cat) and k(cat)/K(m) for enzymic reactions with meta-substituted benzaldehydes have been obtained by using NADH (2)H-labelled in the pro-R C4-H position, and equilibrium constants for the conversion of these aldehydes into the corresponding alcohols (K(eq)) have been measured in the presence of NAD(H) and enzyme. Aldehyde dissociation constants (K(d)) and the hydride transfer rate constant (k(7)) have been calculated from steady-state rate and KIE data. Quantitative structure-activity relationship analysis was used to factor the observed substituent dependence of k(cat)/K(m) into a major electronic effect and a productive positional effect of the para substituent. k(cat)/K(m) (after correction for substituent position) and K(eq) obeyed log-linear correlations over the substituent parameter, Hammett sigma, giving identical slope values (rho) of +1.4 to +1.7, whereas the same Hammett plot for logK(d) yielded rho=-1.5. This leads to the conclusion that electron-withdrawing substituents facilitate the reaction and increase binding to about the same extent. KIE values for k(cat) (1.8) and k(cat)/K(m) (2.7), and likewise k(7), showed no substituent dependence. Therefore, irrespective of the observed changes in reactivity over the substrate series studied no shift in the character of the rate-limiting transition state of hydride transfer occurred. The signs and magnitudes of rho values suggest this transition state to be product

  20. Cloning, expression and characterization of a putative 2,5-diketo-D-gluconic acid reductase in Comamonas testosteroni.

    PubMed

    Chen, Yuanan; Ji, Wei; Zhang, Hao; Zhang, Xiao; Yu, Yuanhua

    2015-06-05

    Aldo-keto reductases (AKRs) are a superfamily of soluble NAD(P)(H) oxidoreductases. The function of the enzymes is to reduce aldehydes and ketones into primary and secondary alcohols. We have cloned a 2,5-diketo-D-gluconic acid reductase (2,5DKGR) gene from Comamonas testosteroni (C. testosteroni) ATCC11996 (a Gram-negative bacterium which can use steroids as carbon and energy source) into plasmid pET-15b and over expressed in Escherichia coli BL21 (DE3). The protein was purified by His-tag Metal chelating affinity chromatography column. The 2,5-diketo-D-gluconic acid reductase (2,5DKGR) gene contains 1062 bp and could be translated into a protein of 353 amino acid residues. Three consensus sequences of the AKR superfamily are found as GxxxxDxAxxY, LxxxGxxxPxxGxG and LxxxxxxxxxDxxxxH. GxxxxDxAxxY is the active site, LxxxGxxxPxxGxG is the Cofactor-binding site for NAD(P)(H), LxxxxxxxxxDxxxxH is used for supporting the 3D structure. 2,5-diketo-D-gluconic acid reductase gene of C. testosteroni was knocked out and a mutant M-AKR was obtained. Compared to wild type C. testosteroni, degradations of testosterone, estradiol, oestrone and methyltestosterone in mutant M-AKR were decreased. Therefore, 2,5-diketo-D-gluconic acid reductase in C. testosteroni is involved in steroid degradation.

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

    PubMed

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

    2014-09-12

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

  2. Structure and Catalytic Mechanism of Human Steroid 5-Reductase (AKR1D1)

    SciTech Connect

    Costanzo, L.; Drury, J; Christianson, D; Penning, T

    2009-01-01

    Human steroid 5{beta}-reductase (aldo-keto reductase (AKR) 1D1) catalyzes reduction of {Delta}{sup 4}-ene double bonds in steroid hormones and bile acid precursors. We have reported the structures of an AKR1D1-NADP{sup +} binary complex, and AKR1D1-NADP{sup +}-cortisone, AKR1D1-NADP{sup +}-progesterone and AKR1D1-NADP{sup +}-testosterone ternary complexes at high resolutions. Recently, structures of AKR1D1-NADP{sup +}-5{beta}-dihydroprogesterone complexes showed that the product is bound unproductively. Two quite different mechanisms of steroid double bond reduction have since been proposed. However, site-directed mutagenesis supports only one mechanism. In this mechanism, the 4-pro-R hydride is transferred from the re-face of the nicotinamide ring to C5 of the steroid substrate. E120, a unique substitution in the AKR catalytic tetrad, permits a deeper penetration of the steroid substrate into the active site to promote optimal reactant positioning. It participates with Y58 to create a 'superacidic' oxyanion hole for polarization of the C3 ketone. A role for K87 in the proton relay proposed using the AKR1D1-NADP{sup +}-5{beta}-dihydroprogesterone structure is not supported.

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

    PubMed

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

    2017-03-01

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

  4. 5β-Reduced steroids and human Δ(4)-3-ketosteroid 5β-reductase (AKR1D1).

    PubMed

    Chen, Mo; Penning, Trevor M

    2014-05-01

    5β-Reduced steroids are non-planar steroids that have a 90° bend in their structure to create an A/B cis-ring junction. This novel property is required for bile-acids to act as emulsifiers, but in addition 5β-reduced steroids have remarkable physiology and may act as potent tocolytic agents, endogenous cardiac glycosides, neurosteroids, and can act as ligands for orphan and membrane bound receptors. In humans there is only a single 5β-reductase gene AKR1D1, which encodes Δ(4)-3-ketosteroid-5β-reductase (AKR1D1). This enzyme is a member of the aldo-keto reductase superfamily, but possesses an altered catalytic tetrad, in which Glu120 replaces the conserved His residue. This predominant liver enzyme generates all 5β-dihydrosteroids in the C19-C27 steroid series. Mutations exist in the AKR1D1 gene, which result in loss of protein stability and are causative in bile-acid deficiency.

  5. Alteration of the specificity of the cofactor-binding pocket of Corynebacterium 2,5-diketo-D-gluconic acid reductase A.

    PubMed

    Banta, Scott; Swanson, Barbara A; Wu, Shan; Jarnagin, Alisha; Anderson, Stephen

    2002-02-01

    The NADPH-dependent 2,5-diketo-D-gluconic acid (2,5-DKG) reductase enzyme is a required component in some novel biosynthetic vitamin C production processes. This enzyme catalyzes the conversion of 2,5-DKG to 2-keto-L-gulonic acid, which is an immediate precursor to L-ascorbic acid. Forty unique site-directed mutations were made at five residues in the cofactor-binding pocket of 2,5-DKG reductase A in an attempt to improve its ability to use NADH as a cofactor. NADH is more stable, less expensive and more prevalent in the cell than is NADPH. To the best of our knowledge, this is the first focused attempt to alter the cofactor specificity of a member of the aldo-keto reductase superfamily by engineering improved activity with NADH into the enzyme. Activity of the mutants with NADH or NADPH was assayed using activity-stained native polyacrylamide gels. Eight of the mutants at three different sites were identified as having improved activity with NADH. These mutants were purified and subjected to a kinetic characterization with NADH as a cofactor. The best mutant obtained, R238H, produced an almost 7-fold improvement in catalysis with NADH compared with the wild-type enzyme. Surprisingly, most of this catalytic improvement appeared to be due to an improvement in the apparent kcat for the reaction rather than a large improvement in the affinity of the enzyme for NADH.

  6. Comparative transcript and alkaloid profiling in Papaver species identifies a short chain dehydrogenase/reductase involved in morphine biosynthesis.

    PubMed

    Ziegler, Jörg; Voigtländer, Susan; Schmidt, Jürgen; Kramell, Robert; Miersch, Otto; Ammer, Christian; Gesell, Andreas; Kutchan, Toni M

    2006-10-01

    Plants of the order Ranunculales, especially members of the species Papaver, accumulate a large variety of benzylisoquinoline alkaloids with about 2500 structures, but only the opium poppy (Papaver somniferum) and Papaver setigerum are able to produce the analgesic and narcotic morphine and the antitussive codeine. In this study, we investigated the molecular basis for this exceptional biosynthetic capability by comparison of alkaloid profiles with gene expression profiles between 16 different Papaver species. Out of 2000 expressed sequence tags obtained from P. somniferum, 69 show increased expression in morphinan alkaloid-containing species. One of these cDNAs, exhibiting an expression pattern very similar to previously isolated cDNAs coding for enzymes in benzylisoquinoline biosynthesis, showed the highest amino acid identity to reductases in menthol biosynthesis. After overexpression, the protein encoded by this cDNA reduced the keto group of salutaridine yielding salutaridinol, an intermediate in morphine biosynthesis. The stereoisomer 7-epi-salutaridinol was not formed. Based on its similarities to a previously purified protein from P. somniferum with respect to the high substrate specificity, molecular mass and kinetic data, the recombinant protein was identified as salutaridine reductase (SalR; EC 1.1.1.248). Unlike codeinone reductase, an enzyme acting later in the pathway that catalyses the reduction of a keto group and which belongs to the family of the aldo-keto reductases, the cDNA identified in this study as SalR belongs to the family of short chain dehydrogenases/reductases and is related to reductases in monoterpene metabolism.

  7. Detoxifying Enzymes at the Cross-Roads of Inflammation, Oxidative Stress, and Drug Hypersensitivity: Role of Glutathione Transferase P1-1 and Aldose Reductase

    PubMed Central

    Sánchez-Gómez, Francisco J.; Díez-Dacal, Beatriz; García-Martín, Elena; Agúndez, José A. G.; Pajares, María A.; Pérez-Sala, Dolores

    2016-01-01

    Phase I and II enzymes are involved in the metabolism of endogenous reactive compounds as well as xenobiotics, including toxicants and drugs. Genotyping studies have established several drug metabolizing enzymes as markers for risk of drug hypersensitivity. However, other candidates are emerging that are involved in drug metabolism but also in the generation of danger or costimulatory signals. Enzymes such as aldo-keto reductases (AKR) and glutathione transferases (GST) metabolize prostaglandins and reactive aldehydes with proinflammatory activity, as well as drugs and/or their reactive metabolites. In addition, their metabolic activity can have important consequences for the cellular redox status, and impacts the inflammatory response as well as the balance of inflammatory mediators, which can modulate epigenetic factors and cooperate or interfere with drug-adduct formation. These enzymes are, in turn, targets for covalent modification and regulation by oxidative stress, inflammatory mediators, and drugs. Therefore, they constitute a platform for a complex set of interactions involving drug metabolism, protein haptenation, modulation of the inflammatory response, and/or generation of danger signals with implications in drug hypersensitivity reactions. Moreover, increasing evidence supports their involvement in allergic processes. Here, we will focus on GSTP1-1 and aldose reductase (AKR1B1) and provide a perspective for their involvement in drug hypersensitivity. PMID:27540362

  8. NADPH-dependent Reductases Involved in the Detoxification of Reactive Carbonyls in Plants*

    PubMed Central

    Yamauchi, Yasuo; Hasegawa, Ayaka; Taninaka, Ai; Mizutani, Masaharu; Sugimoto, Yukihiro

    2011-01-01

    Reactive carbonyls, especially α,β-unsaturated carbonyls produced through lipid peroxidation, damage biomolecules such as proteins and nucleotides; elimination of these carbonyls is therefore essential for maintaining cellular homeostasis. In this study, we focused on an NADPH-dependent detoxification of reactive carbonyls in plants and explored the enzyme system involved in this detoxification process. Using acrolein (CH2 = CHCHO) as a model α,β-unsaturated carbonyl, we purified a predominant NADPH-dependent acrolein-reducing enzyme from cucumber leaves, and we identified the enzyme as an alkenal/one oxidoreductase (AOR) catalyzing reduction of an α,β-unsaturated bond. Cloning of cDNA encoding AORs revealed that cucumber contains two distinct AORs, chloroplastic AOR and cytosolic AOR. Homologs of cucumber AORs were found among various plant species, including Arabidopsis, and we confirmed that a homolog of Arabidopsis (At1g23740) also had AOR activity. Phylogenetic analysis showed that these AORs belong to a novel class of AORs. They preferentially reduced α,β-unsaturated ketones rather than α,β-unsaturated aldehydes. Furthermore, we selected candidates of other classes of enzymes involved in NADPH-dependent reduction of carbonyls based on the bioinformatic information, and we found that an aldo-keto reductase (At2g37770) and aldehyde reductases (At1g54870 and At3g04000) were implicated in the reduction of an aldehyde group of saturated aldehydes and methylglyoxal as well as α,β-unsaturated aldehydes in chloroplasts. These results suggest that different classes of NADPH-dependent reductases cooperatively contribute to the detoxification of reactive carbonyls. PMID:21169366

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

    PubMed

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

    2015-07-01

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

  10. Insights from modeling the 3D structure of NAD(P)H-dependent D-xylose reductase of Pichia stipitis and its binding interactions with NAD and NADP.

    PubMed

    Wang, Jing-Fang; Wei, Dong-Qing; Lin, Ying; Wang, Yong-Hua; Du, Hong-Li; Li, Yi-Xve; Chou, Kuo-Chen

    2007-07-27

    NAD(P)H-dependent d-xylose reductase is a homodimeric oxidoreductase that belongs to the aldo-keto reductase superfamily. The enzyme has the special function to catalyze the first step in the assimilation of xylose into yeast metabolic pathways. Performing this function via reducing the open chain xylose to xylitol, the xylose reductase of Pichia stipitis is one of the most important enzymes that can be used to construct recombinant Saccharomyces cerevisiae strain for utilizing xylose and producing alcohol. To investigate into the interaction mechanism of the enzyme with its ligand NAD and NADP, the 3D structure was developed for the NAD(P)H-dependent d-xylose reductase from P. stipitis. With the 3D structure, the molecular docking operations were conducted to find the most stable bindings of the enzyme with NAD and NADP, respectively. Based on these results, the binding pockets of the enzyme for NAD and NADP have been explicitly defined. It has been found that the residues in forming the binding pockets for both NAD and NADP are almost the same and mainly hydrophilic. These findings may be used to guide mutagenesis studies, providing useful clues to modify the enzyme to improve the utilization of xylose for producing alcohol. Also, because human aldose reductases have the function to reduce the open chain form of glucose to sorbitol, a process physiologically significant for diabetic patients at the time that their blood glucose levels are elevated, the information gained through this study may also stimulate the development of new strategies for therapeutic treatment of diabetes.

  11. Structural alteration of cofactor specificity in Corynebacterium 2,5-diketo-D-gluconic acid reductase

    PubMed Central

    Sanli, Gulsah; Banta, Scott; Anderson, Stephen; Blaber, Michael

    2004-01-01

    Corynebacterium 2,5-Diketo-D-gluconic acid reductase (2,5-DKGR) catalyzes the reduction of 2,5-diketo-D-gluconic acid (2,5-DKG) to 2-Keto-L-gulonic acid (2-KLG). 2-KLG is an immediate precursor to L-ascorbic acid (vitamin C), and 2,5-DKGR is, therefore, an important enzyme in a novel industrial method for the production of vitamin C. 2,5-DKGR, as with most other members of the aldo-keto reductase (AKR) superfamily, exhibits a preference for NADPH compared to NADH as a cofactor in the stereo-specific reduction of substrate. The application of 2,5-DKGR in the industrial production of vitamin C would be greatly enhanced if NADH could be efficiently utilized as a cofactor. A mutant form of 2,5-DKGR has previously been identified that exhibits two orders of magnitude higher activity with NADH in comparison to the wild-type enzyme, while retaining a high level of activity with NADPH. We report here an X-ray crystal structure of the holo form of this mutant in complex with NADH cofactor, as well as thermodynamic stability data. By comparing the results to our previously reported X-ray structure of the holo form of wild-type 2,5-DKGR in complex with NADPH, the structural basis of the differential NAD(P)H selectivity of wild-type and mutant 2,5-DKGR enzymes has been identified. PMID:14718658

  12. Step 1: C3 Flight Demo Data Analysis Plan

    NASA Technical Reports Server (NTRS)

    2005-01-01

    The Data Analysis Plan (DAP) describes the data analysis that the C3 Work Package (WP) will perform in support of the Access 5 Step 1 C3 flight demonstration objectives as well as the processes that will be used by the Flight IPT to gather and distribute the data collected to satisfy those objectives. In addition to C3 requirements, this document will encompass some Human Systems Interface (HSI) requirements in performing the C3 flight demonstrations. The C3 DAP will be used as the primary interface requirements document between the C3 Work Package and Flight Test organizations (Flight IPT and Non-Access 5 Flight Programs). In addition to providing data requirements for Access 5 flight test (piggyback technology demonstration flights, dedicated C3 technology demonstration flights, and Airspace Operations Demonstration flights), the C3 DAP will be used to request flight data from Non- Access 5 flight programs for C3 related data products

  13. Plant progesterone 5beta-reductase is not homologous to the animal enzyme. Molecular evolutionary characterization of P5betaR from Digitalis purpurea.

    PubMed

    Gavidia, Isabel; Tarrío, Rosa; Rodríguez-Trelles, Francisco; Pérez-Bermúdez, Pedro; Seitz, H Ulrich

    2007-03-01

    Plants of the genus Digitalis produce cardiac glycosides, i.e. digoxin, which are widely used for congestive heart failure. Progesterone 5beta-reductase (P5betaR) is a key enzyme in the biosynthesis of these natural products. Here, we have carried out the purification and partial amino acid sequencing of the native P5betaR from foxglove (Digitalis purpurea), and isolated a cDNA encoding this enzyme. Similarly to other steroid 5beta-reductases, the recombinant P5betaR catalyzes the stereospecific reduction of the Delta(4)-double bond of several steroids with a 3-oxo,Delta(4,5) structure. The gene encoding P5betaR is expressed in all plant organs, and maximally transcribed in leaves and mature flowers. P5betaR belongs to the short-chain dehydrogenase/reductase (SDR) superfamily, bearing no structural homology to its mammalian counterpart, which is a member of the aldo-keto reductase (AKR) superfamily. A similar situation occurs with 3beta-hydroxy-Delta(5)-steroid dehydrogenase (3betaHSD), the gene immediately preceding P5betaR in the cardenolide pathway, which suggests that the entire route has evolved independently in animals and plants. P5betaR is retained only in plants, where it is ubiquitous, and a few distantly related bacterial lineages after its diversification from the last universal common ancestor. Evolutionary conserved changes in its putative active site suggest that plant P5betaR is a member of a novel subfamily of extended SDRs, or a new SDR family.

  14. Crustacean oxi-reductases protein sequences derived from a functional genomic project potentially involved in ecdysteroid hormones metabolism - a starting point for function examination.

    PubMed

    Tom, Moshe; Manfrin, Chiara; Giulianini, Piero G; Pallavicini, Alberto

    2013-12-01

    A transcriptomic assembly originated from hypodermis and Y organ of the crustacean Pontastacus leptodactylus is used here for in silico characterization of oxi-reductase enzymes potentially involved in the metabolism of ecdysteroid molting hormones. RNA samples were extracted from male Y organ and its neighboring hypodermis in all stages of the molt cycle. An equimolar RNA mix from all stages was sequenced using next generation sequencing technologies and de novo assembled, resulting with 74,877 unique contigs. These transcript sequences were annotated by examining their resemblance to all GenBank translated transcripts, determining their Gene Ontology terms and their characterizing domains. Based on the present knowledge of arthropod ecdysteroid metabolism and more generally on steroid metabolism in other taxa, transcripts potentially related to ecdysteroid metabolism were identified and their longest possible conceptual protein sequences were constructed in two stages, correct reading frame was deduced from BLASTX resemblances, followed by elongation of the protein sequence by identifying the correct translation frame of the original transcript. The analyzed genes belonged to several oxi-reductase superfamilies including the Rieske non heme iron oxygenases, cytochrome P450s, short-chained hydroxysteroid oxi-reductases, aldo/keto oxireductases, lamin B receptor/sterol reductases and glucose-methanol-cholin oxi-reductatses. A total of 68 proteins were characterized and the most probable participants in the ecdysteroid metabolism where indicated. The study provides transcript and protein structural information, a starting point for further functional studies, using a variety of gene-specific methods to demonstrate or disprove the roles of these proteins in relation to ecdysteroid metabolism in P. leptodactylus.

  15. Probing the substrate binding site of Candida tenuis xylose reductase (AKR2B5) with site-directed mutagenesis.

    PubMed

    Kratzer, Regina; Leitgeb, Stefan; Wilson, David K; Nidetzky, Bernd

    2006-01-01

    Little is known about how substrates bind to CtXR (Candida tenuis xylose reductase; AKR2B5) and other members of the AKR (aldo-keto reductase) protein superfamily. Modelling of xylose into the active site of CtXR suggested that Trp23, Asp50 and Asn309 are the main components of pentose-specific substrate-binding recognition. Kinetic consequences of site-directed substitutions of these residues are reported. The mutants W23F and W23Y catalysed NADH-dependent reduction of xylose with only 4 and 1% of the wild-type efficiency (kcat/K(m)) respectively, but improved the wild-type selectivity for utilization of ketones, relative to xylose, by factors of 156 and 471 respectively. Comparison of multiple sequence alignment with reported specificities of AKR members emphasizes a conserved role of Trp23 in determining aldehyde-versus-ketone substrate selectivity. D50A showed 31 and 18% of the wild-type catalytic-centre activities for xylose reduction and xylitol oxidation respectively, consistent with a decrease in the rates of the chemical steps caused by the mutation, but no change in the apparent substrate binding constants and the pattern of substrate specificities. The 30-fold preference of the wild-type for D-galactose compared with 2-deoxy-D-galactose was lost completely in N309A and N309D mutants. Comparison of the 2.4 A (1 A=0.1 nm) X-ray crystal structure of mutant N309D bound to NAD+ with the previous structure of the wild-type holoenzyme reveals no major structural perturbations. The results suggest that replacement of Asn309 with alanine or aspartic acid disrupts the function of the original side chain in donating a hydrogen atom for bonding with the substrate C-2(R) hydroxy group, thus causing a loss of transition-state stabilization energy of 8-9 kJ/mol.

  16. Inhibition of AKR1C3 Activation Overcomes Resistance to Abiraterone in Advanced Prostate Cancer.

    PubMed

    Liu, Chengfei; Armstrong, Cameron M; Lou, Wei; Lombard, Alan; Evans, Christopher P; Gao, Allen C

    2017-01-01

    Abiraterone suppresses intracrine androgen synthesis via inhibition of CYP17A1. However, clinical evidence suggests that androgen synthesis is not fully inhibited by abiraterone and the sustained androgen production may lead to disease relapse. In the present study, we identified AKR1C3, an important enzyme in the steroidogenesis pathway, as a critical mechanism driving resistance to abiraterone through increasing intracrine androgen synthesis and enhancing androgen signaling. We found that overexpression of AKR1C3 confers resistance to abiraterone while downregulation of AKR1C3 resensitizes resistant cells to abiraterone treatment. In abiraterone-resistant prostate cancer cells, AKR1C3 is overexpressed and the levels of intracrine androgens are elevated. In addition, AKR1C3 activation increases intracrine androgen synthesis and enhances androgen receptor (AR) signaling via activating AR transcriptional activity. Treatment of abiraterone-resistant cells with indomethacin, an AKR1C3 inhibitor, overcomes resistance and enhances abiraterone therapy both in vitro and in vivo by reducing the levels of intracrine androgens and diminishing AR transcriptional activity. These results demonstrate that AKR1C3 activation is a critical mechanism of resistance to abiraterone through increasing intracrine androgen synthesis and enhancing androgen signaling. Furthermore, this study provides a preclinical proof-of-principle for clinical trials investigating the combination of targeting AKR1C3 using indomethacin with abiraterone for advanced prostate cancer. Mol Cancer Ther; 16(1); 35-44. ©2016 AACR.

  17. Identification of a novel polyfluorinated compound as a lead to inhibit the human enzymes aldose reductase and AKR1B10: structure determination of both ternary complexes and implications for drug design.

    PubMed

    Cousido-Siah, Alexandra; Ruiz, Francesc X; Mitschler, André; Porté, Sergio; de Lera, Ángel R; Martín, María J; Manzanaro, Sonia; de la Fuente, Jesús A; Terwesten, Felix; Betz, Michael; Klebe, Gerhard; Farrés, Jaume; Parés, Xavier; Podjarny, Alberto

    2014-03-01

    Aldo-keto reductases (AKRs) are mostly monomeric enzymes which fold into a highly conserved (α/β)8 barrel, while their substrate specificity and inhibitor selectivity are determined by interaction with residues located in three highly variable external loops. The closely related human enzymes aldose reductase (AR or AKR1B1) and AKR1B10 are of biomedical interest because of their involvement in secondary diabetic complications (AR) and in cancer, e.g. hepatocellular carcinoma and smoking-related lung cancer (AKR1B10). After characterization of the IC50 values of both AKRs with a series of polyhalogenated compounds, 2,2',3,3',5,5',6,6'-octafluoro-4,4'-biphenyldiol (JF0064) was identified as a lead inhibitor of both enzymes with a new scaffold (a 1,1'-biphenyl-4,4'-diol). An ultrahigh-resolution X-ray structure of the AR-NADP(+)-JF0064 complex has been determined at 0.85 Å resolution, allowing it to be observed that JF0064 interacts with the catalytic residue Tyr48 through a negatively charged hydroxyl group (i.e. the acidic phenol). The non-competitive inhibition pattern observed for JF0064 with both enzymes suggests that this acidic hydroxyl group is also present in the case of AKR1B10. Moreover, the combination of surface lysine methylation and the introduction of K125R and V301L mutations enabled the determination of the X-ray crystallographic structure of the corresponding AKR1B10-NADP(+)-JF0064 complex. Comparison of the two structures has unveiled some important hints for subsequent structure-based drug-design efforts.

  18. Intracrine androgens and AKR1C3 activation confer resistance to enzalutamide in prostate cancer

    PubMed Central

    Liu, Chengfei; Lou, Wei; Zhu, Yezi; Yang, Joy C.; Nadiminty, Nagalakshmi; Gaikwad, Nilesh W.; Evans, Christopher P.; Gao, Allen C.

    2015-01-01

    The introduction of enzalutamide and abiraterone has led to improvement in the treatment of metastatic castration-resistant prostate cancer (mCRPC). However, acquired resistance to enzalutamide and abiraterone therapies frequently develops within a short period in many patients. In the present study, we developed enzalutamide resistant prostate cancer cells in an effort to understand the mechanisms of resistance. Global gene expression analysis showed that steroid biosynthesis pathway is activated in enzalutamide resistant prostate cancer cells. One of the crucial steroidogenic enzymes, AKR1C3, was significantly elevated in enzalutamide resistant cells. In addition, AKR1C3 is highly expressed in metastatic and recurrent prostate cancer and in enzalutamide resistant prostate xenograft tumors. Liquid Chromatography-Mass Spectrometry (LC-MS) analysis of the steroid metabolites revealed that androgen precursors such as cholesterol, DHEA and progesterone, as well as androgens are highly up regulated in enzalutamide resistant prostate cancer cells compared to the parental cells. Knock down of AKR1C3 expression by shRNA or inhibition of AKR1C3 enzymatic activity by indomethacin resensitized enzalutamide resistant prostate cancer cells to enzalutamide treatment both in vitro and in vivo. In contrast, overexpression of AKR1C3 confers resistance to enzalutamide. Furthermore, the combination of indomethacin and enzalutamide resulted in significant inhibition of enzalutamide-resistant tumor growth. These results suggest that AKR1C3 activation is a critical resistance mechanism associated with enzalutamide resistance, targeting intracrine androgens and AKR1C3 will overcome enzalutamide resistance and improve survival of advanced prostate cancer patients. PMID:25649766

  19. Differential Feedback Regulation of Δ4-3-Oxosteroid 5β-Reductase Expression by Bile Acids

    PubMed Central

    Valanejad, Leila; Nadolny, Christina; Shiffka, Stephanie; Chen, Yuan; You, Sangmin; Deng, Ruitang

    2017-01-01

    Δ4-3-oxosteroid 5β-reductase is member D1 of the aldo-keto reductase family 1 (AKR1D1), which catalyzes 5β-reduction of molecules with a 3-oxo-4-ene structure. Bile acid intermediates and most of the steroid hormones carry the 3-oxo-4-ene structure. Therefore, AKR1D1 plays critical roles in both bile acid synthesis and steroid hormone metabolism. Currently our understanding on transcriptional regulation of AKR1D1 under physiological and pathological conditions is very limited. In this study, we investigated the regulatory effects of primary bile acids, chenodeoxycholic acid (CDCA) and cholic acid (CA), on AKR1D1 expression. The expression levels of AKR1D1 mRNA and protein in vitro and in vivo following bile acid treatments were determined by real-time PCR and Western blotting. We found that CDCA markedly repressed AKR1D1 expression in vitro in human hepatoma HepG2 cells and in vivo in mice. On the contrary, CA significantly upregulated AKR1D1 expression in HepG2 cells and in mice. Further mechanistic investigations revealed that the farnesoid x receptor (FXR) signaling pathway was not involved in regulating AKR1D1 by bile acids. Instead, CDCA and CA regulated AKR1D1 through the mitogen-activated protein kinases/c-Jun N-terminal kinases (MAPK/JNK) signaling pathway. Inhibition of the MAPK/JNK pathway effectively abolished CDCA and CA-mediated regulation of AKR1D1. It was thus determined that AKR1D1 expression was regulated by CDCA and CA through modulating the MAPK/JNK signaling pathway. In conclusion, AKR1D1 expression was differentially regulated by primary bile acids through negative and positive feedback mechanisms. The findings indicated that both bile acid concentrations and compositions play important roles in regulating AKR1D1 expression, and consequently bile acid synthesis and steroid hormone metabolism. PMID:28125709

  20. Bladder cancer risk and genetic variation in AKR1C3 and other metabolizing genes

    PubMed Central

    Figueroa, Jonine D.; Malats, Núria; García-Closas, Montserrat; Real, Francisco X.; Silverman, Debra; Kogevinas, Manolis; Chanock, Stephen; Welch, Robert; Dosemeci, Mustafa; Lan, Qing; Tardón, Adonina; Serra, Consol; Carrato, Alfredo; García-Closas, Reina; Castaño-Vinyals, Gemma; Rothman, Nathaniel

    2008-01-01

    Aromatic amines (AAs) and polycyclic aromatic hydrocarbons (PAHs) are carcinogens present in tobacco smoke and functional polymorphisms in NAT2 and GSTM1 metabolizing genes are associated with increased bladder cancer risk. We evaluated whether genetic variation in other candidate metabolizing genes are also associated with risk. Candidates included genes that control the transcription of metabolizing genes [aryl hydrocarbon receptor (AHR), AHRR and aryl hydrocarbon nuclear translocator (ARNT)] and genes that activate/detoxify AA or PAH (AKR1C3, CYP1A1, CYP1A2, CYP1B1, CYP3A4, EPHX1, EPHX2, NQO1, MPO, UGT1A4, SULT1A1 and SULT1A2). Using genotype data from 1150 cases of urothelial carcinomas and 1149 controls from the Spanish Bladder Cancer Study, we estimated odds ratios (ORs) and 95% confidence intervals (CIs) adjusting for age, gender, region and smoking status. Based on a test for trend, we observed 10 non-redundant single-nucleotide polymorphisms (SNPs) in five genes (AKR1C3, ARNT, CYP1A1, CYP1B1 and SULT1A2) significantly associated with bladder cancer risk. We observed an inverse association with risk for the AKR1C3 promoter SNP rs1937845 [OR (95% CI) for heterozygote and homozygote variant compared with common homozygote genotype were 0.86 (0.70–1.06) and 0.74 (0.57–0.96), respectively; P for trend = 0.02]. Interestingly, genetic variation in this region has been associated with lung, non-Hodgkin lymphoma and prostate cancer risk. Analysis of additional SNPs to capture most (∼90%) of common genetic variation in AKR1C3 and haplotype walking analyses based on all AKR1C3 SNPs (n = 25) suggest two separate regions associated with bladder cancer risk. These results indicate that genetic variation in carcinogen-metabolizing genes, particularly AKR1C3, could be associated with bladder cancer risk. PMID:18632753

  1. Discovery of 2-methyl-1-{1-[(5-methyl-1H-indol-2-yl)carbonyl]piperidin-4-yl}propan-2-ol: a novel, potent and selective type 5 17β-hydroxysteroid dehydrogenase inhibitor.

    PubMed

    Watanabe, Kazushi; Kakefuda, Akio; Yasuda, Minoru; Enjo, Kentaro; Kikuchi, Aya; Furutani, Takashi; Naritomi, Yoichi; Otsuka, Yukio; Okada, Minoru; Ohta, Mitsuaki

    2013-09-01

    Type 5 17β-hydroxysteroid dehydrogenase (17β-HSD5), also known as aldo-keto reductase 1C3 (AKR1C3), is a member of the aldo-keto reductase superfamily of enzymes and is expressed in the human prostate. One of the main functions of 17β-HSD5 is to catalyze the conversion of the weak androgen, androstenedione, to the potent androgen, testosterone. The concentration of intraprostatic 5α-dihydrotestosterone (DHT) in patients following chemical or surgical castration has been reported to remain as high as 39% of that of healthy men, with 17β-HSD5 shown to be involved in this androgen synthesis. Inhibition of 17β-HSD5 therefore represents a promising target for the treatment of castration-resistant prostate cancer (CRPC). To investigate this, we conducted high-throughput screening (HTS) and identified compound 2, which displayed a structure distinct from known 17β-HSD5 inhibitors. To optimize the inhibitory activity of compound 2, we first introduced a primary alcohol group. We then converted the primary alcohol group to a tertiary alcohol, which further enhanced the inhibitory activity, improved metabolic stability, and led to the identification of compound 17. Oral administration of compound 17 to castrated nude mice bearing the CWR22R xenograft resulted in the suppression of androstenedione (AD)-induced intratumoral testosterone production. Compound 17 also demonstrated good isoform selectivity, minimal inhibitory activity against either CYP or hERG, and enhanced pharmacokinetic and physicochemical properties.

  2. AKR1B1 — EDRN Public Portal

    Cancer.gov

    From NCBI Gene: This gene encodes a member of the aldo/keto reductase superfamily, which consists of more than 40 known enzymes and proteins. This member catalyzes the reduction of a number of aldehydes, including the aldehyde form of glucose, and is thereby implicated in the development of diabetic complications by catalyzing the reduction of glucose to sorbitol. Multiple pseudogenes have been identified for this gene. The nomenclature system used by the HUGO Gene Nomenclature Committee to define human aldo-keto reductase family members is known to differ from that used by the Mouse Genome Informatics database. [provided by RefSeq, Feb 2009

  3. Global modeling of the C1-C3 alkyl nitrates using STOCHEM-CRI

    NASA Astrophysics Data System (ADS)

    Khan, M. A. H.; Cooke, M. C.; Utembe, S. R.; Morris, W. C.; Archibald, A. T.; Derwent, R. G.; Jenkin, M. E.; Orr-Ewing, A. J.; Higgins, C. M.; Percival, C. J.; Leather, K. E.; Shallcross, D. E.

    2015-12-01

    The atmospheric global budget and distribution of C1-C3 alkyl nitrates have been investigated using a global three-dimensional chemistry transport model, STOCHEM-CRI. Alkyl nitrates (RONO2) are significant NOx reservoir species and the more detailed VOC oxidation mechanism (CRI v2-R5) leads to greater photochemical production. RONO2 are significant sources of NOx in regions remote from NOx sources. The study shows that the global burden and the atmospheric life-time of C1-C3 alkyl nitrates are 113 Gg and 9-10 days, respectively, which are in excellent agreement with estimates established by previous studies. The abundance of alkyl nitrates have been found to be higher in the continental atmosphere, with CH3ONO2 mixing ratios up to 20 ppt over the Amazon rainforest. Up to 15, 10, 2, and 5 ppt of modelled CH3ONO2, C2H5ONO2, n-C3H7ONO2 and i-C3H7ONO2 have been found in the northern hemisphere over regions with large anthropogenic emissions of NOx and VOCs. The combination of atmospheric production and long-range transport led to high alkyl nitrate levels at high latitudes. The model performance for C1-C3 alkyl nitrates was established using observations from nine flights and nine field campaigns. The comparison shows a tendency towards model under-prediction of the observations, particularly in the southern hemispheric marine boundary layer, possibly due to the absence of oceanic production mechanisms and air-sea exchange processes in the model. The discrepancies between model and observed seasonal cycles, especially of CH3ONO2, in both hemispheres are discussed.

  4. The Steroidogenic Enzyme AKR1C3 Regulates Stability of the Ubiquitin Ligase Siah2 in Prostate Cancer Cells*

    PubMed Central

    Fan, Lingling; Peng, Guihong; Hussain, Arif; Fazli, Ladan; Guns, Emma; Gleave, Martin; Qi, Jianfei

    2015-01-01

    Re-activation of androgen receptor (AR) activity is the main driver for development of castration-resistant prostate cancer. We previously reported that the ubiquitin ligase Siah2 enhanced AR transcriptional activity and prostate cancer cell growth. Among the genes we found to be regulated by Siah2 was AKR1C3, which encodes a key androgen biosynthetic enzyme implicated in castration-resistant prostate cancer development. Here, we found that Siah2 inhibition in CWR22Rv1 prostate cancer cells decreased AKR1C3 expression as well as intracellular androgen levels, concomitant with inhibition of cell growth in vitro and in orthotopic prostate tumors. Re-expression of either wild-type or catalytically inactive forms of AKR1C3 partially rescued AR activity and growth defects in Siah2 knockdown cells, suggesting a nonenzymatic role for AKR1C3 in these outcomes. Unexpectedly, AKR1C3 re-expression in Siah2 knockdown cells elevated Siah2 protein levels, whereas AKR1C3 knockdown had the opposite effect. We further found that AKR1C3 can bind Siah2 and inhibit its self-ubiquitination and degradation, thereby increasing Siah2 protein levels. We observed parallel expression of Siah2 and AKR1C3 in human prostate cancer tissues. Collectively, our findings identify a new role for AKR1C3 in regulating Siah2 stability and thus enhancing Siah2-dependent regulation of AR activity in prostate cancer cells. PMID:26160177

  5. The Steroidogenic Enzyme AKR1C3 Regulates Stability of the Ubiquitin Ligase Siah2 in Prostate Cancer Cells.

    PubMed

    Fan, Lingling; Peng, Guihong; Hussain, Arif; Fazli, Ladan; Guns, Emma; Gleave, Martin; Qi, Jianfei

    2015-08-21

    Re-activation of androgen receptor (AR) activity is the main driver for development of castration-resistant prostate cancer. We previously reported that the ubiquitin ligase Siah2 enhanced AR transcriptional activity and prostate cancer cell growth. Among the genes we found to be regulated by Siah2 was AKR1C3, which encodes a key androgen biosynthetic enzyme implicated in castration-resistant prostate cancer development. Here, we found that Siah2 inhibition in CWR22Rv1 prostate cancer cells decreased AKR1C3 expression as well as intracellular androgen levels, concomitant with inhibition of cell growth in vitro and in orthotopic prostate tumors. Re-expression of either wild-type or catalytically inactive forms of AKR1C3 partially rescued AR activity and growth defects in Siah2 knockdown cells, suggesting a nonenzymatic role for AKR1C3 in these outcomes. Unexpectedly, AKR1C3 re-expression in Siah2 knockdown cells elevated Siah2 protein levels, whereas AKR1C3 knockdown had the opposite effect. We further found that AKR1C3 can bind Siah2 and inhibit its self-ubiquitination and degradation, thereby increasing Siah2 protein levels. We observed parallel expression of Siah2 and AKR1C3 in human prostate cancer tissues. Collectively, our findings identify a new role for AKR1C3 in regulating Siah2 stability and thus enhancing Siah2-dependent regulation of AR activity in prostate cancer cells.

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

    SciTech Connect

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

    2012-10-10

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

  7. SILAC-based quantitative proteomic analysis reveals widespread molecular alterations in human skin keratinocytes upon chronic arsenic exposure.

    PubMed

    Mir, Sartaj Ahmad; Pinto, Sneha M; Paul, Somnath; Raja, Remya; Nanjappa, Vishalakshi; Syed, Nazia; Advani, Jayshree; Renuse, Santosh; Sahasrabuddhe, Nandini A; Prasad, T S Keshava; Giri, Ashok K; Gowda, Harsha; Chatterjee, Aditi

    2017-03-01

    Chronic exposure to arsenic is associated with dermatological and nondermatological disorders. Consumption of arsenic-contaminated drinking water results in accumulation of arsenic in liver, spleen, kidneys, lungs, and gastrointestinal tract. Although arsenic is cleared from these sites, a substantial amount of residual arsenic is left in keratin-rich tissues including skin. Epidemiological studies suggest the association of skin cancer upon arsenic exposure, however, the mechanism of arsenic-induced carcinogenesis is not completely understood. We developed a cell line based model to understand the molecular mechanisms involved in arsenic-mediated toxicity and carcinogenicity. Human skin keratinocyte cell line, HaCaT, was chronically exposed to 100 nM sodium arsenite over a period of 6 months. We observed an increase in basal ROS levels in arsenic-exposed cells. SILAC-based quantitative proteomics approach resulted in identification of 2111 proteins of which 42 proteins were found to be overexpressed and 54 downregulated (twofold) upon chronic arsenic exposure. Our analysis revealed arsenic-induced overexpression of aldo-keto reductase family 1 member C2 (AKR1C2), aldo-keto reductase family 1 member C3 (AKR1C3), glutamate-cysteine ligase catalytic subunit (GCLC), and NAD(P)H dehydrogenase [quinone] 1 (NQO1) among others. We observed downregulation of several members of the plakin family including periplakin (PPL), envoplakin (EVPL), and involucrin (IVL) that are essential for terminal differentiation of keratinocytes. MRM and Western blot analysis confirmed differential expression of several candidate proteins. Our study provides insights into molecular alterations upon chronic arsenic exposure on skin.

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

    EPA Science Inventory

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

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

    EPA Science Inventory

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

  10. LC/MSMS STUDY OF BENZO[A]PYRENE-7,8-QUINONE ADDUCTION TO GLOBIN TRYPTIC PEPTIDES AND N-ACETYLAMINO ACIDS

    EPA Science Inventory

    Benzo[a]pyrene-7,8-quinone (BPQ) is regarded as a reactive genotoxic compound enzymatically formed from a xenobiotic precursor benzo[a]pyrene-7,8-diol by aldo-keto-reductase family of enzymes. Because BPQ, a Michael electrophile, was previously shown to react with oligonucleotide...

  11. ERG/AKR1C3/AR constitutes a feed-forward loop for AR signaling in prostate cancer cells

    PubMed Central

    Powell, Katelyn; Semaan, Louie; Conley-LaComb, M. Katie; Asangani, Irfan; Wu, Yi-Mi; Ginsburg, Kevin B.; Williams, Julia; Squire, Jeremy A.; Maddipati, Krishna R.; Cher, Michael L.; Chinni, Sreenivasa R.

    2015-01-01

    Purpose Intratumoral androgen synthesis in prostate cancer (PCa) contributes to the development of castration-resistant prostate cancer (CRPC). Several enzymes responsible for androgen biosynthesis have been shown to be overexpressed in CRPC, thus contributing to CRPC in a castrated environment. The TMPRSS2-ERG transcription factor has been shown to be present in primary PCa tumors as well as CRPC tumors. We hypothesize that TMPRSS2-ERG fusions regulate androgen biosynthetic enzyme (ABE) gene expression and the production of androgens, which contributes to the development of CRPC. Experimental design We used a panel of assays including lentivirus transduction, gene expression, chromatin immunoprecipitation and sequencing, Liquid chromatography-Mass spectrometric quantitation, immunocytochemistry, immunohistochemistry and bio-informatics analysis of gene microarray data bases to determine ERG regulation of androgen synthesis. Results We found that ERG regulated the expression of the ABE AKR1C3 in PCa cells via direct binding to the AKR1C3 gene. Knockdown of ERG resulted in reduced AKR1C3 expression, which caused a reduction in both DHT synthesis and PSA expression in VCaP PCa cells treated with 5α-androstanedione, a DHT precursor metabolite. Immunohistochemical staining revealed that ERG was co-expressed with AKR1C3 in PCa tissue samples. Conclusions These data suggest that AKR1C3 catalyzes the biochemical reduction of 5α-Androstanedione to DHT in PCa cells, and that ERG regulates this step through upregulation of AKR1C3 expression. Elucidation of ERG regulation of ABEs in CRPC may help to stratify TMPRSS2-ERG fusion-positive PCa patients in the clinic for anti-AR driven therapies; and AKR1C3 may serve as a valuable therapeutic target in the treatment of CRPC. PMID:25754347

  12. Thioredoxin reductase.

    PubMed Central

    Mustacich, D; Powis, G

    2000-01-01

    The mammalian thioredoxin reductases (TrxRs) are a family of selenium-containing pyridine nucleotide-disulphide oxidoreductases with mechanistic and sequence identity, including a conserved -Cys-Val-Asn-Val-Gly-Cys- redox catalytic site, to glutathione reductases. TrxRs catalyse the NADPH-dependent reduction of the redox protein thioredoxin (Trx), as well as of other endogenous and exogenous compounds. The broad substrate specificity of mammalian TrxRs is due to a second redox-active site, a C-terminal -Cys-SeCys- (where SeCys is selenocysteine), that is not found in glutathione reductase or Escherichia coli TrxR. There are currently two confirmed forms of mammalian TrxRs, TrxR1 and TrxR2, and it is possible that other forms will be identified. The availability of Se is a key factor determining TrxR activity both in cell culture and in vivo, and the mechanism(s) for the incorporation of Se into TrxRs, as well as the regulation of TrxR activity, have only recently begun to be investigated. The importance of Trx to many aspects of cell function make it likely that TrxRs also play a role in protection against oxidant injury, cell growth and transformation, and the recycling of ascorbate from its oxidized form. Since TrxRs are able to reduce a number of substrates other than Trx, it is likely that additional biological effects will be discovered for TrxR. Furthermore, inhibiting TrxR with drugs may lead to new treatments for human diseases such as cancer, AIDS and autoimmune diseases. PMID:10657232

  13. Linkage and association of haplotypes at the APOA1/C3/A4/A5 genecluster to familial combined hyperlipidemia

    SciTech Connect

    Eichenbaum-Voline, Sophie; Olivier, Michael; Jones, Emma L.; Naoumova, Rossitza P.; Jones, Bethan; Gau, Brian; Seed, Mary; Betteridge,D. John; Galton, David J.; Rubin, Edward M.; Scott, James; Shoulders,Carol C.; Pennacchio, Len A.

    2002-09-15

    Combined hyperlipidemia (CHL) is a common disorder of lipidmetabolism that leads to an increased risk of cardiovascular disease. Thelipid profile of CHL is characterised by high levels of atherogeniclipoproteins and low levels of high-density-lipoprotein-cholesterol.Apolipoprotein (APO) A5 is a newly discovered gene involved in lipidmetabolism located within 30kbp of the APOA1/C3/A4 gene cluster. Previousstudies have indicated that sequence variants in this cluster areassociated with increased plasma lipid levels. To establish whethervariation at the APOA5 gene contributes to the transmission of CHL, weperformed linkage and linkage disequilibrium (LD) tests on a large cohortof families (n=128) with familial CHL (FCHL). The linkage data producedevidence for linkage of the APOA1/C3/A4/A5 genomic interval to FCHL (NPL= 1.7, P = 0.042). The LD studies substantiated these data. Twoindependent rare alleles, APOA5c.56G and APOC3c.386G of this gene clusterwere over-transmitted in FCHL (P = 0.004 and 0.007, respectively), andthis was associated with a reduced transmission of the most commonAPOA1/C3/A4/A5 haplotype (frequency 0.4425) to affected subjects (P =0.013). The APOA5c.56G allele was associated with increased plasmatriglyceride levels in FCHL probands, whereas the second, andindependent, APOC3c.386G allele was associated with increased plasmatriglyceride levels in FCHL pedigree founders. Thus, this allele (or anallele in LD) may mark a quantitative trait associated with FCHL, as wellas representing a disease susceptibility locus for the condition. Thisstudy establishes that sequence variation in the APOA1/C3/A4/A5 genecluster contributes to the transmission of FCHL in a substantialproportion of affected families, and that these sequence variants mayalso contribute to the lipid abnormalities of the metabolic syndrome,which is present in up to 40 percent of persons with cardiovasculardisease.

  14. Posterior arthrodesis of C1-C3 for the stabilization of multiple unstable upper cervical fractures with spinal cord compromise

    PubMed Central

    Xue, Deting; Chen, Qixin; Chen, Gang; Zhuo, Wenhai; Li, Fangcai

    2017-01-01

    Abstract Background: Multiple fractures of the atlas and axis are rare. The management of multiple fragment axis fractures and unstable atlas fractures is still challenging for the spinal surgeon. There are no published reports of similar fractures with 3-part fracture of axis associated with an unstable atlas fracture. Case summary: We present a patient with concurrent axis and atlas fractures, which have not been reported. The patient suffered hyperextension injury with neck pain and numbness of the bilateral upper extremity associated with weakness after a 2-m fall. The axis fractures included an odontoid type IIA fracture and traumatic spondylolisthesis of C2-C3. The atlas fracture was unstable. The neurological examination manifested as central canal syndrome, which was due to the hyperextension injury of cervical spine and spondylolisthesis of C2-C3. The patient was diagnosed as multiple unstable upper cervical fractures with spinal cord compromise. We performed posterior arthrodesis of C1-C3. Postoperatively, the patient showed neurological improvement, and C1-C3 had fused at the 3-month follow-up. Conclusion: Posterior arthrodesis of C1-C3 could provide a stable fixation for the 3 parts of axis (an odontoid type IIA fracture and traumatic spondylolisthesis of C2-C3) combined an unstable atlas fracture. Both the patient and the doctor were satisfied with the results of the treatment. So posterior arthrodesis of C1-C3 is a suitable treatment option for the treatment of a concurrent unstable atlas fracture and multiple fractures of the axis. PMID:28072744

  15. Variation in AKR1C3, which Encodes the Neuroactive Steroid Synthetic Enzyme 3α-HSD Type 2 (17β-HSD Type 5), Moderates the Subjective Effects of Alcohol

    PubMed Central

    Milivojevic, Verica; Feinn, Richard; Kranzler, Henry R.; Covault, Jonathan

    2014-01-01

    Rationale Animal models suggest that neuroactive steroids contribute to alcohol’s acute effects. We previously reported that a common non-synonymous polymorphism, AKR1C3*2 in the gene encoding the enzyme 3α-HSD2/17β-HSD5 and a synonymous SNP, rs248793, in SRD5A1, which encodes 5α-reductase, were associated with alcohol dependence (AD). Objectives To investigate whether these polymorphisms moderate subjective effects of alcohol in humans and whether AKR1C3*2 affects neuroactive steroid synthesis. Methods 65 Caucasian men (34 lighter and 31 heavier drinkers; mean age 26.2 y) participated in a double-blind laboratory study where they consumed drinks containing no ethanol or 0.8 g/kg of ethanol. Breath alcohol, heart rate (HR), and self-reported alcohol effects were measured at 40-min intervals and genotype was examined as a moderator of alcohol’s effects. Levels of the neuroactive steroid 5α-androstane-3α,17β-diol and its precursors, 3α,5α-androsterone and dihydrotestosterone, were measured at study entry using GC/MS. Results Initially, carriers of the AD-protective AK1C3*2 G-allele had higher levels of 5α-androstane-3α,17β-diol relative to the precursor 3α,5α-androsterone than C-allele homozygotes. AKR1C3*2 G-allele carriers exhibited greater increases in heart rate and stimulant and sedative effects of alcohol than C-allele homozygotes. The genotype effects on sedation were observed only in heavier drinkers. The only effect of the SRD5A1 SNP was to moderate HR. There were no interactive effects of the two SNPs. Conclusions The observed effects of variation in a gene encoding a neuroactive steroid biosynthetic enzyme on the rate of 17p–reduction of androsterone relative to androstanediol and on alcohol’s sedative effects may help to explain the association of AKR1C3*2 with AD. PMID:24838369

  16. AKR1B10 — EDRN Public Portal

    Cancer.gov

    The AKR1B10 protein is a member of the aldo/keto reductase superfamily. This superfamily has more than 40 known enzymes and proteins. AKR1B10 acts as an all-trans-retinaldehyde reductase. It can efficiently reduce aliphatic and aromatic aldehydes, and is less active on hexoses (in vitro). It is highly expressed in adrenal gland, small intestine, and colon, and may play an important role in liver carcinogenesis.

  17. IUPAC-NIST Solubility Data Series. 101. Alcohols + Hydrocarbons + Water Part 3. C1-C3 Alcohols + Aromatic Hydrocarbons

    NASA Astrophysics Data System (ADS)

    Oracz, Paweł; Góral, Marian; Wiśniewska-Gocłowska, Barbara; Shaw, David G.; Mączyński, Andrzej

    2016-09-01

    The mutual solubilities and related liquid-liquid equilibria for 11 ternary systems of C1-C3 alcohols with aromatic hydrocarbons and water are exhaustively and critically reviewed. Reports of experimental determination of solubility that appeared in the primary literature prior to the end of 2012 are compiled. For nine systems, sufficient data are available (two or more independent determinations) to allow critical evaluation. All new data are expressed as mass percent and mole fraction as well as the originally reported units. In addition to the standard evaluation criteria used throughout the Solubility Data Series, an additional criterion was used for each of the evaluated systems. These systems include one binary miscibility gap in the hydrocarbon + water subsystem. The binary tie lines were compared with the recommended values published previously.

  18. IUPAC-NIST Solubility Data Series. 101. Alcohols + Hydrocarbons + Water. Part 2. C1-C3 Alcohols + Aliphatic Hydrocarbons

    NASA Astrophysics Data System (ADS)

    Oracz, Paweł; Góral, Marian; Wiśniewska-Gocłowska, Barbara; Shaw, David G.; Mączyński, Andrzej

    2016-09-01

    The mutual solubilities and related liquid-liquid equilibria for 37 ternary systems of C1-C3 alcohols with aliphatic hydrocarbons and water are exhaustively and critically reviewed. Reports of experimental determination of solubility that appeared in the primary literature prior to the end of 2012 are compiled. For 14 systems, sufficient data are available (two or more independent determinations) to allow critical evaluation. All data are expressed as mass percent and mole fraction as well as the originally reported units. In addition to the standard evaluation criteria used throughout the Solubility Data Series, an additional criterion was used for each of the evaluated systems. These systems include one binary miscibility gap in the hydrocarbon + water subsystem and another one can be in the methanol + hydrocarbon subsystem. The binary tie lines were compared with the recommended values published previously.

  19. A Role of the Reaction Kernel in Propagation and Stabilization of Edge Diffusion Flames of C1-C3 Hydrocarbons

    NASA Technical Reports Server (NTRS)

    Takahashi, Fumiaki; Katta, Viswanath R.

    2003-01-01

    Diffusion flame stabilization is of essential importance in both Earth-bound combustion systems and spacecraft fire safety. Local extinction, re-ignition, and propagation processes may occur as a result of interactions between the flame zone and vortices or fire-extinguishing agents. By using a computational fluid dynamics code with a detailed chemistry model for methane combustion, the authors have revealed the chemical kinetic structure of the stabilizing region of both jet and flat-plate diffusion flames, predicted the flame stability limit, and proposed diffusion flame attachment and detachment mechanisms in normal and microgravity. Because of the unique geometry of the edge of diffusion flames, radical back-diffusion against the oxygen-rich entrainment dramatically enhanced chain reactions, thus forming a peak reactivity spot, i.e., reaction kernel, responsible for flame holding. The new results have been obtained for the edge diffusion flame propagation and attached flame structure using various C1-C3 hydrocarbons.

  20. Mechanism of Austenite Formation from Spheroidized Microstructure in an Intermediate Fe-0.1C-3.5Mn Steel

    NASA Astrophysics Data System (ADS)

    Lai, Qingquan; Gouné, Mohamed; Perlade, Astrid; Pardoen, Thomas; Jacques, Pascal; Bouaziz, Olivier; Bréchet, Yves

    2016-07-01

    The austenitization from a spheroidized microstructure during intercritical annealing was studied in a Fe-0.1C-3.5Mn alloy. The austenite grains preferentially nucleate and grow from intergranular cementite. The nucleation at intragranular cementite is significantly retarded or even suppressed. The DICTRA software, assuming local equilibrium conditions, was used to simulate the austenite growth kinetics at various temperatures and for analyzing the austenite growth mechanism. The results indicate that both the mode and the kinetics of austenite growth strongly depend on cementite composition. With sufficiently high cementite Mn content, the austenite growth is essentially composed of two stages, involving the partitioning growth controlled by Mn diffusion inside ferrite, followed by a stage controlled by Mn diffusion within austenite for final equilibration. The partitioning growth results in a homogeneous distribution of carbon within austenite, which is supported by NanoSIMS carbon mapping.

  1. Sulforaphane Preconditioning Sensitizes Human Colon Cancer Cells towards the Bioreductive Anticancer Prodrug PR-104A

    PubMed Central

    Erzinger, Melanie M.; Bovet, Cédric; Hecht, Katrin M.; Senger, Sabine; Winiker, Pascale; Sobotzki, Nadine; Cristea, Simona; Beerenwinkel, Niko; Shay, Jerry W.; Marra, Giancarlo; Wollscheid, Bernd; Sturla, Shana J.

    2016-01-01

    The chemoprotective properties of sulforaphane (SF), derived from cruciferous vegetables, are widely acknowledged to arise from its potent induction of xenobiotic-metabolizing and antioxidant enzymes. However, much less is known about the impact of SF on the efficacy of cancer therapy through the modulation of drug-metabolizing enzymes. To identify proteins modulated by a low concentration of SF, we treated HT29 colon cancer cells with 2.5 μM SF. Protein abundance changes were detected by stable isotope labeling of amino acids in cell culture. Among 18 proteins found to be significantly up-regulated, aldo-keto reductase 1C3 (AKR1C3), bioactivating the DNA cross-linking prodrug PR-104A, was further characterized. Preconditioning HT29 cells with SF reduced the EC50 of PR-104A 3.6-fold. The increase in PR-104A cytotoxicity was linked to AKR1C3 abundance and activity, both induced by SF in a dose-dependent manner. This effect was reproducible in a second colon cancer cell line, SW620, but not in other colon cancer cell lines where AKR1C3 abundance and activity were absent or barely detectable and could not be induced by SF. Interestingly, SF had no significant influence on PR-104A cytotoxicity in non-cancerous, immortalized human colonic epithelial cell lines expressing either low or high levels of AKR1C3. In conclusion, the enhanced response of PR-104A after preconditioning with SF was apparent only in cancer cells provided that AKR1C3 is expressed, while its expression in non-cancerous cells did not elicit such a response. Therefore, a subset of cancers may be susceptible to combined food-derived component and prodrug treatments with no harm to normal tissues. PMID:26950072

  2. Fundamentals and Bioengineering of Enzymatic Fuel Cells. Part 1. Bioengineering of Enzymes as Electrocatalysts

    DTIC Science & Technology

    2012-01-31

    PAGE. The purified polypeptide was lyophilized after dialysis against water for 3 days. Construction of pQE9HSslac and expression and purification of...after 3 days of dialysis against DI water. Hydrogel preparation. Neat hydrogels of OsHSH-1 and OsHSH-2 were prepared by one of two methods: 1...identities). Structures were analyzed using MolProbity27 and verified against other members of the aldo- keto reductase superfamily. The conserved

  3. Quinone Reductase 2 Is a Catechol Quinone Reductase

    SciTech Connect

    Fu, Yue; Buryanovskyy, Leonid; Zhang, Zhongtao

    2008-09-05

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

  4. Effects of variations in the APOA1/C3/A4/A5 gene cluster on different parameters of postprandial lipid metabolism in healthy young men

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: The APOA1/C3/A4/A5 gene cluster encodes important regulators of fasting lipids, but the majority of lipid metabolism takes place in the postprandial state, and knowledge about gene regulation in this state is scarce. With the aim of characterizing possible regulators of lipid metabolism...

  5. The APOA1/C3/A4/A5 cluster and markers of allostatic load in the Boston Puerto Rican Health Study

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The APOA1/C3/A4/A5 cluster encodes key regulators of plasma lipids. Interactions between dietary factors and single nucleotide polymorphisms (SNPs) in the cluster have been reported. Allostatic load, or physiological dysregulation in response to stress, has been implicated in shaping health disparit...

  6. Biological Role of Aldo–Keto Reductases in Retinoic Acid Biosynthesis and Signaling

    PubMed Central

    Ruiz, F. Xavier; Porté, Sergio; Parés, Xavier; Farrés, Jaume

    2012-01-01

    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. PMID:22529810

  7. The human kidney is a progesterone-metabolizing and androgen-producing organ.

    PubMed

    Quinkler, M; Bumke-Vogt, C; Meyer, B; Bähr, V; Oelkers, W; Diederich, S

    2003-06-01

    Progesterone (P) is a potent antagonist of the human mineralocorticoid receptor (MR) in vitro. We have previously demonstrated effective downstream metabolism of P in the kidney. This mechanism potentially protects the MR from P action. Here, we have investigated the expression and functional activity of steroidogenic enzymes in human kidney. RT-PCR analysis demonstrated the expression of 5 alpha-reductase type 1, 5 beta-reductase, aldo-keto-reductase (AKR) 1C1, AKR1C2, AKR1C3, 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) type 2, and 17 alpha-hydroxylase/17,20-lyase (P450c17). The presence of 3 beta-HSD type 2 and P450c17 indicated that conversion of pregnenolone to dehydroepiandrosterone (DHEA) and to androstenedione may take place effectively in kidney. To investigate this further, we incubated kidney subcellular fractions with radiolabeled pregnenolone. This resulted in efficient formation of DHEA from pregnenolone, indicating both 17 alpha-hydroxylase and 17,20-lyase activities exerted by P450c17. Radiolabeled DHEA was converted via androstenedione, androstenediol, and testosterone, indicating both 3 beta-HSD type 2 activity and 17 beta-HSD activity. In addition, the conversion of testosterone to 5 alpha-dihydrotestosterone was detectable, indicating 5 alpha-reductase activity. In conclusion, we verified the expression and functional activity of several enzymes involved in downstream metabolism of P and androgen synthesis in human kidney. These findings may be critical to the understanding of water balance during the menstrual cycle and pregnancy and of sex differences in hypertension.

  8. Global investigation of potential energy surfaces for the pyrolysis of C(1)-C(3) hydrocarbons: toward the development of detailed kinetic models from first principles.

    PubMed

    Ryazantsev, Mikhail N; Jamal, Adeel; Maeda, Satoshi; Morokuma, Keiji

    2015-11-07

    Detailed kinetic models (DKMs) are the most fundamental "bottom-up" approaches to computational investigation of the pyrolysis and oxidation of fuels. The weakest points of existing DKMs are incomplete information about the reaction types that can be involved in the potential energy surfaces (PESs) in pyrolysis and oxidation processes. Also, the computational thermodynamic parameters available in the literature vary widely with the level of theory employed. More sophisticated models require improvement both in our knowledge of the type of the reactions involved and the consistency of thermodynamic and kinetic parameters. In this paper, we aim to address these issues by developing ab initio models that can be used to describe early stages of pyrolysis of C1-C3 hydrocarbons. We applied a recently developed global reaction route mapping (GRRM) strategy to systematically investigate the PES of the pyrolysis of C1-C3 hydrocarbons at a consistent level of theory. The reactions are classified into 14 reaction types. The critical points on the PES for all reactions in the network are calculated at the highly accurate UCCSD(T)-F12b/cc-pVTZ//UM06-2X/cc-pVTZ level of theory. The data reported in this paper can be used for first principle calculations of kinetic constants and for a subsequent study on modeling the evolution of the species from the reaction network of the pyrolysis and oxidation of C1-C3 hydrocarbons.

  9. Green tea consumption, genetic susceptibility, PAH-rich smoky coal, and the risk of lung cancer.

    PubMed

    Bonner, Matthew R; Rothman, Nathaniel; Mumford, Judy L; He, Xingzhou; Shen, Min; Welch, Robert; Yeager, Meredith; Chanock, Stephen; Caporaso, Neil; Lan, Qing

    2005-04-04

    Experimental evidence suggests that green tea (Camellia sinesis) may reduce the risk of lung cancer through several hypothesized mechanisms including scavenging oxidative radicals, inhibition of tumor initiation, and modulation of detoxification enzymes. However, epidemiologic results have not been consistent as to the relationship between green tea consumption and lung caner prevention. We employed a population-based case-control study of 122 cases and 122 controls to investigate the effect that green tea consumption may have on the risk of lung cancer and whether polymorphisms in 8-oxoguanine-DNA glycosylase (OGG1), glutathione-S-transferase M1 (GSTM1), and aldo-keto reductase 1C3 (AKR1C3) modify such an association. Daily green tea consumption was associated with a non-significant reduction in lung cancer risk. However, the effect of smoky coal exposure was higher for non-drinkers (odds ratio (OR)=4.93; 95% confidence interval (95% CI)=1.27-19.13) than for drinkers (OR=1.88; 95% CI=1.01-3.48). Further, among individuals with the OGG1 Cys(326) allele, daily consumption was associated with a 72% reduction (95% CI=0.09-0.94). Among GSTM1 null homozygotes, those who consumed green tea daily had a non-significant reduction in risk compared with non-consumers. Green tea consumption had no effect among OGG1 Ser(326) homozygotes or GSTM1 carriers. In addition, AKR1C3 genotype did not modulate the effect of green tea consumption. The chemopreventive effects of green tea in this population may be restricted to individuals who are particularly susceptible to oxidative stress and oxidative DNA damage.

  10. The Activity of SN33638, an Inhibitor of AKR1C3, on Testosterone and 17β-Estradiol Production and Function in Castration-Resistant Prostate Cancer and ER-Positive Breast Cancer

    PubMed Central

    Yin, Yarong Diana; Fu, Melissa; Brooke, Darby G.; Heinrich, Daniel M.; Denny, William A.; Jamieson, Stephen M. F.

    2014-01-01

    AKR1C3 is a novel therapeutic target in castration-resistant prostate cancer (CRPC) and estrogen receptor (ER)-positive breast cancer because of its ability to produce testosterone and 17β-estradiol intratumorally, thus promoting nuclear receptor signaling and tumor progression. A panel of CRPC, ER-positive breast cancer and high/low AKR1C3-expressing cell lines were treated with SN33638, a selective inhibitor of AKR1C3, in the presence of hormone or prostaglandin (PG) precursors, prior to evaluation of cell proliferation and levels of 11β-PG F2α (11β-PGF2α), testosterone, 17β-estradiol, and prostate-specific antigen (PSA). A meta-analysis of AKR1C3 mRNA expression in patient samples was also conducted, which revealed that AKR1C3 mRNA was upregulated in CRPC, but downregulated in ER-positive breast cancer. 11β-PGF2α and testosterone levels in the cell line panel correlated with AKR1C3 protein expression. SN33638 prevented 11β-PGF2α formation in cell lines that expressed AKR1C3, but partially inhibited testosterone formation and subsequently cell proliferation and/or PSA expression only in high (LAPC4 AKR1C3-overexpressing cells) or moderate (22RV1) AKR1C3-expressing cell lines. SN33638 had little effect on 17β-estradiol production or estrone-stimulated cell proliferation in ER-positive breast cancer cell lines. Although SN33638 could prevent 11β-PGF2α formation, its ability to prevent testosterone and 17β-estradiol production and their roles in CRPC and ER-positive breast cancer progression was limited due to AKR1C3-independent steroid hormone production, except in LAPC4 AKR1C3 cells where the majority of testosterone was AKR1C3-dependent. These results suggest that inhibition of AKR1C3 is unlikely to produce therapeutic benefit in CRPC and ER-positive breast cancer patients, except possibly in the small subpopulation of CRPC patients with tumors that have upregulated AKR1C3 expression and are dependent on AKR1C3 to produce the testosterone required

  11. Development of Potent and Selective Indomethacin Analogues for the Inhibition of AKR1C3 (Type 5 17β-Hydroxysteroid Dehydrogenase/Prostaglandin F Synthase) in Castrate-Resistant Prostate Cancer

    PubMed Central

    2013-01-01

    Castrate-resistant prostate cancer (CRPC) is a fatal, metastatic form of prostate cancer. CRPC is characterized by reactivation of the androgen axis due to changes in androgen receptor signaling and/or adaptive intratumoral androgen biosynthesis. AKR1C3 is upregulated in CRPC where it catalyzes the formation of potent androgens. This makes AKR1C3 a target for the treatment of CRPC. AKR1C3 inhibitors should not inhibit AKR1C1/AKR1C2, which inactivate 5α-dihydrotestosterone. Indomethacin, used to inhibit cyclooxygenase, also inhibits AKR1C3 and displays selectivity over AKR1C1/AKR1C2. Parallel synthetic strategies were used to generate libraries of indomethacin analogues, which exhibit reduced cyclooxygenase inhibitory activity but retain AKR1C3 inhibitory potency and selectivity. The lead compounds inhibited AKR1C3 with nanomolar potency, displayed >100-fold selectivity over AKR1C1/AKR1C2, and blocked testosterone formation in LNCaP-AKR1C3 cells. The AKR1C3·NADP+·2′-des-methyl-indomethacin crystal structure was determined, and it revealed a unique inhibitor binding mode. The compounds reported are promising agents for the development of therapeutics for CRPC. PMID:23432095

  12. Identification of Lactobacillus brevis using a species-specific AFLP-derived marker.

    PubMed

    Fusco, Vincenzina; Quero, Grazia Marina; Chieffi, Daniele; Franz, Charles M A P

    2016-09-02

    A simple and specific method for the rapid detection and identification of Lactobacillus brevis was developed. A fAFLP (Fluorescent Amplified Fragment Length Polymorphisms) marker for L. brevis was used to design oligonucleotide primers for a species-specific PCR assay, targeting a 125bp fragment of the gene encoding the aldo/keto reductase of the diketogulonate-reductase family of L. brevis. This assay resulted in 100% inclusivity and exclusivity of assignment of strains to the species L. brevis. The analytical specificity of this assay was successfully tested to identify L. brevis isolates from sourdoughs.

  13. Nitrate and periplasmic nitrate reductases

    PubMed Central

    Sparacino-Watkins, Courtney; Stolz, John F.; Basu, Partha

    2014-01-01

    The nitrate anion is a simple, abundant and relatively stable species, yet plays a significant role in global cycling of nitrogen, global climate change, and human health. Although it has been known for quite some time that nitrate is an important species environmentally, recent studies have identified potential medical applications. In this respect the nitrate anion remains an enigmatic species that promises to offer exciting science in years to come. Many bacteria readily reduce nitrate to nitrite via nitrate reductases. Classified into three distinct types – periplasmic nitrate reductase (Nap), respiratory nitrate reductase (Nar) and assimilatory nitrate reductase (Nas), they are defined by their cellular location, operon organization and active site structure. Of these, Nap proteins are the focus of this review. Despite similarities in the catalytic and spectroscopic properties Nap from different Proteobacteria are phylogenetically distinct. This review has two major sections: in the first section, nitrate in the nitrogen cycle and human health, taxonomy of nitrate reductases, assimilatory and dissimilatory nitrate reduction, cellular locations of nitrate reductases, structural and redox chemistry are discussed. The second section focuses on the features of periplasmic nitrate reductase where the catalytic subunit of the Nap and its kinetic properties, auxiliary Nap proteins, operon structure and phylogenetic relationships are discussed. PMID:24141308

  14. Haplotypes in the APOA1-C3-A4-A5 gene cluster affect plasma lipids in both humans and baboons

    SciTech Connect

    Wang, Qian-fei; Liu, Xin; O'Connell, Jeff; Peng, Ze; Krauss, Ronald M.; Rainwater, David L.; VandeBerg, John L.; Rubin, Edward M.; Cheng, Jan-Fang; Pennacchio, Len A.

    2003-09-15

    Genetic studies in non-human primates serve as a potential strategy for identifying genomic intervals where polymorphisms impact upon human disease-related phenotypes. It remains unclear, however, whether independently arising polymorphisms in orthologous regions of non-human primates leads to similar variation in a quantitative trait found in both species. To explore this paradigm, we studied a baboon apolipoprotein gene cluster (APOA1/C3/A4/A5) for which the human gene orthologs have well established roles in influencing plasma HDL-cholesterol and triglyceride concentrations. Our extensive polymorphism analysis of this 68 kb gene cluster in 96 pedigreed baboons identified several haplotype blocks each with limited diversity, consistent with haplotype findings in humans. To determine whether baboons, like humans, also have particular haplotypes associated with lipid phenotypes, we genotyped 634 well characterized baboons using 16 haplotype tagging SNPs. Genetic analysis of single SNPs, as well as haplotypes, revealed an association of APOA5 and APOC3 variants with HDL cholesterol and triglyceride concentrations, respectively. Thus, independent variation in orthologous genomic intervals does associate with similar quantitative lipid traits in both species, supporting the possibility of uncovering human QTL genes in a highly controlled non-human primate model.

  15. Artificial photosynthesis of C1-C3 hydrocarbons from water and CO2 on titanate nanotubes decorated with nanoparticle elemental copper and CdS quantum dots.

    PubMed

    Park, Hyunwoong; Ou, Hsin-Hung; Colussi, Agustín J; Hoffmann, Michael R

    2015-05-14

    The conversion of CO2 and water into value-added fuels with visible light is difficult to achieve in inorganic photocatalytic systems. However, we synthesized a ternary catalyst, CdS/(Cu-TNTs), which is assembled on a core of sodium trititanate nanotubes (TNTs; NaxH2-xTi3O7) decorated with elemental copper deposits followed by an overcoat of CdS quantum dot deposits. This ternary photocatalyst is capable of catalyzing the conversion of CO2 and water into C1-C3 hydrocarbons (e.g., CH4, C2H6, C3H8, C2H4, C3H6) upon irradiation with visible light above 420 nm. With this composite photocatalyst, sacrificial electron donors are not required for the photoreduction of CO2. We have shown that water is the principal photoexcited-state electron donor, while CO2 bound to the composite surface serves as the corresponding electron acceptor. If the photochemical reaction is carried out under an atmosphere of 99.9% (13)CO2, then the product hydrocarbons are built upon a (13)C backbone. However, free molecular H2 is not observed over 5 h of visible light irradiation even though proton reduction in aqueous solution is thermodynamically favored over CO2 reduction. In terms of photocatalytic efficiency, the stoichiometric fraction of Na(+) in TNTs appears to be an important factor that influences the formation of the observed hydrocarbons. The coordination of CO2 to surface exchange sites on the ternary catalyst leads to the formation of surface-bound CO2 and related carbonate species. It appears that the bidentate binding of O═C═O to certain reactive surface sites reduces the energy barrier for conduction band electron transfer to CO2. The methyl radical (CH3(•)), an observed intermediate in the reaction, was positively identified using an ESR spin trapping probe molecule. The copper deposits on the surface of TNTs appear to play a major role in the transient trapping of methyl radical, which in turn self-reacts to produce ethane.

  16. Zeatin reductase in Phaseolus embryos

    SciTech Connect

    Martin, R.C.; Mok, David, W.S.; Mok, M.C. )

    1989-04-01

    Zeatin was converted to O-xylosylzeatin in embryos of Phaseolus vulgaris . O-xylosyldihydrozeatin was also identified as a zeatin metabolite. Incubation of embryo extracts with {sup 14}C-zeatin and {sup 14}C-O-xylosylzeatin revealed that reduction preceeds the O-xylosylation of zeatin. An enzyme responsible for reducing the N{sup 6}-side chain was isolated and partially purified using ammonium sulfate fractionation and affinity, gel filtration and anion exchange chromatography. The NADPH dependent reductase was zeatin specific and did not recognize cis-zeatin, ribosylzeatin, i{sup 6}Ade or i{sup 6}Ado. Two forms of the reductase could be separated by either gel filtration or anion exchange HPLC. The HMW isozyme (Mr. 55,000) eluted from the anion exchange column later than the LMW isozyme (Mr. 25,000). Interspecific differences in zeatin reductase activity were also detected.

  17. Diesel Exhaust Influences Carcinogenic PAH-Induced Genotoxicity and Gene Expression in Human Breast Epithelial Cells in Culture

    PubMed Central

    Courter, Lauren A.; Pereira, Cliff; Baird, William M.

    2009-01-01

    The carcinogenic polycyclic aromatic hydrocarbon ns (PAHs) benzo[a]pyrene (B[a]P) and dibenzo[a,l]pyrene (DB[a,l]P) are widespread environmental pollutants, however their toxicological effects within a mixture is not established. We investigated the influence of diesel exhaust (DE) on B[a]P and DB[a,l]P-induced PAH-DNA adduct formation, metabolic activation, gene expression and 8-oxo-dG adduct levels in human breast epithelial cells (MCF-10A) in culture. Following 24 and 48 h, cells co-exposed to DE plus B[a]P exhibited a significant decrease in PAH-DNA adduct levels, compared with B[a]P alone, as determined by 33P-postlabeling combined with reversed-phase high performance liquid chromatography (HPLC). Cytochrome P450 (CYP) enzyme activity, as measured by the ethoxyresorufin O-deethylase (EROD) assay and CYP1B1 expression, significantly increased with co-exposure of DE plus DB[a,l]P, compared with DB[a,l]P alone. Aldo keto-reductase (AKR)1C1, AKR1C2,and AKR1C3 expression also significantly increased in cells exposed to DE plus PAH, compared with PAH exposure alone. Cell populations exhibiting 8-oxo-dG adducts significantly increased in response to exposure to B[a]P or DE plus B[a]P for 24 h, compared with vehicle control, as quantified by flow cytometry. These results suggest that complex mixtures may modify the carcinogenic potency of PAH by shifting the metabolic activation pathway from the production of PAH diol-epoxides to AKR pathway-derived metabolites. PMID:17612574

  18. DNA Adduct Profiles Predict in Vitro Cell Viability after Treatment with the Experimental Anticancer Prodrug PR104A

    PubMed Central

    2017-01-01

    PR104A is an experimental DNA-alkylating hypoxia-activated prodrug that can also be activated in an oxygen-independent manner by the two-electron aldo-keto reductase 1C3. Nitroreduction leads to the formation of cytotoxic hydroxylamine (PR104H) and amine (PR104M) metabolites, which induce DNA mono and cross-linked adducts in cells. PR104A-derived DNA adducts can be utilized as drug-specific biomarkers of efficacy and as a mechanistic tool to elucidate the cellular and molecular effects of PR104A. Toward this goal, a mass spectrometric bioanalysis approach based on a stable isotope-labeled adduct mixture (SILAM) and selected reaction monitoring (SRM) data acquisition for relative quantitation of PR104A-derived DNA adducts in cells was developed. Use of this SILAM-based approach supported simultaneous relative quantitation of 33 PR104A-derived DNA adducts in the same sample, which allowed testing of the hypothesis that the enhanced cytotoxicity, observed by preconditioning cells with the transcription-activating isothiocyanate sulforaphane, is induced by an increased level of DNA adducts induced by PR104H and PR104M, but not PR104A. By applying the new SILAM-SRM approach, we found a 2.4-fold increase in the level of DNA adducts induced by PR104H and PR104M in HT-29 cells preconditioned with sulforaphane and a corresponding 2.6-fold increase in cytotoxicity. These results suggest that DNA adduct levels correlate with drug potency and underly the possibility of monitoring PR104A-derived DNA adducts as biomarkers of efficacy. PMID:28140568

  19. Isolated menthone reductase and nucleic acid molecules encoding same

    DOEpatents

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

    2013-04-23

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

  20. Nitrate reductase from Rhodopseudomonas sphaeroides.

    PubMed Central

    Kerber, N L; Cardenas, J

    1982-01-01

    The facultative phototroph Rhodopseudomonas sphaeroides DSM158 was incapable of either assimilating or dissimilating nitrate, although the organism could reduce it enzymatically to nitrite either anaerobically in the light or aerobically in the dark. Reduction of nitrate was mediated by a nitrate reductase bound to chromatophores that could be easily solubilized and functioned with chemically reduced viologens or photochemically reduced flavins as electron donors. The enzyme was solubilized, and some of its kinetic and molecular parameters were determined. It seemed to be nonadaptive, ammonia did not repress its synthesis, and its activity underwent a rapid decline when the cells entered the stationary growth phase. Studies with inhibitors and with metal antagonists indicated that molybdenum and possibly iron participate in the enzymatic reduction of nitrate. The conjectural significance of this nitrate reductase in phototrophic bacteria is discussed. PMID:6978883

  1. Fatty acyl-CoA reductase

    SciTech Connect

    Reiser, Steven E.; Somerville, Chris R.

    1998-12-01

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

  2. Nitrate Reductase Regulates Expression of Nitrite Uptake and Nitrite Reductase Activities in Chlamydomonas reinhardtii 1

    PubMed Central

    Galván, Aurora; Cárdenas, Jacobo; Fernández, Emilio

    1992-01-01

    In Chlamydomonas reinhardtii mutants defective at the structural locus for nitrate reductase (nit-1) or at loci for biosynthesis of the molybdopterin cofactor (nit-3, nit-4, or nit-5 and nit-6), both nitrite uptake and nitrite reductase activities were repressed in ammonium-grown cells and expressed at high amounts in nitrogen-free media or in media containing nitrate or nitrite. In contrast, wild-type cells required nitrate induction for expression of high levels of both activities. In mutants defective at the regulatory locus for nitrate reductase (nit-2), very low levels of nitrite uptake and nitrite reductase activities were expressed even in the presence of nitrate or nitrite. Both restoration of nitrate reductase activity in mutants defective at nit-1, nit-3, and nit-4 by isolating diploid strains among them and transformation of a structural mutant upon integration of the wild-type nit-1 gene gave rise to the wild-type expression pattern for nitrite uptake and nitrite reductase activities. Conversely, inactivation of nitrate reductase by tungstate treatment in nitrate, nitrite, or nitrogen-free media made wild-type cells respond like nitrate reductase-deficient mutants with respect to the expression of nitrite uptake and nitrite reductase activities. Our results indicate that nit-2 is a regulatory locus for both the nitrite uptake system and nitrite reductase, and that the nitrate reductase enzyme plays an important role in the regulation of the expression of both enzyme activities. PMID:16668656

  3. Neuroprotective role for carbonyl reductase?

    PubMed

    Maser, Edmund

    2006-02-24

    Oxidative stress is increasingly implicated in neurodegenerative disorders including Alzheimer's, Parkinson's, Huntington's, and Creutzfeld-Jakob diseases or amyotrophic lateral sclerosis. Reactive oxygen species seem to play a significant role in neuronal cell death in that they generate reactive aldehydes from membrane lipid peroxidation. Several neuronal diseases are associated with increased accumulation of abnormal protein adducts of reactive aldehydes, which mediate oxidative stress-linked pathological events, including cellular growth inhibition and apoptosis induction. Combining findings on neurodegeneration and oxidative stress in Drosophila with studies on the metabolic characteristics of the human enzyme carbonyl reductase (CR), it is clear now that CR has a potential physiological role for neuroprotection in humans. Several lines of evidence suggest that CR represents a significant pathway for the detoxification of reactive aldehydes derived from lipid peroxidation and that CR in humans is essential for neuronal cell survival and to confer protection against oxidative stress-induced brain degeneration.

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

    PubMed

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

    2012-06-26

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

  5. Genetics Home Reference: 5-alpha reductase deficiency

    MedlinePlus

    ... About half of these individuals adopt a male gender role in adolescence or early adulthood. Related Information ... 1730-5. Citation on PubMed Cohen-Kettenis PT. Gender change in 46,XY persons with 5alpha-reductase- ...

  6. A dissimilatory nitrite reductase in Paracoccus halodenitrificans

    NASA Technical Reports Server (NTRS)

    Grant, M. A.; Hochstein, L. I.

    1984-01-01

    Paracoccus halodenitrificans produced a membrane-associated nitrite reductase. Spectrophotometric analysis showed it to be associated with a cd-cytochrome and located on the inner side of the cytoplasmic membrane. When supplied with nitrite, membrane preparations produced nitrous oxide and nitric oxide in different ratios depending on the electron donor employed. The nitrite reductase was maximally active at relatively low concentrations of sodium chloride and remained attached to the membranes at 100 mM sodium chloride.

  7. Characterization of thyroidal glutathione reductase

    SciTech Connect

    Raasch, R.J.

    1989-01-01

    Glutathione levels were determined in bovine and rat thyroid tissue by enzymatic conjugation with 1-chloro-2,4-dinitrobenzene using glutathione S-transferase. Bovine thyroid tissue contained 1.31 {+-} 0.04 mM reduced glutathione (GSH) and 0.14 {+-} 0.02 mM oxidized glutathione (GSSG). In the rat, the concentration of GSH was 2.50 {+-} 0.05 mM while GSSG was 0.21 {+-} 0.03 mM. Glutathione reductase (GR) was purified from bovine thyroid to electrophoretic homogeneity by ion exchange, affinity and molecular exclusion chromatography. A molecular weight range of 102-109 kDa and subunit size of 55 kDa were determined for GR. Thyroidal GR was shown to be a favoprotein with one FAD per subunit. The Michaelis constants of bovine thyroidal GR were determined to be 21.8 {mu}M for NADPH and 58.8 {mu}M for GSSG. The effect of thyroid stimulating hormone (TSH) and thyroxine (T{sub 4}) on in vivo levels of GR and glucose 6-phosphate dehydrogenase were determined in rat thyroid homogenates. Both enzymes were stimulated by TSH treatment and markedly reduced following T{sub 4} treatment. Lysosomal hydrolysis of ({sup 125}I)-labeled and unlabeled thyroglobulin was examined using size exclusion HPLC.

  8. Respiratory arsenate reductase as a bidirectional enzyme

    USGS Publications Warehouse

    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.

  9. The tyrosyl free radical in ribonucleotide reductase.

    PubMed Central

    Gräslund, A; Sahlin, M; Sjöberg, B M

    1985-01-01

    The enzyme, ribonucleotide reductase, catalyses the formation of deoxyribonucleotides from ribonucleotides, a reaction essential for DNA synthesis in all living cells. The Escherichia coli ribonucleotide reductase, which is the prototype of all known eukaryotic and virus-coded enzymes, consists of two nonidentical subunits, proteins B1 and B2. The B2 subunit contains an antiferromagnetically coupled pair of ferric ions and a stable tyrosyl free radical. EPR studies show that the tyrosyl radical, formed by loss of ferric ions and a stable tyrosyl free radical. EPR studies show that the tyrosyl radical, formed by loss of an electron, has its unpaired spin density delocalized in the aromatic ring of tyrosine. Effects of iron-radical interaction indicate a relatively close proximity between the iron center and the radical. The EPR signal of the radical can be studied directly in frozen packed cells of E. coli or mammalian origin, if the cells are made to overproduce ribonucleotide reductase. The hypothetic role of the tyrosyl free radical in the enzymatic reaction is not yet elucidated, except in the reaction with the inhibiting substrate analogue 2'-azido-CDP. In this case, the normal tyrosyl radical is destroyed with concomitant appearance of a 2'-azido-CDP-localized radical intermediate. Attempts at spin trapping of radical reaction intermediates have turned out negative. In E. coli the activity of ribonucleotide reductase may be regulated by enzymatic activities that interconvert a nonradical containing form and the fully active protein B2. In synchronized mammalian cells, however, the cell cycle variation of ribonucleotide reductase, studied by EPR, was shown to be due to de novo protein synthesis. Inhibitors of ribonucleotide reductase are of medical interest because of their ability to control DNA synthesis. One example is hydroxyurea, used in cancer therapy, which selectively destroys the tyrosyl free radical. PMID:3007085

  10. Evaluation of nitrate reductase activity in Rhizobium japonicum

    SciTech Connect

    Streeter, J.G.; DeVine, P.J.

    1983-08-01

    Nitrate reductase activity was evaluated by four approaches, using four strains of Rhizobium japonicum and 11 chlorate-resistant mutants of the four strains. It was concluded that in vitro assays with bacteria or bacteroids provide the most simple and reliable assessment of the presence or absence of nitrate reductase. Nitrite reductase activity with methyl viologen and dithionite was found, but the enzyme activity does not confound the assay of nitrate reductase. 18 references

  11. Isolation, sequence identification and tissue expression profile of two novel soybean (glycine max) genes-vestitone reductase and chalcone reductase.

    PubMed

    Liu, G Y

    2009-09-01

    The complete mRNA sequences of two soybean (glycine max) genes-vestitone reductase and chalcone reductase, were amplified using the rapid amplification of cDNA ends methods. The sequence analysis of these two genes revealed that soybean vestitone reductase gene encodes a protein of 327 amino acids which has high homology with the vestitone reductase of Medicago sativa (77%). The soybean chalcone reductase gene encodes a protein of 314 amino acids that has high homology with the chalcone reductase of kudzu vine (88%) and medicago sativa (83%). The expression profiles of the soybean vestitone reductase and chalcone reductase genes were studied and the results indicated that these two soybean genes were differentially expressed in detected soybean tissues including leaves, stems, roots, inflorescences, embryos and endosperm. Our experiment established the foundation for further research on these two soybean genes.

  12. Human hydroxysteroid dehydrogenases and pre-receptor regulation: Insights into inhibitor design and evaluation

    PubMed Central

    Penning, Trevor M.

    2011-01-01

    Hydroxysteroid dehydrogenases (HSDs) represent a major class of NAD(P)(H) dependent steroid hormone oxidoreductases involved in the pre-receptor regulation of hormone action. This is achieved by HSDs working in pairs so that they can interconvert ketosteroids with hydroxysteroids resulting in a change in ligand potency for nuclear receptors. HSDs belong to two protein superfamilies the aldo-keto reductases and the short-chain dehydrogenase/reductases. In humans, many of the important enzymes have been thoroughly characterized including the elucidation of their three-dimensional structures. Because these enzymes play fundamental roles in steroid hormone action they can be considered to be drug targets for a variety of steroid driven diseases: e.g. metabolic syndrome and obesity, inflammation, and hormone dependent malignancies of the endometrium, prostate and breast. This article will review how fundamental knowledge of these enzymes can be exploited in the development of isoform specific HSD inhibitors from both protein superfamilies. PMID:21272640

  13. Post-translational Regulation of Nitrate Reductase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Nitrate reductase (NR) catalyzes the reduction of nitrate to nitrite, which is the first step in the nitrate assimilation pathway, but can also reduce nitrite to nitric oxide (NO), an important signaling molecule that is thought to mediate a wide array of of developmental and physiological processes...

  14. Fumarate Reductase Activity of Streptococcus faecalis

    PubMed Central

    Aue, B. J.; Diebel, R. H.

    1967-01-01

    Some characteristics of a fumarate reductase from Streptococcus faecalis are described. The enzyme had a pH optimum of 7.4; optimal activity was observed when the ionic strength of the phosphate buffer was adjusted to 0.088. The Km value of the enzyme for reduced flavin mononucleotide was 2 × 10−4 m as determined with a 26-fold preparation. In addition to fumarate, the enzyme reduced maleate and mesaconate. No succinate dehydrogenase activity was detected, but succinate did act as an inhibitor of the fumarate reductase activity. Other inhibitors were malonate, citraconate, and trans-, trans-muconate. Metal-chelating agents did not inhibit the enzyme. A limited inhibition by sulfhydryl-binding agents was observed, and the preparations were sensitive to air oxidation and storage. Glycine, alanine, histidine, and possibly lysine stimulated fumarate reductase activity in the cell-free extracts. However, growth in media supplemented with glycine did not enhance fumarate reductase activity. The enzymatic activity appears to be constitutive. PMID:4960892

  15. Control of dihydrofolate reductase messenger ribonucleic acid production

    SciTech Connect

    Leys, E.J.; Kellems, R.E.

    1981-11-01

    The authors used methotrexate-resistant mouse cells in which dihydrofolate reductase levels are approximately 500 times normal to study the effect of growth stimulation on dihydrofolate reductase gene expression. As a result of growth stimulation, the relative rate of dihydrofolate reductase protein synthesis increased threefold, reaching a maximum between 25 and 30 h after stimulation. The relative rate of dihydrofolate reductase messenger ribonucleic acid production (i.e., the appearance of dihydrofolate reductase messenger ribonucleic acid in the cytoplasm) increased threefold after growth stimulation and was accompanied by a corresponding increase in the relative steady-state level of dihydrofolate reductase ribonucleic acid in the nucleus. However, the increase in the nuclear level of dihydrofolate reductase ribonucleic acid was not accompanied by a significant increase in the relative rate of transcription of the dihydrofolate reductase genes. These data indicated that the relative rate of appearance of dihydrofolate reductase messenger ribonucleic acid in the cytoplasm depends on the relative stability of the dihydrofolate reductase ribonucleic acid sequences in the nucleus and is not dependent on the relative rate of transcription of the dihydrofolate reductase genes.

  16. Augmentation of CFTR maturation by S-nitrosoglutathione reductase

    PubMed Central

    Sawczak, Victoria; Zaidi, Atiya; Butler, Maya; Bennett, Deric; Getsy, Paulina; Zeinomar, Maryam; Greenberg, Zivi; Forbes, Michael; Rehman, Shagufta; Jyothikumar, Vinod; DeRonde, Kim; Sattar, Abdus; Smith, Laura; Corey, Deborah; Straub, Adam; Sun, Fei; Palmer, Lisa; Periasamy, Ammasi; Randell, Scott; Kelley, Thomas J.; Lewis, Stephen J.

    2015-01-01

    S-nitrosoglutathione (GSNO) reductase regulates novel endogenous S-nitrosothiol signaling pathways, and mice deficient in GSNO reductase are protected from airways hyperreactivity. S-nitrosothiols are present in the airway, and patients with cystic fibrosis (CF) tend to have low S-nitrosothiol levels that may be attributed to upregulation of GSNO reductase activity. The present study demonstrates that 1) GSNO reductase activity is increased in the cystic fibrosis bronchial epithelial (CFBE41o−) cells expressing mutant F508del-cystic fibrosis transmembrane regulator (CFTR) compared with the wild-type CFBE41o− cells, 2) GSNO reductase expression level is increased in the primary human bronchial epithelial cells expressing mutant F508del-CFTR compared with the wild-type cells, 3) GSNO reductase colocalizes with cochaperone Hsp70/Hsp90 organizing protein (Hop; Stip1) in human airway epithelial cells, 4) GSNO reductase knockdown with siRNA increases the expression and maturation of CFTR and decreases Stip1 expression in human airway epithelial cells, 5) increased levels of GSNO reductase cause a decrease in maturation of CFTR, and 6) a GSNO reductase inhibitor effectively reverses the effects of GSNO reductase on CFTR maturation. These studies provide a novel approach to define the subcellular location of the interactions between Stip1 and GSNO reductase and the role of S-nitrosothiols in these interactions. PMID:26637637

  17. Characterization of the cancer chemopreventive NRF2-dependent gene battery in human keratinocytes: demonstration that the KEAP1-NRF2 pathway, and not the BACH1-NRF2 pathway, controls cytoprotection against electrophiles as well as redox-cycling compounds.

    PubMed

    MacLeod, A Kenneth; McMahon, Michael; Plummer, Simon M; Higgins, Larry G; Penning, Trevor M; Igarashi, Kazuhiko; Hayes, John D

    2009-09-01

    To better understand the role of transcription factor NF-E2-related factor (NRF) 2 in the human and its contribution to cancer chemoprevention, we have knocked down its negative regulators, Kelch-like ECH-associated protein 1 (KEAP1) and broad-complex, tramtrack and bric à brac and cap'n'collar homology 1 (BACH1), in HaCaT keratinocytes. Whole-genome microarray revealed that knockdown of KEAP1 resulted in 23 messenger RNAs (mRNAs) being up-regulated > or = 2.0-fold. mRNA for aldo-keto reductase (AKR) 1B10, AKR1C1, AKR1C2 and AKR1C3 were induced to the greatest extent, showing increases of between 12- and 16-fold, whereas mRNA for glutamate-cysteine ligase catalytic and modifier subunits, NAD(P)H:quinone oxidoreductase-1 and haem oxygenase-1 (HMOX1) were induced between 2.0- and 4.8-fold. Knockdown of BACH1 increased HMOX1 135-fold but induced the other genes examined to a maximum of only 2.7-fold. Activation of NRF2, by KEAP1 knockdown, caused a 75% increase in the amount of glutathione in HaCaT cells and a 1.4- to 1.6-fold increase in their resistance to the electrophiles acrolein, chlorambucil and cumene hydroperoxide (CuOOH), as well as the redox-cycling agent menadione. Inhibition of glutathione synthesis during KEAP1 knockdown, by treatment with buthionine sulfoximine, abrogated resistance to acrolein, chlorambucil and CuOOH, but not to menadione. In contrast, knockdown of BACH1 did not increase glutathione levels or resistance to xenobiotics. Knockdown of NRF2 in HaCaT cells decreased glutathione to approximately 80% of normal homeostatic levels and similarly reduced their tolerance of electrophiles. Thus, the KEAP1-NRF2 pathway determines resistance to electrophiles and redox-cycling compounds in human keratinocytes through glutathione-dependent and glutathione-independent mechanisms. This study also shows that AKR1B10, AKR1C1 and AKR1C2 proteins have potential utility as biomarkers for NRF2 activation in the human.

  18. FRUCTOSE-6-PHOSPHATE REDUCTASE FROM SALMONELLA GALLINARUM

    PubMed Central

    Zancan, Glaci T.; Bacila, Metry

    1964-01-01

    Zancan, Glaci T. (Universidade do Paraná, Curitiba, Paraná, Brazil), and Metry Bacila. Fructose-6-phosphate reductase from Salmonella gallinarum. J. Bacteriol. 87:614–618. 1964.—A fructose-6-phosphate reductase present in cell-free extracts of Salmonella gallinarum was purified approximately 42 times. The optimal pH for this enzyme is 8.0. The enzyme is specific for fructose-6-phosphate and reduced nicotinamide adenine dinucleotide (NADH). The dissociation constants are 1.78 × 10−4m for fructose-6-phosphate and 8.3 × 10−5m for NADH. The Q10, reaction order, and equilibrium constant were determined. The enzyme is sensitive to p-chloromercuribenzoic acid, but not to o-iodosobenzoic acid nor to N-ethylmaleimide. PMID:14127579

  19. Characterization of erythrose reductases from filamentous fungi

    PubMed Central

    2013-01-01

    Proteins with putative erythrose reductase activity have been identified in the filamentous fungi Trichoderma reesei, Aspergillus niger, and Fusarium graminearum by in silico analysis. The proteins found in T. reesei and A. niger had earlier been characterized as glycerol dehydrogenase and aldehyde reductase, respectively. Corresponding genes from all three fungi were cloned, heterologously expressed in Escherichia coli, and purified. Subsequently, they were used to establish optimal enzyme assay conditions. All three enzymes strictly require NADPH as cofactor, whereas with NADH no activity could be observed. The enzymatic characterization of the three enzymes using ten substrates revealed high substrate specificity and activity with D-erythrose and D-threose. The enzymes from T. reesei and A. niger herein showed comparable activities, whereas the one from F. graminearum reached only about a tenth of it for all tested substrates. In order to proof in vivo the proposed enzyme function, we overexpressed the erythrose reductase-encoding gene in T. reesei. An increased production of erythritol by the recombinant strain compared to the parental strain could be detected. PMID:23924507

  20. A Ferredoxin Disulfide Reductase Delivers Electrons to the Methanosarcina barkeri Class III Ribonucleotide Reductase

    PubMed Central

    2015-01-01

    Two subtypes of class III anaerobic ribonucleotide reductases (RNRs) studied so far couple the reduction of ribonucleotides to the oxidation of formate, or the oxidation of NADPH via thioredoxin and thioredoxin reductase. Certain methanogenic archaea contain a phylogenetically distinct third subtype of class III RNR, with distinct active-site residues. Here we report the cloning and recombinant expression of the Methanosarcina barkeri class III RNR and show that the electrons required for ribonucleotide reduction can be delivered by a [4Fe-4S] protein ferredoxin disulfide reductase, and a conserved thioredoxin-like protein NrdH present in the RNR operon. The diversity of class III RNRs reflects the diversity of electron carriers used in anaerobic metabolism. PMID:26536144

  1. Methionine sulfoxide reductase contributes to meeting dietary methionine requirements

    PubMed Central

    Zhao, Hang; Kim, Geumsoo; Levine, Rodney L.

    2012-01-01

    Methionine sulfoxide reductases are present in all aerobic organisms. They contribute to antioxidant defenses by reducing methionine sulfoxide in proteins back to methionine. However, the actual in vivo roles of these reductases are not well defined. Since methionine is an essential amino acid in mammals, we hypothesized that methionine sulfoxide reductases may provide a portion of the dietary methionine requirement by recycling methionine sulfoxide. We used a classical bioassay, the growth of weanling mice fed diets varying in methionine, and applied it to mice genetically engineered to alter the levels of methionine sulfoxide reductase A or B1. Mice of all genotypes were growth retarded when raised on chow containing 0.10% methionine instead of the standard 0.45% methionine. Retardation was significantly greater in knockout mice lacking both reductases. We conclude that the methionine sulfoxide reductases can provide methionine for growth in mice with limited intake of methionine, such as may occur in the wild. PMID:22521563

  2. Limited proteolysis of the nitrate reductase from spinach leaves.

    PubMed

    Kubo, Y; Ogura, N; Nakagawa, H

    1988-12-25

    The functional structure of assimilatory NADH-nitrate reductase from spinach leaves was studied by limited proteolysis experiments. After incubation of purified nitrate reductase with trypsin, two stable products of 59 and 45 kDa were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The fragment of 45 kDa was purified by Blue Sepharose chromatography. NADH-ferricyanide reductase and NADH-cytochrome c reductase activities were associated with this 45-kDa fragment which contains FAD, heme, and NADH binding fragment. After incubation of purified nitrate reductase with Staphylococcus aureus V8 protease, two major peaks were observed by high performance liquid chromatography size exclusion gel filtration. FMNH2-nitrate reductase and reduced methyl viologen-nitrate reductase activities were associated with the first peak of 170 kDa which consists of two noncovalently associated (75-90-kDa) fragments. NADH-ferricyanide reductase activity, however, was associated with the second peak which consisted of FAD and NADH binding sites. Incubation of the 45-kDa fragment with S. aureus V8 protease produced two major fragments of 28 and 14 kDa which contained FAD and heme, respectively. These results indicate that the molybdenum, heme, and FAD components of spinach nitrate reductase are contained in distinct domains which are covalently linked by exposed hinge regions. The molybdenum domain appears to be important in the maintenance of subunit interactions in the enzyme complex.

  3. AKR1C2 — EDRN Public Portal

    Cancer.gov

    From NCBI Gene: This gene encodes a member of the aldo/keto reductase superfamily, which consists of more than 40 known enzymes and proteins. These enzymes catalyze the conversion of aldehydes and ketones to their corresponding alcohols using NADH and/or NADPH as cofactors. The enzymes display overlapping but distinct substrate specificity. This enzyme binds bile acid with high affinity, and shows minimal 3-alpha-hydroxysteroid dehydrogenase activity. This gene shares high sequence identity with three other gene members and is clustered with those three genes at chromosome 10p15-p14. Three transcript variants encoding two different isoforms have been found for this gene. [provided by RefSeq, Dec 2011

  4. Plant science. Morphinan biosynthesis in opium poppy requires a P450-oxidoreductase fusion protein.

    PubMed

    Winzer, Thilo; Kern, Marcelo; King, Andrew J; Larson, Tony R; Teodor, Roxana I; Donninger, Samantha L; Li, Yi; Dowle, Adam A; Cartwright, Jared; Bates, Rachel; Ashford, David; Thomas, Jerry; Walker, Carol; Bowser, Tim A; Graham, Ian A

    2015-07-17

    Morphinan alkaloids from the opium poppy are used for pain relief. The direction of metabolites to morphinan biosynthesis requires isomerization of (S)- to (R)-reticuline. Characterization of high-reticuline poppy mutants revealed a genetic locus, designated STORR [(S)- to (R)-reticuline] that encodes both cytochrome P450 and oxidoreductase modules, the latter belonging to the aldo-keto reductase family. Metabolite analysis of mutant alleles and heterologous expression demonstrate that the P450 module is responsible for the conversion of (S)-reticuline to 1,2-dehydroreticuline, whereas the oxidoreductase module converts 1,2-dehydroreticuline to (R)-reticuline rather than functioning as a P450 redox partner. Proteomic analysis confirmed that these two modules are contained on a single polypeptide in vivo. This modular assembly implies a selection pressure favoring substrate channeling. The fusion protein STORR may enable microbial-based morphinan production.

  5. Integrating protein-protein interaction networks with phenotypes reveals signs of interactions

    PubMed Central

    Vinayagam, Arunachalam; Zirin, Jonathan; Roesel, Charles; Hu, Yanhui; Yilmazel, Bahar; Samsonova, Anastasia A.; Neumüller, Ralph A.; Mohr, Stephanie E.; Perrimon, Norbert

    2013-01-01

    A major objective of systems biology is to organize molecular interactions as networks and to characterize information-flow within networks. We describe a computational framework to integrate protein-protein interaction (PPI) networks and genetic screens to predict the “signs” of interactions (i.e. activation/inhibition relationships). We constructed a Drosophila melanogaster signed PPI network, consisting of 6,125 signed PPIs connecting 3,352 proteins that can be used to identify positive and negative regulators of signaling pathways and protein complexes. We identified an unexpected role for the metabolic enzymes Enolase and Aldo-keto reductase as positive and negative regulators of proteolysis, respectively. Characterization of the activation/inhibition relationships between physically interacting proteins within signaling pathways will impact our understanding of many biological functions, including signal transduction and mechanisms of disease. PMID:24240319

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

    PubMed

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

    2014-12-01

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

  7. Enzyme toolbox: novel enantiocomplementary imine reductases.

    PubMed

    Scheller, Philipp N; Fademrecht, Silvia; Hofelzer, Sebastian; Pleiss, Jürgen; Leipold, Friedemann; Turner, Nicholas J; Nestl, Bettina M; Hauer, Bernhard

    2014-10-13

    Reducing reactions are among the most useful transformations for the generation of chiral compounds in the fine-chemical industry. Because of their exquisite selectivities, enzymatic approaches have emerged as the method of choice for the reduction of C=O and activated C=C bonds. However, stereoselective enzymatic reduction of C=N bonds is still in its infancy-it was only recently described after the discovery of enzymes capable of imine reduction. In our work, we increased the spectrum of imine-reducing enzymes by database analysis. By combining the currently available knowledge about the function of imine reductases with the experimentally uncharacterized diversity stored in protein sequence databases, three novel imine reductases with complementary enantiopreference were identified along with amino acids important for catalysis. Furthermore, their reducing capability was demonstrated by the reduction of the pharmaceutically relevant prochiral imine 2-methylpyrroline. These novel enzymes exhibited comparable to higher catalytic efficiencies than previously described enzymes, and their biosynthetic potential is highlighted by the full conversion of 2-methylpyrroline in whole cells with excellent selectivities.

  8. Soluble ascorbate free radical reductase in the human lens.

    PubMed

    Bando, M; Obazawa, H

    1994-01-01

    A major and a minor ascorbate free radical (AFR) reductase were separated from the soluble fraction in the human lens cortex by DEAE-cellulose ion-exchange column chromatography. These AFR reductases also exhibited diaphorase activity using dichlorophenolindophenol and ferricyanide as electron acceptors. The major AFR reductase was partially purified by 5'AMP-Sepharose 4B affinity column chromatography. This partially purified AFR reductase showed a single band of diaphorase activity in native polyacrylamide disc gel electrophoresis. This activity band corresponded to the major protein observed in protein staining by Coomassie Brilliant Blue. However, the protein staining by Coomassie Brilliant Blue showed this activity band surrounded by diffused staining. Molecular weight of the partially purified AFR reductase was determined to be 32 kDa by gel filtration, and the apparent Km value for AFR was about 15 microM. This major lens AFR reductase could be distinguished from soluble Neurospora, Euglena and cucumber AFR reductases, and from two ubiquitous enzymes with reduction activity of AFR and/or foreign compounds, ie, NADH-cytochrome b5 reductase and DT-diaphorase, by their molecular weights, Km values and/or ion-exchange chromatographic behaviors.

  9. Functional and Phylogenetic Divergence of Fungal Adenylate-Forming Reductases

    PubMed Central

    Kalb, Daniel; Lackner, Gerald

    2014-01-01

    A key step in fungal l-lysine biosynthesis is catalyzed by adenylate-forming l-α-aminoadipic acid reductases, organized in domains for adenylation, thiolation, and the reduction step. However, the genomes of numerous ascomycetes and basidiomycetes contain an unexpectedly large number of additional genes encoding similar but functionally distinct enzymes. Here, we describe the functional in vitro characterization of four reductases which were heterologously produced in Escherichia coli. The Ceriporiopsis subvermispora serine reductase Nps1 features a terminal ferredoxin-NADP+ reductase (FNR) domain and thus belongs to a hitherto undescribed class of fungal multidomain enzymes. The second major class is characterized by the canonical terminal short-chain dehydrogenase/reductase domain and represented by Ceriporiopsis subvermispora Nps3 as the first biochemically characterized l-α-aminoadipic acid reductase of basidiomycete origin. Aspergillus flavus l-tyrosine reductases LnaA and LnbA are members of a distinct phylogenetic clade. Phylogenetic analysis supports the view that fungal adenylate-forming reductases are more diverse than previously recognized and belong to four distinct classes. PMID:25085485

  10. Proteome Screening of Pleural Effusions Identifies Galectin 1 as a Diagnostic Biomarker and Highlights Several Prognostic Biomarkers for Malignant Mesothelioma*

    PubMed Central

    Mundt, Filip; Johansson, Henrik J.; Forshed, Jenny; Arslan, Sertaç; Metintas, Muzaffer; Dobra, Katalin; Lehtiö, Janne; Hjerpe, Anders

    2014-01-01

    Malignant mesothelioma is an aggressive asbestos-induced cancer, and affected patients have a median survival of approximately one year after diagnosis. It is often difficult to reach a conclusive diagnosis, and ancillary measurements of soluble biomarkers could increase diagnostic accuracy. Unfortunately, few soluble mesothelioma biomarkers are suitable for clinical application. Here we screened the effusion proteomes of mesothelioma and lung adenocarcinoma patients to identify novel soluble mesothelioma biomarkers. We performed quantitative mass-spectrometry-based proteomics using isobaric tags for quantification and used narrow-range immobilized pH gradient/high-resolution isoelectric focusing (pH 4–4.25) prior to analysis by means of nano liquid chromatography coupled to MS/MS. More than 1,300 proteins were identified in pleural effusions from patients with malignant mesothelioma (n = 6), lung adenocarcinoma (n = 6), or benign mesotheliosis (n = 7). Data are available via ProteomeXchange with identifier PXD000531. The identified proteins included a set of known mesothelioma markers and proteins that regulate hallmarks of cancer such as invasion, angiogenesis, and immune evasion, plus several new candidate proteins. Seven candidates (aldo-keto reductase 1B10, apolipoprotein C-I, galectin 1, myosin-VIIb, superoxide dismutase 2, tenascin C, and thrombospondin 1) were validated by enzyme-linked immunosorbent assays in a larger group of patients with mesothelioma (n = 37) or metastatic carcinomas (n = 25) and in effusions from patients with benign, reactive conditions (n = 16). Galectin 1 was identified as overexpressed in effusions from lung adenocarcinoma relative to mesothelioma and was validated as an excellent predictor for metastatic carcinomas against malignant mesothelioma. Galectin 1, aldo-keto reductase 1B10, and apolipoprotein C-I were all identified as potential prognostic biomarkers for malignant mesothelioma. This analysis of the effusion proteome

  11. Transcripts of anthocyanidin reductase and leucoanthocyanidin reductase and measurement of catechin and epicatechin in tartary buckwheat.

    PubMed

    Kim, Yeon Bok; Thwe, Aye Aye; Kim, Yeji; Li, Xiaohua; Cho, Jin Woong; Park, Phun Bum; Valan Arasu, Mariadhas; Abdullah Al-Dhabi, Naif; Kim, Sun-Ju; Suzuki, Tastsuro; Hyun Jho, Kwang; Park, Sang Un

    2014-01-01

    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.

  12. Docking and molecular dynamics studies at trypanothione reductase and glutathione reductase active sites.

    PubMed

    Iribarne, Federico; Paulino, Margot; Aguilera, Sara; Murphy, Miguel; Tapia, Orlando

    2002-05-01

    A theoretical docking study on the active sites of trypanothione reductase (TR) and glutathione reductase (GR) with the corresponding natural substrates, trypanothione disulfide (T[S]2) and glutathione disulfide (GSSG), is reported. Molecular dynamics simulations were carried out in order to check the robustness of the docking results. The energetic results are in agreement with previous experimental findings and show the crossed complexes have lower stabilization energies than the natural ones. To test DOCK3.5, four nitro furanic compounds, previously designed as potentially active anti-chagasic molecules, were docked at the GR and TR active sites with the DOCK3.5 procedure. A good correlation was found between differential inhibitory activity and relative interaction energy (affinity). The results provide a validation test for the use of DOCK3.5 in connection with the design of anti-chagasic drugs.

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

    PubMed Central

    Kim, Yeon Bok; Thwe, Aye Aye; Kim, YeJi; Li, Xiaohua; Cho, Jin Woong; Park, Phun Bum; Valan Arasu, Mariadhas; Abdullah Al-Dhabi, Naif; Kim, Sun-Ju; Suzuki, Tastsuro; Hyun Jho, Kwang; Park, Sang Un

    2014-01-01

    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. PMID:24605062

  14. Methylenetetrahydrofolate reductase: biochemical characterization and medical significance.

    PubMed

    Trimmer, Elizabeth E

    2013-01-01

    Methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction of 5,10-methylenetetrahydofolate (CH2-H4folate) to 5-methyltetrahydrofolate (CH3-H4folate). The enzyme employs a noncovalently-bound flavin adenine dinucleotide (FAD), which accepts reducing equivalents from NAD(P)H and transfers them to CH2-H4folate. The reaction provides the sole source of CH3-H4folate, which is utilized by methionine synthase in the synthesis of methionine from homocysteine. MTHFR plays a key role in folate metabolism and in the homeostasis of homocysteine; mutations in the enzyme lead to hyperhomocyst(e)inemia. A common C677T polymorphism in MTHFR has been associated with an increased risk for the development of cardiovascular disease, Alzheimer's disease, and depression in adults, and of neural tube defects in the fetus. The mutation also confers protection for certain types of cancers. This review presents the current knowledge of the enzyme, its biochemical characterization, and medical significance.

  15. Enhanced silver nanoparticle synthesis by optimization of nitrate reductase activity.

    PubMed

    Vaidyanathan, Ramanathan; Gopalram, Shubaash; Kalishwaralal, Kalimuthu; Deepak, Venkataraman; Pandian, Sureshbabu Ram Kumar; Gurunathan, Sangiliyandi

    2010-01-01

    Nanostructure materials are attracting a great deal of attention because of their potential for achieving specific processes and selectivity, especially in biological and pharmaceutical applications. The generation of silver nanoparticles using optimized nitrate reductase for the reduction of Ag(+) with the retention of enzymatic activity in the complex is being reported. This report involves the optimization of enzyme activity to bring about enhanced nanoparticle synthesis. Response surface methodology and central composite rotary design (CCRD) were employed to optimize a fermentation medium for the production of nitrate reductase by Bacillus licheniformis at pH 8. The four variables involved in the study of nitrate reductase were Glucose, Peptone, Yeast extract and KNO(3). Glucose had a significant effect on nitrate reductase production. The optimized medium containing (%) Glucose: 1.5, Peptone: 1, Yeast extract: 0.35 and KNO(3): 0.35 resulted in a nitrate reductase activity of 452.206 U/ml which is same as that of the central level. The medium A (showing least nitrate reductase activity) and the medium B (showing maximum nitrate reductase activity) were compared for the synthesis. Spectrophotometric analysis revealed that the particles exhibited a peak at 431 nm and the A(431) for the medium B was 2-fold greater than that of the medium A. The particles were also characterized using TEM. The particles synthesized using the optimized enzyme activity ranged from 10 to 80 nm and therefore can be extended to various medicinal applications.

  16. Solubilization and Resolution of the Membrane-Bound Nitrite Reductase from Paracoccus Halodenitrificans into Nitrite and Nitric Oxide Reductases

    NASA Technical Reports Server (NTRS)

    Grant, Michael A.; Cronin, Sonja E.; Hochstein, Lawrence I.

    1984-01-01

    Membranes prepared from Paracoccus halodenitrificans reduced nitrite or nitric oxide to nitrous oxide. Extraction of these membranes with the detergent CHAPSO [3-(3-Chlolamidoporopyldimethylammonio)-1-(2- hydroxy-1-propanesulfonate)], followed by ammonium sulfate fractionation of the solubilized proteins, resulted in the separation of nitrite and nitric oxide reductase activities. The fraction containing nitrite reductase activity spectrally resembled a cd-type cytochrome. Several cytochromes were detected in the nitric oxide reductase fraction. Which, if any, of these cytochromes is associated with the reduction of nitric oxide is not clear at this time.

  17. Enantioselective imine reduction catalyzed by imine reductases and artificial metalloenzymes.

    PubMed

    Gamenara, Daniela; Domínguez de María, Pablo

    2014-05-21

    Adding value to organic synthesis. Novel imine reductases enable the enantioselective reduction of imines to afford optically active amines. Likewise, novel bioinspired artificial metalloenzymes can perform the same reaction as well. Emerging proof-of-concepts are herein discussed.

  18. Exploration of Nitrate Reductase Metabolic Pathway in Corynebacterium pseudotuberculosis

    PubMed Central

    Abreu, Vinícius; Diniz, Carlos; Dorneles, Elaine M. S.; Barh, Debmalya

    2017-01-01

    Based on the ability of nitrate reductase synthesis, Corynebacterium pseudotuberculosis is classified into two biovars: Ovis and Equi. Due to the presence of nitrate reductase, the Equi biovar can survive in absence of oxygen. On the other hand, Ovis biovar that does not have nitrate reductase is able to adapt to various ecological niches and can grow on certain carbon sources. Apart from these two biovars, some other strains are also able to carry out the reduction of nitrate. The enzymes that are involved in electron transport chain are also identified by in silico methods. Findings about pathogen metabolism can contribute to the identification of relationship between nitrate reductase and the C. pseudotuberculosis pathogenicity, virulence factors, and discovery of drug targets. PMID:28316974

  19. 21 CFR 864.7375 - Glutathione reductase assay.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... activity of the enzyme glutathione reductase in serum, plasma, or erythrocytes by such techniques as fluorescence and photometry. The results of this assay are used in the diagnosis of liver disease,...

  20. Purification and characterization of assimilatory nitrite reductase from Candida utilis.

    PubMed

    Sengupta, S; Shaila, M S; Rao, G R

    1996-07-01

    Nitrate assimilation in many plants, algae, yeasts and bacteria is mediated by two enzymes, nitrate reductase (EC 1.6.6.2) and nitrite reductase (EC 1.7.7.1). They catalyse the stepwise reduction of nitrate to nitrite and nitrite to ammonia respectively. The nitrite reductase from an industrially important yeast, Candida utilis, has been purified to homogeneity. Purified nitrite reductase is a heterodimer and the molecular masses of the two subunits are 58 and 66 kDa. The native enzyme exhibits a molecular mass of 126 kDa as analysed by gel filtration. The identify of the two subunits of nitrite reductase was confirmed by immunoblotting using antibody for Cucurbita pepo leaf nitrite reductase. The presence of two different sized transcripts coding for the two subunits was confirmed by (a) in vitro translation of mRNA from nitrate-induced C. utilis followed by immunoprecipitation of the in vitro translated products with heterologous nitrite reductase antibody and (b) Northern-blot analysis. The 66 kDa subunit is acidic in nature which is probably due to its phosphorylated status. The enzyme is stable over a range of temperatures. Both subunits can catalyse nitrite reduction, and the reconstituted enzyme, at a higher protein concentration, shows an activity similar to that of the purified enzyme. Each of these subunits has been shown to contain a few unique peptides in addition to a large number of common peptides. Reduced Methyl Viologen has been found to be as effective an electron donor as NADPH in the catalytic process, a phenomenon not commonly seen for nitrite reductases from other systems.

  1. Molybdenum effector of fumarate reductase repression and nitrate reductase induction in Escherichia coli.

    PubMed Central

    Iuchi, S; Lin, E C

    1987-01-01

    In Escherichia coli the presence of nitrate prevents the utilization of fumarate as an anaerobic electron acceptor. The induction of the narC operon encoding the nitrate reductase is coupled to the repression of the frd operon encoding the fumarate reductase. This coupling is mediated by nitrate as an effector and the narL product as the regulatory protein (S. Iuchi and E. C. C. Lin, Proc. Natl. Acad. Sci. USA 84:3901-3905, 1987). The protein-ligand complex appears to control narC positively but frd negatively. In the present study we found that a molybdenum coeffector acted synergistically with nitrate in the regulation of frd and narC. In chlD mutants believed to be impaired in molybdate transport (or processing), full repression of phi(frd-lac) and full induction of phi(narC-lac) by nitrate did not occur unless the growth medium was directly supplemented with molybdate (1 microM). This requirement was not clearly manifested in wild-type cells, apparently because it was met by the trace quantities of molybdate present as a contaminant in the mineral medium. In chlB mutants, which are known to accumulate the Mo cofactor because of its failure to be inserted as a prosthetic group into proteins such as nitrate reductase, nitrate repression of frd and induction of narC were also intensified by molybdate supplementation. In this case a deficiency of the molybdenum coeffector might have resulted from enhanced feedback inhibition of molybdate transport (or processing) by the elevated level of the unutilized Mo cofactor. In addition, mutations in chlE, which are known to block the synthesis of the organic moiety of the Mo cofactor, lowered the threshold concentration of nitrate (< 1 micromole) necessary for frd repression and narC induction. These changes could be explained simply by the higher intracellular nitrate attainable in cells lacking the ability to destroy the effector. PMID:3301812

  2. Distribution of Prx-linked hydroperoxide reductase activity among microorganisms.

    PubMed

    Takeda, Kouji; Nishiyama, Yoshitaka; Yoda, Koji; Watanabe, Toshihiro; Nimura-Matsune, Kaori; Mura, Kiyoshi; Tokue, Chiyoko; Katoh, Tetzuya; Kawasaki, Shinji; Niimura, Youichi

    2004-01-01

    Peroxiredoxin (Prx) constitutes a large family of enzymes found in microorganisms, animals, and plants, but the detection of the activities of Prx-linked hydroperoxide reductases (peroxiredoxin reductases) in cell extracts, and the purification based on peroxide reductase activity, have only been done in bacteria and Trypanosomatidae. A peroxiredoxin reductase (NADH oxidase) from a bacterium, Amphibacillus, displayed only poor activities in the presence of purified Prx from Saccharomyces or Synechocystis, while it is highly active in the presence of bacterial Prx. These results suggested that an enzyme system different from that in bacteria might exist for the reduction of Prx in yeast and cyanobacteria. Prx-linked hydroperoxide reductase activities were detected in cell extracts of Saccharomyces, Synechocystis, and Chlorella, and the enzyme activities of Saccharomyces and Chlorella were induced under vigorously aerated culture conditions and intensive light exposure conditions, respectively. Partial purification of Prx-linked peroxidase from the induced yeast cells indicated that the Prx-linked peroxidase system consists of two protein components, namely, thioredoxin and thioredoxin reductase. This finding is consistent with the previous report on its purification based on its protein protection activity against oxidation [Chae et al., J. Biol. Chem., 269, 27670-27678 (1994)]. In this study we have confirmed that Prx-linked peroxidase activity are widely distributed, not only in bacteria species and Trypanosomatidae, but also in yeast and photosynthetic microorganisms, and showed reconstitution of the activity from partially purified interspecies components.

  3. Carbon-carbon double-bond reductases in nature.

    PubMed

    Huang, Minmin; Hu, Haihong; Ma, Li; Zhou, Quan; Yu, Lushan; Zeng, Su

    2014-08-01

    Reduction of C = C bonds by reductases, found in a variety of microorganisms (e.g. yeasts, bacteria, and lower fungi), animals, and plants has applications in the production of metabolites that include pharmacologically active drugs and other chemicals. Therefore, the reductase enzymes that mediate this transformation have become important therapeutic targets and biotechnological tools. These reductases are broad-spectrum, in that, they can act on isolation/conjugation C = C-bond compounds, α,β-unsaturated carbonyl compounds, carboxylic acids, acid derivatives, and nitro compounds. In addition, several mutations in the reductase gene have been identified, some associated with diseases. Several of these reductases have been cloned and/or purified, and studies to further characterize them and determine their structure in order to identify potential industrial biocatalysts are still in progress. In this study, crucial reductases for bioreduction of C = C bonds have been reviewed with emphasis on their principal substrates and effective inhibitors, their distribution, genetic polymorphisms, and implications in human disease and treatment.

  4. Microsecond subdomain folding in dihydrofolate reductase.

    PubMed

    Arai, Munehito; Iwakura, Masahiro; Matthews, C Robert; Bilsel, Osman

    2011-07-08

    The characterization of microsecond dynamics in the folding of multisubdomain proteins has been a major challenge in understanding their often complex folding mechanisms. Using a continuous-flow mixing device coupled with fluorescence lifetime detection, we report the microsecond folding dynamics of dihydrofolate reductase (DHFR), a two-subdomain α/β/α sandwich protein known to begin folding in this time range. The global dimensions of early intermediates were monitored by Förster resonance energy transfer, and the dynamic properties of the local Trp environments were monitored by fluorescence lifetime detection. We found that substantial collapse occurs in both the locally connected adenosine binding subdomain and the discontinuous loop subdomain within 35 μs of initiation of folding from the urea unfolded state. During the fastest observable ∼550 μs phase, the discontinuous loop subdomain further contracts, concomitant with the burial of Trp residue(s), as both subdomains achieve a similar degree of compactness. Taken together with previous studies in the millisecond time range, a hierarchical assembly of DHFR--in which each subdomain independently folds, subsequently docks, and then anneals into the native conformation after an initial heterogeneous global collapse--emerges. The progressive acquisition of structure, beginning with a continuously connected subdomain and spreading to distal regions, shows that chain entropy is a significant organizing principle in the folding of multisubdomain proteins and single-domain proteins. Subdomain folding also provides a rationale for the complex kinetics often observed.

  5. Active sites of thioredoxin reductases: why selenoproteins?

    PubMed

    Gromer, Stephan; Johansson, Linda; Bauer, Holger; Arscott, L David; Rauch, Susanne; Ballou, David P; Williams, Charles H; Schirmer, R Heiner; Arnér, Elias S J

    2003-10-28

    Selenium, an essential trace element for mammals, is incorporated into a selected class of selenoproteins as selenocysteine. All known isoenzymes of mammalian thioredoxin (Trx) reductases (TrxRs) employ selenium in the C-terminal redox center -Gly-Cys-Sec-Gly-COOH for reduction of Trx and other substrates, whereas the corresponding sequence in Drosophila melanogaster TrxR is -Ser-Cys-Cys-Ser-COOH. Surprisingly, the catalytic competence of these orthologous enzymes is similar, whereas direct Sec-to-Cys substitution of mammalian TrxR, or other selenoenzymes, yields almost inactive enzyme. TrxRs are therefore ideal for studying the biology of selenocysteine by comparative enzymology. Here we show that the serine residues flanking the C-terminal Cys residues of Drosophila TrxRs are responsible for activating the cysteines to match the catalytic efficiency of a selenocysteine-cysteine pair as in mammalian TrxR, obviating the need for selenium. This finding suggests that the occurrence of selenoenzymes, which implies that the organism is selenium-dependent, is not necessarily associated with improved enzyme efficiency. Our data suggest that the selective advantage of selenoenzymes is a broader range of substrates and a broader range of microenvironmental conditions in which enzyme activity is possible.

  6. Alterations in estrogen signalling pathways upon acquisition of anthracycline resistance in breast tumor cells

    PubMed Central

    Chewchuk, Simon; Guo, Baoqing; Parissenti, Amadeo Mark

    2017-01-01

    Intrinsic or acquired drug resistance is a major impediment to the successful treatment of women with breast cancer using chemotherapy. We have observed that MCF-7 breast tumor cells selected for resistance to doxorubicin or epirubicin (MCF-7DOX2 and MCF-7EPI cells, respectively) exhibited increased expression of several members of the aldo-keto reductase (AKR) gene family (in particular AKR1C3 and AKR1B10) relative to control MCF-7CC cells selected by propagation in the absence of drug. Normal cellular roles for the AKRs include the promotion of estrogen (E2) synthesis from estrone (E1) and the hydroxylation and detoxification of exogenous xenobiotics such as anthracycline chemotherapy drugs. While hydroxylation of anthracyclines strongly attenuates their cytotoxicity, it is unclear whether the enhanced AKR expression in the above anthracycline-resistant cells promotes E2 synthesis and/or alterations in E2 signalling pathways and whether such changes contribute to enhanced survival and anthracycline resistance. To determine the role of AKRs and E2 pathways in doxorubicin resistance, we examined changes in the expression of E2-related genes and proteins upon acquisition of doxorubicin resistance. We also assessed the effects of AKR overexpression or downregulation or the effects of activators or inhibitors of E2-dependent pathways on previously acquired resistance to doxorubicin. In this study we observed that the enhanced AKR expression upon acquisition of anthracycline resistance was, in fact, associated with enhanced E2 production. However, the expression of estrogen receptor α (ERα) was reduced by 2- to 5-fold at the gene transcript level and 2- to 20-fold at the protein level upon acquisition of anthracycline resistance. This was accompanied by an even stronger reduction in ERα phosphorylation and activity, including highly suppressed expression of two proteins under E2-dependent control (Bcl-2 and cyclin D1). The diminished Bcl-2 and cyclin D1 expression

  7. Sulfite reductase protects plants against sulfite toxicity.

    PubMed

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

    2013-02-01

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

  8. Uterine glutathione reductase activity: modulation by estrogens and progesterone.

    PubMed

    Díaz-Flores, M; Baiza-Gutman, L A; Pedrón, N N; Hicks, J J

    1999-10-29

    The aim of this study was to determine whether glutathione reductase activity in uterine tissue is regulated by sex hormones. In spayed rats uterine glutathione reductase was significantly increased by exogenous estrogen (P< 0.01), progesterone (P< 0.01) or estrogen plus progesterone (P<0.01). When enzyme activity is expressed per mg protein, daily administration of estrogen or progesterone induces a progressive increase of this enzyme between 24 to 48 h or 24 to 72 h of treatment, respectively. Whereas the combination of both steroids causes an earlier and higher increase in glutathione reductase activity at 24 h of treatment. Estradiol singly or in combination with progesterone induced the highest protein concentration in the uterus. Whereas uterine DNA concentration is only significantly affected by estradiol. Our results suggest that uterine glutathione reductase is regulated by estradiol and progesterone and may be involved in maintaining levels of reduced glutathione in the uterus. This compound may be required for control of the redox state of thiol groups and in detoxification reactions involving H2O2 and electrophylic substances. The antioxidant action of estrogens is partially due to the stimulation of glutathione reductase.

  9. HMG-CoA reductase guides migrating primordial germ cells.

    PubMed

    Van Doren, M; Broihier, H T; Moore, L A; Lehmann, R

    1998-12-03

    The enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase is best known for catalysing a rate-limiting step in cholesterol biosynthesis, but it also participates in the production of a wide variety of other compounds. Some clinical benefits attributed to inhibitors of HMG-CoA reductase are now thought to be independent of any serum cholesterol-lowering effect. Here we describe a new cholesterol-independent role for HMG-CoA reductase, in regulating a developmental process: primordial germ cell migration. We show that in Drosophila this enzyme is highly expressed in the somatic gonad and that it is necessary for primordial germ cells to migrate to this tissue. Misexpression of HMG-CoA reductase is sufficient to attract primordial germ cells to tissues other than the gonadal mesoderm. We conclude that the regulated expression of HMG-CoA reductase has a critical developmental function in providing spatial information to guide migrating primordial germ cells.

  10. Bacterial morphinone reductase is related to Old Yellow Enzyme.

    PubMed Central

    French, C E; Bruce, N C

    1995-01-01

    Morphinone reductase, produced by Pseudomonas putida M10, catalyses the NADH-dependent saturation of the carbon-carbon double bond of morphinone and codeinone, and is believed to be involved in the metabolism of morphine and codeine. The structural gene encoding morphinone reductase, designated morB, was cloned from Ps. putida M10 genomic DNA by the use of degenerate oligonucleotide probes based on elements of the amino acid sequence of the purified enzyme. Sequence analysis and structural characteristics indicated that morphinone reductase is related to the flavoprotein alpha/beta-barrel oxidoreductases, and is particularly similar to Old Yellow Enzyme of Saccharomyces spp. and the related oestrogen-binding protein of Candida albicans. Expressed sequence tags from several plant species show high homology to these enzymes, suggesting the presence of a family of enzymes conserved in plants and fungi. Although related bacterial proteins are known, morphinone reductase appears to be more similar to the eukaryotic proteins. Morphinone reductase was overexpressed in Escherichia coli, and has potential applications for the industrial preparation of semisynthetic opiates. Images Figure 1 Figure 5 PMID:8554504

  11. Purification and properties of proline reductase from Clostridium sticklandii.

    PubMed

    Seto, B; Stadtman, T C

    1976-04-25

    Proline reductase of Clostridium sticklandii is a membrane-bound protein and is released by treatment with detergents. The enzyme has been purified to homogeneity and is estimated by gel filtration and sedimentation equilibrium centrifugation to have a molecular weight of 298,000 to 327,000. A minimum molecular weight of 30,000 to 31,000 was calculated on the basis of sodium dodecyl sulfate-acrylamide gel electrophoresis and amino acid composition. Amino acid analysis showed a preponderance of acidic amino acids. No tryptophan was detected in the protein either spectrophotometrically or by amino acid analysis. A total of 20 sulfhydryl groups measured by titration of the reduced protein with 5,5'-dithiobis(2-nitrobenzoic acid) is in agreement with 20 cystic acid residues determined in hydrolysates of performic acid-oxidized protein. No molybdenum, iron, or selenium was found in the pure protein. Although NADH is the physiological electron donor for the proline reductase complex, the purified 300,000 molecular weight reductase component is inactive in the presence of NADH in vitro. Dithiothreitol, in contrast, can serve as electron donor both for unpurified (putative proline reductase complex) and purified proline reductase in vitro.

  12. Potential use of aldose reductase inhibitors to prevent diabetic complications.

    PubMed

    Zenon, G J; Abobo, C V; Carter, B L; Ball, D W

    1990-06-01

    Reviewed are (1) the biochemical basis and pathophysiology of diabetic complications and (2) the structure-activity relationships, pharmacology, pharmacokinetics, clinical trials, and adverse effects of aldose reductase inhibitors (ARIs). ARIs are a new class of drugs potentially useful in preventing diabetic complications, the most widely studied of which have been cataracts and neuropathy. ARIs inhibit aldose reductase, the first, rate-limiting enzyme in the polyol metabolic pathway. In nonphysiological hyperglycemia the activity of hexokinase becomes saturated while that of aldose reductase is enhanced, resulting in intracellular accumulation of sorbitol. Because sorbitol does not readily penetrate the cell membrane it can persist within cells, which may lead to diabetic complications. ARIs are a class of structurally dissimilar compounds that include carboxylic acid derivatives, flavonoids, and spirohydantoins. The major pharmacologic action of an ARI involves competitive binding to aldose reductase and consequent blocking of sorbitol production. ARIs delay cataract formation in animals, but the role of aldose reductase in cataract formation in human diabetics has not been established. The adverse effects of ARIs include hypersensitivity reactions. Although the polyol pathway may not be solely responsible for diabetic complications, studies suggest that therapy with ARIs could be beneficial. Further research is needed to determine the long-term impact and adverse effects of ARIs in the treatment of diabetic complications.

  13. Sulfur Isotope Effects of Dissimilatory Sulfite Reductase

    PubMed Central

    Leavitt, William D.; Bradley, Alexander S.; Santos, André A.; Pereira, Inês A. C.; Johnston, David T.

    2015-01-01

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

  14. Sulfur Isotope Effects of Dissimilatory Sulfite Reductase.

    PubMed

    Leavitt, William D; Bradley, Alexander S; Santos, André A; Pereira, Inês A C; Johnston, David T

    2015-01-01

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

  15. Selenium in thioredoxin reductase: a mechanistic perspective.

    PubMed

    Lacey, Brian M; Eckenroth, Brian E; Flemer, Stevenson; Hondal, Robert J

    2008-12-02

    Most high M(r) thioredoxin reductases (TRs) have the unusual feature of utilizing a vicinal disulfide bond (Cys(1)-Cys(2)) which forms an eight-membered ring during the catalytic cycle. Many eukaryotic TRs have replaced the Cys(2) position of the dyad with the rare amino acid selenocysteine (Sec). Here we demonstrate that Cys- and Sec-containing TRs are distinguished by the importance each class of enzymes places on the eight-membered ring structure in the catalytic cycle. This hypothesis was explored by studying the truncated enzyme missing the C-terminal ring structure in conjunction with oxidized peptide substrates to investigate the reduction and opening of this dyad. The peptide substrates were identical in sequence to the missing part of the enzyme, containing either a disulfide or selenylsulfide linkage, but were differentiated by the presence (cyclic) and absence (acyclic) of the ring structure. The ratio of these turnover rates informs that the ring is only of modest importance for the truncated mouse mitochondrial Sec-TR (ring/no ring = 32), while the ring structure is highly important for the truncated Cys-TRs from Drosophila melanogaster and Caenorhabditis elegans (ring/no ring > 1000). All three enzymes exhibit a similar dependence upon leaving group pK(a) as shown by the use of the acyclic peptides as substrates. These two factors can be reconciled for Cys-TRs if the ring functions to simultaneously allow for attack by a nearby thiolate while correctly positioning the leaving group sulfur atom to accept a proton from the enzymic general acid. For Sec-TRs the ring is unimportant because the lower pK(a) of the selenol relative to a thiol obviates its need to be protonated upon S-Se bond scission and permits physical separation of the selenol and the general acid. Further study of the biochemical properties of the truncated Cys and Sec TR enzymes demonstrates that the chemical advantage conferred on the eukaryotic enzyme by a selenol is the ability to

  16. A detoxifying oxygen reductase in the anaerobic protozoan Entamoeba histolytica.

    PubMed

    Vicente, João B; Tran, Vy; Pinto, Liliana; Teixeira, Miguel; Singh, Upinder

    2012-09-01

    We report the characterization of a bacterial-type oxygen reductase abundant in the cytoplasm of the anaerobic protozoan parasite Entamoeba histolytica. Upon host infection, E. histolytica is confronted with various oxygen tensions in the host intestine, as well as increased reactive oxygen and nitrogen species at the site of local tissue inflammation. Resistance to oxygen-derived stress thus plays an important role in the pathogenic potential of E. histolytica. The genome of E. histolytica has four genes that encode flavodiiron proteins, which are bacterial-type oxygen or nitric oxide reductases and were likely acquired by lateral gene transfer from prokaryotes. The EhFdp1 gene has higher expression in virulent than in nonvirulent Entamoeba strains and species, hinting that the response to oxidative stress may be one correlate of virulence potential. We demonstrate that EhFdp1 is abundantly expressed in the cytoplasm of E. histolytica and that the protein levels are markedly increased (up to ~5-fold) upon oxygen exposure. Additionally, we produced fully functional recombinant EhFdp1 and demonstrated that this enzyme is a specific and robust oxygen reductase but has poor nitric oxide reductase activity. This observation represents a new mechanism of oxygen resistance in the anaerobic protozoan pathogen E. histolytica.

  17. 21 CFR 864.7375 - Glutathione reductase assay.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 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...

  18. Thioredoxin and NADP-thioredoxin reductase from cultured carrot cells

    NASA Technical Reports Server (NTRS)

    Johnson, T. C.; Cao, R. Q.; Kung, J. E.; Buchanan, B. B.

    1987-01-01

    Dark-grown carrot (Daucus carota L.) tissue cultures were found to contain both protein components of the NADP/thioredoxin system--NADP-thioredoxin reductase and the thioredoxin characteristic of heterotrophic systems, thioredoxin h. Thioredoxin h was purified to apparent homogeneity and, like typical bacterial counterparts, was a 12-kdalton (kDa) acidic protein capable of activating chloroplast NADP-malate dehydrogenase (EC 1.1.1.82) more effectively than fructose-1,6-bisphosphatase (EC 3.1.3.11). NADP-thioredoxin reductase (EC 1.6.4.5) was partially purified and found to be an arsenite-sensitive enzyme composed of two 34-kDa subunits. Carrot NADP-thioredoxin reductase resembled more closely its counterpart from bacteria rather than animal cells in acceptor (thioredoxin) specificity. Upon greening of the cells, the content of NADP-thioredoxin-reductase activity, and, to a lesser extent, thioredoxin h decreased. The results confirm the presence of a heterotrophic-type thioredoxin system in plant cells and raise the question of its physiological function.

  19. Obtaining partial purified xylose reductase from Candida guilliermondii

    PubMed Central

    Tomotani, Ester Junko; de Arruda, Priscila Vaz; Vitolo, Michele; de Almeida Felipe, Maria das Graças

    2009-01-01

    The enzymatic bioconversion of xylose into xylitol by xylose reductase (XR) is an alternative for chemical and microbiological processes. The partial purified XR was obtained by using the following three procedures: an agarose column, a membrane reactor or an Amicon Ultra-15 50K Centrifugal Filter device at yields of 40%, 7% and 67%, respectively. PMID:24031408

  20. Dissimilatory Nitrite Reductase Genes from Autotrophic Ammonia-Oxidizing Bacteria

    PubMed Central

    Casciotti, Karen L.; Ward, Bess B.

    2001-01-01

    The presence of a copper-containing dissimilatory nitrite reductase gene (nirK) was discovered in several isolates of β-subdivision ammonia-oxidizing bacteria using PCR and DNA sequencing. PCR primers Cunir3 and Cunir4 were designed based on published nirK sequences from denitrifying bacteria and used to amplify a 540-bp fragment of the nirK gene from Nitrosomonas marina and five additional isolates of ammonia-oxidizing bacteria. Amplification products of the expected size were cloned and sequenced. Alignment of the nucleic acid and deduced amino acid (AA) sequences shows significant similarity (62 to 75% DNA, 58 to 76% AA) between nitrite reductases present in these nitrifiers and the copper-containing nitrite reductase found in classic heterotrophic denitrifiers. While the presence of a nitrite reductase in Nitrosomonas europaea is known from early biochemical work, preliminary sequence data from its genome indicate a rather low similarity to the denitrifier nirKs. Phylogenetic analysis of the partial nitrifier nirK sequences indicates that the topology of the nirK tree corresponds to the 16S rRNA and amoA trees. While the role of nitrite reduction in the metabolism of nitrifying bacteria is still uncertain, these data show that the nirK gene is present in closely related nitrifying isolates from many oceanographic regions and suggest that nirK sequences retrieved from the environment may include sequences from ammonia-oxidizing bacteria. PMID:11319103

  1. 21 CFR 864.7375 - Glutathione reductase assay.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 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...

  2. 21 CFR 864.7375 - Glutathione reductase assay.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 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...

  3. 21 CFR 864.7375 - Glutathione reductase assay.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 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...

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

    NASA Astrophysics Data System (ADS)

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

    1998-05-01

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

  5. The Kinetics and Inhibition of the Enzyme Methemoglobin Reductase

    ERIC Educational Resources Information Center

    Splittgerber, A. G.; And Others

    1975-01-01

    Describes an undergraduate biochemistry experiment which involves the preparation and kinetics of an oxidation-reduction enzyme system, methemoglobin reductase. A crude enzyme extract is prepared and assayed spectrophotometrically. The enzyme system obeys Michaelis-Menton kinetics with respect to both substrate and the NADH cofactor. (MLH)

  6. [Malate oxidation by mitochondrial succinate:ubiquinone-reductase].

    PubMed

    Belikova, Iu O; Kotliar, A B

    1988-04-01

    Succinate:ubiquinone reductase was shown to catalyze the oxidation of L- and D-stereoisomers of malate by artificial electron acceptors and ubiquinone. The rate of malate oxidation by succinate:ubiquinone reductase is by two orders of magnitude lower than that for the natural substrate--succinate. The values of kinetic constants for the oxidation of D- and L-stereoisomers of malate are equal to: V infinity = 0.1 mumol/min/mg protein, Km = 2 mM and V infinity = 0.05 mumol/min/mg protein, Km = 2 mM, respectively. The malate dehydrogenase activity is fully inhibited by the inhibitors of the dicarboxylate-binding site of the enzyme, i.e., N-ethylmaleimide and malonate and is practically insensitive to carboxin, a specific inhibitor of the ubiquinone-binding center. The enol form of oxaloacetate was shown to be the product of malate oxidation by succinate:ubiquinone reductase. The kinetics of inhibition of the enzyme activity by the ketone and enol forms of oxaloacetate was studied. Both forms of oxaloacetate effectively inhibit the succinate:ubiquinone reductase reaction.

  7. The arsenic hyperaccumulating Pteris vittata expresses two arsenate reductases

    PubMed Central

    Cesaro, Patrizia; Cattaneo, Chiara; Bona, Elisa; Berta, Graziella; Cavaletto, Maria

    2015-01-01

    Enzymatic reduction of arsenate to arsenite is the first known step in arsenate metabolism in all organisms. Although the presence of one mRNA arsenate reductase (PvACR2) has been characterized in gametophytes of P. vittata, no arsenate reductase protein has been directly observed in this arsenic hyperaccumulating fern, yet. In order to assess the possible presence of arsenate reductase in P. vittata, two recombinant proteins, ACR2-His6 and Trx-His6-S-Pv2.5–8 were prepared in Escherichia coli, purified and used to produce polyclonal antibodies. The presence of these two enzymes was evaluated by qRT-PCR, immunoblotting and direct MS analysis. Enzymatic activity was detected in crude extracts. For the first time we detected and identified two arsenate reductase proteins (PvACR2 and Pv2.5–8) in sporophytes and gametophytes of P. vittata. Despite an increase of the mRNA levels for both proteins in roots, no difference was observed at the protein level after arsenic treatment. Overall, our data demonstrate the constitutive protein expression of PvACR2 and Pv2.5–8 in P. vittata tissues and propose their specific role in the complex metabolic network of arsenic reduction. PMID:26412036

  8. The arsenic hyperaccumulating Pteris vittata expresses two arsenate reductases.

    PubMed

    Cesaro, Patrizia; Cattaneo, Chiara; Bona, Elisa; Berta, Graziella; Cavaletto, Maria

    2015-09-28

    Enzymatic reduction of arsenate to arsenite is the first known step in arsenate metabolism in all organisms. Although the presence of one mRNA arsenate reductase (PvACR2) has been characterized in gametophytes of P. vittata, no arsenate reductase protein has been directly observed in this arsenic hyperaccumulating fern, yet. In order to assess the possible presence of arsenate reductase in P. vittata, two recombinant proteins, ACR2-His6 and Trx-His6-S-Pv2.5-8 were prepared in Escherichia coli, purified and used to produce polyclonal antibodies. The presence of these two enzymes was evaluated by qRT-PCR, immunoblotting and direct MS analysis. Enzymatic activity was detected in crude extracts. For the first time we detected and identified two arsenate reductase proteins (PvACR2 and Pv2.5-8) in sporophytes and gametophytes of P. vittata. Despite an increase of the mRNA levels for both proteins in roots, no difference was observed at the protein level after arsenic treatment. Overall, our data demonstrate the constitutive protein expression of PvACR2 and Pv2.5-8 in P. vittata tissues and propose their specific role in the complex metabolic network of arsenic reduction.

  9. [Inhibition of aldose reductase by Chinese herbal medicine].

    PubMed

    Mao, X M; Zhang, J Q

    1993-10-01

    Seven Chinese herbal drugs were screened for experimental inhibition of lens aldose reductase activity, among which quercetin exhibited potent enzyme-inhibitory activities in vitro. Its IC50 value was 3.44 x 10(-7) mol/L. It may be helpful in the prophylaxis and treatment of diabetic complications.

  10. Characterization of mitochondrial thioredoxin reductase from C. elegans

    SciTech Connect

    Lacey, Brian M.; Hondal, Robert J. . E-mail: Robert.Hondal@uvm.edu

    2006-08-04

    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 {sub cat} of 610 min{sup -1} and a K {sub m} of 610 {mu}M using E. coli thioredoxin as substrate. The reported k {sub cat} is 25% of the k {sub 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.

  11. The arsenic hyperaccumulating Pteris vittata expresses two arsenate reductases

    NASA Astrophysics Data System (ADS)

    Cesaro, Patrizia; Cattaneo, Chiara; Bona, Elisa; Berta, Graziella; Cavaletto, Maria

    2015-09-01

    Enzymatic reduction of arsenate to arsenite is the first known step in arsenate metabolism in all organisms. Although the presence of one mRNA arsenate reductase (PvACR2) has been characterized in gametophytes of P. vittata, no arsenate reductase protein has been directly observed in this arsenic hyperaccumulating fern, yet. In order to assess the possible presence of arsenate reductase in P. vittata, two recombinant proteins, ACR2-His6 and Trx-His6-S-Pv2.5-8 were prepared in Escherichia coli, purified and used to produce polyclonal antibodies. The presence of these two enzymes was evaluated by qRT-PCR, immunoblotting and direct MS analysis. Enzymatic activity was detected in crude extracts. For the first time we detected and identified two arsenate reductase proteins (PvACR2 and Pv2.5-8) in sporophytes and gametophytes of P. vittata. Despite an increase of the mRNA levels for both proteins in roots, no difference was observed at the protein level after arsenic treatment. Overall, our data demonstrate the constitutive protein expression of PvACR2 and Pv2.5-8 in P. vittata tissues and propose their specific role in the complex metabolic network of arsenic reduction.

  12. The polymorphisms in methylenetetrahydrofolate reductase, methionine synthase, methionine synthase reductase, and the risk of colorectal cancer.

    PubMed

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

    2012-01-01

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

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

    PubMed Central

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

    2012-01-01

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

  14. Treatment of hirsutism with 5 alpha-reductase inhibitors.

    PubMed

    Brooks, J R

    1986-05-01

    Much os the evidence gathered from studies of 5 alpha-reductase activity levels and androgen metabolism in the skin of hirsute women and the excretion of androgen metabolites by hirsute women indicates that 5 alpha-reduced androgens are probably of primary importance in hirsutism. Unfortunately, until very recently, the lack of a suitable 5 alpha-reductase inhibitor made it very difficult to adequately test the hypothesis that such an inhibitor might be useful in the treatment of hirsutism and certain other androgen-related diseases. No substance was available which had good, unambiguous activity in vivo as a 5 alpha-reductase inhibitor. A number of 4-azasteroids have now been found to possess excellent 5 alpha-reductase inhibitory activity both in vitro and in vivo. Among other properties, several of these compounds show little or no affinity for the androgen receptor of rat prostate cytosol, they attenuate the growth promoting effect of T, but not DHT, on the ventral prostate of castrated male rats, they cause a marked reduction in prostatic DHT concentration in acutely treated rats and dogs and they bring about a significant decline in prostate size in chronically treated rats and dogs. It is expected that, in the near future, one or more of these highly active 5 alpha-reductase inhibitors will be tested in the clinic as a treatment for hirsutism. The results of those studies will be awaited with a great deal of interest since they should considerably advance our understanding of this disease and possibly contribute to its control.

  15. Assimilatory nitrate reductase from the green alga Ankistrodesmus braunii.

    PubMed

    De la Rosa, M A

    1983-01-01

    Assimilatory nitrate reductase (NAD(P)H-nitrate oxidoreductase, EC 1.6.6.2) from the green alga Ankistrodesmus braunii can be purified to homogeneity by dye-ligand chromatography on blue-Sepharose. The purified enzyme, whose turnover number is 623 s-1, presents an optimum pH of 7.5 and Km values of 13 microM, 23 microM and 0.15 mM for NADH, NADPH and nitrate, respectively. The NADH-nitrate reductase activity exhibits an iso ping pong bi bi kinetic mechanism. The molecular weight of the native nitrate reductase is 467 400, while that of its subunits is 58 750. These values suggest an octameric structure for the enzyme, which has been confirmed by electron microscopy. As deduced from spectrophotometric and fluorimetric studies, the enzyme contains FAD and cytochrome b-557 as prosthetic groups. FAD is not covalently bound to the protein and is easily dissociated in diluted solutions from the enzyme. Its apparent Km value is 4 nM, indicative of a high affinity of the enzyme for FAD. The results of the quantitative analyses of prosthetic groups indicate that nitrate reductase contains four molecules of flavin, four heme irons, and two atoms of molybdenum. The three components act sequentially transferring electrons from reduced pyridine nucleotides to nitrate, thus forming a short electron transport chain along the protein. A mechanism is proposed for the redox interconversion of the nitrate reductase activity. Inactivation seems to occur by formation of a stable complex of reduced enzyme with cyanide or superoxide, while reactivation is a consequence of reoxidation of the inactive enzyme. Both reactions imply the transfer of only one electron.

  16. Measurement of nitrous oxide reductase activity in aquatic sediments

    SciTech Connect

    Miller, L.G.; Oremland, R.S.; Paulsen, S.

    1986-01-01

    Denitrification in aquatic sediments was measured by an N/sub 2/O reductase assay. Sediments consumed small added quantities of N/sub 2/O over short periods (a few hours). In experiments with sediment slurries, N/sub 2/O reductase activity was inhibited by 0/sub 2/, C/sub 2/H/sub 2/, heat treatment, and by high levels of nitrate (1 mM) or sulfide (10 mM). However, ambient levels of nitrate (<100 ..mu..M) did not influence activity, and moderate levels (about 150 ..mu..M) induced only a short lag before reductase activity began. Moderate levels of sulfide (<1 mM) had no effect on N/sub 2/O reductase activity. Nitrous oxide reductase displayed Michaelis-Menten kinetics in sediments from freshwater, estuarine, and alkaline-saline environments. An in situ assay was devised in which a solution of N/sub 2/O was injected into sealed glass cores containing intact sediment. Two estimates of net rates of denitrification in San Francisco Bay under approximated in situ conditions were 0.009 and 0.041 mmol of N/sub 2/O per m/sup 2/ per h. Addition of chlorate to inhibit denitrification in these intact-core experiments (to estimate gross rates of N/sub 2/O consumption) resulted in approximately a 14% upward revision of estimates of net rates. These results were comparable to an in situ estimate of 0.022 mmol of N/sub 2/O per m/sup 2/ per h made with the acetylene block assay.

  17. Measurement of nitrous oxide reductase activity in aquatic sediments

    USGS Publications Warehouse

    Miller, L.G.; Oremland, R.S.; Paulsen, S.

    1986-01-01

    Denitrification in aquatic sediments was measured by an N2O reductase assay. Sediments consumed small added quantities of N2O over short periods (a few hours). In experiments with sediment slurries, N2O reductase activity was inhibited by O2, C2H2, heat treatment, and by high levels of nitrate (1 mM) or sulfide (10 mM). However, ambient levels of nitrate (<100 μM) did not influence activity, and moderate levels (about 150 μM) induced only a short lag before reductase activity began. Moderate levels of sulfide (<1 mM) had no effect on N2O reductase activity. Nitrous oxide reductase displayed Michaelis-Menten kinetics in sediments from freshwater (Km = 2.17 μM), estuarine (Km = 14.5 μM), and alkaline-saline (Km = 501 μM) environments. An in situ assay was devised in which a solution of N2O was injected into sealed glass cores containing intact sediment. Two estimates of net rates of denitrification in San Francisco Bay under approximated in situ conditions were 0.009 and 0.041 mmol of N2O per m2 per h. Addition of chlorate to inhibit denitrification in these intact-core experiments (to estimate gross rates of N2O consumption) resulted in approximately a 14% upward revision of estimates of net rates. These results were comparable to an in situ estimate of 0.022 mmol of N2O per m2 per h made with the acetylene block assay.

  18. Identification and characterization of 2-naphthoyl-coenzyme A reductase, the prototype of a novel class of dearomatizing reductases.

    PubMed

    Eberlein, Christian; Estelmann, Sebastian; Seifert, Jana; von Bergen, Martin; Müller, Michael; Meckenstock, Rainer U; Boll, Matthias

    2013-06-01

    The enzymatic dearomatization of aromatic ring systems by reduction represents a highly challenging redox reaction in biology and plays a key role in the degradation of aromatic compounds under anoxic conditions. In anaerobic bacteria, most monocyclic aromatic growth substrates are converted to benzoyl-coenzyme A (CoA), which is then dearomatized to a conjugated dienoyl-CoA by ATP-dependent or -independent benzoyl-CoA reductases. It was unresolved whether or not related enzymes are involved in the anaerobic degradation of environmentally relevant polycyclic aromatic hydrocarbons (PAHs). In this work, a previously unknown dearomatizing 2-naphthoyl-CoA reductase was purified from extracts of the naphthalene-degrading, sulphidogenic enrichment culture N47. The oxygen-tolerant enzyme dearomatized the non-activated ring of 2-naphthoyl-CoA by a four-electron reduction to 5,6,7,8-tetrahydro-2-naphthoyl-CoA. The dimeric 150 kDa enzyme complex was composed of a 72 kDa subunit showing sequence similarity to members of the flavin-containing 'old yellow enzyme' family. NCR contained FAD, FMN, and an iron-sulphur cluster as cofactors. Extracts of Escherichia coli expressing the encoding gene catalysed 2-naphthoyl-CoA reduction. The identified NCR is a prototypical enzyme of a previously unknown class of dearomatizing arylcarboxyl-CoA reductases that are involved in anaerobic PAH degradation; it fundamentally differs from known benzoyl-CoA reductases.

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

    NASA Technical Reports Server (NTRS)

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

    2003-01-01

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

  20. Immunological approach to the regulation of nitrate reductase in Monoraphidium braunii.

    PubMed

    Díez, J; López-Ruiz, A

    1989-02-01

    The effects of different culture conditions on nitrate reductase activity and nitrate reductase protein from Monoraphidium braunii have been studied, using two different immunological techniques, rocket immunoelectrophoresis and an enzyme-linked immunosorbent assay, to determine nitrate reductase protein. The nitrogen sources ammonium and glutamine repressed nitrate reductase synthesis, while nitrite, alanine, and glutamate acted as derepressors. There was a four- to eightfold increase of nitrate reductase activity and a twofold increase of nitrate reductase protein under conditions of nitrogen starvation versus growth on nitrate. Nitrate reductase synthesis was repressed in darkness. However, when Monoraphidium was grown under heterotrophic conditions with glucose as the carbon and energy source, the synthesis of nitrate reductase was maintained. With ammonium or darkness, changes in nitrate reductase activity correlated fairly well with changes in nitrate reductase protein, indicating that in both cases loss of activity was due to repression and not to inactivation of the enzyme. Experiments using methionine sulfoximine, to inhibit ammonium assimilation, showed that ammonium per se and not a product of its metabolism was the corepressor of the enzyme. The appearance of nitrate reductase activity after transferring the cells to induction media was prevented by cycloheximide and by 6-methylpurine, although in this latter case the effect was observed only in cells preincubated with the inhibitor for 1 h before the induction period.

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

    DOEpatents

    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.

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

    DOEpatents

    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.

  3. New nonsteroidal steroid 5 alpha-reductase inhibitors. Syntheses and structure-activity studies on carboxamide phenylalkyl-substituted pyridones and piperidones.

    PubMed

    Hartmann, R W; Reichert, M

    2000-05-01

    In the search for nonsteroidal inhibitors of 5 alpha-reductase for the treatment of benign prostatic hyperplasia (BPH), we synthesized diisopropyl (1a-8a) and tert-butyl (1b-8b) benzamides, as well as ethyl benzoates (1c, 3c), which were substituted in 4 position via variable alkyl spacer (n = 0: 1-4, n = 1: 5, 7 and n = 3: 6, 8) with a 1-methyl-2-pyridone (1, 2, 5, 6) or a 1-methyl-2-piperidone (3, 4, 7, 8) moiety mimicking steroidal ring A. The directly connected benzamides (1a-4a, 1b-4b) and benzoates (1c, 3c) were obtained by palladium-catalysed coupling reaction of diethyl(3-pyridyl)-borane with 4-bromobenzoic acid derivatives, followed by alpha-oxidation of the 1-methyl-pyridinium salt and subsequent separation of the regioisomers. Catalytic hydrogenation of the pyridones (1, 2) led to the piperidones (3, 4). The preparation of the benzamides with a methylene (5, 7) and a propylene spacer (6, 8), respectively, started with the reduction of the keto group of 5-benzoyl-1,2-dihydro-1-methyl-2(1H)-pyridone and catalytic hydrogenation of the alkene obtained by Wittig reaction of 5-formyl-1,2-dihydro-1-methyl-2(1H)-pyridone with (2-phenylethyl)triphenylphosphonium bromide, respectively. The phenyl ring was functionalized by Friedel-Crafts reaction, haloform cleavage to give the acid, formation of the acid chloride, and subsequent treatment with the appropriate amines. Again, catalytic hydrogenation of the pyridones (5, 6) led to the piperidones (7, 8). The 5 alpha-reductase inhibitory properties were determined using rat ventral prostate, as well as human BPH tissue as enzyme source, 1 beta-2 beta-[3H]testosterone as substrate and a HPLC procedure for the separation of dihydrotestosterone (DHT). Tested at a concentration of 100 microM, the inhibition values of 1-8 ranged from 0-79%. Significant differences were observed between rat and human enzyme. The most active compound was ethyl 4-(1-methyl-2-oxopiperid-5-yl)benzoate 3c (68%) for the human enzyme and N,N-bis(1

  4. The X-ray crystal structure of APR-B, an atypical adenosine 5'-phosphosulfate reductase from Physcomitrella patens.

    PubMed

    Stevenson, Clare E M; Hughes, Richard K; McManus, Michael T; Lawson, David M; Kopriva, Stanislav

    2013-11-15

    Sulfonucleotide reductases catalyse the first reductive step of sulfate assimilation. Their substrate specificities generally correlate with the requirement for a [Fe4S4] cluster, where adenosine 5'-phosphosulfate (APS) reductases possess a cluster and 3'-phosphoadenosine 5'-phosphosulfate reductases do not. The exception is the APR-B isoform of APS reductase from the moss Physcomitrella patens, which lacks a cluster. The crystal structure of APR-B, the first for a plant sulfonucleotide reductase, is consistent with a preference for APS. Structural conservation with bacterial APS reductase rules out a structural role for the cluster, but supports the contention that it enhances the activity of conventional APS reductases.

  5. The respiratory arsenate reductase from Bacillus selenitireducens strain MLS10

    USGS Publications Warehouse

    Afkar, E.; Lisak, J.; Saltikov, C.; Basu, P.; Oremland, R.S.; Stolz, J.F.

    2003-01-01

    The respiratory arsenate reductase from the Gram-positive, haloalkaliphile, Bacillus selenitireducens strain MLS10 was purified and characterized. It is a membrane bound heterodimer (150 kDa) composed of two subunits ArrA (110 kDa) and ArrB (34 kDa), with an apparent Km for arsenate of 34 ??M and Vmax of 2.5 ??mol min-1 mg-1. Optimal activity occurred at pH 9.5 and 150 g l-1 of NaCl. Metal analysis (inductively coupled plasma mass spectrometry) of the holoenzyme and sequence analysis of the catalytic subunit (ArrA; the gene for which was cloned and sequenced) indicate it is a member of the DMSO reductase family of molybdoproteins. ?? 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

  6. Pyrroline-5-Carboxylate Reductase in Soybean Nodules 1

    PubMed Central

    Chilson, Oscar P.; Kelly-Chilson, Anne E.; Schneider, Julie D.

    1992-01-01

    Characteristics of pyrroline-5-carboxylate reductase (P5CR) from Bradyrhizobium japonicum bacteroids and cultured rhizobia were compared with those of the enzyme in soybean nodule host cytosol. Reductase from host cytosol differed from that in bacteroids in: (a) the effect of pH on enzymic activity, (b) the capacity to catalyze both reduction of pyrroline-5-carboxylic acid and NAD+-dependent proline oxidation, (c) apparent affinities for pyrroline-5-carboxylic acid, and (d) sensitivities to inhibition by NADP+ and proline. The K1 for proline inhibition of P5CR in bacteroid cytosol was 1.8 millimolar. The properties of P5CR in B. japonicum and bacteroid cytosol were similar. The specific activities of P5CR in the cytosolic fractions of the nodule host and the bacteroid compartment were also comparable. PMID:16668837

  7. Characterization of 12-Oxo-Phytodienoic Acid Reductase in Corn

    PubMed Central

    Vick, Brady A.; Zimmerman, Don C.

    1986-01-01

    12-Oxo-phytodienoic acid reductase, an enzyme of the biosynthetic pathway that converts linolenic acid to jasmonic acid, has been characterized from the kernel and seedlings of corn (Zea mays L.). The molecular weight of the enzyme, estimated by gel filtration, was 54,000. Optimum enzyme activity was observed over a broad pH range, from pH 6.8 to 9.0. The enzyme had a Km of 190 micromolar for its substrate, 12-oxo-phytodienoic acid. The preferred reductant was NADPH, for which the enzyme exhibited a Km of 13 micromolar, compared with 4.2 millimolar for NADH. Reductase activity was low in the corn kernel but increased five-fold by the fifth day after germination and then gradually declined. PMID:16664582

  8. [Properties of a nitrite reductase inhibitor protein from Pseudomonas aeruginosa].

    PubMed

    Karapetian, A V; Nalbandian, R M

    1993-08-01

    The amino acid composition and major physico-chemical properties of the "nonblue" copper protein isolated earlier from Pseudomonas aeruginosa have been determined. It has been found that the azurin oxidase, cytochrome c551 oxidase and superoxide dismutase activities of the enzyme are inhibited by this protein. The inhibition seems to be due to the protein interaction with the electron-accepting center of nitrite reductase.

  9. Aldose and aldehyde reductases : structure-function studies on the coenzyme and inhibitor-binding sites.

    SciTech Connect

    El-Kabbani, O.; Old, S. E.; Ginell, S. L.; Carper, D. A.; Biosciences Division; Monash Univ.; NIH

    1999-09-03

    PURPOSE: To identify the structural features responsible for the differences in coenzyme and inhibitor specificities of aldose and aldehyde reductases. METHODS: The crystal structure of porcine aldehyde reductase in complex with NADPH and the aldose reductase inhibitor sorbinil was determined. The contribution of each amino acid lining the coenzyme-binding site to the binding of NADPH was calculated using the Discover package. In human aldose reductase, the role of the non-conserved Pro 216 (Ser in aldehyde reductase) in the binding of coenzyme was examined by site-directed mutagenesis. RESULTS: Sorbinil binds to the active site of aldehyde reductase and is hydrogen-bonded to Trp 22, Tyr 50, His 113, and the non-conserved Arg 312. Unlike tolrestat, the binding of sorbinil does not induce a change in the side chain conformation of Arg 312. Mutation of Pro 216 to Ser in aldose reductase makes the binding of coenzyme more similar to that of aldehyde reductase. CONCLUSIONS: The participation of non-conserved active site residues in the binding of inhibitors and the differences in the structural changes required for the binding to occur are responsible for the differences in the potency of inhibition of aldose and aldehyde reductases. We report that the non-conserved Pro 216 in aldose reductase contributes to the tight binding of NADPH.

  10. Early diagnosis and management of 5 alpha-reductase deficiency.

    PubMed Central

    Odame, I; Donaldson, M D; Wallace, A M; Cochran, W; Smith, P J

    1992-01-01

    Two siblings of Pakistani origin, karyotype 46 XY, were born with predominantly female external genitalia with minute phallus, bifid scrotum, urogenital sinus, and palpable gonads. The older sibling at the age of 8 days showed an adequate testosterone response to human chorionic gonadotrophin (hCG) stimulation. The diagnosis of 5 alpha-reductase deficiency was made at age 6 years when no 5 alpha-reduced glucocorticoid metabolites were detectable in urine even after tetracosactrin (Synacthen) stimulation. In the younger sibling the diagnosis of 5 alpha-reductase deficiency was provisionally made at the early age of 3 days on the basis of high urinary tetrahydrocortisol (THF)/allotetrahydrocortisol (5 alpha-THF) ratio and this ratio increased with age confirming the diagnosis. Plasma testosterone: dihydrotestosterone (DHT) ratio before and after hCG stimulation was within normal limits at age 3 days but was raised at age 9 months. Topical DHT cream application to the external genitalia promoted significant phallic growth in both siblings and in the older sibling corrective surgery was facilitated. In prepubertal male pseudohermaphrodites with normal or raised testosterone concentrations, phallic growth in response to DHT cream treatment could be an indirect confirmation of 5 alpha-reductase deficiency. Images Figure 1 PMID:1626992

  11. Cloning and Sequence Analysis of Two Pseudomonas Flavoprotein Xenobiotic Reductases

    PubMed Central

    Blehert, David S.; Fox, Brian G.; Chambliss, Glenn H.

    1999-01-01

    The genes encoding flavin mononucleotide-containing oxidoreductases, designated xenobiotic reductases, from Pseudomonas putida II-B and P. fluorescens I-C that removed nitrite from nitroglycerin (NG) by cleavage of the nitroester bond were cloned, sequenced, and characterized. The P. putida gene, xenA, encodes a 39,702-Da monomeric, NAD(P)H-dependent flavoprotein that removes either the terminal or central nitro groups from NG and that reduces 2-cyclohexen-1-one but did not readily reduce 2,4,6-trinitrotoluene (TNT). The P. fluorescens gene, xenB, encodes a 37,441-Da monomeric, NAD(P)H-dependent flavoprotein that exhibits fivefold regioselectivity for removal of the central nitro group from NG and that transforms TNT but did not readily react with 2-cyclohexen-1-one. Heterologous expression of xenA and xenB was demonstrated in Escherichia coli DH5α. The transcription initiation sites of both xenA and xenB were identified by primer extension analysis. BLAST analyses conducted with the P. putida xenA and the P. fluorescens xenB sequences demonstrated that these genes are similar to several other bacterial genes that encode broad-specificity flavoprotein reductases. The prokaryotic flavoprotein reductases described herein likely shared a common ancestor with old yellow enzyme of yeast, a broad-specificity enzyme which may serve a detoxification role in antioxidant defense systems. PMID:10515912

  12. The existence and significance of a mitochondrial nitrite reductase.

    PubMed

    Nohl, Hans; Staniek, Katrin; Kozlov, Andrey V

    2005-01-01

    The physiological functions of nitric oxide (NO) are well established. The finding that the endothelium-derived relaxing factor (EDRF) is NO was totally unexpected. It was shown that NO is a reaction product of an enzymatically catalyzed, overall, 5-electron oxidation of guanidinium nitrogen from L-arginine followed by the release of the free radical species NO. NO is synthesized by a single protein complex supported by cofactors, coenzymes (such as tetrahydrobiopterin) and cytochrome P450. The latter can uncouple from substrate oxidation producing O2*- radicals. The research groups of Richter [Ghafourifar P, Richter C. Nitric oxide synthase activity in mitochondria. FEBS Lett 1997; 418: 291-296.] and Boveris [Giulivi C, Poderoso JJ, Boveris A. Production of nitric oxide by mitochondria. J Biol Chem 1998; 273: 11038-11043.] identified a mitochondrial NO synthase (NOS). There are, however, increasing reports demonstrating that mitochondrial NO is derived from cytosolic NOS belonging to the Ca2+-dependent enzymes. NO was thought to control cytochrome oxidase. This assumption is controversial due to the life-time of NO in biological systems (millisecond range). We found a nitrite reductase in mitochondria which is of major interest. Any increase of nitrite in the tissue which is the first oxidation product of NO, for instance following NO donors, will stimulate NO-recycling via mitochondrial nitrite reductase. In this paper, we describe the identity and the function of mitochondrial nitrite reductase and the consequences of NO-recycling in the metabolic compartment of mitochondria.

  13. Phosphoglycerate kinase acts in tumour angiogenesis as a disulphide reductase

    NASA Astrophysics Data System (ADS)

    Lay, Angelina J.; Jiang, Xing-Mai; Kisker, Oliver; Flynn, Evelyn; Underwood, Anne; Condron, Rosemary; Hogg, Philip J.

    2000-12-01

    Disulphide bonds in secreted proteins are considered to be inert because of the oxidizing nature of the extracellular milieu. An exception to this rule is a reductase secreted by tumour cells that reduces disulphide bonds in the serine proteinase plasmin. Reduction of plasmin initiates proteolytic cleavage in the kringle 5 domain and release of the tumour blood vessel inhibitor angiostatin. New blood vessel formation or angiogenesis is critical for tumour expansion and metastasis. Here we show that the plasmin reductase isolated from conditioned medium of fibrosarcoma cells is the glycolytic enzyme phosphoglycerate kinase. Recombinant phosphoglycerate kinase had the same specific activity as the fibrosarcoma-derived protein. Plasma of mice bearing fibrosarcoma tumours contained several-fold more phosphoglycerate kinase, as compared with mice without tumours. Administration of phosphoglycerate kinase to tumour-bearing mice caused an increase in plasma levels of angiostatin, and a decrease in tumour vascularity and rate of tumour growth. Our findings indicate that phosphoglycerate kinase not only functions in glycolysis but is secreted by tumour cells and participates in the angiogenic process as a disulphide reductase.

  14. The effect of quercetin and galangin on glutathione reductase.

    PubMed

    Paulíková, Helena; Berczeliová, Elena

    2005-12-01

    Quercetin and galangin can change the activity of glutathione reductase. Quercetin (a catechol structure in the B-ring) and galangin (any hydroxyl group in the B-ring) have different biological activities but, both possess high antioxidant abilities. Quercetin during the antioxidative action, is converted into an oxidized products (o-semiquinone and o-quinone), and subsequently glutathionyl adducts may be formed or SH-enzyme can be inhibited. We have tried to see whether inhibition of glutathione reductase (GR) can be influenced by preincubation of enzyme with NADPH (a creation of reduced form of enzyme, GRH(2)) and whether diaphorase activity of the enzyme is decreased by these flavonoids. The results confirmed that quercetin inhibits GRH(2) and inhibition is reduced by addition of EDTA or N-acetylcysteine. Both of flavonoids have no effect on diaphorase activity of glutathione reductase and this enzyme could increase the production of free radicals by catalysis of reduction of o-quinone during action of quercetin in vivo.

  15. Methionine Sulfoxide Reductases Are Essential for Virulence of Salmonella Typhimurium

    PubMed Central

    Rouf, Syed Fazle; Kitowski, Vera; Böhm, Oliver M.; Rhen, Mikael; Jäger, Timo; Bange, Franz-Christoph

    2011-01-01

    Production of reactive oxygen species represents a fundamental innate defense against microbes in a diversity of host organisms. Oxidative stress, amongst others, converts peptidyl and free methionine to a mixture of methionine-S- (Met-S-SO) and methionine-R-sulfoxides (Met-R-SO). To cope with such oxidative damage, methionine sulfoxide reductases MsrA and MsrB are known to reduce MetSOs, the former being specific for the S-form and the latter being specific for the R-form. However, at present the role of methionine sulfoxide reductases in the pathogenesis of intracellular bacterial pathogens has not been fully detailed. Here we show that deletion of msrA in the facultative intracellular pathogen Salmonella (S.) enterica serovar Typhimurium increased susceptibility to exogenous H2O2, and reduced bacterial replication inside activated macrophages, and in mice. In contrast, a ΔmsrB mutant showed the wild type phenotype. Recombinant MsrA was active against free and peptidyl Met-S-SO, whereas recombinant MsrB was only weakly active and specific for peptidyl Met-R-SO. This raised the question of whether an additional Met-R-SO reductase could play a role in the oxidative stress response of S. Typhimurium. MsrC is a methionine sulfoxide reductase previously shown to be specific for free Met-R-SO in Escherichia (E.) coli. We tested a ΔmsrC single mutant and a ΔmsrBΔmsrC double mutant under various stress conditions, and found that MsrC is essential for survival of S. Typhimurium following exposure to H2O2, as well as for growth in macrophages, and in mice. Hence, this study demonstrates that all three methionine sulfoxide reductases, MsrA, MsrB and MsrC, facilitate growth of a canonical intracellular pathogen during infection. Interestingly MsrC is specific for the repair of free methionine sulfoxide, pointing to an important role of this pathway in the oxidative stress response of Salmonella Typhimurium. PMID:22073230

  16. Kinetic characteristics of ZENECA ZD5522, a potent inhibitor of human and bovine lens aldose reductase.

    PubMed

    Cook, P N; Ward, W H; Petrash, J M; Mirrlees, D J; Sennitt, C M; Carey, F; Preston, J; Brittain, D R; Tuffin, D P; Howe, R

    1995-04-18

    Aldose reductase (aldehyde reductase 2) catalyses the conversion of glucose to sorbitol, and methylglyoxal to acetol. Treatment with aldose reductase inhibitors (ARIs) is a potential approach to decrease the development of diabetic complications. The sulphonylnitromethanes are a recently discovered class of aldose reductase inhibitors, first exemplified by ICI215918. We now describe enzyme kinetic characterization of a second sulphonylnitromethane, 3',5'-dimethyl-4'-nitromethylsulphonyl-2-(2-tolyl)acetanilide (ZD5522), which is at least 10-fold more potent against bovine lens aldose reductase in vitro and which also has a greater efficacy for reduction of rat nerve sorbitol levels in vivo (ED95 = 2.8 mg kg-1 for ZD5522 and 20 mg kg-1 for ICI 215918). ZD5522 follows pure noncompetitive kinetics against bovine lens aldose reductase when either glucose or methylglyoxal is varied (K(is) = K(ii) = 7.2 and 4.3 nM, respectively). This contrasts with ICI 215918 which is an uncompetitive inhibitor (K(ii) = 100 nM) of bovine lens aldose reductase when glucose is varied. Against human recombinant aldose reductase, ZD5522 displays mixed noncompetitive kinetics with respect to both substrates (K(is) = 41 nM, K(ii) = 8 nM with glucose and K(is) = 52 nM, K(ii) = 3.8 nM with methylglyoxal). This is the first report of the effects of a sulphonylnitromethane on either human aldose reductase or utilization of methylglyoxal. These results are discussed with reference to a Di Iso Ordered Bi Bi mechanism for aldose reductase, where the inhibitors compete with binding of both the aldehyde substrate and alcohol product. This model may explain why aldose reductase inhibitors follow noncompetitive or uncompetitive kinetics with respect to aldehyde substrates, and X-ray crystallography paradoxically locates an ARI within the substrate binding site. Aldehyde reductase (aldehyde reductase 1) is closely related to aldose reductase. Inhibition of bovine kidney aldehyde reductase by ZD5522

  17. Regulation of Nitrate Reductase Activity in Corn (Zea mays L.) Seedlings by Endogenous Metabolites 1

    PubMed Central

    Schrader, L. E.; Hageman, R. H.

    1967-01-01

    Primary and secondary metabolites of inorganic nitrogen metabolism were evaluated as inhibitors of nitrate reductase (EC 1.6.6.1) induction in green leaf tissue of corn seedlings. Nitrite, nitropropionic acid, ammonium ions, and amino acids were not effective as inhibitors of nitrate reductase activity or synthesis. Increasing α-amino nitrogen and protein content of intact corn seedlings by culture techniques significantly enhanced rather than decreased the potential for induction of nitrate reductase activity in excised seedlings. Secondary metabolites, derived from phenylalanine and tyrosine, were tested as inhibitors of induction of nitrate reductase. Of the 9 different phenylpropanoid compounds tested, only coumarin, trans-cinnamic and trans-o-hydroxycinnamic acids inhibited induction of nitrate reductase. While coumarin alone exhibited a relatively greater inhibitory effect on enzyme induction than on general protein synthesis (the latter measured by incorporation of labeled amino acids), this differential effect may have been dependent upon unequal rates of synthesis and accumulation with respect to the initial levels of nitrate reductase and general proteins. Because of the short half-life of nitrate reductase, inhibitors of protein synthesis in general could still achieve differential regulation of nitrogen metabolism. Coumarin did not inhibit nitrate reductase activity when added directly to the assay mixture at 5 mm. Carbamyl phosphate and its chemical derivative, cyanate, were found to be competitive (with nitrate) inhibitors of nitrate reductase. The data suggest that cyanate is the active inhibitor in the carbamyl phosphate preparations. PMID:16656715

  18. A flavone from Manilkara indica as a specific inhibitor against aldose reductase in vitro.

    PubMed

    Haraguchi, Hiroyuki; Hayashi, Ryosuke; Ishizu, Takashi; Yagi, Akira

    2003-09-01

    Isoaffinetin (5,7,3',4',5'-pentahydroxyflavone-6-C-glucoside) was isolated from Manilkara indica as a potent inhibitor of lens aldose reductase by bioassay-directed fractionation. This C-glucosyl flavone showed specific inhibition against aldose reductases (rat lens, porcine lens and recombinant human) with no inhibition against aldehyde reductase and NADH oxidase. Kinetic analysis showed that isoaffinetin exhibited uncompetitive inhibition against both dl-glyceraldehyde and NADPH. A structure-activity relationship study revealed that the increasing number of hydroxy groups in the B-ring contributes to the increase in aldose reductase inhibition by C-glucosyl flavones.

  19. Ammonification in Bacillus subtilis Utilizing Dissimilatory Nitrite Reductase Is Dependent on resDE

    PubMed Central

    Hoffmann, Tamara; Frankenberg, Nicole; Marino, Marco; Jahn, Dieter

    1998-01-01

    During anaerobic nitrate respiration Bacillus subtilis reduces nitrate via nitrite to ammonia. No denitrification products were observed. B. subtilis wild-type cells and a nitrate reductase mutant grew anaerobically with nitrite as an electron acceptor. Oxygen-sensitive dissimilatory nitrite reductase activity was demonstrated in cell extracts prepared from both strains with benzyl viologen as an electron donor and nitrite as an electron acceptor. The anaerobic expression of the discovered nitrite reductase activity was dependent on the regulatory system encoded by resDE. Mutation of the gene encoding the regulatory Fnr had no negative effect on dissimilatory nitrite reductase formation. PMID:9422613

  20. Structure of the Molybdenum Site of EEcherichia Coli Trimethylamine N-Oxide Reductase

    SciTech Connect

    Zhang, L.; Nelson, K.Johnson; Rajagopalan, K.V.; George, G.N.

    2009-05-28

    We report a structural characterization of the molybdenum site of recombinant Escherichia coli trimethylamine N-oxide (TMAO) reductase using X-ray absorption spectroscopy. The enzyme active site shows considerable similarity to that of dimethyl sulfoxide (DMSO) reductase, in that, like DMSO reductase, the TMAO reductase active site can exist in multiple forms. Examination of the published crystal structure of TMAO oxidase from Shewanella massilia indicates that the postulated Mo coordination structure is chemically impossible. The presence of multiple active site structures provides a potential explanation for the anomalous features reported from the crystal structure.

  1. Components of glycine reductase from Eubacterium acidaminophilum. Cloning, sequencing and identification of the genes for thioredoxin reductase, thioredoxin and selenoprotein PA.

    PubMed

    Lübbers, M; Andreesen, J R

    1993-10-15

    The genes encoding thioredoxin reductase (trxB), thioredoxin (trxA), protein PA of glycine reductase (grdA) and the first 23 amino acids of the large subunit of protein PC of glycine reductase (grdC) belonging to the reductive deamination systems present in Eubacterium acidaminophilum were cloned and sequenced. The proteins were products of closely linked genes with 314 codons (thioredoxin reductase), 110 codons (thioredoxin), and 158 codons (protein PA). The protein previously called 'atypically small lipoamide dehydrogenase' or 'electron transferring flavoprotein' could now conclusively be identified as a thioredoxin reductase (subunit mass of 34781 Da) by the alignment with the enzyme of Escherichia coli showing the same typical order of the corresponding domains. The thioredoxin (molecular mass of 11742 Da) deviated considerably from the known consensus sequence, even in the most strongly conserved redox-active segment WCGPC that was now GCVPC. The selenocysteine of protein PA (molecular mass of 16609 Da) was encoded by TGA. The protein was highly similar to those of Clostridium purinolyticum and Clostridium sticklandii involved in glycine reductase. Thioredoxin reductase and thioredoxin of E. acidaminophilum could be successfully expressed in E. coli.

  2. Mutation of the Inducible ARABIDOPSIS THALIANA CYTOCHROME P450 REDUCTASE2 Alters Lignin Composition and Improves Saccharification1[W][OPEN

    PubMed Central

    Sundin, Lisa; Vanholme, Ruben; Geerinck, Jan; Goeminne, Geert; Höfer, René; Kim, Hoon; Ralph, John; Boerjan, Wout

    2014-01-01

    ARABIDOPSIS THALIANA CYTOCHROME P450 REDUCTASE1 (ATR1) and ATR2 provide electrons from NADPH to a large number of CYTOCHROME P450 (CYP450) enzymes in Arabidopsis (Arabidopsis thaliana). Whereas ATR1 is constitutively expressed, the expression of ATR2 appears to be induced during lignin biosynthesis and upon stresses. Therefore, ATR2 was hypothesized to be preferentially involved in providing electrons to the three CYP450s involved in lignin biosynthesis: CINNAMATE 4-HYDROXYLASE (C4H), p-COUMARATE 3-HYDROXYLASE1 (C3H1), and FERULATE 5-HYDROXYLASE1 (F5H1). Here, we show that the atr2 mutation resulted in a 6% reduction in total lignin amount in the main inflorescence stem and a compositional shift of the remaining lignin to a 10-fold higher fraction of p-hydroxyphenyl units at the expense of syringyl units. Phenolic profiling revealed shifts in lignin-related phenolic metabolites, in particular with the substrates of C4H, C3H1 and F5H1 accumulating in atr2 mutants. Glucosinolate and flavonol glycoside biosynthesis, both of which also rely on CYP450 activities, appeared less affected. The cellulose in the atr2 inflorescence stems was more susceptible to enzymatic hydrolysis after alkaline pretreatment, making ATR2 a potential target for engineering plant cell walls for biofuel production. PMID:25315601

  3. Structural and Biochemical Characterization of Cinnamoyl-CoA Reductases.

    PubMed

    Sattler, Steven A; Walker, Alexander M; Vermerris, Wilfred; Sattler, Scott E; Kang, ChulHee

    2017-02-01

    Cinnamoyl-coenzyme A reductase (CCR) catalyzes the reduction of hydroxycinnamoyl-coenzyme A (CoA) esters using NADPH to produce hydroxycinnamyl aldehyde precursors in lignin synthesis. The catalytic mechanism and substrate specificity of cinnamoyl-CoA reductases from sorghum (Sorghum bicolor), a strategic plant for bioenergy production, were deduced from crystal structures, site-directed mutagenesis, and kinetic and thermodynamic analyses. Although SbCCR1 displayed higher affinity for caffeoyl-CoA or p-coumaroyl-CoA than for feruloyl-CoA, the enzyme showed significantly higher activity for the latter substrate. Through molecular docking and comparisons between the crystal structures of the Vitis vinifera dihydroflavonol reductase and SbCCR1, residues threonine-154 and tyrosine-310 were pinpointed as being involved in binding CoA-conjugated phenylpropanoids. Threonine-154 of SbCCR1 and other CCRs likely confers strong substrate specificity for feruloyl-CoA over other cinnamoyl-CoA thioesters, and the T154Y mutation in SbCCR1 led to broader substrate specificity and faster turnover. Through data mining using our structural and biochemical information, four additional putative CCR genes were discovered from sorghum genomic data. One of these, SbCCR2, displayed greater activity toward p-coumaroyl-CoA than did SbCCR1, which could imply a role in the synthesis of defense-related lignin. Taken together, these findings provide knowledge about critical residues and substrate preference among CCRs and provide, to our knowledge, the first three-dimensional structure information for a CCR from a monocot species.

  4. Thioredoxin Glutathione Reductase-Dependent Redox Networks in Platyhelminth Parasites

    PubMed Central

    Bonilla, Mariana; Gladyshev, Vadim N.

    2013-01-01

    Abstract Significance: Platyhelminth parasites cause chronic infections that are a major cause of disability, mortality, and economic losses in developing countries. Maintaining redox homeostasis is a major adaptive problem faced by parasites and its disruption can shift the biochemical balance toward the host. Platyhelminth parasites possess a streamlined thiol-based redox system in which a single enzyme, thioredoxin glutathione reductase (TGR), a fusion of a glutaredoxin (Grx) domain to canonical thioredoxin reductase (TR) domains, supplies electrons to oxidized glutathione (GSSG) and thioredoxin (Trx). TGR has been validated as a drug target for schistosomiasis. Recent Advances: In addition to glutathione (GSH) and Trx reduction, TGR supports GSH-independent deglutathionylation conferring an additional advantage to the TGR redox array. Biochemical and structural studies have shown that the TR activity does not require the Grx domain, while the glutathione reductase and deglutathionylase activities depend on the Grx domain, which receives electrons from the TR domains. The search for TGR inhibitors has identified promising drug leads, notably oxadiazole N-oxides. Critical Issues: A conspicuous feature of platyhelminth TGRs is that their Grx-dependent activities are temporarily inhibited at high GSSG concentrations. The mechanism underlying the phenomenon and its biological relevance are not completely understood. Future Directions: The functional diversity of Trxs and Grxs encoded in platyhelminth genomes remains to be further assessed to thoroughly understand the TGR-dependent redox network. Optimization of TGR inhibitors and identification of compounds targeting other parasite redox enzymes are good options to clinically develop relevant drugs for these neglected, but important diseases. Antioxid. Redox Signal. 19, 735–745. PMID:22909029

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

    PubMed

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

    2015-10-01

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

  6. Terpenoids from Diplophyllum taxifolium with quinone reductase-inducing activity.

    PubMed

    Wang, Xiao; Zhang, Jiao-Zhen; Zhou, Jin-Chuan; Shen, Tao; Lou, Hong-Xiang

    2016-03-01

    Two new ent-prenylaromadendrane-type diterpenoids, diplotaxifols A (1) and B (2), a new ent-eudesmol, ent-eudesma-4(15),11(13)-dien-6α,12-diol (3), eight new eudesmanolides enantiomers (4-11) of the corresponding compounds from higher plants along with four known ent-eudesmanolides (12-15) were isolated from the 95% EtOH extract of Chinese liverwort Diplophyllum taxifolium. Their structures were elucidated on the basis of MS, NMR and IR spectral data, and confirmed by single-crystal X-ray diffraction analysis. The quinone reductase-inducing activity of the compounds was evaluated.

  7. Applications of Carboxylic Acid Reductases in Oleaginous Microbes

    SciTech Connect

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

    2016-04-24

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

  8. A Novel NADPH-dependent flavoprotein reductase from Bacillus megaterium acts as an efficient cytochrome P450 reductase.

    PubMed

    Milhim, Mohammed; Gerber, Adrian; Neunzig, Jens; Hannemann, Frank; Bernhardt, Rita

    2016-08-10

    Cytochromes P450 (P450s) require electron transfer partners to catalyze substrate conversions. With regard to biotechnological approaches, the elucidation of novel electron transfer proteins is of special interest, as they can influence the enzymatic activity and specificity of the P450s. In the current work we present the identification and characterization of a novel soluble NADPH-dependent diflavin reductase from Bacillus megaterium with activity towards a bacterial (CYP106A1) and a microsomal (CYP21A2) P450 and, therefore, we referred to it as B. megaterium cytochrome P450 reductase (BmCPR). Sequence analysis of the protein revealed besides the conserved FMN-, FAD- and NADPH-binding motifs, the presence of negatively charged cluster, which is thought to represent the interaction domain with P450s and/or cytochrome c. BmCPR was expressed and purified to homogeneity in Escherichia coli. The purified BmCPR exhibited a characteristic diflavin reductase spectrum, and showed a cytochrome c reducing activity. Furthermore, in an in vitro reconstituted system, the BmCPR was able to support the hydroxylation of testosterone and progesterone with CYP106A1 and CYP21A2, respectively. Moreover, in view of the biotechnological application, the BmCPR is very promising, as it could be successfully utilized to establish CYP106A1- and CYP21A2-based whole-cell biotransformation systems, which yielded 0.3g/L hydroxy-testosterone products within 8h and 0.16g/L 21-hydroxyprogesterone within 6h, respectively. In conclusion, the BmCPR reported herein owns a great potential for further applications and studies and should be taken into consideration for bacterial and/or microsomal CYP-dependent bioconversions.

  9. Aldose Reductase-catalyzed Reduction of Aldehyde Phospholipids

    PubMed Central

    Srivastava, Sanjay; Spite, Matthew; Trent, John O.; West, Matthew B.; Ahmed, Yonis; Bhatnagar, Aruni

    2012-01-01

    SUMMARY Oxidation of unsaturated phospholipids results in the generation of aldehyde side chains that remain esterified to the phospholipid backbone. Such “core” aldehydes elicit immune responses and promote inflammation. However, the biochemical mechanisms by which phospholipid aldehydes are metabolized or detoxified are not well understood. In the studies reported here, we examined whether aldose reductase (AR), which reduces hydrophobic aldehydes, metabolizes phospholipid aldehydes. Incubation with AR led to the reduction of 5-oxovaleroyl, 7-oxo-5-heptenoyl, 5-hydroxy-6-oxo-caproyl, and 5-hydroxy-8-oxo-6-octenoyl phospholipids generated upon oxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC). The enzyme also catalyzed the reduction of phospholipid aldehydes generated from the oxidation of 1-alkyl, and 1-alkenyl analogs of PAPC, and 1-palmitoyl-2-arachidonoyl phosphatidic acid or phosphoglycerol. Aldose reductase catalyzed the reduction of chemically synthesized 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphatidylcholine (POVPC) with a Km of 10 μM. Addition of POVPC to the culture medium led to incorporation and reduction of the aldehyde in COS-7 and THP-1 cells. Reduction of POVPC in these cells was prevented by the AR inhibitors sorbinil and tolrestat and was increased in COS-7 cells overexpressing AR. Together, these observations suggest that AR may be a significant participant in the metabolism of several structurally diverse phospholipid aldehydes. This metabolism may be a critical regulator of the pro-inflammatory and immunogenic effects of oxidized phospholipids. PMID:15465833

  10. Two fatty acyl reductases involved in moth pheromone biosynthesis

    PubMed Central

    Antony, Binu; Ding, Bao-Jian; Moto, Ken’Ichi; Aldosari, Saleh A.; Aldawood, Abdulrahman S.

    2016-01-01

    Fatty acyl reductases (FARs) constitute an evolutionarily conserved gene family found in all kingdoms of life. Members of the FAR gene family play diverse roles, including seed oil synthesis, insect pheromone biosynthesis, and mammalian wax biosynthesis. In insects, FAR genes dedicated to sex pheromone biosynthesis (pheromone-gland-specific fatty acyl reductase, pgFAR) form a unique clade that exhibits substantial modifications in gene structure and possesses unique specificity and selectivity for fatty acyl substrates. Highly selective and semi-selective ‘single pgFARs’ produce single and multicomponent pheromone signals in bombycid, pyralid, yponomeutid and noctuid moths. An intriguing question is how a ‘single reductase’ can direct the synthesis of several fatty alcohols of various chain lengths and isomeric forms. Here, we report two active pgFARs in the pheromone gland of Spodoptera, namely a semi-selective, C14:acyl-specific pgFAR and a highly selective, C16:acyl-specific pgFAR, and demonstrate that these pgFARs play a pivotal role in the formation of species-specific signals, a finding that is strongly supported by functional gene expression data. The study envisages a new area of research for disclosing evolutionary changes associated with C14- and C16-specific FARs in moth pheromone biosynthesis. PMID:27427355

  11. Human Neuroglobin Functions as a Redox-regulated Nitrite Reductase*

    PubMed Central

    Tiso, Mauro; Tejero, Jesús; Basu, Swati; Azarov, Ivan; Wang, Xunde; Simplaceanu, Virgil; Frizzell, Sheila; Jayaraman, Thottala; Geary, Lisa; Shapiro, Calli; Ho, Chien; Shiva, Sruti; Kim-Shapiro, Daniel B.; Gladwin, Mark T.

    2011-01-01

    Neuroglobin is a highly conserved hemoprotein of uncertain physiological function that evolved from a common ancestor to hemoglobin and myoglobin. It possesses a six-coordinate heme geometry with proximal and distal histidines directly bound to the heme iron, although coordination of the sixth ligand is reversible. We show that deoxygenated human neuroglobin reacts with nitrite to form nitric oxide (NO). This reaction is regulated by redox-sensitive surface thiols, cysteine 55 and 46, which regulate the fraction of the five-coordinated heme, nitrite binding, and NO formation. Replacement of the distal histidine by leucine or glutamine leads to a stable five-coordinated geometry; these neuroglobin mutants reduce nitrite to NO ∼2000 times faster than the wild type, whereas mutation of either Cys-55 or Cys-46 to alanine stabilizes the six-coordinate structure and slows the reaction. Using lentivirus expression systems, we show that the nitrite reductase activity of neuroglobin inhibits cellular respiration via NO binding to cytochrome c oxidase and confirm that the six-to-five-coordinate status of neuroglobin regulates intracellular hypoxic NO-signaling pathways. These studies suggest that neuroglobin may function as a physiological oxidative stress sensor and a post-translationally redox-regulated nitrite reductase that generates NO under six-to-five-coordinate heme pocket control. We hypothesize that the six-coordinate heme globin superfamily may subserve a function as primordial hypoxic and redox-regulated NO-signaling proteins. PMID:21296891

  12. A mutant of barley lacking NADH-hydroxypyruvate reductase

    SciTech Connect

    Blackwell, R.; Lea, P. )

    1989-04-01

    A mutant of barley, LaPr 88/29, deficient in peroxisomal NADH-hydroxypyruvate reductase (HPR) activity has been identified. Compared to the wild type the activities of NADH-HPR and NADPH-HPR were severely reduced but the mutant was still capable of fixing CO{sub 2} at rates equivalent to 75% of that of the wild type in air. Although lacking an enzyme in the main photorespiratory pathway, there appeared to be little disruption to photorespiratory metabolism as ammonia release, CO{sub 2} efflux and {sup 14}CO{sub 2} release from L-(U-{sup 14}C) serine were similar in both mutant and wild type. LaPr 88/29 has been used to show that NADH-glyoxylate reductase (GR) and NADH-HPR are probably not catalyzed by the same enzyme in barley and that over 80% of the NADPH-HPR activity is due to the NADH-HPR enzyme. Immunological studies, using antibodies raised against spinach HPR, have shown that the NADH-dependent enzyme protein is absent in LaPr 88/29 but there appears to be enhanced synthesis of the NADPH-dependent enzyme protein.

  13. Fluorescent analogues of methotrexate: characterization and interaction with dihydrofolate reductase.

    PubMed

    Kumar, A A; Kempton, R J; Anstead, G M; Freisheim, J H

    1983-01-18

    The dansylated derivatives of lysine and ornithine analogues of methotrexate exhibit fluorescence properties characteristic of the dansyl moiety with an excitation at 328 nm and an emission maximum at 580 nm in aqueous media. As in the case of dansyl amino acids, the fluorescence emission is dependent upon the polarity of the medium. In solvents of low dielectric constant there is an enhancement of the dansyl fluorescence intensity as well as a shift to shorter wavelengths. The dansylated analogues show a reduction in the quantum yields as compared to N epsilon-dansyl-L-lysine and 5-(N,N-dimethylamino)-1-naphthalenesulfonic acid. The absorption spectra of the two dansyl analogues are similar to the spectra of the parent basic amino acid precursors but with reduced molar extinction values. The two fluorescent analogues of methotrexate were found to be potent inhibitors of purified dihydrofolate reductases from Lactobacillus casei and from chicken liver. The binding of these fluorescent analogues to either dihydrofolate reductase resulted in 10-15-nm blue shift of the ligand emission maxima and a 2-5-fold enhancement of the emission. These fluorescent properties of the bound ligands indicate a possible interaction of the dansyl moiety with a region on the enzyme molecule which is more hydrophobic relative to the surrounding solvent.

  14. ADP-ribosylation of dinitrogenase reductase in Rhodobacter capsulatus

    SciTech Connect

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

    1989-07-25

    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 {sup 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.

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

    SciTech Connect

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

    1986-11-01

    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 (/sup 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 (/sup 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.

  16. Increased nitrite reductase activity of fetal versus adult ovine hemoglobin

    PubMed Central

    Blood, Arlin B.; Tiso, Mauro; Verma, Shilpa T.; Lo, Jennifer; Joshi, Mahesh S.; Azarov, Ivan; Longo, Lawrence D.; Gladwin, Mark T.; Kim-Shapiro, Daniel B.; Power, Gordon G.

    2009-01-01

    Growing evidence indicates that nitrite, NO2−, serves as a circulating reservoir of nitric oxide (NO) bioactivity that is activated during physiological and pathological hypoxia. One of the intravascular mechanisms for nitrite conversion to NO is a chemical nitrite reductase activity of deoxyhemoglobin. The rate of NO production from this reaction is increased when hemoglobin is in the R conformation. Because the mammalian fetus exists in a low-oxygen environment compared with the adult and is exposed to episodes of severe ischemia during the normal birthing process, and because fetal hemoglobin assumes the R conformation more readily than adult hemoglobin, we hypothesized that nitrite reduction to NO may be enhanced in the fetal circulation. We found that the reaction was faster for fetal than maternal hemoglobin or blood and that the reactions were fastest at 50–80% oxygen saturation, consistent with an R-state catalysis that is predominant for fetal hemoglobin. Nitrite concentrations were similar in blood taken from chronically instrumented normoxic ewes and their fetuses but were elevated in response to chronic hypoxia. The findings suggest an augmented nitrite reductase activity of fetal hemoglobin and that the production of nitrite may participate in the regulation of vascular NO homeostasis in the fetus. PMID:19028797

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

    PubMed Central

    Hoffmann, Christina; Dietrich, Michael; Herrmann, Ann-Kathrin; Schacht, Teresa

    2017-01-01

    Neuronal degeneration in multiple sclerosis has been linked to oxidative stress. Dimethyl fumarate (DMF) is an effective oral therapeutic option shown to reduce disease activity and progression in patients with relapsing-remitting multiple sclerosis. DMF activates the transcription factor nuclear factor erythroid 2-related factor 2 (NRF2) leading to increased synthesis of the major cellular antioxidant glutathione (GSH) and prominent neuroprotection in vitro. We previously demonstrated that DMF is capable of raising GSH levels even when glutathione synthesis is inhibited, suggesting enhanced GSH recycling. Here, we found that DMF indeed induces glutathione reductase (GSR), a homodimeric flavoprotein that catalyzes GSSG reduction to GSH by using NADPH as a reducing cofactor. Knockdown of GSR using a pool of E. coli RNase III-digested siRNAs or pharmacological inhibition of GSR, however, also induced the antioxidant response rendering it impossible to verify the suspected attenuation of DMF-mediated neuroprotection. However, in cystine-free medium, where GSH synthesis is abolished, pharmacological inhibition of GSR drastically reduced the effect of DMF on glutathione recycling. We conclude that DMF increases glutathione recycling through induction of glutathione reductase. PMID:28116039

  18. Nitrate metabolism in tobacco leaves overexpressing Arabidopsis nitrite reductase.

    PubMed

    Davenport, Susie; Le Lay, Pascaline; Sanchez-Tamburrrino, Juan Pablo

    2015-12-01

    Primary nitrogen assimilation in plants includes the reduction of nitrite to ammonium in the chloroplasts by the enzyme nitrite reductase (NiR EC:1.7.7.1) or in the plastids of non-photosynthetic organs. Here we report on a study overexpressing the Arabidopsis thaliana NiR (AtNiR) gene in tobacco plants under the control of a constitutive promoter (CERV - Carnation Etched Ring Virus). The aim was to overexpress AtNiR in an attempt to alter the level of residual nitrite in the leaf which can act as precursor to the formation of nitrosamines. The impact of increasing the activity of AtNiR produced an increase in leaf protein and a stay-green phenotype in the primary transformed AtNiR population. Investigation of the T1 homozygous population demonstrated elevated nitrate reductase (NR) activity, reductions in leaf nitrite and nitrate and the amino acids proline, glutamine and glutamate. Chlorophyl content of the transgenic lines was increased, as evidenced by the stay-green phenotype. This reveals the importance of NiR in primary nitrogen assimilation and how modification of this key enzyme affects both the nitrogen and carbon metabolism of tobacco plants.

  19. Properties of the arsenate reductase of plasmid R773.

    PubMed

    Gladysheva, T B; Oden, K L; Rosen, B P

    1994-06-14

    Resistance to toxic oxyanions in Escherichia coli is conferred by the ars operon carried on plasmid R773. The gene products of this operon catalyze extrusion of antimonials and arsenicals from cells of E. coli, thus providing resistance to those toxic oxyanions. In addition, resistance to arsenate is conferred by the product of the arsC gene. In this report, purified ArsC protein was shown to catalyze reduction of arsenate to arsenite. The enzymatic activity of the ArsC protein required glutaredoxin as a source of reducing equivalents. Other reductants, including glutathione and thioredoxin, were not effective electron donors. A spectrophotometric assay was devised in which arsenate reduction was coupled to NADPH oxidation. The results obtained with the coupled assay corresponded to those found by direct reduction of radioactive arsenate to arsenite. The only substrate of the reaction was arsenate (Km = 8 mM); other oxyanions including phosphate, sulfate, and antimonate were not reduced. Phosphate and sulfate were weak inhibitors, while the product, arsenite, was a stronger inhibitor (Ki = 0.1 mM). Arsenate reductase activity exhibited a pH optimum of 6.3-6.8. These results indicate that the ArsC protein is a novel reductase, and elucidation of its enzymatic mechanism should be of interest.

  20. Sequence and properties of pentaerythritol tetranitrate reductase from Enterobacter cloacae PB2.

    PubMed

    French, C E; Nicklin, S; Bruce, N C

    1996-11-01

    Pentaerythritol tetranitrate reductase, which reductively liberates nitrite from nitrate esters, is related to old yellow enzyme. Pentaerythritol tetranitrate reductase follows a ping-pong mechanism with competitive substrate inhibition by NADPH, is strongly inhibited by steroids, and is capable of reducing the unsaturated bond of 2-cyclohexen-1-one.

  1. Determination of the specific activities of methionine sulfoxide reductase A and B by capillary electrophoresis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A capillary electrophoresis (CE) method for the determination of methionine sulfoxide reductase A and methionine sulfoxide reductase B activities in mouse liver is described. The method is based on detection of the 4-(dimethylamino)azobenzene-4’-sulfonyl derivative of L-methionine (dabsyl Met), the ...

  2. QTL analysis of ferric reductase activity in the model legume lotus japonicus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Physiological and molecular studies have demonstrated that iron accumulation from the soil into Strategy I plants can be limited by ferric reductase activity. An initial study of Lotus japonicus ecotypes Miyakojima MG-20 and Gifu B-129 identified significant leaf chlorosis and ferric reductase activ...

  3. Biliverdin Reductase Mediates Hypoxia-Induced EMT via PI3-Kinase and Akt

    PubMed Central

    Zeng, Rui; Yao, Ying; Han, Min; Zhao, Xiaoqin; Liu, Xiao-Cheng; Wei, Juncheng; Luo, Yun; Zhang, Juan; Zhou, Jianfeng; Wang, Shixuan; Ma, Ding; Xu, Gang

    2008-01-01

    Chronic hypoxia in the renal parenchyma is thought to induce epithelial-to-mesenchymal transition (EMT), leading to fibrogenesis and ultimately end-stage renal failure. Biliverdin reductase, recently identified as a serine/threonine/tyrosine kinase that may activate phosphatidylinositol 3-kinase (PI3K) and Akt, is upregulated in response to reactive oxygen species that may accompany hypoxia. We investigated this potential role of biliverdin reductase in hypoxia-induced renal tubular EMT. Expression of biliverdin reductase was upregulated in a human proximal tubule cell line (HK-2) cultured in hypoxic conditions (1% O2), and this was accompanied by reduced expression of E-cadherin and increased expression of the mesenchymal marker vimentin. Inhibiting PI3K reversed these changes, consistent with EMT. In normoxic conditions, overexpression of biliverdin reductase promoted similar characteristics of EMT, which were also reversed by inhibiting PI3K. Furthermore, using small interfering RNA (siRNA) to knockdown biliverdin reductase, we demonstrated that the enzyme associates with phosphorylated Akt and mediates the hypoxia-induced EMT phenotype. In vivo, expression of biliverdin reductase increased in the tubular epithelia of 5/6-nephrectomized rats, and immunohistochemistry of serial sections demonstrated similar localization of phosphorylated Akt and biliverdin reductase. In conclusion, biliverdin reductase mediates hypoxia-induced EMT through a PI3K/Akt-dependent pathway. PMID:18184861

  4. Nitrate transport is independent of NADH and NAD(P)H nitrate reductases in barley seedlings

    NASA Technical Reports Server (NTRS)

    Warner, R. L.; Huffaker, R. C.

    1989-01-01

    Barley (Hordeum vulgare L.) has NADH-specific and NAD(P)H-bispecific nitrate reductase isozymes. Four isogenic lines with different nitrate reductase isozyme combinations were used to determine the role of NADH and NAD(P)H nitrate reductases on nitrate transport and assimilation in barley seedlings. Both nitrate reductase isozymes were induced by nitrate and were required for maximum nitrate assimilation in barley seedlings. Genotypes lacking the NADH isozyme (Az12) or the NAD(P)H isozyme (Az70) assimilated 65 or 85%, respectively, as much nitrate as the wild type. Nitrate assimilation by genotype (Az12;Az70) which is deficient in both nitrate reductases, was only 13% of the wild type indicating that the NADH and NAD(P)H nitrate reductase isozymes are responsible for most of the nitrate reduction in barley seedlings. For all genotypes, nitrate assimilation rates in the dark were about 55% of the rates in light. Hypotheses that nitrate reductase has direct or indirect roles in nitrate uptake were not supported by this study. Induction of nitrate transporters and the kinetics of net nitrate uptake were the same for all four genotypes indicating that neither nitrate reductase isozyme has a direct role in nitrate uptake in barley seedlings.

  5. Comparison of finasteride (Proscar), a 5 alpha reductase inhibitor, and various commercial plant extracts in in vitro and in vivo 5 alpha reductase inhibition.

    PubMed

    Rhodes, L; Primka, R L; Berman, C; Vergult, G; Gabriel, M; Pierre-Malice, M; Gibelin, B

    1993-01-01

    Human prostate was used as a source of 5 alpha reductase. Compounds were incubated with an enzyme preparation and [3H]testosterone. [3H]-dihydrotestosterone production was measured to calculate 5 alpha reductase activity. IC50 values (ng/ml) were finasteride = 1; Permixon = 5,600; Talso = 7,000; Strogen Forte = 31,000; Prostagutt = 40,000; and Tadenan = 63,000. Bazoton and Harzol had no activity at concentrations up to 500,000 ng/ml. In castrate rats stimulated with testosterone (T) or dihydrotestosterone (DHT), finasteride, but not Permixon or Bazoton, inhibited T stimulated prostate growth, while none of the three compounds inhibited DHT stimulated growth. These results demonstrate that finasteride inhibits 5 alpha reductase, while Permixon and Bazoton have neither anti-androgen nor 5 alpha reductase inhibitory activity. In addition, in a 7 day human clinical trial, finasteride, but not Permixon or placebo, decreased serum DHT in men, further confirming the lack of 5 alpha reductase inhibition by Permixon. Finasteride and the plant extracts listed above do not inhibit the binding of DHT to the rat prostatic androgen receptor (concentrations to 100 micrograms/ml). Based on these results, it is unlikely that these plant extracts would shrink the prostate by inhibiting androgen action or 5 alpha reductase.

  6. Isolation of ascorbate free radical reductase from rabbit lens soluble fraction.

    PubMed

    Bando, Masayasu; Inoue, Takashi; Oka, Mikako; Nakamura, Kayako; Kawai, Kenji; Obazawa, Hajime; Kobayashi, Shizuko; Takehana, Makoto

    2004-12-01

    Ascorbate free radical (AFR) reductase with diaphorase activity was isolated from the rabbit lens soluble fraction to characterise some molecular properties of the enzyme. The isolation was accomplished using gel filtration (Sephadex G-75 superfine or Sephacryl S-200 HR), affinity chromatography (Affi-Gel Blue), native isoelectric focusing and two-dimensional gel electrophoresis. A major soluble AFR reductase was found at an isoelectric point of 8.4 and a molecular weight of 31 kDa, and a few minor enzymes were also detected in the range of pI 7.0-8.6. An unknown N-terminal partial amino acid sequence was determined in one peptide fragment prepared from the major enzyme fraction. From the sequence analysis, it is discussed that the lens soluble AFR reductase may differ from NADH-cytochrome b5 reductase reported to be involved in the membrane-bound AFR reductase activity of mitochondria, microsomes and plasma membrane.

  7. Trypanothione Reductase: A Viable Chemotherapeutic Target for Antitrypanosomal and Antileishmanial Drug Design

    PubMed Central

    Khan, M. Omar F.

    2007-01-01

    Trypanosomiasis and leishmaniasis are two debilitating disease groups caused by parasites of Trypanosoma and Leishmania spp. and affecting millions of people worldwide. A brief outline of the potential targets for rational drug design against these diseases are presented, with an emphasis placed on the enzyme trypanothione reductase. Trypanothione reductase was identified as unique to parasites and proposed to be an effective target against trypanosomiasis and leishmaniasis. The biochemical basis of selecting this enzyme as a target, with reference to the simile and contrast to human analogous enzyme glutathione reductase, and the structural aspects of its active site are presented. The process of designing selective inhibitors for the enzyme trypanothione reductase has been discussed. An overview of the different chemical classes of inhibitors of trypanothione reductase with their inhibitory activities against the parasites and their prospects as future chemotherapeutic agents are briefly revealed. PMID:21901070

  8. Epigallocatechin-3-gallate potently inhibits the in vitro activity of hydroxy-3-methyl-glutaryl-CoA reductase[S

    PubMed Central

    Cuccioloni, Massimiliano; Mozzicafreddo, Matteo; Spina, Michele; Tran, Chi Nhan; Falconi, Maurizio; Eleuteri, Anna Maria; Angeletti, Mauro

    2011-01-01

    Hydroxy-3-methyl-glutaryl-CoA reductase (HMGR) is the rate-controlling enzyme of cholesterol synthesis, and owing to its biological and pharmacological relevance, researchers have investigated several compounds capable of modulating its activity with the hope of developing new hypocholesterolemic drugs. In particular, polyphenol-rich extracts were extensively tested for their cholesterol-lowering effect as alternatives, or adjuvants, to the conventional statin therapies, but a full understanding of the mechanism of their action has yet to be reached. Our work reports on a detailed kinetic and equilibrium study on the modulation of HMGR by the most-abundant catechin in green tea, epigallocatechin-3-gallate (EGCG). Using a concerted approach involving spectrophotometric, optical biosensor, and chromatographic analyses, molecular docking, and site-directed mutagenesis on the cofactor site of HMGR, we have demonstrated that EGCG potently inhibits the in vitro activity of HMGR (Ki in the nanomolar range) by competitively binding to the cofactor site of the reductase. Finally, we evaluated the effect of combined EGCG-statin administration. PMID:21357570

  9. A new cotton SDR family gene encodes a polypeptide possessing aldehyde reductase and 3-ketoacyl-CoA reductase activities.

    PubMed

    Pang, Yu; Song, Wen-Qiang; Chen, Fang-Yuan; Qin, Yong-Mei

    2010-03-01

    To understand regulatory mechanisms of cotton fiber development, microarray analysis has been performed for upland cotton (Gossypium hirsutum). Based on this, a cDNA (GhKCR3) encoding a polypeptide belonging to short-chain alcohol dehydrogenase/reductase family was isolated and cloned. It contains an open reading frame of 987 bp encoding a polypeptide of 328 amino acid residues. Following its overexpression in bacterial cells, the purified recombinant protein specifically uses NADPH to reduce a variety of short-chain aldehydes. A fragment between Gly180 and Gly191 was found to be essential for its catalytic activity. Though the GhKCR3 gene shares low sequence similarities to the ortholog of Saccharomyces cerevisiae YBR159w that encodes 3-ketoacyl-CoA reductase (KCR) catalyzing the second step of fatty acid elongation, it was surprisingly able to complement the yeast ybr159wDelta mutant. Gas chromatography-mass spectrometry analysis showed that very long-chain fatty acids, especially C26:0, were produced in the ybr159wDelta mutant cells expressing GhKCR3. Applying palmitoyl-CoA and malonyl-CoA as substrates, GhKCR3 showed KCR activity in vitro. Quantitative real time-PCR analysis indicated GhKCR3 transcripts accumulated in rapidly elongating fibers, roots, and stems. Our results suggest that GhKCR3 is probably a novel KCR contributing to very long-chain fatty acid biosynthesis in plants.

  10. Polymorphisms of methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MTR), methionine synthase reductase (MTRR), and thymidylate synthase (TYMS) in multiple myeloma risk.

    PubMed

    Lima, Carmen S P; Ortega, Manoela M; Ozelo, Margareth C; Araujo, Renato C; De Souza, Cármino A; Lorand-Metze, Irene; Annichino-Bizzacchi, Joyce M; Costa, Fernando F

    2008-03-01

    We tested whether the polymorphisms of the methylenetetrahydrofolate reductase gene, MTHFR C677T and A1298C, the methionine synthase gene, MTR A2756G, the methionine synthase reductase gene, MTRR A66G, and the thymidylate synthase gene, TYMS 2R-->3R, involved in folate and methionine metabolism, altered the risk for multiple myeloma (MM). Genomic DNA from 123MM patients and 188 controls was analysed by polymerase chain reaction and restriction digestion for the polymorphism analyses. The frequency of the MTR 2756 AG plus GG genotype was higher in patients than in controls (39.8% versus 23.4%, P=0.001). Individual carriers of the variant allele G had a 2.31 (95% CI: 1.38-3.87)-fold increased risk for MM compared with others. In contrast, similar frequencies of the MTHFR, the MTRR and the TYMS genotypes were seen in patients and controls. These results suggest, for the first time, a role for the MTR A2756G polymorphism in MM risk in our country, but should be confirmed by large-scale epidemiological studies with patients and controls age matched.

  11. 5,10-Methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MTRR), and methionine synthase reductase (MTR) gene polymorphisms and adult meningioma risk.

    PubMed

    Zhang, Jun; Zhou, Yan-Wen; Shi, Hua-Ping; Wang, Yan-Zhong; Li, Gui-Ling; Yu, Hai-Tao; Xie, Xin-You

    2013-11-01

    The causes of meningiomas are not well understood. Folate metabolism gene polymorphisms have been shown to be associated with various human cancers. It is still controversial and ambiguous between the functional polymorphisms of folate metabolism genes 5,10-methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MTRR), and methionine synthase reductase (MTR) and risk of adult meningioma. A population-based case–control study involving 600 meningioma patients (World Health Organization [WHO] Grade I, 391 cases; WHO Grade II, 167 cases; WHO Grade III, 42 cases) and 600 controls was done for the MTHFR C677T and A1298C, MTRR A66G, and MTR A2756G variants in Chinese Han population. The folate metabolism gene polymorphisms were determined by using a polymerase chain reaction–restriction fragment length polymorphism assay. Meningioma cases had a significantly lower frequency of MTHFR 677 TT genotype [odds ratio (OR) = 0.49, 95 % confidence interval (CI) 0.33–0.74; P = 0.001] and T allele (OR = 0.80, 95 % CI 0.67–0.95; P = 0.01) than controls. A significant association between risk of meningioma and MTRR 66 GG (OR = 1.41, 95 % CI 1.02–1.96; P = 0.04) was also observed. When stratifying by the WHO grade of meningioma, no association was found. Our study suggested that MTHFR C677T and MTRR A66G variants may affect the risk of adult meningioma in Chinese Han population.

  12. Curcumin is a tight-binding inhibitor of the most efficient human daunorubicin reductase--Carbonyl reductase 1.

    PubMed

    Hintzpeter, Jan; Hornung, Jan; Ebert, Bettina; Martin, Hans-Jörg; Maser, Edmund

    2015-06-05

    Curcumin is a major component of the plant Curcuma longa L. It is traditionally used as a spice and coloring in foods and is an important ingredient in curry. Curcuminoids have anti-oxidant and anti-inflammatory properties and gained increasing attention as potential neuroprotective and cancer preventive compounds. In the present study, we report that curcumin is a potent tight-binding inhibitor of human carbonyl reductase 1 (CBR1, Ki=223 nM). Curcumin acts as a non-competitive inhibitor with respect to the substrate 2,3-hexandione as revealed by plotting IC50-values against various substrate concentrations and most likely as a competitive inhibitor with respect to NADPH. Molecular modeling supports the finding that curcumin occupies the cofactor binding site of CBR1. Interestingly, CBR1 is one of the most effective human reductases in converting the anthracycline anti-tumor drug daunorubicin to daunorubicinol. The secondary alcohol metabolite daunorubicinol has significantly reduced anti-tumor activity and shows increased cardiotoxicity, thereby limiting the clinical use of daunorubicin. Thus, inhibition of CBR1 may increase the efficacy of daunorubicin in cancer tissue and simultaneously decrease its cardiotoxicity. Western-blots demonstrated basal expression of CBR1 in several cell lines. Significantly less daunorubicin reduction was detected after incubating A549 cell lysates with increasing concentrations of curcumin (up to 60% less with 50 μM curcumin), suggesting a beneficial effect in the co-treatment of anthracycline anti-tumor drugs together with curcumin.

  13. Naegleria fowleri: a free-living highly pathogenic amoeba contains trypanothione/trypanothione reductase and glutathione/glutathione reductase systems.

    PubMed

    Ondarza, Raúl N; Hurtado, Gerardo; Tamayo, Elsa; Iturbe, Angélica; Hernández, Eva

    2006-11-01

    This paper presents definitive data showing that the thiol-bimane compound isolated and purified by HPLC from Naegleria fowleri trophozoites unequivocally corresponds by matrix assisted laser-desorption ionization-time-of-flight MS, to the characteristic monoprotonated ion of trypanothione-(bimane)(2) [M(+)H(+)] of m/z 1104.57 and to the trypanothione-(bimane) of m/z 914.46. The trypanothione disulfide T(S)(2) was also found to have a molecular ion of m/z 723.37. Additionally HPLC demonstrated that thiol-bimane compounds corresponding to cysteine and glutathione were present in Naegleria. The ion patterns of the thiol-bimane compounds prepared from commercial trypanothione standard, Entamoeba histolytica and Crithidia luciliae are identical to the Naegleria thiol-bimane compound. Partially purified extracts from N. fowleri showed the coexistence of glutathione and trypanothione reductases activities. There is not doubt that the thiol compound trypanothione, which was previously thought to occur only in Kinetoplastida, is also present in the human pathogens E. histolytica and N. fowleri, as well as in the non-pathogenic euglenozoan E. gracilis. The presence of the trypanothione/trypanothione reductase system in N. fowleri creates the possibility of using this enzyme as a new "drug target" for rationally designed drugs to eliminate the parasite, without affecting the human host.

  14. Thioredoxin-thioredoxin reductase system of Streptomyces clavuligerus: sequences, expression, and organization of the genes.

    PubMed Central

    Cohen, G; Yanko, M; Mislovati, M; Argaman, A; Schreiber, R; Av-Gay, Y; Aharonowitz, Y

    1993-01-01

    The genes that encode thioredoxin and thioredoxin reductase of Streptomyces clavuligerus were cloned, and their DNA sequences were determined. Previously, we showed that S. clavuligerus possesses a disulfide reductase with broad substrate specificity that biochemically resembles the thioredoxin oxidoreductase system and may play a role in the biosynthesis of beta-lactam antibiotics. It consists consists of two components, a 70-kDa NADPH-dependent flavoprotein disulfide reductase with two identical subunits and a 12-kDa heat-stable protein general disulfide reductant. In this study, we found, by comparative analysis of their predicted amino acid sequences, that the 35-kDa protein is in fact thioredoxin reductase; it shares 48.7% amino acid sequence identity with Escherichia coli thioredoxin reductase, the 12-kDa protein is thioredoxin, and it shares 28 to 56% amino acid sequence identity with other thioredoxins. The streptomycete thioredoxin reductase has the identical cysteine redox-active region--Cys-Ala-Thr-Cys--and essentially the same flavin adenine dinucleotide- and NADPH dinucleotide-binding sites as E. coli thioredoxin reductase and is partially able to accept E. coli thioredoxin as a substrate. The streptomycete thioredoxin has the same cysteine redox-active segment--Trp-Cys-Gly-Pro-Cys--that is present in virtually all eucaryotic and procaryotic thioredoxins. However, in vivo it is unable to donate electrons to E. coli methionine sulfoxide reductase and does not serve as a substrate in vitro for E. coli thioredoxin reductase. The S. clavuligerus thioredoxin (trxA) and thioredoxin reductase (trxB) genes are organized in a cluster. They are transcribed in the same direction and separated by 33 nucleotides. In contrast, the trxA and trxB genes of E. coli, the only other organism in which both genes have been characterized, are physically widely separated. Images PMID:8349555

  15. Flavin reductase: sequence of cDNA from bovine liver and tissue distribution.

    PubMed Central

    Quandt, K S; Hultquist, D E

    1994-01-01

    Flavin reductase catalyzes electron transfer from reduced pyridine nucleotides to methylene blue or riboflavin, and this catalysis is the basis of the therapeutic use of methylene blue or riboflavin in the treatment of methemoglobinemia. A cDNA for a mammalian flavin reductase has been isolated and sequenced. Degenerate oligonucleotides, with sequences based on amino acid sequences of peptides derived from bovine erythrocyte flavin reductase, were used as primers in PCR to selectively amplify a partial cDNA that encodes the bovine reductase. The template used in the PCR was first strand cDNA synthesized from bovine liver total RNA using oligo(dT) primers. A PCR product was used as a specific probe to screen a bovine liver cDNA library. The sequence determined from two overlapping clones contains an open reading frame of 621 nucleotides and encodes 206 amino acids. The amino acid sequence deduced from the bovine liver flavin reductase cDNA matches the amino acid sequences determined for erythrocyte reductase-derived peptides, and the predicted molecular mass of 22,001 Da for the liver reductase agrees well with the molecular mass of 21,994 Da determined for the erythrocyte reductase by electrospray mass spectrometry. The amino acid sequence at the N terminus of the reductase has homology to sequences of pyridine nucleotide-dependent enzymes, and the predicted secondary structure, beta alpha beta, resembles the common nucleotide-binding structural motif. RNA blot analysis indicates a single 1-kilobase reductase transcript in human heart, kidney, liver, lung, pancreas, placenta, and skeletal muscle. Images PMID:7937764

  16. Purification and characterization of dimeric dihydrodiol dehydrogenase from dog liver.

    PubMed

    Sato, K; Nakanishi, M; Deyashiki, Y; Hara, A; Matsuura, K; Ohya, I

    1994-09-01

    High NADP(+)-linked dihydrodiol dehydrogenase activity was detected in dog liver cytosol, from which a dimeric enzyme composed of M(r) 39,000 subunits was purified to homogeneity. The enzyme oxidized trans-cyclohexanediol, and trans-dihydrodiols of benzene and naphthalene, the [1R,2R]-isomers of which were selectively oxidized. In the reverse reaction in the presence of NADPH as a coenzyme, the enzyme reduced alpha-dicarbonyl compounds, such as methylglyoxal, 3-deoxyglucosone, and diacetyl, and some compounds with a carbonyl group, such as glyceraldehyde, lactaldehyde, and acetoin. 4-Hydroxyphenylketones and ascorbates inhibited the enzyme. The results of steady-state kinetic analyses indicated that the reaction proceeds through an ordered bi bi mechanism with the coenzyme binding to the free enzyme, and suggested that the inhibitors bind to the enzyme-NADP+ binary complex. The dimeric enzyme was detected in liver and kidney of dog, and was immunochemically similar to the dimeric enzymes from monkey kidney, rabbit lens, and pig liver. The sequences (total 127 amino acid residues) of eight peptides derived on enzymatic digestion of the dog liver enzyme did not show significant similarity with the primary structures of members of the aldo-keto reductase and short chain dehydrogenase superfamilies, which include monomeric dihydrodiol dehydrogenases and carbonyl reductase, respectively.

  17. Multiple abiotic stress tolerance in Vigna mungo is altered by overexpression of ALDRXV4 gene via reactive carbonyl detoxification.

    PubMed

    Singh, Preeti; Kumar, Deepak; Sarin, Neera Bhalla

    2016-06-01

    Vigna mungo (blackgram) is an important leguminous pulse crop, which is grown for its protein rich edible seeds. Drought and salinity are the major abiotic stresses which adversely affect the growth and productivity of crop plants including blackgram. The ALDRXV4 belongs to the aldo-keto reductase superfamily of enzymes that catalyze the reduction of carbonyl metabolites in the cells and plays an important role in the osmoprotection and detoxification of the reactive carbonyl species. In the present study, we developed transgenic plants of V. mungo using Agrobacterium mediated transformation. The transgene integration was confirmed by Southern blot analysis whereas the expression was confirmed by RT-PCR, Western blot and enzyme activity. The T1 generation transgenic plants displayed improved tolerance to various environmental stresses, including drought, salt, methyl viologen and H2O2 induced oxidative stress. The increased aldose reductase activity, higher sorbitol content and less accumulation of the toxic metabolite, methylglyoxal in the transgenic lines under non-stress and stress (drought and salinity) conditions resulted in increased protection through maintenance of better photosynthetic efficiency, higher relative water content and less photooxidative damage. The accumulation of reactive oxygen species was remarkably decreased in the transgenic lines as compared with the wild type plants. This study of engineering multiple stress tolerance in blackgram, is the first report to date and this strategy for trait improvement is proposed to provide a novel germplasm for blackgram production on marginal lands.

  18. The role of Cercospora zeae-maydis homologs of Rhodobacter sphaeroides 1O2-resistance genes in resistance to the photoactivated toxin cercosporin.

    PubMed

    Beseli, Aydin; Goulart da Silva, Marilia; Daub, Margaret E

    2015-01-01

    The photosynthetic bacterium Rhodobacter sphaeroides and plant pathogenic fungus Cercospora nicotianae have been used as models for understanding resistance to singlet oxygen ((1)O(2)), a highly toxic reactive oxygen species. In Rhodobacter and Cercospora, (1)O(2) is derived, respectively, from photosynthesis and from the (1)O(2)-generating toxin cercosporin which the fungus produces to parasitize plants. We identified common genes recovered in transcriptome studies of putative (1)O(2)-resistance genes in these two systems, suggesting common (1)O(2)-resistance mechanisms. To determine if the Cercospora homologs of R. sphaeroides (1)O(2)-resistance genes are involved in resistance to cercosporin, we expressed the genes in the cercosporin-sensitive fungus Neurospora crassa and assayed for increases in cercosporin resistance. Neurospora crassa transformants expressing genes encoding aldo/keto reductase, succinyl-CoA ligase, O-acetylhomoserine (thiol) lyase, peptide methionine sulphoxide reductase and glutathione S-transferase did not have elevated levels of cercosporin resistance. Several transformants expressing aldehyde dehydrogenase were significantly more resistant to cercosporin. Expression of the transgene and enzyme activity did not correlate with resistance, however. We conclude that although the genes tested in this study are important in (1)O(2) resistance in R. sphaeroides, their Cercospora homologs are not involved in resistance to (1)O(2) generated from cercosporin.

  19. Genes and proteins of the alternative steroid backdoor pathway for dihydrotestosterone synthesis are expressed in the human ovary and seem enhanced in the polycystic ovary syndrome.

    PubMed

    Marti, Nesa; Galván, José A; Pandey, Amit V; Trippel, Mafalda; Tapia, Coya; Müller, Michel; Perren, Aurel; Flück, Christa E

    2017-02-05

    Recently, dihydrotestosterone biosynthesis through the backdoor pathway has been implicated for the human testis in addition to the classic pathway for testosterone (T) synthesis. In the human ovary, androgen precursors are crucial for estrogen synthesis and hyperandrogenism in pathologies such as the polycystic ovary syndrome is partially due to ovarian overproduction. However, a role for the backdoor pathway is only established for the testis and the adrenal, but not for the human ovary. To investigate whether the backdoor pathway exists in normal and PCOS ovaries, we performed specific gene and protein expression studies on ovarian tissues. We found aldo-keto reductases (AKR1C1-1C4), 5α-reductases (SRD5A1/2) and retinol dehydrogenase (RoDH) expressed in the human ovary, indicating that the ovary might produce dihydrotestosterone via the backdoor pathway. Immunohistochemical studies showed specific localization of these proteins to the theca cells. PCOS ovaries show enhanced expression, what may account for the hyperandrogenism.

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

    PubMed

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

    2011-07-01

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

  1. A ribonucleotide reductase inhibitor with deoxyribonucleoside-reversible cytotoxicity.

    PubMed

    Crona, Mikael; Codó, Paula; Jonna, Venkateswara Rao; Hofer, Anders; Fernandes, Aristi P; Tholander, Fredrik

    2016-11-01

    Ribonucleotide Reductase (RNR) is the sole enzyme that catalyzes the reduction of ribonucleotides into deoxyribonucleotides. Even though RNR is a recognized target for antiproliferative molecules, and the main target of the approved drug hydroxyurea, few new leads targeted to this enzyme have been developed. We have evaluated a recently identified set of RNR inhibitors with respect to inhibition of the human enzyme and cellular toxicity. One compound, NSC73735, is particularly interesting; it is specific for leukemia cells and is the first identified compound that hinders oligomerization of the mammalian large RNR subunit. Similar to hydroxyurea, it caused a disruption of the cell cycle distribution of cultured HL-60 cells. In contrast to hydroxyurea, the disruption was reversible, indicating higher specificity. NSC73735 thus defines a potential lead candidate for RNR-targeted anticancer drugs, as well as a chemical probe with better selectivity for RNR inhibition than hydroxyurea.

  2. Crystal structure of isoflavone reductase from alfalfa (Medicago sativa L.).

    PubMed

    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.

  3. Vitamin K epoxide reductase: homology, active site and catalytic mechanism.

    PubMed

    Goodstadt, Leo; Ponting, Chris P

    2004-06-01

    Vitamin K epoxide reductase (VKOR) recycles reduced vitamin K, which is used subsequently as a co-factor in the gamma-carboxylation of glutamic acid residues in blood coagulation enzymes. VKORC1, a subunit of the VKOR complex, has recently been shown to possess this activity. Here, we show that VKORC1 is a member of a large family of predicted enzymes that are present in vertebrates, Drosophila, plants, bacteria and archaea. Four cysteine residues and one residue, which is either serine or threonine, are identified as likely active-site residues. In some plant and bacterial homologues the VKORC1 homologous domain is fused with domains of the thioredoxin family of oxidoreductases. These might reduce disulfide bonds of VKORC1-like enzymes as a prerequisite for their catalytic activities.

  4. Thioredoxin reductase 1 suppresses adipocyte differentiation and insulin responsiveness

    PubMed Central

    Peng, Xiaoxiao; Giménez-Cassina, Alfredo; Petrus, Paul; Conrad, Marcus; Rydén, Mikael; Arnér, Elias S. J.

    2016-01-01

    Recently thioredoxin reductase 1 (TrxR1), encoded by Txnrd1, was suggested to modulate glucose and lipid metabolism in mice. Here we discovered that TrxR1 suppresses insulin responsiveness, anabolic metabolism and adipocyte differentiation. Immortalized mouse embryonic fibroblasts (MEFs) lacking Txnrd1 (Txnrd1−/−) displayed increased metabolic flux, glycogen storage, lipogenesis and adipogenesis. This phenotype coincided with upregulated PPARγ expression, promotion of mitotic clonal expansion and downregulation of p27 and p53. Enhanced Akt activation also contributed to augmented adipogenesis and insulin sensitivity. Knockdown of TXNRD1 transcripts accelerated adipocyte differentiation also in human primary preadipocytes. Furthermore, TXNRD1 transcript levels in subcutaneous adipose tissue from 56 women were inversely associated with insulin sensitivity in vivo and lipogenesis in their isolated adipocytes. These results suggest that TrxR1 suppresses anabolic metabolism and adipogenesis by inhibition of intracellular signaling pathways downstream of insulin stimulation. PMID:27346647

  5. B-factor Analysis and Conformational Rearrangement of Aldose Reductase.

    PubMed

    Balendiran, Ganesaratnam K; Pandian, J Rajendran; Drake, Evin; Vinayak, Anubhav; Verma, Malkhey; Cascio, Duilio

    2014-01-01

    The NADPH-dependent reduction of glucose reaction that is catalyzed by Aldose Reductase (AR) follows a sequential ordered kinetic mechanism in which the co-factor NADPH binds to the enzyme prior to the aldehyde substrate. The kinetic/structural experiments have found a conformational change involving a hinge-like movement of a surface loop (residues 213-224) which is anticipated to take place upon the binding of the diphosphate moiety of NADPH. The reorientation of this loop, expected to permit the release of NADP(+), represents the rate-limiting step of the catalytic mechanism. This study reveals: 1) The Translation/Libration/Screw (TLS) analysis of absolute B-factors of apo AR crystal structures indicates that the 212-224 loop might move as a rigid group. 2) Residues that make the flexible loop slide in the AR binary and ternary complexes. 3) The normalized B-factors separate this segment into three different clusters with fewer residues.

  6. Purification and characterization of 5-ketofructose reductase from Erwinia citreus.

    PubMed Central

    Schrimsher, J L; Wingfield, P T; Bernard, A; Mattaliano, R; Payton, M A

    1988-01-01

    5-Ketofructose reductase [D(-)fructose:(NADP+) 5-oxidoreductase] was purified to homogeneity from Erwinia citreus and demonstrated to catalyse the reversible NADPH-dependent reduction of 5-ketofructose (D-threo-2,5-hexodiulose) to D-fructose. The enzyme appeared as a single species upon analyses by SDS/polyacrylamide-gel electrophoresis and isoelectric focusing with an apparent relative molecular mass of 40,000 and an isoelectric point of 4.4. The amino acid composition of the enzyme and the N-terminal sequence of the first 39 residues are described. The steady-state kinetic mechanism was an ordered one with NADPH binding first to the enzyme and then to 5-ketofructose, and the order of product release was D-fructose followed by NADP+. The reversible nature of the reaction offers the possibility of using this enzyme for the determination of D-fructose. Images Fig. 1. Fig. 2. PMID:3178725

  7. Mechanism of inhibition of ribonucleotide reductase with motexafin gadolinium (MGd)

    SciTech Connect

    Zahedi Avval, Farnaz; Berndt, Carsten; Pramanik, Aladdin; Holmgren, Arne

    2009-02-13

    Motexafin gadolinium (MGd) is an expanded porphyrin anticancer agent which selectively targets tumor cells and works as a radiation enhancer, with promising results in clinical trials. Its mechanism of action is oxidation of intracellular reducing molecules and acting as a direct inhibitor of mammalian ribonucleotide reductase (RNR). This paper focuses on the mechanism of inhibition of RNR by MGd. Our experimental data present at least two pathways for inhibition of RNR; one precluding subunits oligomerization and the other direct inhibition of the large catalytic subunit of the enzyme. Co-localization of MGd and RNR in the cytoplasm particularly in the S-phase may account for its inhibitory properties. These data can elucidate an important effect of MGd on the cancer cells with overproduction of RNR and its efficacy as an anticancer agent and not only as a general radiosensitizer.

  8. Structure of a bacterial homologue of vitamin K epoxide reductase

    SciTech Connect

    Li, Weikai; Schulman, Sol; Dutton, Rachel J.; Boyd, Dana; Beckwith, Jon; Rapoport, Tom A.

    2010-03-19

    Vitamin K epoxide reductase (VKOR) generates vitamin K hydroquinone to sustain {gamma}-carboxylation of many blood coagulation factors. Here, we report the 3.6 {angstrom} crystal structure of a bacterial homologue of VKOR from Synechococcus sp. The structure shows VKOR in complex with its naturally fused redox partner, a thioredoxin-like domain, and corresponds to an arrested state of electron transfer. The catalytic core of VKOR is a four transmembrane helix bundle that surrounds a quinone, connected through an additional transmembrane segment with the periplasmic thioredoxin-like domain. We propose a pathway for how VKOR uses electrons from cysteines of newly synthesized proteins to reduce a quinone, a mechanism confirmed by in vitro reconstitution of vitamin K-dependent disulphide bridge formation. Our results have implications for the mechanism of the mammalian VKOR and explain how mutations can cause resistance to the VKOR inhibitor warfarin, the most commonly used oral anticoagulant.

  9. Go green: the anti-inflammatory effects of biliverdin reductase.

    PubMed

    Wegiel, Barbara; Otterbein, Leo E

    2012-01-01

    Biliverdin (BV) has emerged as a cytoprotective and important anti-inflammatory molecule. Conversion of BV to bilirubin (BR) is catalyzed by biliverdin reductase (BVR) and is required for the downstream signaling and nuclear localization of BVR. Recent data by others and us make clear that BVR is a critical regulator of innate immune responses resulting from acute insult and injury and moreover, that a lack of BVR results in an enhanced proinflammatory phenotype. In macrophages, BVR is regulated by its substrate BV which leads to activation of the PI3K-Akt-IL-10 axis and inhibition of TLR4 expression via direct binding of BVR to the TLR4 promoter. In this review, we will summarize recent findings on the role of BVR and the bile pigments in inflammation in context with its activity as an enzyme, receptor, and transcriptional regulator.

  10. Identification of imine reductase-specific sequence motifs.

    PubMed

    Fademrecht, Silvia; Scheller, Philipp N; Nestl, Bettina M; Hauer, Bernhard; Pleiss, Jürgen

    2016-05-01

    Chiral amines are valuable building blocks for the production of a variety of pharmaceuticals, agrochemicals and other specialty chemicals. Only recently, imine reductases (IREDs) were discovered which catalyze the stereoselective reduction of imines to chiral amines. Although several IREDs were biochemically characterized in the last few years, knowledge of the reaction mechanism and the molecular basis of substrate specificity and stereoselectivity is limited. To gain further insights into the sequence-function relationships, the Imine Reductase Engineering Database (www.IRED.BioCatNet.de) was established and a systematic analysis of 530 putative IREDs was performed. A standard numbering scheme based on R-IRED-Sk was introduced to facilitate the identification and communication of structurally equivalent positions in different proteins. A conservation analysis revealed a highly conserved cofactor binding region and a predominantly hydrophobic substrate binding cleft. Two IRED-specific motifs were identified, the cofactor binding motif GLGxMGx(5 )[ATS]x(4) Gx(4) [VIL]WNR[TS]x(2) [KR] and the active site motif Gx[DE]x[GDA]x[APS]x(3){K}x[ASL]x[LMVIAG]. Our results indicate a preference toward NADPH for all IREDs and explain why, despite their sequence similarity to β-hydroxyacid dehydrogenases (β-HADs), no conversion of β-hydroxyacids has been observed. Superfamily-specific conservations were investigated to explore the molecular basis of their stereopreference. Based on our analysis and previous experimental results on IRED mutants, an exclusive role of standard position 187 for stereoselectivity is excluded. Alternatively, two standard positions 139 and 194 were identified which are superfamily-specifically conserved and differ in R- and S-selective enzymes.

  11. Evidence for a Hexaheteromeric Methylenetetrahydrofolate Reductase in Moorella thermoacetica

    PubMed Central

    Mock, Johanna; Wang, Shuning; Huang, Haiyan; Kahnt, Jörg

    2014-01-01

    Moorella thermoacetica can grow with H2 and CO2, forming acetic acid from 2 CO2 via the Wood-Ljungdahl pathway. All enzymes involved in this pathway have been characterized to date, except for methylenetetrahydrofolate reductase (MetF). We report here that the M. thermoacetica gene that putatively encodes this enzyme, metF, is part of a transcription unit also containing the genes hdrCBA, mvhD, and metV. MetF copurified with the other five proteins encoded in the unit in a hexaheteromeric complex with an apparent molecular mass in the 320-kDa range. The 40-fold-enriched preparation contained per mg protein 3.1 nmol flavin adenine dinucleotide (FAD), 3.4 nmol flavin mononucleotide (FMN), and 110 nmol iron, almost as predicted from the primary structure of the six subunits. It catalyzed the reduction of methylenetetrahydrofolate with reduced benzyl viologen but not with NAD(P)H in either the absence or presence of oxidized ferredoxin. It also catalyzed the reversible reduction of benzyl viologen with NADH (diaphorase activity). Heterologous expression of the metF gene in Escherichia coli revealed that the subunit MetF contains one FMN rather than FAD. MetF exhibited 70-fold-higher methylenetetrahydrofolate reductase activity with benzyl viologen when produced together with MetV, which in part shows sequence similarity to MetF. Heterologously produced HdrA contained 2 FADs and had NAD-specific diaphorase activity. Our results suggested that the physiological electron donor for methylenetetrahydrofolate reduction in M. thermoacetica is NADH and that the exergonic reduction of methylenetetrahydrofolate with NADH is coupled via flavin-based electron bifurcation with the endergonic reduction of an electron acceptor, whose identity remains unknown. PMID:25002540

  12. The modulation of carbonyl reductase 1 by polyphenols.

    PubMed

    Boušová, Iva; Skálová, Lenka; Souček, Pavel; Matoušková, Petra

    2015-01-01

    Carbonyl reductase 1 (CBR1), an enzyme belonging to the short-chain dehydrogenases/reductases family, has been detected in all human tissues. CBR1 catalyzes the reduction of many xenobiotics, including important drugs (e.g. anthracyclines, nabumetone, bupropion, dolasetron) and harmful carbonyls and quinones. Moreover, it participates in the metabolism of a number of endogenous compounds and it may play a role in certain pathologies. Plant polyphenols are not only present in many human food sources, but are also a component of many popular dietary supplements and herbal medicines. Many studies reviewed herein have demonstrated the potency of certain flavonoids, stilbenes and curcuminoids in the inhibition of the activity of CBR1. Interactions of these polyphenols with transcriptional factors, which regulate CBR1 expression, have also been reported in several studies. As CBR1 plays an important role in drug metabolism as well as in the protection of the organism against potentially harmful carbonyls, the modulation of its expression/activity may have significant pharmacological and/or toxicological consequences. Some polyphenols (e.g. luteolin, apigenin and curcumin) have been shown to be very potent CBR1 inhibitors. The inhibition of CBR1 seems useful regarding the increased efficacy of anthracycline therapy, but it may cause the worse detoxification of reactive carbonyls. Nevertheless, all known information about the interactions of polyphenols with CBR1 have only been based on the results of in vitro studies. With respect to the high importance of CBR1 and the frequent consumption of polyphenols, in vivo studies would be very helpful for the evaluation of risks/benefits of polyphenol interactions with CBR1.

  13. d-Apiose Reductase from Aerobacter aerogenes1

    PubMed Central

    Neal, Donna L.; Kindel, Paul K.

    1970-01-01

    A strain of Aerobacter aerogenes PRL-R3 has been isolated which utilizes d-apiose as its sole source of carbon. A new enzyme, d-apiose reductase, was discovered in this strain. The enzyme was not present when the strain was grown on d-glucose. d-Apiose reductase catalyzes the nicotinamide adenine dinucleotide-dependent interconversion of d-apiose and d-apiitol. The enzyme is specific for d-apiose and d-apiitol, with a few possible exceptions. The Km for d-apiose is 0.02 m. The Km for d-apiitol is 0.01 m. The enzyme is almost completely specific for the reduced and oxidized forms of nicotinamide adenine dinucleotide. When cell-free extracts were centrifuged at 100,000 × g for 1 hr, the enzyme remained in solution. Optimal activity for the reduction of d-apiose was obtained at pH 7.5 in glycylglycine buffer, whereas for the oxidation of d-apiitol it was obtained at pH 10.5 in glycine buffer. Enzymatic reduction of d-apiose was not appreciably affected by the presence of 0.02 m ethylenediaminetetraacetate. Paper chromatography and specific spray reagents were used to identify d-apiitol and d-apiose as the products of this reversible reaction. d-Apiose and d-apiitol did not serve as substrates for ribitol dehydrogenase and d-arabitol dehydrogenase from A. aerogenes PRL-R3. PMID:4314545

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

    PubMed

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

    2015-11-25

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

  15. Purification, properties, and sequence of glycerol trinitrate reductase from Agrobacterium radiobacter.

    PubMed Central

    Snape, J R; Walkley, N A; Morby, A P; Nicklin, S; White, G F

    1997-01-01

    Glycerol trinitrate (GTN) reductase, which enables Agrobacterium radiobacter to utilize GTN and related explosives as sources of nitrogen for growth, was purified and characterized, and its gene was cloned and sequenced. The enzyme was a 39-kDa monomeric protein which catalyzed the NADH-dependent reductive scission of GTN (Km = 23 microM) to glycerol dinitrates (mainly the 1,3-isomer) with a pH optimum of 6.5, a temperature optimum of 35 degrees C, and no dependence on metal ions for activity. It was also active on pentaerythritol tetranitrate (PETN), on isosorbide dinitrate, and, very weakly, on ethyleneglycol dinitrate, but it was inactive on isopropyl nitrate, hexahydro-1,3,5-trinitro-1,3,5-triazine, 2,4,6-trinitrotoluene, ammonium ions, nitrate, or nitrite. The amino acid sequence deduced from the DNA sequence was homologous (42 to 51% identity and 61 to 69% similarity) to those of PETN reductase from Enterobacter cloacae, N-ethylmaleimide reductase from Escherichia coli, morphinone reductase from Pseudomonas putida, and old yellow enzyme from Saccharomyces cerevisiae, placing the GTN reductase in the alpha/beta barrel flavoprotein group of proteins. GTN reductase and PETN reductase were very similar in many respects except in their distinct preferences for NADH and NADPH cofactors, respectively. PMID:9401040

  16. Reduction of mitochondrial protein mitoNEET [2Fe-2S] clusters by human glutathione reductase

    PubMed Central

    Landry, Aaron P.; Cheng, Zishuo; Ding, Huangen

    2015-01-01

    Human mitochondrial outer membrane protein mitoNEET is a newly discovered target of type II diabetes drug pioglitazone. Structurally, mitoNEET is a homodimer with each monomer containing an N-terminal transmembrane alpha helix tethered to mitochondrial outer membrane and a C-terminal cytosolic domain hosting a redox active [2Fe-2S] cluster. Genetic studies have shown that mitoNEET has a central role in regulating energy metabolism in mitochondria. However, specific function of mitoNEET remains largely elusive. Here we find that the mitoNEET [2Fe-2S] clusters can be efficiently reduced by Escherichia coli thioredoxin reductase and glutathione reductase in an NADPH-dependent reaction. Purified human glutathione reductase has the same activity as E. coli thioredoxin reductase and glutathione reductase to reduce the mitoNEET [2Fe-2S] clusters. However, rat thioredoxin reductase, a human thioredoxin reductase homolog that contains selenocysteine in the catalytic center, has very little or no activity to reduce the mitoNEET [2Fe-2S] clusters. N-ethylmaleimide, a potent thiol modifier, completely inhibits human glutathione reductase to reduce the mitoNEET [2Fe-2S] clusters, indicating that the redox active disulfide in the catalytic center of human glutathione reductase may be directly involved in reducing the mitoNEET [2Fe-2S] clusters. Additional studies reveal that the reduced mitoNEET [2Fe-2S] clusters in mouse heart cell extracts can be reversibly oxidized by hydrogen peroxide without disruption of the clusters, suggesting that the mitoNEET [2Fe-2S] clusters may undergo redox transition to regulate energy metabolism in mitochondria in response to oxidative signals. PMID:25645953

  17. Pyrroline-5-Carboxylate Reductase in Chlorella autotrophica and Chlorella saccharophila in Relation to Osmoregulation.

    PubMed

    Laliberté, G; Hellebust, J A

    1989-11-01

    Pyrroline-5-carboxylate (P5C) reductase (EC 1.5.1.2), which catalyzes the reduction of P5C to proline, was partially purified from two Chlorella species; Chlorella autotrophica, a euryhaline marine alga that responds to increases in salinity by accumulating proline and ions, and Chlorella saccharophila, which does not accumulate proline for osmoregulation. From the elution profile of this enzyme from an anion exchange column in Tris-HCl buffer (pH 7.6), containing sorbitol and glycine betaine, it was shown that P5C reductase from C. autotrophica was a neutral protein whereas the enzyme from C. saccharophila was negatively charged. The kinetic mechanisms of the reductase was characteristic of a ping-pong mechanism with double competitive substrate inhibition. Both enzymes showed high specificity for NADH as cofactor. The affinities of the reductases for their substrates did not change when the cells were grown at different salinities. In both algae, the apparent K(m) values of the reductase for P5C and NADH were 0.17 and 0.10 millimolar, respectively. A fourfold increase in maximal velocity of the reductase was observed when C. autotrophica was transferred from 50 to 150% artificial sea water. Even though the reductase was inhibited by NaCl, KCl, and proline, it still showed appreciable activity in the presence of these compounds at molar concentrations. A possible role for the regulation of proline synthesis at the step catalyzed by P5C reductase is discussed in relation to the specificity of P5C reductase for NADH and its responses to salt treatments.

  18. Steroidal pyrazolines evaluated as aromatase and quinone reductase-2 inhibitors for chemoprevention of cancer.

    PubMed

    Abdalla, Mohamed M; Al-Omar, Mohamed A; Bhat, Mashooq A; Amr, Abdel-Galil E; Al-Mohizea, Abdullah M

    2012-05-01

    The aromatase and quinone reductase-2 inhibition of synthesized heterocyclic pyrazole derivatives fused with steroidal structure for chemoprevention of cancer is reported herein. All compounds were interestingly less toxic than the reference drug (Cyproterone(®)). The aromatase inhibitory activities of these compounds were much more potent than the lead compound resveratrol, which has an IC(50) of 80 μM. In addition, all the compounds displayed potent quinone reductase-2 inhibition. Initially the acute toxicity of the compounds was assayed via the determination of their LD(50). The aromatase and quinone reductase-2 inhibitors resulting from this study have potential value in the treatment and prevention of cancer.

  19. Comparison of the Stereospecificity and Immunoreactivity of NADH-Ferricyanide Reductases in Plant Membranes.

    PubMed Central

    Fredlund, K. M.; Struglics, A.; Widell, S.; Askerlund, P.; Kader, J. C.; Moller, I. M.

    1994-01-01

    The substrate stereospecificity of NADH-ferricyanide reductase activities in the inner mitochondrial membrane and peroxisomal membrane of potato (Solanum tuberosum L.) tubers, spinach (Spinacea oleracea L.) leaf plasma membrane, and red beetroot (Beta vulgaris L.) tonoplast were all specific for the [beta]-hydrogen of NADH, whereas the reductases in wheat root (Triticum aestivum L.) endoplasmic reticulum and potato tuber outer mitochondrial membrane were both [alpha]-hydrogen specific. In all isolated membrane fractions one or several polypeptides with an apparent size of 45 to 55 kD cross-reacted with antibodies raised against a microsomal NADH-ferricyanide reductase on western blots. PMID:12232391

  20. Cloning and characterization of the methyl coenzyme M reductase genes from Methanobacterium thermoautotrophicum.

    PubMed Central

    Bokranz, M; Bäumner, G; Allmansberger, R; Ankel-Fuchs, D; Klein, A

    1988-01-01

    The genes coding for methyl coenzyme M reductase were cloned from a genomic library of Methanobacterium thermoautotrophicum Marburg into Escherichia coli by using plasmid expression vectors. When introduced into E. coli, the reductase genes were expressed, yielding polypeptides identical in size to the three known subunits of the isolated enzyme, alpha, beta, and gamma. The polypeptides also reacted with the antibodies raised against the respective enzyme subunits. In M. thermoautotrophicum, the subunits are encoded by a gene cluster whose transcript boundaries were mapped. Sequence analysis revealed two more open reading frames of unknown function located between two of the methyl coenzyme M reductase genes. Images PMID:2448287

  1. Proanthocyanidin synthesis and expression of genes encoding leucoanthocyanidin reductase and anthocyanidin reductase in developing grape berries and grapevine leaves.

    PubMed

    Bogs, Jochen; Downey, Mark O; Harvey, John S; Ashton, Anthony R; Tanner, Gregory J; Robinson, Simon P

    2005-10-01

    Proanthocyanidins (PAs), also called condensed tannins, can protect plants against herbivores and are important quality components of many fruits. Two enzymes, leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR), can produce the flavan-3-ol monomers required for formation of PA polymers. We isolated and functionally characterized genes encoding both enzymes from grapevine (Vitis vinifera L. cv Shiraz). ANR was encoded by a single gene, but we found two highly related genes encoding LAR. We measured PA content and expression of genes encoding ANR, LAR, and leucoanthocyanidin dioxygenase in grape berries during development and in grapevine leaves, which accumulated PA throughout leaf expansion. Grape flowers had high levels of PA, and accumulation continued in skin and seeds from fruit set until the onset of ripening. VvANR was expressed throughout early flower and berry development, with expression increasing after fertilization. It was expressed in berry skin and seeds until the onset of ripening, and in expanding leaves. The genes encoding LAR were expressed in developing fruit, particularly in seeds, but had low expression in leaves. The two LAR genes had different patterns of expression in skin and seeds. During grape ripening, PA levels decreased in both skin and seeds, and expression of genes encoding ANR and LAR were no longer detected. The results indicate that PA accumulation occurs early in grape development and is completed when ripening starts. Both ANR and LAR contribute to PA synthesis in fruit, and the tissue and temporal-specific regulation of the genes encoding ANR and LAR determines PA accumulation and composition during grape berry development.

  2. A cytochrome cd1-type nitrite reductase mediates the first step of denitrification in Alcaligenes eutrophus.

    PubMed

    Sann, R; Kostka, S; Friedrich, B

    1994-01-01

    Respiratory nitrite reductase (NIR) has been purified from the soluble extract of denitrifying cells of Alcaligenes eutrophus strain H16 to apparent electrophoretic homogeneity. The enzyme was induced under anoxic conditions in the presence of nitrite. Purified NIR showed typical features of a cytochrome cd1-type nitrite reductase. It appeared to be a dimer of kDa subunits, its activity was only weakly inhibited by the copper chelator diethyldithiocarbamate, and spectral analysis revealed absorption maxima which were characteristic for the presence of heme c and heme d1. The isoelectric point of 8.6 was considerably higher than the pI determined for cd1 nitrite reductases from pseudomonads. Eighteen amino acids at the N-terminus of the A. eutrophus NIR, obtained by protein sequencing, showed no significant homology to the N-terminal region of nitrite reductases from Pseudomonas stutzeri and Pseudomonas aeruginosa.

  3. Inhibition of carbonyl reductase activity in pig heart by alkyl phenyl ketones.

    PubMed

    Imamura, Yorishige; Narumi, Rika; Shimada, Hideaki

    2007-02-01

    The inhibitory effects of alkyl phenyl ketones on carbonyl reductase activity were examined in pig heart. In this study, carbonyl reductase activity was estimated as the ability to reduce 4-benzoylpyridine to S(-)-alpha-phenyl-4-pyridylmethanol in the cytosolic fraction from pig heart (pig heart cytosol). The order of their inhibitory potencies was hexanophenone > valerophenone > heptanophenone > butyrophenone > propiophenone. The inhibitory potencies of acetophenone and nonanophenone were much lower. A significant relationship was observed between Vmax/Km values for the reduction of alkyl phenyl ketones and their inhibitory potencies for carbonyl reductase activity in pig heart cytosol. Furthermore, hexanophenone was a competitive inhibitor for the enzyme activity. These results indicate that several alkyl phenyl ketones including hexanophenone inhibit carbonyl reductase activity in pig heart cytosol, by acting as substrate inhibitors.

  4. Amplification and loss of dihydrofolate reductase genes in a Chinese hamster ovary cell line

    SciTech Connect

    Kaufman, R.J.; Schimke, R.T.

    1981-12-01

    During stepwise increases in the methotrexate concentration in culture medium, the authors selected Chinese hamster ovary cells that contained elevated dihydrofolate reductase levels which were proportional to the number of dihydrofolate reductase gene copies (i.e., gene amplification). The authors studied the dihydrofolate reductase levels in individual cells that underwent the initial steps of methotrexate resistance by using the fluorescence-activated cell sorter technique. Such cells constituted a heterogeneous population with differing dihydrofolate reductase levels, and they characteristically lost the elevated enzyme levels when they were grown in the absence of methotrexate. The progeny of individual cells with high enzyme levels behaved differently and could lose all or variable numbers of the amplified genes.

  5. An electron transport system in maize roots for reactions of glutamate synthase and nitrite reductase : physiological and immunochemical properties of the electron carrier and pyridine nucleotide reductase.

    PubMed

    Suzuki, A; Oaks, A; Jacquot, J P; Vidal, J; Gadal, P

    1985-06-01

    A non-heme iron containing protein which bears an antigenic similarity to ferredoxin from spinach leaves (Spinacia oleracea L.) has been identified in extracts prepared from young roots of maize (Zea mays L., hybrid W64A x W182E). The ferredoxin-like root electron carrier could substitute for ferredoxin in a cytochrome c reduction system in which pyridine nucleotide (NADPH) reduces the root electron carrier in a reaction catalyzed by ferredoxin-NADP(+) reductase (EC 1.6.7.1) from spinach leaves. However, the root electron carrier did not mediate the photoreduction of NADP(+) in an illuminated reconstituted chloroplast system.A pyridine nucleotide reductase which shares identical immunological determinants with the ferredoxin-NADP(+) reductase from spinach leaves has also been characterized from maize roots. Root pyridine nucleotide reductase mediated the transfer of electrons from either NADPH or NADH to cytochrome c via ferredoxin or the root electron carrier. Under chemical reducing conditions with sodium dithionite and bicarbonate, the ferredoxin-like root electron carrier served as an electron carrier for the ferredoxin-requiring glutamate synthase (EC 1.4.7.1) and nitrite reductase (EC 1.7.7.1) obtained from maize roots or leaves. In the presence of root pyridine nucleotide reductase and root electron carrier, either NADPH or NADH served as the primary electron donor for glutamate synthesis in extracts from maize roots or leaves. The electron transport system originating with NADH or NADPH, was, however, not able to mediate the reduction of NO(2) (-) to NH(3).

  6. In vitro inhibition of human erythrocyte glutathione reductase by some new organic nitrates.

    PubMed

    Sentürk, Murat; Talaz, Oktay; Ekinci, Deniz; Cavdar, Hüseyin; Küfrevioğlu, Omer Irfan

    2009-07-01

    Glutathione reductase (GR), is responsible for the existence of GSH molecule, a crucial antioxidant against oxidative stress reagents. The antimalarial activities of some redox active compounds are attributed to their inhibition of antioxidant flavoenzyme glutathione reductase, and inhibitors are therefore expected to be useful for the treatment of malaria. Twelve organic nitrate derivatives were synthesized and treated with human erythrocyte GR. The molecules were identified as strong GR inhibitors and novel antimalaria candidates.

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

    SciTech Connect

    Kuriyan, J.; Xiangpeng Kong; Krishna, T.S.R.; Murgolo, N.J.; Field, H.; Cerami, A.; Henderson, G.B. ); Sweet, R.M. )

    1991-10-01

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

  8. Genetic and Physiologic Characterization of Ferric/Cupric Reductase Constitutive Mutants of Cryptococcus neoformans

    PubMed Central

    Nyhus, Karin J.; Jacobson, Eric S.

    1999-01-01

    Cryptococcus neoformans is a pathogenic yeast that causes meningitis in immunocompromised patients. Because iron acquisition is critical for growth of a pathogen in a host, we studied the regulation of the ferric reductase and ferrous uptake system of this organism. We isolated 18 mutants, representing four independent loci, with dysregulated ferric reductase. The mutant strains had >10-fold higher than wild-type WT reductase activity in the presence of iron. Two of the strains also had >7-fold higher than WT iron uptake in the presence of iron but were not markedly iron sensitive. Both were sensitive to the oxidative stresses associated with superoxide and hydrogen peroxide. One strain exhibited only 23% of the WT level of iron uptake in the absence of iron and grew poorly without iron supplementation of the medium, phenotypes consistent with an iron transport deficiency; it was sensitive to superoxide but not to hydrogen peroxide. The fourth strain had high reductase activity but normal iron uptake; it was not very sensitive to oxidative stress. We also demonstrated that the ferric reductase was regulated by copper and could act as a cupric reductase. Sensitivity to oxidants may be related to iron acquisition by a variety of mechanisms and may model the interaction of the yeast with the immune system. PMID:10225895

  9. Ascorbate free radical reductases and diaphorases in soluble fractions of the human lens.

    PubMed

    Bando, M; Obazawa, H

    1995-12-01

    Major and minor ascorbate free radical (AFR) reductases, with diaphorase activity, and three other diaphorases were separated from the human lens soluble fraction by DEAE-cellulose ion-exchange column chromatography. They were characterized for adsorptivity to ion-exchange and 5'AMP-Sepharose 4B affinity columns, kinetic properties, and substrate specificity. The latter diaphorases were closely correlated with NADH-cytochrome beta 5 reductase. The major and minor AFR reductases were regarded as a major diaphorase group different from two ubiquitous diaphorases, i.e., NADH-cytochrome beta 5 reductase and DT-diaphorase. A major AFR reductase was partially purified approximately 50 fold over the lens soluble fraction by ion-exchange, affinity, and gel filtration (Sephacryl S-200 HR) column chromatography. From the partially purified enzyme, 2 bands, one sharp and one diffuse, were obtained by native polyacrylamide gel electrophoresis. Two proteins, of 20 and 24 kDa, were identified in the active enzyme bands by SDS-polyacrylamide gel electrophoresis. This suggests that the 20 and/or 24 kDa proteins may be components of the major AFR reductase.

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

    PubMed Central

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

    2014-01-01

    In situ reduction of selenite to elemental selenium (Se(0)), by microorganisms in sediments and soils is an important process and greatly affects the environmental distribution and the biological effects of selenium. However, the mechanism behind such a biological process remains unrevealed yet. Here we use Shewanella oneidensis MR-1, a widely-distributed dissimilatory metal-reducing bacterium with a powerful and diverse respiration capability, to evaluate the involvement of anaerobic respiration system in the microbial selenite reduction. With mutants analysis, we identify fumarate reductase FccA as the terminal reductase of selenite in periplasm. Moreover, we find that such a reduction is dependent on central respiration c-type cytochrome CymA. In contrast, nitrate reductase, nitrite reductase, and the Mtr electron transfer pathway do not work as selenite reductases. These findings reveal a previously unrecognized role of anaerobic respiration reductases of S. oneidensis MR-1 in selenite reduction and geochemical cycles of selenium in sediments and soils. PMID:24435070

  11. The role of glutathione reductase and related enzymes on cellular redox homoeostasis network.

    PubMed

    Couto, Narciso; Wood, Jennifer; Barber, Jill

    2016-06-01

    In this review article we examine the role of glutathione reductase in the regulation, modulation and maintenance of cellular redox homoeostasis. Glutathione reductase is responsible for maintaining the supply of reduced glutathione; one of the most abundant reducing thiols in the majority of cells. In its reduced form, glutathione plays key roles in the cellular control of reactive oxygen species. Reactive oxygen species act as intracellular and extracellular signalling molecules and complex cross talk between levels of reactive oxygen species, levels of oxidised and reduced glutathione and other thiols, and antioxidant enzymes such as glutathione reductase determine the most suitable conditions for redox control within a cell or for activation of programmed cell death. Additionally, we discuss the translation and expression of glutathione reductase in a number of organisms including yeast and humans. In yeast and human cells, a single gene expresses more than one form of glutathione reductase, destined for residence in the cytoplasm or for translocation to different organelles; in plants, however, two genes encoding this protein have been described. In general, insects and kinetoplastids (a group of protozoa, including Plasmodia and Trypanosoma) do not express glutathione reductase or glutathione biosynthetic enzymes. Instead, they express either the thioredoxin system or the trypanothione system. The thioredoxin system is also present in organisms that have the glutathione system and there may be overlapping functions with cross-talk between the two systems. Finally we evaluate therapeutic targets to overcome oxidative stress associated cellular disorders.

  12. Androgen Regulation of 5α-Reductase Isoenzymes in Prostate Cancer: Implications for Prostate Cancer Prevention

    PubMed Central

    Li, Jin; Ding, Zhiyong; Wang, Zhengxin; Lu, Jing-Fang; Maity, Sankar N.; Navone, Nora M.; Logothetis, Christopher J.; Mills, Gordon B.; Kim, Jeri

    2011-01-01

    The enzyme 5α-reductase, which converts testosterone to dihydrotestosterone (DHT), performs key functions in the androgen receptor (AR) signaling pathway. The three isoenzymes of 5α-reductase identified to date are encoded by different genes: SRD5A1, SRD5A2, and SRD5A3. In this study, we investigated mechanisms underlying androgen regulation of 5α-reductase isoenzyme expression in human prostate cells. We found that androgen regulates the mRNA level of 5α-reductase isoenzymes in a cell type–specific manner, that such regulation occurs at the transcriptional level, and that AR is necessary for this regulation. In addition, our results suggest that AR is recruited to a negative androgen response element (nARE) on the promoter of SRD5A3 in vivo and directly binds to the nARE in vitro. The different expression levels of 5α-reductase isoenzymes may confer response or resistance to 5α-reductase inhibitors and thus may have importance in prostate cancer prevention. PMID:22194926

  13. Cyclohexanol and methylcyclohexanols. A family of inhibitors of hepatic HMGCoA reductase in vivo.

    PubMed

    Miciak, A; White, D A; Middleton, B

    1986-10-15

    Oral dosing of rats with cyclohexanol and methylcyclohexanols resulted in the inhibition of hepatic HMGCoA reductase. Neither cyclohexane or cyclohexane diols exerted any effects. Inhibition was not due to alcohol dehydrogenase mediated changes in redox state since 3,3',5-trimethylcyclohexanol (TMC), a non substrate for alcohol dehydrogenase, was a potent inhibitor of HMGCoA reductase. Following a single dose of TMC there was no alteration in total hepatic HMGCoA reductase activity for more than 6 hr after which the enzyme activity was depressed in a dose-dependent manner. The normal diurnal rhythm of HMGCoA reductase was reduced in amplitude following TMC administration but the phase was unaltered and the t 1/2 for activity decay following the peak of activity was unaffected. Prior to the inhibitory effect of a TMC dose becoming apparent in total HMGCoA reductase activity we found that the expressed activity of the enzyme (after isolation in F- medium to suppress endogenous protein phosphatase) was depressed by 43%. The inhibitory effect of TMC on total HMGCoA reductase activity seen 8 hr or more after dosing was reflected by inhibition of sterol synthesis in liver measured in vivo after [3H]-H2O administration.

  14. A novel L-xylulose reductase essential for L-arabinose catabolism in Trichoderma reesei.

    PubMed

    Metz, Benjamin; Mojzita, Dominik; Herold, Silvia; Kubicek, Christian P; Richard, Peter; Seiboth, Bernhard

    2013-04-09

    L-Xylulose reductases belong to the superfamily of short chain dehydrogenases and reductases (SDRs) and catalyze the NAD(P)H-dependent reduction of L-xylulose to xylitol in L-arabinose and glucuronic acid catabolism. Here we report the identification of a novel L-xylulose reductase LXR3 in the fungus Trichoderma reesei by a bioinformatic approach in combination with a functional analysis. LXR3, a 31 kDa protein, catalyzes the reduction of L-xylulose to xylitol via NADPH and is also able to convert D-xylulose, D-ribulose, L-sorbose, and D-fructose to their corresponding polyols. Transcription of lxr3 is specifically induced by L-arabinose and L-arabitol. Deletion of lxr3 affects growth on L-arabinose and L-arabitol and reduces total NADPH-dependent LXR activity in cell free extracts. A phylogenetic analysis of known L-xylulose reductases shows that LXR3 is phylogenetically different from the Aspergillus niger L-xylulose reductase LxrA and, moreover, that all identified true L-xylulose reductases belong to different clades within the superfamily of SDRs. This indicates that the enzymes responsible for the reduction of L-xylulose in L-arabinose and glucuronic acid catabolic pathways have evolved independently and that even the fungal LXRs of the L-arabinose catabolic pathway have evolved in different clades of the superfamily of SDRs.

  15. A Novel l-Xylulose Reductase Essential for l-Arabinose Catabolism in Trichoderma reesei

    PubMed Central

    2013-01-01

    l-Xylulose reductases belong to the superfamily of short chain dehydrogenases and reductases (SDRs) and catalyze the NAD(P)H-dependent reduction of l-xylulose to xylitol in l-arabinose and glucuronic acid catabolism. Here we report the identification of a novel l-xylulose reductase LXR3 in the fungus Trichoderma reesei by a bioinformatic approach in combination with a functional analysis. LXR3, a 31 kDa protein, catalyzes the reduction of l-xylulose to xylitol via NADPH and is also able to convert d-xylulose, d-ribulose, l-sorbose, and d-fructose to their corresponding polyols. Transcription of lxr3 is specifically induced by l-arabinose and l-arabitol. Deletion of lxr3 affects growth on l-arabinose and l-arabitol and reduces total NADPH-dependent LXR activity in cell free extracts. A phylogenetic analysis of known l-xylulose reductases shows that LXR3 is phylogenetically different from the Aspergillus nigerl-xylulose reductase LxrA and, moreover, that all identified true l-xylulose reductases belong to different clades within the superfamily of SDRs. This indicates that the enzymes responsible for the reduction of l-xylulose in l-arabinose and glucuronic acid catabolic pathways have evolved independently and that even the fungal LXRs of the l-arabinose catabolic pathway have evolved in different clades of the superfamily of SDRs. PMID:23506391

  16. Characterization of anaerobic sulfite reduction by Salmonella typhimurium and purification of the anaerobically induced sulfite reductase

    SciTech Connect

    Hallenbeck, P.C. ); Clark, M.A.; Barrett, E.L. )

    1989-06-01

    Mutants of Salmonella typhimurium that lack the biosynthetic sulfite reductase (cysI and cysJ mutants) retain the ability to reduce sulfite for growth under anaerobic conditions. Here we report studies of sulfite reduction by a cysI mutant of S. typhimurium and purification of the associated anaerobic sulfite reductase. Sulfite reduction for anaerobic growth did not require a reducing atmosphere but was prevented by an argon atmosphere contaminated with air (<0.33%). It was also prevented by the presence of 0.1 mM nitrate. Anaerobic growth in liquid minimal medium, but not on agar, was found to require additions of trace amounts (10{sup {minus}7} M) of cysteine. Spontaneous mutants that grew under the argon contaminated with air also lost the requirement for 10{sup {minus}7}M cysteine for anaerobic growth in liquid. A role for sulfite reduction in anaerobic energy generation was contraindicated by the findings that sulfite reduction did not improve cell yields, and anaerobic sulfite reductase activity was greatest during the stationary phase of growth. Sulfite reductase was purified from the cytoplasmic fraction of the anaerobically grown cysI mutant and was purified 190-fold. The most effective donor in crude extracts was NADH. NADHP and methyl viologen were, respectively, 40 and 30% as effective as NADH. Oxygen reversibly inhibited the enzyme. The anaerobic sulfite reductase showed some resemblance to the biosynthetic sulfite reductase, but apparently it has a unique, as yet unidentified function.

  17. Catalytic cycle of human glutathione reductase near 1 Å resolution

    PubMed Central

    Berkholz, Donald S.; Faber, H. Richard; Savvides, Savvas N.; Karplus, P. Andrew

    2008-01-01

    Summary Efficient enzyme catalysis depends on exquisite details of structure beyond those resolvable in typical medium- and high-resolution crystallographic analyses. Here we report synchrotron-based cryocrystallographic studies of natural substrate complexes of the flavoenzyme human glutathione reductase (GR) at nominal resolutions between 1.1 and 0.95 Å that reveal new aspects of its mechanism. Compression in the active site causes overlapping van der Waals radii and distortion in the nicotinamide ring of the NADPH substrate, which enhances catalysis via stereoelectronic effects. The bound NADPH and redox-active disulfide are positioned optimally on opposite sides of the flavin for a 1,2-addition across a flavin double bond. The new structures extend earlier observations to reveal that the redox-active disulfide loop in GR is an extreme case of sequential peptide bonds systematically deviating from planarity, a net deviation of 53° across 5 residues. But this apparent strain is not a factor in catalysis as it is present in both oxidized and reduced structures. Intriguingly, the flavin bond lengths in oxidized GR are intermediate between those expected for oxidized and reduced flavin, but we present evidence that this may not be due to the protein environment but instead to partial synchrotron reduction of the flavin by the synchrotron beam. Finally, of more general relevance, we present evidence that the structures of synchrotron-reduced disulfide bonds cannot generally be used as reliable models for naturally reduced disulfide bonds. PMID:18638483

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

    PubMed

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

    2009-10-01

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

  19. Erythrocyte aldose reductase activity and sorbitol levels in diabetic retinopathy

    PubMed Central

    Satyanarayana, A.; Balakrishna, N.; Ayyagari, Radha; Padma, M.; Viswanath, K.; Petrash, J. Mark

    2008-01-01

    Purpose Activation of polyol pathway due to increased aldose reductase (ALR2) activity has been implicated in the development of diabetic complications including diabetic retinopathy (DR), a leading cause of blindness. However, the relationship between hyperglycemia-induced activation of polyol pathway in retina and DR is still uncertain. We investigated the relationship between ALR2 levels and human DR by measuring ALR2 activity and its product, sorbitol, in erythrocytes. Methods We enrolled 362 type 2 diabetic subjects (T2D) with and without DR and 66 normal subjects in this clinical case-control study. Clinical evaluation of DR in T2D patients was done by fundus examination. ALR2 activity and sorbitol levels along with glucose and glycosylated hemoglobin (HbA1C) levels in erythrocytes were determined. Results T2D patients with DR showed significantly higher specific activity of ALR2 as compared to T2D patients without DR. Elevated levels of sorbitol in T2D patients with DR, as compared to T2D patients without DR, corroborated the increased ALR2 activity in erythrocytes of DR patients. However, the increased ALR2 activity was not significantly associated with diabetes duration, age, and HbA1C in both the DR group and total T2D subjects. Conclusions Levels of ALR2 activity as well as sorbitol in erythrocytes may have value as a quantitative trait to be included among other markers to establish a risk profile for development of DR. PMID:18385795

  20. [Molecular characterizations of two dehydroascorbate reductases from Selaginella moellendorffii].

    PubMed

    Cheng, Zishuo; Lan, Ting; Li, Di; Yang, Hailing; Zeng, Qingyin

    2011-01-01

    Plant dehydroascorbate reductase (DHAR) is a physiologically important reducing enzyme in the ascorbate-glutathione recycling reaction. In this study, two DHARs genes (SmDHAR1 and SmDHAR2) were isolated from Selaginella moellendorffii. The SmDHAR1 and SmDHAR2 genes encode two proteins of 218 and 241 amino acid residues, with a calculated molecular mass of 23.97 kDa and 27.33 kDa, respectively. The genomic sequence analysis showed SmDHAR1 and SmDHAR2 contained five and six introns, respectively. Reverse transcription PCR revealed that the SmDHAR1 and SmDHAR2 were constitutive expression genes in S. moellendorffii. The recombinant SmDHAR1 and SmDHAR2 proteins were overexpressed in E. coli, and were purified by Ni-affinity chromatography. The recombinant SmDHAR1 showed 116-fold higher enzymatic activity towards the substrate dehydroascorbate than recombinant SmDHAR2. The recombinant SmDHAR1 showed higher thermal stability than recombinant SmDHAR2. These results indicated obvious functional divergence between the duplicate genes SmDHAR1 and SmDHAR2.

  1. The superoxide reductase from the early diverging eukaryote Giardia intestinalis.

    PubMed

    Testa, Fabrizio; Mastronicola, Daniela; Cabelli, Diane E; Bordi, Eugenio; Pucillo, Leopoldo P; Sarti, Paolo; Saraiva, Lígia M; Giuffrè, Alessandro; Teixeira, Miguel

    2011-10-15

    Unlike superoxide dismutases (SODs), superoxide reductases (SORs) eliminate superoxide anion (O(2)(•-)) not through its dismutation, but via reduction to hydrogen peroxide (H(2)O(2)) in the presence of an electron donor. The microaerobic protist Giardia intestinalis, responsible for a common intestinal disease in humans, though lacking SOD and other canonical reactive oxygen species-detoxifying systems, is among the very few eukaryotes encoding a SOR yet identified. In this study, the recombinant SOR from Giardia (SOR(Gi)) was purified and characterized by pulse radiolysis and stopped-flow spectrophotometry. The protein, isolated in the reduced state, after oxidation by superoxide or hexachloroiridate(IV), yields a resting species (T(final)) with Fe(3+) ligated to glutamate or hydroxide depending on pH (apparent pK(a)=8.7). Although showing negligible SOD activity, reduced SOR(Gi) reacts with O(2)(•-) with a pH-independent second-order rate constant k(1)=1.0×10(9) M(-1) s(-1) and yields the ferric-(hydro)peroxo intermediate T(1); this in turn rapidly decays to the T(final) state with pH-dependent rates, without populating other detectable intermediates. Immunoblotting assays show that SOR(Gi) is expressed in the disease-causing trophozoite of Giardia. We propose that the superoxide-scavenging activity of SOR in Giardia may promote the survival of this air-sensitive parasite in the fairly aerobic proximal human small intestine during infection.

  2. Correlated Protein Motion Measurements of Dihydrofolate Reductase Crystals

    NASA Astrophysics Data System (ADS)

    Xu, Mengyang; Niessen, Katherine; Pace, James; Cody, Vivian; Markelz, Andrea

    2014-03-01

    We report the first direct measurements of the long range structural vibrational modes in dihydrofolate reductase (DHFR). DHFR is a universal housekeeping enzyme that catalyzes the reduction of 7,8-dihydrofolate to 5,6,7,8-tetra-hydrofolate, with the aid of coenzyme nicotinamide adenine dinucleotide phosphate (NADPH). This crucial enzymatic role as the target for anti-cancer [methotrexate (MTX)], and other clinically useful drugs, has made DHFR a long-standing target of enzymological studies. The terahertz (THz) frequency range (5-100 cm-1), corresponds to global correlated protein motions. In our lab we have developed Crystal Anisotropy Terahertz Microscopy (CATM), which directly measures these large scale intra-molecular protein vibrations, by removing the relaxational background of the solvent and residue side chain librational motions. We demonstrate narrowband features in the anisotropic absorbance for mouse DHFR with the ligand binding of NADPH and MTX single crystals as well as Escherichia coli DHFR with the ligand binding of NADPH and MTX single crystals. This work is supported by NSF grant MRI2 grant DBI2959989.

  3. A second target of benzamide riboside: dihydrofolate reductase.

    PubMed

    Roussel, Breton; Johnson-Farley, Nadine; Kerrigan, John E; Scotto, Kathleen W; Banerjee, Debabrata; Felczak, Krzysztof; Pankiewicz, Krzysztof W; Gounder, Murugesan; Lin, HongXia; Abali, Emine Ercikan; Bertino, Joseph R

    2012-11-01

    Dihydrofolate reductase (DHFR) is an essential enzyme involved in de novo purine and thymidine biosynthesis. For several decades, selective inhibition of DHFR has proven to be a potent therapeutic approach in the treatment of various cancers including acute lymphoblastic leukemia, non-Hodgkin's lymphoma, osteogenic sarcoma, carcinoma of the breast, and head and neck cancer. Therapeutic success with DHFR inhibitor methotrexate (MTX) has been compromised in the clinic, which limits the success of MTX treatment by both acquired and intrinsic resistance mechanisms. We report that benzamide riboside (BR), via anabolism to benzamide adenine dinucleotide (BAD) known to potently inhibit inosine monophosphate dehydrogenase (IMPDH), also inhibits cell growth through a mechanism involving downregulation of DHFR protein. Evidence to support this second site of action of BR includes the finding that CCRF-CEM/R human T-cell lymphoblasic leukemia cells, resistant to MTX as a consequence of gene amplification and overexpression of DHFR, are more resistant to BR than are parental cells. Studies of the mechanism by which BR lowers DHFR showed that BR, through its metabolite BAD, reduced NADP and NADPH cellular levels by inhibiting nicotinamide adenine dinucleotide kinase (NADK). As consequence of the lack of NADPH, DHFR was shown to be destabilized. We suggest that, inhibition of NADK is a new approach to downregulate DHFR and to inhibit cell growth.

  4. Chromate reductase activity in Streptomyces sp. MC1.

    PubMed

    Polti, Marta A; Amoroso, María J; Abate, Carlos M

    2010-02-01

    Biological transformation of Cr(VI) to Cr(III) by enzymatic reduction may provide a less costly and more environmentally friendly approach to remediation. In a previous report a Cr(VI) resistant actinomycete strain, Streptomyces sp. MC1, was able to reduce Cr(VI) present in a synthetic medium, soil extract and soil samples. This is the first time optimal conditions such as pH, temperature, growth phase and electron donor have been elucidated in vitro for Cr(VI) reduction by a streptomycete. Chromate reductase of Streptomyces sp. MC1 is a constitutive enzyme which was mainly associated with biomass and required NAD(P)H as an electron donor. It was active over a broad temperature (19-39 degrees C) and pH (5-8) range, and optimum conditions were 30 degrees C and pH 7. The enzyme was present in supernatant, pellet and cell free extract. Bioremediation with the enzyme was observed in non-compatible cell reproduction systems, conditions frequently found in contaminated environments.

  5. Identification of activators of methionine sulfoxide reductases A and B

    PubMed Central

    Cudic, Predrag; Joshi, Neelambari; Sagher, Daphna; Williams, Brandon T.; Stawikowski, Maciej J.; Weissbach, Herbert

    2016-01-01

    The methionine sulfoxide reductase (Msr) family of enzymes has been shown to protect cells against oxidative damage. The two major Msr enzymes, MsrA and MsrB, can repair oxidative damage to proteins due to reactive oxygen species, by reducing the methionine sulfoxide in proteins back to methionine. A role of MsrA in animal aging was first demonstrated in D. melanogaster where transgenic flies over-expressing recombinant bovine MsrA had a markedly extended life span. Subsequently, MsrA was also shown to be involved in the life span extension in C. elegans. These results supported other studies that indicated up-regulation, or activation, of the normal cellular protective mechanisms that cells use to defend against oxidative damage could be an approach to treat age related diseases and slow the aging process. In this study we have identified, for the first time, compounds structurally related to the natural products fusaricidins that markedly activate recombinant bovine and human MsrA and human MsrB. PMID:26718410

  6. Structural Basis for Activation of Class Ib Ribonucleotide Reductase

    SciTech Connect

    Boal, Amie K.; Cotruvo, Jr., Joseph A.; Stubbe, JoAnne; Rosenzweig, Amy C.

    2010-12-03

    The class Ib ribonucleotide reductase of Escherichia coli can initiate reduction of nucleotides to deoxynucleotides with either a Mn{sub 2}{sup III}-tyrosyl radical (Y{sm_bullet}) or a Fe{sub 2}{sup III}-Y{sm_bullet} cofactor in the NrdF subunit. Whereas Fe{sub 2}{sup III}-Y{sm_bullet} can self-assemble from Fe{sub 2}{sup II}-NrdF and O{sub 2}, activation of Mn{sub 2}{sup II}-NrdF requires a reduced flavoprotein, NrdI, proposed to form the oxidant for cofactor assembly by reduction of O{sub 2}. The crystal structures reported here of E. coli Mn{sub 2}{sup II}-NrdF and Fe{sub 2}{sup II}-NrdF reveal different coordination environments, suggesting distinct initial binding sites for the oxidants during cofactor activation. In the structures of Mn{sub 2}{sup II}-NrdF in complex with reduced and oxidized NrdI, a continuous channel connects the NrdI flavin cofactor to the NrdF Mn{sub 2}{sup II} active site. Crystallographic detection of a putative peroxide in this channel supports the proposed mechanism of Mn{sub 2}{sup III}-Y{sm_bullet} cofactor assembly.

  7. Structure and kinetics assays of recombinant Schistosoma mansoni dihydrofolate reductase.

    PubMed

    Serrão, Vitor Hugo Balasco; Romanello, Larissa; Cassago, Alexandre; de Souza, Juliana Roberta Torini; Cheleski, Juliana; DeMarco, Ricardo; Brandão-Neto, José; Pereira, Humberto D'Muniz

    2017-03-11

    The parasite Schistosoma mansoni possesses all pathways for pyrimidine biosynthesis, in which dihydrofolate reductase (DHFR), thymidylate cycle participants, is essential for nucleotide metabolism to obtain energy and structural nucleic acids. Thus, DHFRs have been widely suggested as therapeutic targets for the treatment of infectious diseases. In this study, we expressed recombinant SmDHFR in a heterologous manner to obtain structural, biochemical and kinetic information. X-ray diffraction of recombinant SmDHFR at 1.95Å resolution showed that the structure exhibited the canonical DHFR fold. Isothermal titration calorimetry was used to determine the kinetic constants for NADP(+) and dihydrofolate. Moreover, inhibition assays were performed using the commercial folate analogs methotrexate and aminopterin; these analogs are recognized as folate competitors and are used as chemotherapeutic agents in cancer and autoimmune diseases. This study provides information that may prove useful for the future discovery of novel drugs and for understanding these metabolic steps from this pathway of S. mansoni, thus aiding in our understanding of the function of these essential pathways for parasite metabolism.

  8. Fasciola gigantica thioredoxin glutathione reductase: Biochemical properties and structural modeling.

    PubMed

    Gupta, Ankita; Kesherwani, Manish; Velmurugan, Devadasan; Tripathi, Timir

    2016-08-01

    Platyhelminth thioredoxin glutathione reductase (TGR) is a multifunctional enzyme that crosstalk between the conventional thioredoxin (Trx) and glutathione (GSH) system. It has been validated as a potential drug target in blood flukes. In the present study, we have performed a biochemical study on Fasciola gigantica TGR with substrates DTNB and GSSG. The Michaelis constant (Km) with DTNB was found to be 4.34±0.12μM while it was 61.15±1.50μM with GSSG. The kinetic results were compared with the TGR activities of other helminths. FgTGR showed typical hysteretic behavior with GSSG as other TGRs. We also described a homology-based structure of FgTGR. The cofactors (NADPH and FAD) and substrates (GSSG and DTNB) were docked, and two possible binding sites for substrates were identified in a single chain. The substrates were found to bind more favorably in the second site of TrxR domains. We also presented the first report on binding interaction of DTNB with a TGR. DTNB forms H-bond with His204 and Arg450 of chain A, Sec597, and Gly598 from chain B, salt-bridge with Lys124, and numerous other hydrophobic interactions. Helminth TGR represents an important enzyme in the redox and antioxidant system; hence, its inhibition can be used as an effective strategy against liver flukes.

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

    NASA Astrophysics Data System (ADS)

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

    2017-02-01

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

  10. Hydrogenases in sulfate-reducing bacteria function as chromium reductase.

    PubMed

    Chardin, B; Giudici-Orticoni, M-T; De Luca, G; Guigliarelli, B; Bruschi, M

    2003-12-01

    The ability of sulfate-reducing bacteria (SRB) to reduce chromate VI has been studied for possible application to the decontamination of polluted environments. Metal reduction can be achieved both chemically, by H(2)S produced by the bacteria, and enzymatically, by polyhemic cytochromes c(3). We demonstrate that, in addition to low potential polyheme c-type cytochromes, the ability to reduce chromate is widespread among [Fe], [NiFe], and [NiFeSe] hydrogenases isolated from SRB of the genera Desulfovibrio and Desulfomicrobium. Among them, the [Fe] hydrogenase from Desulfovibrio vulgaris strain Hildenborough reduces Cr(VI) with the highest rate. Both [Fe] and [NiFeSe] enzymes exhibit the same K(m) towards Cr(VI), suggesting that Cr(VI) reduction rates are directly correlated with hydrogen consumption rates. Electron paramagnetic resonance spectroscopy enabled us to probe the oxidation by Cr(VI) of the various metal centers in both [NiFe] and [Fe] hydrogenases. These experiments showed that Cr(VI) is reduced to paramagnetic Cr(III), and revealed inhibition of the enzyme at high Cr(VI) concentrations. The significant decrease of both hydrogenase and Cr(VI)-reductase activities in a mutant lacking [Fe] hydrogenase demonstrated the involvement of this enzyme in Cr(VI) reduction in vivo. Experiments with [3Fe-4S] ferredoxin from Desulfovibrio gigas demonstrated that the low redox [Fe-S] (non-heme iron) clusters are involved in the mechanism of metal reduction by hydrogenases.

  11. Transgenic overexpression of ribonucleotide reductase improves cardiac performance

    PubMed Central

    Nowakowski, Sarah G.; Kolwicz, Stephen C.; Korte, Frederick Steven; Luo, Zhaoxiong; Robinson-Hamm, Jacqueline N.; Page, Jennifer L.; Brozovich, Frank; Weiss, Robert S.; Tian, Rong; Murry, Charles E.; Regnier, Michael

    2013-01-01

    We previously demonstrated that cardiac myosin can use 2-deoxy-ATP (dATP) as an energy substrate, that it enhances contraction and relaxation with minimal effect on calcium-handling properties in vitro, and that contractile enhancement occurs with only minor elevation of cellular [dATP]. Here, we report the effect of chronically enhanced dATP concentration on cardiac function using a transgenic mouse that overexpresses the enzyme ribonucleotide reductase (TgRR), which catalyzes the rate-limiting step in de novo deoxyribonucleotide biosynthesis. Hearts from TgRR mice had elevated left ventricular systolic function compared with wild-type (WT) mice, both in vivo and in vitro, without signs of hypertrophy or altered diastolic function. Isolated cardiomyocytes from TgRR mice had enhanced contraction and relaxation, with no change in Ca2+ transients, suggesting targeted improvement of myofilament function. TgRR hearts had normal ATP and only slightly decreased phosphocreatine levels by 31P NMR spectroscopy, and they maintained rate responsiveness to dobutamine challenge. These data demonstrate long-term (at least 5-mo) elevation of cardiac [dATP] results in sustained elevation of basal left ventricular performance, with maintained β-adrenergic responsiveness and energetic reserves. Combined with results from previous studies, we conclude that this occurs primarily via enhanced myofilament activation and contraction, with similar or faster ability to relax. The data are sufficiently compelling to consider elevated cardiac [dATP] as a therapeutic option to treat systolic dysfunction. PMID:23530224

  12. Effects of galactose feeding on aldose reductase gene expression.

    PubMed Central

    Wu, R R; Lyons, P A; Wang, A; Sainsbury, A J; Chung, S; Palmer, T N

    1993-01-01

    Aldose reductase (AR) is implicated in the pathogenesis of the diabetic complications and osmotic cataract. AR has been identified as an osmoregulatory protein, at least in the renal medulla. An outstanding question relates to the response of AR gene expression to diet-induced galactosemia in extrarenal tissues. This paper shows that AR gene expression in different tissues is regulated by a complex multifactorial mechanism. Galactose feeding in the rat is associated with a complex and, on occasions, multiphasic pattern of changes in AR mRNA levels in kidney, testis, skeletal muscle, and brain. These changes are not in synchrony with the temporal sequence of changes in tissue galactitol, galactose, and myoinositol concentrations. Moreover, galactose feeding results in changes in tissue AR activities that are not related, temporally or quantitatively, to the alterations in tissue AR mRNA or galactitol levels. It is concluded that AR gene expression and tissue AR activities are regulated by mechanisms that are not purely dependent on nonspecific alterations in intracellular metabolite concentrations. This conclusion is supported by the finding that chronic xylose feeding, despite being associated with intracellular xylitol accumulation, does not result in alterations in AR mRNA levels, at least in the kidney. PMID:8325980

  13. Nitrate Reductase of Primary Roots of Red Spruce Seedlings 1

    PubMed Central

    Yandow, Tim S.; Klein, Richard M.

    1986-01-01

    Nitrate reductase activity (NRA) was found in primary roots, but not in foliage of red spruce (Picea rubens Sarg.) seedlings. Nitrate induced NRA:NH4+ did not induce and slightly depressed NRA in older seedlings. Induction required 8 hours and, once induced, NRA decreased slowly in the absence of exogenous NO3−. Seedlings were grown in perlite with a complete nutrient solution containing NH4+ to limit NR induction. Established seedlings were stressed with nutrient solutions at pH 3, 4, or 5 supplemented with Cl− salts of Al, Cd, Pb, or Zn each at two concentrations. NRA in primary root tips was measured at 2, 14, 28, and 42 days. NRA induction was greatest at pH 3, and remained high during the period of study. NRA induction at pH 4 was lower. Metal ions suppressed NRA at pH 3 and 5, but enhanced NRA at pH 4. It is concluded that acidity and soluble metals in the root environment of red spruce are unlikely to be important factors in nitrogen transformations in red spruce roots. PMID:16664891

  14. Structure of Escherichia coli Flavodiiron Nitric Oxide Reductase.

    PubMed

    Romão, Célia V; Vicente, João B; Borges, Patrícia T; Victor, Bruno L; Lamosa, Pedro; Silva, Elísio; Pereira, Luís; Bandeiras, Tiago M; Soares, Cláudio M; Carrondo, Maria A; Turner, David; Teixeira, Miguel; Frazão, Carlos

    2016-11-20

    Flavodiiron proteins (FDPs) are present in organisms from all domains of life and have been described so far to be involved in the detoxification of oxygen or nitric oxide (NO), acting as O2 and/or NO reductases. The Escherichia coli FDP, named flavorubredoxin (FlRd), is the most extensively studied FDP. Biochemical and in vivo studies revealed that FlRd is involved in NO detoxification as part of the bacterial defense mechanisms against reactive nitrogen species. E. coli FlRd has a clear preference for NO as a substrate in vitro, exhibiting a very low reactivity toward O2. To contribute to the understanding of the structural features defining this substrate selectivity, we determined the crystallographic structure of E. coli FlRd, both in the isolated and reduced states. The overall tetrameric structure revealed a highly conserved flavodiiron core domain, with a metallo-β-lactamase-like domain containing a diiron center, and a flavodoxin domain with a flavin mononucleotide cofactor. The metal center in the oxidized state has a μ-hydroxo bridge coordinating the two irons, while in the reduced state, this moiety is not detected. Since only the flavodiiron domain was observed in these crystal structures, the structure of the rubredoxin domain was determined by NMR. Tunnels for the substrates were identified, and through molecular dynamics simulations, no differences for O2 or NO permeation were found. The present data represent the first structure for a NO-selective FDP.

  15. Prognostic Relevance of Methylenetetrahydrofolate Reductase Polymorphisms for Prostate Cancer

    PubMed Central

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

    2016-01-01

    Folate metabolism has been associated with cancers via alterations in nucleotide synthesis, DNA methylation, and DNA repair. We hypothesized that genetic variants in methylenetetrahydrofolate reductase (MTHFR), a key enzyme of folate metabolism, would affect the prognosis of prostate cancer. Three haplotype-tagging single-nucleotide polymorphisms (SNPs) across the MTHFR gene region were genotyped in a cohort of 458 patients with clinically localized prostate cancer treated with radical prostatectomy. One SNP, rs9651118, was associated with disease recurrence, and the association persisted after multivariate analyses adjusting for known risk factors. Public dataset analyses suggested that rs9651118 affects MTHFR expression. Quantitative real-time polymerase chain reaction analysis revealed that MTHFR expression is significantly upregulated in prostate tumor tissues when compared with adjacent normal tissues. Furthermore, overexpression of MTHFR correlates with cancer recurrence and death in two independent publicly available prostate cancer datasets. In conclusion, our data provide rationale to further validate the clinical utility of MTHFR rs9651118 as a biomarker for prognosis in prostate cancer. PMID:27916838

  16. CHARACTERIZATION OF THE METHIONINE SULFOXIDE REDUCTASES OF SCHISTOSOMA MANSONI

    PubMed Central

    Oke, Tolulope T.; Moskovitz, Jackob; Williams, David L.

    2013-01-01

    Schistosomiasis, also known as Bilharzia, is an infectious disease caused by several species of Schistosoma. Twenty million individuals suffer severe symptoms and 200,000 people die annually from the disease. The host responds to the presence of S. mansoni by producing reactive oxygen species that cause oxidative stress. We hypothesized that schistosomes produce antioxidants in response to oxidative stress. A known antioxidant protein is methionine sulfoxide reductase (Msr). Methionine residues can be oxidized to methionine sulfoxide in the presence of oxidizing agents, which is readily reversed by the action of the Msr system. Two S. mansoni MsrB genes (MsrB1 and MsrB2) were cloned and the recombinant proteins expressed in bacteria and purified. The S. mansoni MsrB proteins contained the common conserved catalytic and zinc coordinating cysteines. Analysis of the proteins showed that both proteins promote the reduction of both free methionine sulfoxide (Met[O]) and dabsyl-Met(O) to free methionine (Met) and dabsyl-Met, respectively, while exhibiting differences in their specific activities towards these substrates. Using real-time PCR, both proteins were found to be expressed in all stages of the parasite’s life cycle with the highest level of expression of both proteins in the egg stage. This is the first description of MsrB proteins from a parasite. PMID:19604033

  17. Lausannevirus Encodes a Functional Dihydrofolate Reductase Susceptible to Proguanil

    PubMed Central

    Mueller, L.; Hauser, P. M.; Gauye, F.

    2017-01-01

    ABSTRACT Lausannevirus belongs to the family Marseilleviridae within the group of nucleocytoplasmic large DNA viruses (NCLDVs). These giant viruses exhibit unique features, including a large genome, ranging from 100 kb to 2.5 Mb and including from 150 to more than 2,500 genes, as well as the presence of genes coding for proteins involved in transcription and translation. The large majority of Lausannevirus open reading frames have unknown functions. Interestingly, a bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) is encoded in the Lausannevirus genome. The enzyme plays central roles in DNA precursor biosynthesis. DHFR is the pharmacological target of antifolates, such as trimethoprim, pyrimethamine, and proguanil. First, the functionality of Lausannevirus DHFR-TS was demonstrated by the successful complementation of a DHFR-deficient Saccharomyces cerevisiae strain with a plasmid expressing the heterologous gene. Additionally, using this heterologous expression system, we demonstrated the in vitro susceptibility of Lausannevirus DHFR-TS to proguanil and its resistance to pyrimethamine and trimethoprim. Proguanil may provide a unique and useful treatment if Lausannevirus proves to be a human pathogen. To our knowledge, this is the first time that a DHFR-TS has been described and characterized in an NCLDV. PMID:28137801

  18. Short-chain dehydrogenases/reductases (SDR): the 2002 update.

    PubMed

    Oppermann, Udo; Filling, Charlotta; Hult, Malin; Shafqat, Naeem; Wu, Xiaoqiu; Lindh, Monica; Shafqat, Jawed; Nordling, Erik; Kallberg, Yvonne; Persson, Bengt; Jörnvall, Hans

    2003-02-01

    Short-chain dehydrogenases/reductases (SDR) form a large, functionally heterogeneous protein family presently with about 3000 primary and about 30 3D structures deposited in databases. Despite low sequence identities between different forms (about 15-30%), the 3D structures display highly similar alpha/beta folding patterns with a central beta-sheet, typical of the Rossmann-fold. Based on distinct sequence motifs functional assignments and classifications are possible, making it possible to build a general nomenclature system. Recent mutagenetic and structural studies considerably extend the knowledge on the general reaction mechanism, thereby establishing a catalytic tetrad of Asn-Ser-Tyr-Lys residues, which presumably form the framework for a proton relay system including the 2'-OH of the nicotinamide ribose, similar to the mechanism found in horse liver ADH. Based on their cellular functions, several SDR enzymes appear as possible and promising pharmacological targets with application areas spanning hormone-dependent cancer forms or metabolic diseases such as obesity and diabetes, and infectious diseases.

  19. Solvent effects on catalysis by Escherichia coli dihydrofolate reductase.

    PubMed

    Loveridge, E Joel; Tey, Lai-Hock; Allemann, Rudolf K

    2010-01-27

    Hydride transfer catalyzed by dihydrofolate reductase (DHFR) has been described previously within an environmentally coupled model of hydrogen tunneling, where protein motions control binding of substrate and cofactor to generate a tunneling ready conformation and modulate the width of the activation barrier and hence the reaction rate. Changes to the composition of the reaction medium are known to perturb protein motions. We have measured kinetic parameters of the reaction catalyzed by DHFR from Escherichia coli in the presence of various cosolvents and cosolutes and show that the dielectric constant, but not the viscosity, of the reaction medium affects the rate of reaction. Neither the primary kinetic isotope effect on the reaction nor its temperature dependence were affected by changes to the bulk solvent properties. These results are in agreement with our previous report on the effect of solvent composition on catalysis by DHFR from the hyperthermophile Thermotoga maritima. However, the effect of solvent on the temperature dependence of the kinetic isotope effect on hydride transfer catalyzed by E. coli DHFR is difficult to explain within a model, in which long-range motions couple to the chemical step of the reaction, but may indicate the existence of a short-range promoting vibration or the presence of multiple nearly isoenergetic conformational substates of enzymes with similar but distinct catalytic properties.

  20. Constitutive nitrate reductase expression and inhibition in winged bean

    SciTech Connect

    Wu, Shenchuan; Harper, J.E. )

    1990-05-01

    It was found that NO{sub 3}{sup {minus}} had no effect on winged bean nitrate reductase activity (NRA). Similar NRA was expressed in plants grown on NO{sub 3}{sup {minus}}, urea, NH{sub 4}{sup +}, and nil N. This indicated that the primary NR expressed in winged bean was constitutive, rather than substrate-inducible. Maximum NRA in winged bean was obtained in the light. KClO{sub 3} was capable of inhibiting NRA of leaves if added to the root growth medium or to the NR assay medium, indicating possible competition with NO{sub 3}{sup {minus}} at the reduction site. While it has previously been shown that either cycloheximide alone, or both cycloheximide and chloramphenicol impair the synthesis of NR protein, our data unexpectedly demonstrated that cycloheximide had little effect on NRA, whereas chloramphenicol greatly inhibited the expression of NRA in winged bean. One interpretation is that chloroplasts may influence the activity and/or synthesis of constitutive NR proteins.

  1. Optical observation of correlated motions in dihydrofolate reductase

    NASA Astrophysics Data System (ADS)

    Xu, Mengyang; Niessen, Katherine; Pace, James; Cody, Vivian; Markelz, Andrea

    2015-03-01

    Enzyme function relies on its structural flexibility to make conformational changes for substrate binding and product release. An example of a metabolic enzyme where such structural changes are vital is dihydrofolate reductase (DHFR). DHFR is essential in both prokaryotes and eukaryotes for the nucleotide biosynthesis by catalyzing the reduction of dihydrofolate to tetrahydrofolate. NMR dynamical measurements found large amplitude fast dynamics that could indicate rigid-body, twisting-hinge motion for ecDHFR that may mediate flux. The role of such long-range correlated motions in function was suggested by the observed sharp decrease in enzyme activity for the single point mutation G121V, which is remote from active sites. This decrease in activity may be caused by the mutation interfering with the long-range intramolecular vibrations necessary for rapid access to functional configurations. We use our new technique of crystal anisotropy terahertz microscopy (CATM), to observe correlated motions in ecDHFR crystals with the bonding of NADPH and methotrexate. We compare the measured intramolecular vibrational spectrum with calculations using normal mode analysis.

  2. Loss of quinone reductase 2 function selectively facilitates learning behaviors.

    PubMed

    Benoit, Charles-Etienne; Bastianetto, Stephane; Brouillette, Jonathan; Tse, YiuChung; Boutin, Jean A; Delagrange, Philippe; Wong, TakPan; Sarret, Philippe; Quirion, Rémi

    2010-09-22

    High levels of reactive oxygen species (ROS) are associated with deficits in learning and memory with age as well as in Alzheimer's disease. Using DNA microarray, we demonstrated the overexpression of quinone reductase 2 (QR2) in the hippocampus in two models of learning deficits, namely the aged memory impaired rats and the scopolamine-induced amnesia model. QR2 is a cytosolic flavoprotein that catalyzes the reduction of its substrate and enhances the production of damaging activated quinone and ROS. QR2-like immunostaining is enriched in cerebral structures associated with learning behaviors, such as the hippocampal formation and the temporofrontal cortex of rat, mouse, and human brains. In cultured rat embryonic hippocampal neurons, selective inhibitors of QR2, namely S26695 and S29434, protected against menadione-induced cell death by reversing its proapoptotic action. S26695 (8 mg/kg) also significantly inhibited scopolamine-induced amnesia. Interestingly, adult QR2 knock-out mice demonstrated enhanced learning abilities in various tasks, including Morris water maze, object recognition, and rotarod performance test. Other behaviors related to anxiety (elevated plus maze), depression (forced swim), and schizophrenia (prepulse inhibition) were not affected in QR2-deficient mice. Together, these data suggest a role for QR2 in cognitive behaviors with QR2 inhibitors possibly representing a novel therapeutic strategy toward the treatment of learning deficits especially observed in the aged brain.

  3. Severe scoliosis in a patient with severe methylenetetrahydrofolate reductase deficiency.

    PubMed

    Munoz, Tatiana; Patel, Jinesh; Badilla-Porras, Ramses; Kronick, Jonathan; Mercimek-Mahmutoglu, Saadet

    2015-01-01

    Severe methylenetetrahydrofolate reductase (MTHFR) deficiency is a rare autosomal recessively inherited inborn error of folate metabolism. We report a new patient with severe MTHFR deficiency who presented at age 4 months with early onset severe scoliosis associated with severe hypotonia. Markedly decreased MTHFR enzyme activity (0.3 nmoles CHO/mg protein/h; reference range>9) and compound heterozygous mutations (c. 1304T>C; p.Phe435Ser and c.1539dup; p.Glu514Argfs∗24) in the MTHFR gene confirmed the diagnosis. She was treated with vitamin B12, folic acid and betaine supplementation and showed improvements in her developmental milestones and hypotonia. To the best of our knowledge, this is the first patient with MTHFR deficiency reported with severe early onset scoliosis. Despite the late diagnosis and treatment initiation, she showed favorable short-term neurodevelopmental outcome. This case suggests that homocysteine measurement should be included in the investigations of patients with developmental delay, hypotonia and scoliosis within first year of life prior to organizing genetic investigations.

  4. Mutation Update and Review of Severe Methylenetetrahydrofolate Reductase Deficiency.

    PubMed

    Froese, D Sean; Huemer, Martina; Suormala, Terttu; Burda, Patricie; Coelho, David; Guéant, Jean-Louis; Landolt, Markus A; Kožich, Viktor; Fowler, Brian; Baumgartner, Matthias R

    2016-05-01

    Severe 5,10-methylenetetrahydrofolate reductase (MTHFR) deficiency is caused by mutations in the MTHFR gene and results in hyperhomocysteinemia and varying severity of disease, ranging from neonatal lethal to adult onset. Including those described here, 109 MTHFR mutations have been reported in 171 families, consisting of 70 missense mutations, 17 that primarily affect splicing, 11 nonsense mutations, seven small deletions, two no-stop mutations, one small duplication, and one large duplication. Only 36% of mutations recur in unrelated families, indicating that most are "private." The most common mutation is c.1530A>G (numbered from NM_005957.4, p.Lys510 = ) causing a splicing defect, found in 13 families; the most common missense mutation is c.1129C>T (p.Arg377Cys) identified in 10 families. To increase disease understanding, we report enzymatic activity, detected mutations, and clinical onset information (early, <1 year; or late, >1 year) for all published patients available, demonstrating that patients with early onset have less residual enzyme activity than those presenting later. We also review animal models, diagnostic approaches, clinical presentations, and treatment options. This is the first large review of mutations in MTHFR, highlighting the wide spectrum of disease-causing mutations.

  5. Composition and structure of assimilatory nitrate reductase from Ankistrodesmus braunii.

    PubMed

    De la Rosa, M A; Vega, J M; Zumft, W G

    1981-06-10

    Assimilatory NAD(P)H-nitrate reductase (EC 1.6.6.2) from Ankistrodesmus braunii has been purified to homogeneity by affinity chromatography on blue Sepharose. The specific activity of the purified enzyme is in the range of 72 to 80 units/mg of protein. The electronic spectrum of the native enzyme shows absorption maxima at 278, 414 (Soret), 532 (beta), 562 (alpha), and 669 nm and shoulders at 455 and 484 nm, with an A278/A414 ratio of 2.56. The reduced enzyme shows absorption maxima at 424 (Soret), 528 (beta), 557 (alpha),and 669 n. The enzyme complex (Mr = 467,400) is composed of eight similar subunits (Mr = 58,750) and contains 4 molecules of FAD, 4 heme groups, and 2 atoms of molybdenum. Labile sulfide and nonheme iron were not detected. Electron micrographs show the eight subunits arranged alternately in two planes, and an 8-fold rotational symmetry was deduced from highly magnified images processed by optical superposition.

  6. Converting a Sulfenic Acid Reductase into a Disulfide Bond Isomerase

    PubMed Central

    Chatelle, Claire; Kraemer, Stéphanie; Ren, Guoping; Chmura, Hannah; Marechal, Nils; Boyd, Dana; Roggemans, Caroline; Ke, Na; Riggs, Paul; Bardwell, James

    2015-01-01

    Abstract Aims: Posttranslational formation of disulfide bonds is essential for the folding of many secreted proteins. Formation of disulfide bonds in a protein with more than two cysteines is inherently fraught with error and can result in incorrect disulfide bond pairing and, consequently, misfolded protein. Protein disulfide bond isomerases, such as DsbC of Escherichia coli, can recognize mis-oxidized proteins and shuffle the disulfide bonds of the substrate protein into their native folded state. Results: We have developed a simple blue/white screen that can detect disulfide bond isomerization in vivo, using a mutant alkaline phosphatase (PhoA*) in E. coli. We utilized this screen to isolate mutants of the sulfenic acid reductase (DsbG) that allowed this protein to act as a disulfide bond isomerase. Characterization of the isolated mutants in vivo and in vitro allowed us to identify key amino acid residues responsible for oxidoreductase properties of thioredoxin-like proteins such as DsbC or DsbG. Innovation and Conclusions: Using these key residues, we also identified and characterized interesting environmental homologs of DsbG with novel properties, thus demonstrating the capacity of this screen to discover and elucidate mechanistic details of in vivo disulfide bond isomerization. Antioxid. Redox Signal. 23, 945–957. PMID:26191605

  7. Role of Helicobacter pylori methionine sulfoxide reductase in urease maturation

    PubMed Central

    Kuhns, Lisa G.; Mahawar, Manish; Sharp, Joshua S.; Benoit, Stéphane; Maier, Robert J.

    2014-01-01

    The persistence of the gastric pathogen Helicobacter pylori is due in part to urease and Msr (methionine sulfoxide reductase). Upon exposure to relatively mild (21% partial pressure of O2) oxidative stress, a Δmsr mutant showed both decreased urease specific activity in cell-free extracts and decreased nickel associated with the partially purified urease fraction as compared with the parent strain, yet urease apoprotein levels were the same for the Δmsr and wild-type extracts. Urease activity of the Δmsr mutant was not significantly different from the wild-type upon non-stress microaerobic incubation of strains. Urease maturation occurs through nickel mobilization via a suite of known accessory proteins, one being the GTPase UreG. Treatment of UreG with H2O2 resulted in oxidation of MS-identified methionine residues and loss of up to 70% of its GTPase activity. Incubation of pure H2O2-treated UreG with Msr led to reductive repair of nine methionine residues and recovery of up to full enzyme activity. Binding of Msr to both oxidized and non-oxidized UreG was observed by cross-linking. Therefore we conclude Msr aids the survival of H. pylori in part by ensuring continual UreG-mediated urease maturation under stress conditions. PMID:23181726

  8. Light regulates alternative splicing of hydroxypyruvate reductase in pumpkin.

    PubMed

    Mano, S; Hayashi, M; Nishimura, M

    1999-02-01

    Hydroxypyruvate reductase (HPR) is a leaf peroxisomal enzyme that functions in the glycolate pathway of photorespiration in plants. We have obtained two highly similar cDNAs for pumpkin HPR (HPR1 and HPR2). It has been revealed that two HPR mRNAs might be produced by alternative splicing from a single type of pre-mRNA. The HPR1 protein, but not the HPR2 protein, was found to have a targeting sequence into leaf peroxisomes at the C-terminus, suggesting that alternative splicing controls the subcellular localization of the two HPR proteins. Immunoblot analysis and subcellular fractionation experiments showed that HPR1 and HPR2 proteins are localized in leaf peroxisomes and the cytosol, respectively. Moreover, indirect fluorescence microscopy and analyses of transgenic tobacco cultured cells and Arabidopsis thaliana expressing fusion proteins with green fluorescent protein (GFP) revealed the different subcellular localizations of the two HPR proteins. Both mRNAs were induced developmentally and by light, but with quantitative differences. Almost equal amounts of the mRNAs were detected in pumpkin cotyledons grown in darkness, but treatment with light greatly enhanced the production of HPR2 mRNA. These findings indicate that light regulates alternative splicing of HPR mRNA, suggesting the presence of a novel mechanism of mRNA maturation, namely light-regulated alternative splicing, in higher plants.

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

    PubMed Central

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

    2017-01-01

    Dehydroascorbate reductase (DHAR) catalyzes the glutathione (GSH)-dependent reduction of dehydroascorbate and plays a direct role in regenerating ascorbic acid, an essential plant antioxidant vital for defense against oxidative stress. DHAR enzymes bear close structural homology to the glutathione transferase (GST) superfamily of enzymes and contain the same active site motif, but most GSTs do not exhibit DHAR activity. The presence of a cysteine at the active site is essential for the catalytic functioning of DHAR, as mutation of this cysteine abolishes the activity. Here we present the crystal structure of DHAR2 from Arabidopsis thaliana with GSH bound to the catalytic cysteine. This structure reveals localized conformational differences around the active site which distinguishes the GSH-bound DHAR2 structure from that of DHAR1. We also unraveled the enzymatic step in which DHAR releases oxidized glutathione (GSSG). To consolidate our structural and kinetic findings, we investigated potential conformational flexibility in DHAR2 by normal mode analysis and found that subdomain mobility could be linked to GSH binding or GSSG release. PMID:28195196

  10. Atypical features of Thermus thermophilus succinate:quinone reductase.

    PubMed

    Kolaj-Robin, Olga; Noor, Mohamed R; O'Kane, Sarah R; Baymann, Frauke; Soulimane, Tewfik

    2013-01-01

    The Thermus thermophilus succinate:quinone reductase (SQR), serving as the respiratory complex II, has been homologously produced under the control of a constitutive promoter and subsequently purified. The detailed biochemical characterization of the resulting wild type (wt-rcII) and His-tagged (rcII-His(8)-SdhB and rcII-SdhB-His(6)) complex II variants showed the same properties as the native enzyme with respect to the subunit composition, redox cofactor content and sensitivity to the inhibitors malonate, oxaloacetate, 3-nitropropionic acid and nonyl-4-hydroxyquinoline-N-oxide (NQNO). The position of the His-tag determined whether the enzyme retained its native trimeric conformation or whether it was present in a monomeric form. Only the trimer exhibited positive cooperativity at high temperatures. The EPR signal of the [2Fe-2S] cluster was sensitive to the presence of substrate and showed an increased rhombicity in the presence of succinate in the native and in all recombinant forms of the enzyme. The detailed analysis of the shape of this signal as a function of pH, substrate concentration and in the presence of various inhibitors and quinones is presented, leading to a model for the molecular mechanism that underlies the influence of succinate on the rhombicity of the EPR signal of the proximal iron-sulfur cluster.

  11. Phosphorylation and proteolysis of HMGCoA reductase

    SciTech Connect

    Parker, R.A.; Lanier, T.L.; Miller, S.J.; Gibson, D.M.

    1987-05-01

    The phosphorylation of rat liver microsomal 97 kDa HMGCoA reductase (HMGR) was examined by immunoprecipitation and SDS-PAGE using antibodies to 53 kDa HMGR. MgATP preincubation decreased expressed HMGR activity from 10.1 +/- 2.4 to 0.81 +/- 0.2 U/mg. Concomitant incorporation of TSP from el-TSP-ATP into 97 kDa HMGR protein was observed. Competitive antibody binding by affinity-purified 53 kDa HMGR showed that the 97 kDa TSP band was authentic HMGR. HMGR was reactivated and the TSP label was removed by protein phosphatase in a concentration-dependent manner: the increase in expressed/total activity ratio (E/T) correlated linearly with a decrease in 97 kDa TSP. Therefore, the E/T ratio provides a valid index of the phosphorylation state of microsomal 97 kDa HMGR. Protease cleavage patterns of HMGR mass and TSP were compared using calpain: a 52-56 kDa doublet of HMGR mass was observed in immunoblots under conditions in which only the 56 kDa band contained TSP. Further proteolysis decreased the TSP label as the 52 kDa mass product increased. The data suggest that the major phosphorylation site in 97 kDa HMGR lies between two main calpain cleavage sites in the linker region joining the cytoplasmic domain to the membrane-spanning domain of the native enzyme.

  12. Molecular Cloning of Complementary DNA Encoding Maize Nitrite Reductase

    PubMed Central

    Lahners, Kristine; Kramer, Vance; Back, Eduard; Privalle, Laura; Rothstein, Steven

    1988-01-01

    Complementary DNA has been isolated that codes for maize nitrite reductase (NiR) by using the corresponding spinach gene (E Back et al. 1988 Mol Gen Genet 212:20-26) as a heterologous probe. The sequences of the complementary DNAs from the two species are 66% homologous while the deduced amino acid sequences are 86% similar when analogous amino acids are included. A high percentage of the differences in the DNA sequences is due to the extremely strong bias in the corn gene to have a G/C base in the third codon position with 559/569 codons ending in a G or C. Using a hydroponic system, maize seedlings grown in the absence of an exogenous nitrogen source were induced with nitrate or nitrite. Nitrate stimulated a rapid induction of the NiR mRNA in both roots and leaves. There is also a considerable induction of this gene in roots upon the addition of nitrite, although under the conditions used the final mRNA level was not as high as when nitrate was the inducer. There is a small but detectable level of NiR mRNA in leaves prior to induction, but no constitutive NiR mRNA can be seen in the roots. Analysis of genomic DNA supports the notion that there are at least two NiR genes in maize. Images Fig. 3 Fig. 4 Fig. 5 PMID:16666376

  13. Drug susceptibility testing of Mycobacterium tuberculosis with nitrate reductase assay.

    PubMed

    Coban, Ahmet Yilmaz; Birinci, Asuman; Ekinci, Bora; Durupinar, Belma

    2004-09-01

    The nitrate reductase assay (NRA) was evaluated for susceptibility testing of Mycobacterium tuberculosis using 80 clinical isolates of M. tuberculosis and H37Rv as a control strain. All isolates were tested by the proportion method and the NRA for isoniazid (INH), rifampicin (RIF), streptomycin (STR) and ethambutol (ETM). The proportion method was carried out according to NCCLS on Löwenstein-Jensen (LJ) medium and the NRA on LJ medium containing 1000 microg/ml potassium nitrate (KNO(3)). After incubation for 7, 10, 14 and 21 days, Griess reagent was added to each LJ medium and nitrate reduction was determined by a colour change. Comparing the NRA with the proportion method, sensitivities were 100, 100, 82.1 and 92.2% for INH, RIF, STR and ETM, respectively. Specificities were 100, 100, 92.3 and 100% for INH, RIF, STR and ETM, respectively. The results of 2, 22 and 56 isolates were obtained after 7, 10 and 14 days, respectively. The proportion method result were read at 21-28 days. The NRA is rapid, inexpensive and easy to perform. Our results indicated that the NRA is suitable for the early determination of INH and RIF resistance in countries where sophisticated procedures are not always available.

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

    PubMed

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

    2008-11-01

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

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

    SciTech Connect

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

    1995-05-01

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

  16. Molecular basis for thermoprotection in Bemisia: structural differences between whitefly ketose reductase and other medium-chain dehydrogenases/reductases.

    PubMed

    Wolfe, G R; Smith, C A; Hendrix, D L; Salvucci, M E

    1999-02-01

    The silverleaf whitefly (Bemisia argentifolii, Bellows and Perring) accumulates sorbitol as a thermoprotectant in response to elevated temperature. Sorbitol synthesis in this insect is catalyzed by an unconventional ketose reductase (KR) that uses NADPH to reduce fructose. A cDNA encoding the NADPH-KR from adult B. argentifolii was cloned and sequenced to determine the primary structure of this enzyme. The cDNA encoded a protein of 352 amino acids with a calculated molecular mass of 38.2 kDa. The deduced amino acid sequence of the cDNA shared 60% identity with sheep NAD(+)-dependent sorbitol dehydrogenase (SDH). Residues in SDH involved in substrate binding were conserved in the whitefly NADPH-KR. An important structural difference between the whitefly NADPH-KR and NAD(+)-SDHs occurred in the nucleotide-binding site. The Asp residue that coordinates the adenosyl ribose hydroxyls in NAD(+)-dependent dehydrogenases (including NAD(+)-SDH), was replaced by an Ala in the whitefly NADPH-KR. The whitefly NADPH-KR also contained two neutral to Arg substitutions within four residues of the Asp to Ala substitution. Molecular modeling indicated that addition of the Arg residues and loss of the Asp decreased the electric potential of the adenosine ribose-binding pocket, creating an environment favorable for NADPH-binding. Because of the ability to use NADPH, the whitefly NADPH-KR synthesizes sorbitol under physiological conditions, unlike NAD(+)-SDHs, which function in sorbitol catabolism.

  17. Aerobic Degradation of 2,4,6-Trinitrotoluene by Enterobacter cloacae PB2 and by Pentaerythritol Tetranitrate Reductase

    PubMed Central

    French, Christopher E.; Nicklin, Stephen; Bruce, Neil C.

    1998-01-01

    Enterobacter cloacae PB2 was originally isolated on the basis of its ability to utilize nitrate esters, such as pentaerythritol tetranitrate (PETN) and glycerol trinitrate, as the sole nitrogen source for growth. The enzyme responsible is an NADPH-dependent reductase designated PETN reductase. E. cloacae PB2 was found to be capable of slow aerobic growth with 2,4,6-trinitrotoluene (TNT) as the sole nitrogen source. Dinitrotoluenes were not produced and could not be used as nitrogen sources. Purified PETN reductase was found to reduce TNT to its hydride-Meisenheimer complex, which was further reduced to the dihydride-Meisenheimer complex. Purified PETN reductase and recombinant Escherichia coli expressing PETN reductase were able to liberate nitrogen as nitrite from TNT. The ability to remove nitrogen from TNT suggests that PB2 or recombinant organisms expressing PETN reductase may be useful for bioremediation of TNT-contaminated soil and water. PMID:9687442

  18. Aerobic degradation of 2,4,6-trinitrotoluene by Enterobacter cloacae PB2 and by pentaerythritol tetranitrate reductase

    SciTech Connect

    French, C.E.; Bruce, N.C.; Nicklin, S.

    1998-08-01

    Enterobacter cloacae PB2 was originally isolated on the basis of its ability to utilize nitrate esters, such as pentaerythritol tetranitrate (PETN) and glycerol trinitrate, as the sole nitrogen source for growth. The enzyme responsible is an NADPH-dependent reductase designated PETN reductase. E. cloacae PB2 was found to be capable of slow aerobic growth with 2,4,6-trinitrotoluene (TNT) as the sole nitrogen source. Dinitrotoluenes were not produced and could not be used as nitrogen sources. Purified PETN reductase was found to reduce TNT to its hydride-Meisenheimer complex, which was further reduced to the dihydride-Meisenheimer complex. Purified PETN reductase and recombinant Escherichia coli expressing PETN reductase were able to liberate nitrogen as nitrite from TNT. The ability to remove nitrogen from TNT suggests that PB2 or recombinant organisms expressing PETN reductase may be useful for bioremediation of TNT-contaminated soil and water.

  19. Oxidative and reductive metabolism of lipid-peroxidation derived carbonyls

    PubMed Central

    Singh, Mahavir; Kapoor, Aniruddh; Bhatnagar, Aruni

    2015-01-01

    Extensive research has shown that increased production of reactive oxygen species (ROS) results in tissue injury under a variety of pathological conditions and chronic degenerative diseases. While ROS are highly reactive and can incite significant injury, polyunsaturated lipids in membranes and lipoproteins are their main targets. ROS-triggered lipid peroxidation reactions generate a range of reactive carbonyl species (RCS), and these RCS spread and amplify ROS-related injury. Several RCS generated in oxidizing lipids, such as 4-hydroxy trans-2-nonenal (HNE), 4-oxo-2-(E)-nonenal (ONE), acrolein, malondialdehyde (MDA) and phospholipid aldehydes have been shown to be produced under conditions of oxidative stress and contribute to tissue injury and dysfunction by depleting glutathione and other reductants leading to the modification of proteins, lipids, and DNA. To prevent tissue injury, these RCS are metabolized by several oxidoreductases, including members of the aldo-keto reductase (AKR) superfamily, aldehyde dehydrogenases (ALDHs), and alcohol dehydrogenases (ADHs). Metabolism via these enzymes results in RCS inactivation and detoxification, although under some conditions, it can also lead to the generation of signaling molecules that trigger adaptive responses. Metabolic transformation and detoxification of RCS by oxidoreductases prevent indiscriminate ROS toxicity, while at the same time, preserving ROS signaling. A better understanding of RCS metabolism by oxidoreductases could lead to the development of novel therapeutic interventions to decrease oxidative injury in several disease states and to enhance resistance to ROS-induced toxicity. PMID:25559856

  20. NF-E2-related factor 2 regulates the stress response to UVA-1-oxidized phospholipids in skin cells.

    PubMed

    Gruber, Florian; Mayer, Herbert; Lengauer, Barbara; Mlitz, Veronika; Sanders, John M; Kadl, Alexandra; Bilban, Martin; de Martin, Rainer; Wagner, Oswald; Kensler, Thomas W; Yamamoto, Masayuki; Leitinger, Norbert; Tschachler, Erwin

    2010-01-01

    Long-wavelength ultraviolet (UVA-1) radiation causes oxidative stress that modifies cellular molecules. To defend themselves against noxious oxidation products, skin cells produce detoxifying enzymes and antioxidants. We have recently shown that UVA-1 oxidized the abundant membrane phospholipid 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (PAPC), which then induced the stress-response protein heme oxygenase 1 (HO-1) in dermal fibroblasts. Here we examined the effects of UVA-1- and UV-oxidized phospholipids on global gene expression in human dermal fibroblasts and keratinocytes. We identified a cluster of genes that were coinduced by UVA-1-oxidized PAPC and UVA-1 radiation. The cluster included HO-1, glutamate-cysteine ligase modifier subunit, aldo-keto reductases-1-C1 and -C2, and IL-8. These genes are members of the cellular stress response system termed "antioxidant response." Accordingly, the regulatory regions of all of these genes contain binding sites for NF-E2-related factor 2 (NRF2), a major regulator of the antioxidant response. Both UVA-1 irradiation and treatment with oxidized lipids led to increased nuclear accumulation and DNA binding of NRF2. Silencing and deficiency of NRF2 suppressed the antioxidant response. Taken together, our data show that UVA-1-mediated lipid oxidation induces expression of antioxidant response genes, which is dependent on the redox-regulated transcription factor NRF2. Our findings suggest a different view on UV-generated lipid mediators that were commonly regarded as detrimental

  1. Facile fabrication of CdSe/CdS quantum dots and their application on the screening of colorectal cancer

    NASA Astrophysics Data System (ADS)

    Cao, Hongfeng; Dong, Quanjin; Hu, Li; Tu, Shiliang; Chai, Rui; Dai, Qiaoqiong

    2015-11-01

    In this paper, a facile aqueous route to water-soluble CdSe/CdS quantum dots (QDs) under mild conditions has been developed. The samples were characterized by means of transmission electron microscopy, energy-dispersive X-ray spectroscopy, and photoluminescence (PL) spectroscopy. The PL property of the QDs can be controlled by adjusting the reaction time. The CdSe/CdS QDs after 48-h reaction with size of 5 nm have the strongest PL intensity located at 553 nm, and the highest quantum yield of 19.9 %. The obtained QDs were applied for the colorectal cancer screening. The QDs could be conjugated with antibody of aldo-keto reductase family 1, member B10 (AKR1B10) for the detection of AKR1B10. The AKR1B10 in PBS/5 % serum solution with concentration of 1 ng/mL could be well calibrated, and the limit of detection could be lower than 0.05 ng/mL.

  2. De Novo Sequencing and Analysis of Lemongrass Transcriptome Provide First Insights into the Essential Oil Biosynthesis of Aromatic Grasses

    PubMed Central

    Meena, Seema; Kumar, Sarma R.; Venkata Rao, D. K.; Dwivedi, Varun; Shilpashree, H. B.; Rastogi, Shubhra; Shasany, Ajit K.; Nagegowda, Dinesh A.

    2016-01-01

    Aromatic grasses of the genus Cymbopogon (Poaceae family) represent unique group of plants that produce diverse composition of monoterpene rich essential oils, which have great value in flavor, fragrance, cosmetic, and aromatherapy industries. Despite the commercial importance of these natural aromatic oils, their biosynthesis at the molecular level remains unexplored. As the first step toward understanding the essential oil biosynthesis, we performed de novo transcriptome assembly and analysis of C. flexuosus (lemongrass) by employing Illumina sequencing. Mining of transcriptome data and subsequent phylogenetic analysis led to identification of terpene synthases, pyrophosphatases, alcohol dehydrogenases, aldo-keto reductases, carotenoid cleavage dioxygenases, alcohol acetyltransferases, and aldehyde dehydrogenases, which are potentially involved in essential oil biosynthesis. Comparative essential oil profiling and mRNA expression analysis in three Cymbopogon species (C. flexuosus, aldehyde type; C. martinii, alcohol type; and C. winterianus, intermediate type) with varying essential oil composition indicated the involvement of identified candidate genes in the formation of alcohols, aldehydes, and acetates. Molecular modeling and docking further supported the role of identified protein sequences in aroma formation in Cymbopogon. Also, simple sequence repeats were found in the transcriptome with many linked to terpene pathway genes including the genes potentially involved in aroma biosynthesis. This work provides the first insights into the essential oil biosynthesis of aromatic grasses, and the identified candidate genes and markers can be a great resource for biotechnological and molecular breeding approaches to modulate the essential oil composition. PMID:27516768

  3. Identification and Validation of HCC-specific Gene Transcriptional Signature for Tumor Antigen Discovery

    PubMed Central

    Petrizzo, Annacarmen; Caruso, Francesca Pia; Tagliamonte, Maria; Tornesello, Maria Lina; Ceccarelli, Michele; Costa, Valerio; Aprile, Marianna; Esposito, Roberta; Ciliberto, Gennaro; Buonaguro, Franco M.; Buonaguro, Luigi

    2016-01-01

    A novel two-step bioinformatics strategy was applied for identification of signatures with therapeutic implications in hepatitis-associated HCC. Transcriptional profiles from HBV- and HCV-associated HCC samples were compared with non-tumor liver controls. Resulting HCC modulated genes were subsequently compared with different non-tumor tissue samples. Two related signatures were identified, namely “HCC-associated” and “HCC-specific”. Expression data were validated by RNA-Seq analysis carried out on unrelated HCC samples and protein expression was confirmed according to The Human Protein Atlas" (http://proteinatlas.org/), a public repository of immunohistochemistry data. Among all, aldo-keto reductase family 1 member B10, and IGF2 mRNA-binding protein 3 were found strictly HCC-specific with no expression in 18/20 normal tissues. Target peptides for vaccine design were predicted for both proteins associated with the most prevalent HLA-class I and II alleles. The described novel strategy showed to be feasible for identification of HCC-specific proteins as highly potential target for HCC immunotherapy. PMID:27387388

  4. Methyl Jasmonate: Putative Mechanisms of Action on Cancer Cells Cycle, Metabolism, and Apoptosis

    PubMed Central

    Cesari, Italo Mario; Figueiredo Rodrigues, Mariana; Mendonça, Bruna dos Santos; Amôedo, Nivea Dias; Rumjanek, Franklin David

    2014-01-01

    Methyl jasmonate (MJ), an oxylipid that induces defense-related mechanisms in plants, has been shown to be active against cancer cells both in vitro and in vivo, without affecting normal cells. Here we review most of the described MJ activities in an attempt to get an integrated view and better understanding of its multifaceted modes of action. MJ (1) arrests cell cycle, inhibiting cell growth and proliferation, (2) causes cell death through the intrinsic/extrinsic proapoptotic, p53-independent apoptotic, and nonapoptotic (necrosis) pathways, (3) detaches hexokinase from the voltage-dependent anion channel, dissociating glycolytic and mitochondrial functions, decreasing the mitochondrial membrane potential, favoring cytochrome c release and ATP depletion, activating pro-apoptotic, and inactivating antiapoptotic proteins, (4) induces reactive oxygen species mediated responses, (5) stimulates MAPK-stress signaling and redifferentiation in leukemia cells, (6) inhibits overexpressed proinflammatory enzymes in cancer cells such as aldo-keto reductase 1 and 5-lipoxygenase, and (7) inhibits cell migration and shows antiangiogenic and antimetastatic activities. Finally, MJ may act as a chemosensitizer to some chemotherapics helping to overcome drug resistant. The complete lack of toxicity to normal cells and the rapidity by which MJ causes damage to cancer cells turn MJ into a promising anticancer agent that can be used alone or in combination with other agents. PMID:24648844

  5. Transcriptomic responses of European flounder (Platichthys flesus) liver to a brominated flame retardant mixture.

    PubMed

    Williams, Tim D; Diab, Amer M; Gubbins, Matt; Collins, Catherine; Matejusova, Iveta; Kerr, Rose; Chipman, James K; Kuiper, Raoul; Vethaak, A Dick; George, Stephen G

    2013-10-15

    Male European flounder (Platichthys flesus) were exposed to a technical mixture of brominated diphenyl ethers (PDBEs, DE-71, Pentamix) that had been purified to remove contaminating dioxins. Controls were exposed to carrier solvent alone. Fish were exposed to decadally increasing concentrations of Pentamix via both sediment and spiked food. The GENIPOL P. flesus cDNA microarray, differentially expressed gene profiling (DEG) and quantitative PCR were employed to detect hepatic transcriptional differences between exposed fish and controls. Gene transcriptional changes were more sensitive to Pentamix exposure than biomarkers measured previously. Pentamix exposure induced transcripts coding for enzymes of xenobiotic metabolism (CYP1A, aldo-keto reductases) and elicited endocrine disruption (vitellogenin and thyroid hormone receptor alpha), with effects on CYP1A and VTG occurring at the highest exposure. Ontology analysis clearly showed dose-responsive changes indicative of oxidative stress, induction of mitochondrial dysfunction, and apoptosis. We conclude that exposure to PBDEs in both sediment and food has a significant adverse effect on a broad range of crucial biochemical processes in the livers of this widely distributed estuarine fish species, the flounder.

  6. Endogenous alpha-oxoaldehydes and formation of protein and nucleotide advanced glycation endproducts in tissue damage.

    PubMed

    Thornalley, Paul J

    2007-01-01

    Human and other biological tissues face a continual threat of damage by alpha-oxoaldehydes formed endogenously. Glyoxal, methylglyoxal and 3-deoxyglucosone are formed by the degradation of glycolytic intermediates, glycated proteins and lipid peroxidation. They are potent glycating agents of protein and nucleotides leading to the formation of advanced glycation endproducts (AGEs). With proteins, they are arginine residue-directed glycating agents forming mainly hydroimidazolones, found at 0.1-1% of total arginine residues in tissues (2-20% of proteins modified). With nucleotides, imidazopurinone- and N2-carboxyalkyl- derivatives of deoxyguanosine are formed, found at 0.1-0.8 per 10(6) nucleotides in DNA. Glycation occurs in all tissues and body fluids. Cellular proteolysis of AGE-modified proteins and DNA releases glycated amino acids and nucleosides. Glycated amino acids and nucleosides are released into plasma, undergo glomerular filtration and are excreted in urine. The damage to tissue protein and nucleotides by alpha-oxoaldehydes is suppressed by the metabolism of alpha-oxoaldehyde glycating agents by the glutathione-dependent enzyme, glyoxalase I, and aldo-keto reductases. These enzymatic activities are part of the enzymatic defence against glycation. Tissue damage by alpha-oxoaldehyde glycation is implicated in diabetic and non-diabetic vascular disease, renal failure, cirrhosis, Alzheimer's disease, arthritis and ageing.

  7. Utilization of digital differential display to identify differentially expressed genes related to rumen development.

    PubMed

    Kato, Daichi; Suzuki, Yutaka; Haga, Satoshi; So, KyoungHa; Yamauchi, Eri; Nakano, Miwa; Ishizaki, Hiroshi; Choi, Kichoon; Katoh, Kazuo; Roh, Sang-Gun

    2016-04-01

    This study aimed to identify the genes associated with the development of the rumen epithelium by screening for candidate genes by digital differential display (DDD) in silico. Using DDD in NCBI's UniGene database, expressed sequence tag (EST)-based gene expression profiles were analyzed in rumen, reticulum, omasum, abomasum and other tissues in cattle. One hundred and ten candidate genes with high expression in the rumen were derived from a library of all tissues. The expression levels of 11 genes in all candidate genes were analyzed in the rumen, reticulum, omasum and abomasum of nine Japanese Black male calves (5-week-old pre-weaning: n = 3; 15-week-old weaned calves: n = 6). Among the 11 genes, only 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), aldo-keto reductase family 1, member C1-like (AKR1C1), and fatty acid binding protein 3 (FABP3) showed significant changes in the levels of gene expression in the rumen between the pre- and post-weaning of calves. These results indicate that DDD analysis in silico can be useful for screening candidate genes related to rumen development, and that the changes in expression levels of three genes in the rumen may have been caused by weaning, aging or both.

  8. Modulation of Cell Metabolic Pathways and Oxidative Stress Signaling Contribute to Acquired Melphalan Resistance in Multiple Myeloma Cells

    PubMed Central

    Zub, Kamila Anna; de Sousa, Mirta Mittelstedt Leal; Sarno, Antonio; Sharma, Animesh; Demirovic, Aida; Rao, Shalini; Young, Clifford; Aas, Per Arne; Ericsson, Ida; Sundan, Anders; Jensen, Ole Nørregaard; Slupphaug, Geir

    2015-01-01

    Alkylating agents are widely used chemotherapeutics in the treatment of many cancers, including leukemia, lymphoma, multiple myeloma, sarcoma, lung, breast and ovarian cancer. Melphalan is the most commonly used chemotherapeutic agent against multiple myeloma. However, despite a 70–80% initial response rate, virtually all patients eventually relapse due to the emergence of drug-resistant tumour cells. By using global proteomic and transcriptomic profiling on melphalan sensitive and resistant RPMI8226 cell lines followed by functional assays, we discovered changes in cellular processes and pathways not previously associated with melphalan resistance in multiple myeloma cells, including a metabolic switch conforming to the Warburg effect (aerobic glycolysis), and an elevated oxidative stress response mediated by VEGF/IL8-signaling. In addition, up-regulated aldo-keto reductase levels of the AKR1C family involved in prostaglandin synthesis contribute to the resistant phenotype. Finally, selected metabolic and oxidative stress response enzymes were targeted by inhibitors, several of which displayed a selective cytotoxicity against the melphalan-resistant cells and should be further explored to elucidate their potential to overcome melphalan resistance. PMID:25769101

  9. The SUI-homologous translation initiation factor eIF-1 is involved in regulation of ion homeostasis in rice.

    PubMed

    Diédhiou, C J; Popova, O V; Dietz, K-J; Golldack, D

    2008-05-01

    Halophytes survive high salinity by using complex adaptive mechanisms. In a search for novel molecular mechanisms involved in salt acclimation, transcript analyses revealed increased expression of a SUI-homologous translation initiation factor eIF-1 in the salt-tolerant grass species Festuca rubra ssp. littoralis but not in rice. Upon analysis of the cell specificity of eIF-1 transcription by in situ polymerase chain reaction (PCR), predominant signals were detected in rice leaf mesophyll. To further examine the role of eIF-1 in salt tolerance, transgenic rice plants were generated that over-express this factor under the control of the CaMV-35S promoter. The eIF-1 over-expressing lines showed improved growth under salt stress that was correlated with maintenance of photosynthetic activity and reduced Na(+) and Cl(-) accumulation in leaves. The transgenic rice lines also activated expression of the vacuolar H(+)-ATPase. In addition, an oxidoreductase that belongs to the aldo/keto reductase family was identified as a gene with modified expression in the eIF-1 over-expressing lines, compared with wild-type rice. Our data suggest that eIF-1 has a central function in salt-stress adaptation in rice by regulating ion accumulation and the intracellular redox status.

  10. Modulation of cell metabolic pathways and oxidative stress signaling contribute to acquired melphalan resistance in multiple myeloma cells.

    PubMed

    Zub, Kamila Anna; Sousa, Mirta Mittelstedt Leal de; Sarno, Antonio; Sharma, Animesh; Demirovic, Aida; Rao, Shalini; Young, Clifford; Aas, Per Arne; Ericsson, Ida; Sundan, Anders; Jensen, Ole Nørregaard; Slupphaug, Geir

    2015-01-01

    Alkylating agents are widely used chemotherapeutics in the treatment of many cancers, including leukemia, lymphoma, multiple myeloma, sarcoma, lung, breast and ovarian cancer. Melphalan is the most commonly used chemotherapeutic agent against multiple myeloma. However, despite a 70-80% initial response rate, virtually all patients eventually relapse due to the emergence of drug-resistant tumour cells. By using global proteomic and transcriptomic profiling on melphalan sensitive and resistant RPMI8226 cell lines followed by functional assays, we discovered changes in cellular processes and pathways not previously associated with melphalan resistance in multiple myeloma cells, including a metabolic switch conforming to the Warburg effect (aerobic glycolysis), and an elevated oxidative stress response mediated by VEGF/IL8-signaling. In addition, up-regulated aldo-keto reductase levels of the AKR1C family involved in prostaglandin synthesis contribute to the resistant phenotype. Finally, selected metabolic and oxidative stress response enzymes were targeted by inhibitors, several of which displayed a selective cytotoxicity against the melphalan-resistant cells and should be further explored to elucidate their potential to overcome melphalan resistance.

  11. High expression of AKR1B10 predicts low risk of early tumor recurrence in patients with hepatitis B virus-related hepatocellular carcinoma

    PubMed Central

    Wang, Yan-Yan; Qi, Lu-Nan; Zhong, Jian-Hong; Qin, Hong-Gui; Ye, Jia-Zhou; Lu, Shi-Dong; Ma, Liang; Xiang, Bang-De; Li, Le-Qun; You, Xue-Mei

    2017-01-01

    To clarify the relationship between aldo-keto reductase family 1 member B10 (AKR1B10) expression and early hepatocellular carcinoma (HCC) recurrence, this study detected AKR1B10 expression in tumor and adjacent non-tumor tissues from 110 patients with hepatitis B virus (HBV)-related HCC underwent liver resection and analyzed its correlations with clinicopathological characteristics and prognosis of these patients. Detected by quantitative reverse transcription polymerase chain reaction, AKR1B10 mRNA expression showed significantly higher in HCC tissues than in adjacent non-tumor tissues, with a low level in normal liver tissues. Similar results was confirmed at the protein level using immunohistochemistry and Western blotting. High AKR1B10 expression was negatively correlated with serum alpha-fetoprotein level and positively correlated with HBV-DNA level. Patients with high AKR1B10 expression had significantly higher disease-free survival than those with low expression within 2 years after liver resection. Multivariate analysis also confirmed high AKR1B10 expression to be a predictor of low risk of early HCC recurrence. In addition, high AKR1B10 expression was found to be a favorable factor of overall survival. These results suggest that AKR1B10 is involved in HBV-related hepatocarcinogenesis, but its high expression could predict low risk of early tumor recurrence in patients with HBV-related HCC after liver resection. PMID:28181486

  12. Oxidative Stress and Carbonyl Lesions in Ulcerative Colitis and Associated Colorectal Cancer

    PubMed Central

    Wang, Zhiqi; Li, Sai; Cao, Yu; Tian, Xuefei; Zeng, Rong; Liao, Duan-Fang; Cao, Deliang

    2016-01-01

    Oxidative stress has long been known as a pathogenic factor of ulcerative colitis (UC) and colitis-associated colorectal cancer (CAC), but the effects of secondary carbonyl lesions receive less emphasis. In inflammatory conditions, reactive oxygen species (ROS), such as superoxide anion free radical (O2∙−), hydrogen peroxide (H2O2), and hydroxyl radical (HO∙), are produced at high levels and accumulated to cause oxidative stress (OS). In oxidative status, accumulated ROS can cause protein dysfunction and DNA damage, leading to gene mutations and cell death. Accumulated ROS could also act as chemical messengers to activate signaling pathways, such as NF-κB and p38 MAPK, to affect cell proliferation, differentiation, and apoptosis. More importantly, electrophilic carbonyl compounds produced by lipid peroxidation may function as secondary pathogenic factors, causing further protein and membrane lesions. This may in turn exaggerate oxidative stress, forming a vicious cycle. Electrophilic carbonyls could also cause DNA mutations and breaks, driving malignant progression of UC. The secondary lesions caused by carbonyl compounds may be exceptionally important in the case of host carbonyl defensive system deficit, such as aldo-keto reductase 1B10 deficiency. This review article updates the current understanding of oxidative stress and carbonyl lesions in the development and progression of UC and CAC. PMID:26823956

  13. Stereochemical inversion of (S)-reticuline by a cytochrome P450 fusion in opium poppy.

    PubMed

    Farrow, Scott C; Hagel, Jillian M; Beaudoin, Guillaume A W; Burns, Darcy C; Facchini, Peter J

    2015-09-01

    The gateway to morphine biosynthesis in opium poppy (Papaver somniferum) is the stereochemical inversion of (S)-reticuline since the enzyme yielding the first committed intermediate salutaridine is specific for (R)-reticuline. A fusion between a cytochrome P450 (CYP) and an aldo-keto reductase (AKR) catalyzes the S-to-R epimerization of reticuline via 1,2-dehydroreticuline. The reticuline epimerase (REPI) fusion was detected in opium poppy and in Papaver bracteatum, which accumulates thebaine. In contrast, orthologs encoding independent CYP and AKR enzymes catalyzing the respective synthesis and reduction of 1,2-dehydroreticuline were isolated from Papaver rhoeas, which does not accumulate morphinan alkaloids. An ancestral relationship between these enzymes is supported by a conservation of introns in the gene fusions and independent orthologs. Suppression of REPI transcripts using virus-induced gene silencing in opium poppy reduced levels of (R)-reticuline and morphinan alkaloids and increased the overall abundance of (S)-reticuline and its O-methylated derivatives. Discovery of REPI completes the isolation of genes responsible for known steps of morphine biosynthesis.

  14. Thermostable alcohol dehydrogenase from Thermococcus kodakarensis KOD1 for enantioselective bioconversion of aromatic secondary alcohols.

    PubMed

    Wu, Xi; Zhang, Chong; Orita, Izumi; Imanaka, Tadayuki; Fukui, Toshiaki; Xing, Xin-Hui

    2013-04-01

    A novel thermostable alcohol dehydrogenase (ADH) showing activity toward aromatic secondary alcohols was identified from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1 (TkADH). The gene, tk0845, which encodes an aldo-keto reductase, was heterologously expressed in Escherichia coli. The enzyme was found to be a monomer with a molecular mass of 31 kDa. It was highly thermostable with an optimal temperature of 90°C and a half-life of 4.5 h at 95°C. The apparent K(m) values for the cofactors NAD(P)(+) and NADPH were similar within a range of 66 to 127 μM. TkADH preferred secondary alcohols and accepted various ketones and aldehydes as substrates. Interestingly, the enzyme could oxidize 1-phenylethanol and its derivatives having substituents at the meta and para positions with high enantioselectivity, yielding the corresponding (R)-alcohols with optical purities of greater than 99.8% enantiomeric excess (ee). TkADH could also reduce 2,2,2-trifluoroacetophenone to (R)-2,2,2-trifluoro-1-phenylethanol with high enantioselectivity (>99.6% ee). Furthermore, the enzyme showed high resistance to organic solvents and was particularly highly active in the presence of H2O-20% 2-propanol and H2O-50% n-hexane or n-octane. This ADH is expected to be a useful tool for the production of aromatic chiral alcohols.

  15. De Novo Sequencing and Analysis of Lemongrass Transcriptome Provide First Insights into the Essential Oil Biosynthesis of Aromatic Grasses.

    PubMed

    Meena, Seema; Kumar, Sarma R; Venkata Rao, D K; Dwivedi, Varun; Shilpashree, H B; Rastogi, Shubhra; Shasany, Ajit K; Nagegowda, Dinesh A

    2016-01-01

    Aromatic grasses of the genus Cymbopogon (Poaceae family) represent unique group of plants that produce diverse composition of monoterpene rich essential oils, which have great value in flavor, fragrance, cosmetic, and aromatherapy industries. Despite the commercial importance of these natural aromatic oils, their biosynthesis at the molecular level remains unexplored. As the first step toward understanding the essential oil biosynthesis, we performed de novo transcriptome assembly and analysis of C. flexuosus (lemongrass) by employing Illumina sequencing. Mining of transcriptome data and subsequent phylogenetic analysis led to identification of terpene synthases, pyrophosphatases, alcohol dehydrogenases, aldo-keto reductases, carotenoid cleavage dioxygenases, alcohol acetyltransferases, and aldehyde dehydrogenases, which are potentially involved in essential oil biosynthesis. Comparative essential oil profiling and mRNA expression analysis in three Cymbopogon species (C. flexuosus, aldehyde type; C. martinii, alcohol type; and C. winterianus, intermediate type) with varying essential oil composition indicated the involvement of identified candidate genes in the formation of alcohols, aldehydes, and acetates. Molecular modeling and docking further supported the role of identified protein sequences in aroma formation in Cymbopogon. Also, simple sequence repeats were found in the transcriptome with many linked to terpene pathway genes including the genes potentially involved in aroma biosynthesis. This work provides the first insights into the essential oil biosynthesis of aromatic grasses, and the identified candidate genes and markers can be a great resource for biotechnological and molecular breeding approaches to modulate the essential oil composition.

  16. Involvement of an octose ketoreductase and two acyltransferases in the biosynthesis of paulomycins

    NASA Astrophysics Data System (ADS)

    Li, Jine; Wang, Min; Ding, Yong; Tang, Yue; Zhang, Zhiguo; Chen, Yihua

    2016-02-01

    C-4 hydroxyethyl branched octoses have been observed in polysaccharides of several genera of gram negative bacteria and in various antibiotics produced by gram positive bacteria. The C-4 hydroxyethyl branch was proposed to be converted from C-4 acetyl branch by an uncharacterized ketoreduction step. Paulomycins (PAUs) are glycosylated antibiotics with potent inhibitory activity against gram positive bacteria and are structurally defined by its unique C-4‧ hydroxyethyl branched paulomycose moiety. A novel aldo-keto-reductase, Pau7 was characterized as the enzyme catalyzing the stereospecific ketoreduction of 7‧-keto of PAU E (1) to give the C-4‧ hydroxyethyl branched paulomycose moiety of PAU F (2). An acyltransferase Pau6 further decorates the C-4‧ hydroxyethyl branch of paulomycose moiety of 2 by attaching various fatty acyl chains to 7‧-OH to generate diverse PAUs. In addition, another acyltransferase Pau24 was proposed to be responsible for the 13-O-acetylation of PAUs.

  17. Methylglyoxal functions as Hill oxidant and stimulates the photoreduction of O(2) at photosystem I: a symptom of plant diabetes.

    PubMed

    Saito, Ryota; Yamamoto, Hiroshi; Makino, Amane; Sugimoto, Toshio; Miyake, Chikahiro

    2011-09-01

    We elucidated the metabolism of methylglyoxal (MG) in chloroplasts of higher plants. Spinach chloroplasts showed MG-dependent NADPH oxidation because of aldo-keto reductase (AKR) activity. K(m) for MG and V(max) of AKR activity were 6.5 mm and 3.3 µmol NADPH (mg Chl)(-1) h(-1) , respectively. Addition of MG to illuminated chloroplasts induced photochemical quenching (Qp) of Chl fluorescence, indicating that MG stimulated photosynthetic electron transport (PET). Furthermore, MG enhanced the light-dependent uptake of O(2) into chloroplasts. After illumination of chloroplasts, accumulation of H(2) O(2) was observed. K(m) for MG and V(max) of O(2) uptake were about 100 µm and 200 µmol O(2) (mg Chl)(-1) h(-1) , respectively. MG-dependent O(2) uptake was inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB). Under anaerobic conditions, the Qp of Chl fluorescence was suppressed. These results indicate that MG was reduced as a Hill oxidant by the photosystem I (PSI), and that O(2) was reduced to O(2) (-) by the reduced MG. In other words, MG produced in chloroplasts is preferentially reduced by PSI rather than through AKR. This triggers a type of oxidative stress that may be referred to as 'plant diabetes', because it ultimately originates from a common metabolite of the primary pathways of sugar anabolism and catabolism.

  18. Rapid emergence and mechanisms of resistance by U87 glioblastoma cells to doxorubicin in an in vitro tumor microfluidic ecology

    PubMed Central

    Han, Jeonghun; Jun, Yukyung; Kim, So Hyun; Hoang, Hong-Hoa; Jung, Yeonjoo; Kim, Suyeon; Kim, Jaesang; Austin, Robert H.; Lee, Sanghyuk; Park, Sungsu

    2016-01-01

    In vitro prediction of the probable rapid emergence of resistance to a drug in tumors could act to winnow out potential candidates for further costly development. We have developed a microfluidic device consisting of ∼500 hexagonal microcompartments that provides a complex ecology with wide ranges of drug and nutrient gradients and local populations. This ecology of a fragmented metapopulation induced the drug resistance in stage IV U87 glioblastoma cells to doxorubicin in 7 d. Exome and transcriptome sequencing of the resistant cells identified mutations and differentially expressed genes. Gene ontology and pathway analyses of the genes identified showed that they were functionally relevant to the established mechanisms of doxorubicin action. Specifically, we identified (i) a frame-shift insertion in the filamin-A gene, which regulates the influx and efflux of topoisomerase II poisons; (ii) the overexpression of aldo-keto reductase enzymes, which convert doxorubicin into doxorubicinol; and (iii) activation of NF-κB via alterations in the nucleotide-binding oligomerization domain (NOD)-like receptor signaling pathway from mutations in three genes (CARD6, NSD1, and NLRP13) and the overexpression of inflammatory cytokines. Functional experiments support the in silico analyses and, together, demonstrate the effects of these genetic changes. Our findings suggest that, given the rapid evolution of resistance and the focused response, this technology could act as a rapid screening modality for genetic aberrations leading to resistance to chemotherapy as well as counter selection of drugs unlikely to be successful ultimately. PMID:27911816

  19. Thermostable Alcohol Dehydrogenase from Thermococcus kodakarensis KOD1 for Enantioselective Bioconversion of Aromatic Secondary Alcohols

    PubMed Central

    Wu, Xi; Zhang, Chong; Orita, Izumi; Imanaka, Tadayuki

    2013-01-01

    A novel thermostable alcohol dehydrogenase (ADH) showing activity toward aromatic secondary alcohols was identified from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1 (TkADH). The gene, tk0845, which encodes an aldo-keto reductase, was heterologously expressed in Escherichia coli. The enzyme was found to be a monomer with a molecular mass of 31 kDa. It was highly thermostable with an optimal temperature of 90°C and a half-life of 4.5 h at 95°C. The apparent Km values for the cofactors NAD(P)+ and NADPH were similar within a range of 66 to 127 μM. TkADH preferred secondary alcohols and accepted various ketones and aldehydes as substrates. Interestingly, the enzyme could oxidize 1-phenylethanol and its derivatives having substituents at the meta and para positions with high enantioselectivity, yielding the corresponding (R)-alcohols with optical purities of greater than 99.8% enantiomeric excess (ee). TkADH could also reduce 2,2,2-trifluoroacetophenone to (R)-2,2,2-trifluoro-1-phenylethanol with high enantioselectivity (>99.6% ee). Furthermore, the enzyme showed high resistance to organic solvents and was particularly highly active in the presence of H2O–20% 2-propanol and H2O–50% n-hexane or n-octane. This ADH is expected to be a useful tool for the production of aromatic chiral alcohols. PMID:23354700

  20. Genetic Diversity of Benzoyl Coenzyme A Reductase Genes Detected in Denitrifying Isolates and Estuarine Sediment Communities

    PubMed Central

    Song, Bongkeun; Ward, Bess B.

    2005-01-01

    Benzoyl coenzyme A (benzoyl-CoA) reductase is a central enzyme in the anaerobic degradation of organic carbon, which utilizes a common intermediate (benzoyl-CoA) in the metabolism of many aromatic compounds. The diversity of benzoyl-CoA reductase genes in denitrifying bacterial isolates capable of degrading aromatic compounds and in river and estuarine sediment samples from the Arthur Kill in New Jersey and the Chesapeake Bay in Maryland was investigated. Degenerate primers were developed from the known benzoyl-CoA reductase genes from Thauera aromatica, Rhodopseudomonas palustris, and Azoarcus evansii. PCR amplification detected benzoyl-CoA reductase genes in the denitrifying isolates belonging to α-, β-, or γ-Proteobacteria as well as in the sediment samples. Phylogenetic analysis, sequence similarity comparison, and conserved indel determination grouped the new sequences into either the bcr type (found in T. aromatica and R. palustris) or the bzd type (found in A. evansii). All the Thauera strains and the isolates from the genera Acidovorax, Bradyrhizobium, Paracoccus, Ensifer, and Pseudomonas had bcr-type benzoyl-CoA reductases with amino acid sequence similarities of more than 97%. The genes detected from Azarocus strains were assigned to the bzd type. A total of 50 environmental clones were detected from denitrifying consortium and sediment samples, and 28 clones were assigned to either the bcr or the bzd type of benzoyl-CoA reductase genes. Thus, we could determine the genetic capabilities for anaerobic degradation of aromatic compounds in sediment communities of the Chesapeake Bay and the Arthur Kill on the basis of the detection of two types of benzoyl-CoA reductase genes. The detected genes have future applications as genetic markers to monitor aromatic compound degradation in natural and engineered ecosystems. PMID:15812036

  1. Rapid Identification of Aldose Reductase Inhibitory Compounds from Perilla frutescens

    PubMed Central

    Paek, Ji Hun; Shin, Kuk Hyun; Kang, Young-Hee; Lee, Jae-Yong; Lim, Soon Sung

    2013-01-01

    The ethyl acetate (EtOAc) soluble fraction of methanol extracts of Perilla frutescens (P. frutescens) inhibits aldose reductase (AR), the key enzyme in the polyol pathway. Our investigation of inhibitory compounds from the EtOAc soluble fraction of P. frutescens was followed by identification of the inhibitory compounds by a combination of HPLC microfractionation and a 96-well enzyme assay. This allowed the biological activities to be efficiently matched with selected HPLC peaks. Structural analyses of the active compounds were performed by LC-MSn. The main AR inhibiting compounds were tentatively identified as chlorogenic acid and rosmarinic acid by LC-MSn. A two-step high speed counter current chromatography (HSCCC) isolation method was developed with a solvent system of n-hexane-ethyl acetate-methanol-water at 1.5 : 5 : 1 : 5, v/v and 3 : 7 : 5 : 5, v/v. The chemical structures of the isolated compounds were determined by 1H- and 13C-nuclear magnetic resonance spectrometry (NMR). The main compounds inhibiting AR in the EtOAc fraction of methanol extracts of P. frutescens were identified as chlorogenic acid (2) (IC50 = 3.16 μM), rosmarinic acid (4) (IC50 = 2.77 μM), luteolin (5) (IC50 = 6.34 μM), and methyl rosmarinic acid (6) (IC50 = 4.03 μM). PMID:24308003

  2. Increased 5. cap alpha. -reductase activity in idiopathic hirsutism

    SciTech Connect

    Serafini, P.; Lobo, R.A.

    1985-01-01

    In vitro, genital skin 5..cap alpha..-reductase activity (5..cap alpha..-RA) was measured in ten hirsute women with normal androgen levels (idiopathic hirsutism (IH)) and in ten hirsute women with elevated androgen levels (polycystic ovary syndrome (PCO)) in order to determine the influence of secreted androgens on 5..cap alpha..-RA. In vitro 5..cap alpha..-RA was assessed by incubations of skin with /sup 14/C-testosterone (T) for 2 hours, after which steroids were separated and the radioactivity of dihydrotestosterone (DHT) and 5..cap alpha..-androstane 3..cap alpha..-17..beta..-estradiol (3..cap alpha..-diol) in specific eluates were determined. All androgens were normal in IH with the exception of higher levels of 3..cap alpha..-diol glucuronide which were similar to the levels of PCO. The conversion ratio (CR) of T to DHT in IH and PCO were similar, yet significantly greater than the CR of control subjects. The CR of T to 3..cap alpha..-diol in IH and PCO were similar, yet higher than in control subjects. Serum androgens showed no correlation with 5..cap alpha..-RA, while the CR of T to DHT showed a significant positive correlation with the Ferriman and Gallwey score. The increased 5..cap alpha..-RA in IH appears to be independent of serum androgen levels and is, therefore, an inherent abnormality. The term idiopathic is a misnomer, because hirsutism in these patients may be explained on the basis of increased skin 5..cap alpha..-RA.

  3. Metabolism of bupropion by carbonyl reductases in liver and intestine.

    PubMed

    Connarn, Jamie N; Zhang, Xinyuan; Babiskin, Andrew; Sun, Duxin

    2015-07-01

    Bupropion's metabolism and the formation of hydroxybupropion in the liver by cytochrome P450 2B6 (CYP2B6) has been extensively studied; however, the metabolism and formation of erythro/threohydrobupropion in the liver and intestine by carbonyl reductases (CR) has not been well characterized. The purpose of this investigation was to compare the relative contribution of the two metabolism pathways of bupropion (by CYP2B6 and CR) in the subcellular fractions of liver and intestine and to identify the CRs responsible for erythro/threohydrobupropion formation in the liver and the intestine. The results showed that the liver microsome generated the highest amount of hydroxybupropion (Vmax = 131 pmol/min per milligram, Km = 87 μM). In addition, liver microsome and S9 fractions formed similar levels of threohydrobupropion by CR (Vmax = 98-99 pmol/min per milligram and Km = 186-265 μM). Interestingly, the liver has similar capability to form hydroxybupropion (by CYP2B6) and threohydrobupropion (by CR). In contrast, none of the intestinal fractions generate hydroxybupropion, suggesting that the intestine does not have CYP2B6 available for metabolism of bupropion. However, intestinal S9 fraction formed threohydrobupropion to the extent of 25% of the amount of threohydrobupropion formed by liver S9 fraction. Enzyme inhibition and Western blots identified that 11β-dehydrogenase isozyme 1 in the liver microsome fraction is mainly responsible for the formation of threohydrobupropion, and in the intestine AKR7 may be responsible for the same metabolite formation. These quantitative comparisons of bupropion metabolism by CR in the liver and intestine may provide new insight into its efficacy and side effects with respect to these metabolites.

  4. Human endothelial dihydrofolate reductase low activity limits vascular tetrahydrobiopterin recycling.

    PubMed

    Whitsett, Jennifer; Rangel Filho, Artur; Sethumadhavan, Savitha; Celinska, Joanna; Widlansky, Michael; Vasquez-Vivar, Jeannette

    2013-10-01

    Tetrahydrobiopterin (BH₄) is required for NO synthesis and inhibition of superoxide release from endothelial NO synthase. Clinical trials using BH₄ to treat endothelial dysfunction have produced mixed results. Poor outcomes may be explained by the rapid systemic and cellular oxidation of BH₄. One of the oxidation products of BH₄, 7,8-dihydrobiopterin (7,8-BH₂), is recycled back to BH₄ by dihydrofolate reductase (DHFR). This enzyme is ubiquitously distributed and shows a wide range of activity depending on species-specific factors and cell type. Information about the kinetics and efficiency of BH4 recycling in human endothelial cells receiving BH₄ treatment is lacking. To characterize this reaction, we applied a novel multielectrode coulometric HPLC method that enabled the direct quantification of 7,8-BH₂ and BH₄, which is not possible with fluorescence-based methodologies. We found that basal untreated BH₄ and 7,8-BH₂ concentrations in human endothelial cells (ECs) are lower than in bovine and murine endothelioma cells. Treatment of human ECs with BH₄ transiently increased intracellular BH₄ while accumulating the more stable 7,8-BH₂. This was different from bovine or murine ECs, which resulted in preferential BH₄ increase. Using BH₄ diastereomers, 6S-BH₄ and 6R-BH₄, the narrow contribution of enzymatic DHFR recycling to total intracellular BH₄ was demonstrated. Reduction of 7,8-BH₂ to BH₄ occurs at very slow rates in cells and needs supraphysiological levels of 7,8-BH₂, indicating this reaction is kinetically limited. Activity assays verified that human DHFR has very low affinity for 7,8-BH₂ (DHF7,8-BH₂) and folic acid inhibits 7,8-BH₂ recycling. We conclude that low activity of endothelial DHFR is an important factor limiting the benefits of BH4 therapies, which may be further aggravated by folate supplements.

  5. The Reaction Mechanism of Methyl-Coenzyme M Reductase

    PubMed Central

    Wongnate, Thanyaporn; Ragsdale, Stephen W.

    2015-01-01

    Methyl-coenzyme M reductase (MCR) is a nickel tetrahydrocorphinoid (coenzyme F430) containing enzyme involved in the biological synthesis and anaerobic oxidation of methane. MCR catalyzes the conversion of methyl-2-mercaptoethanesulfonate (methyl-SCoM) and N-7-mercaptoheptanoylthreonine phosphate (CoB7SH) to CH4 and the mixed disulfide CoBS-SCoM. In this study, the reaction of MCR from Methanothermobacter marburgensis, with its native substrates was investigated using static binding, chemical quench, and stopped-flow techniques. Rate constants were measured for each step in this strictly ordered ternary complex catalytic mechanism. Surprisingly, in the absence of the other substrate, MCR can bind either substrate; however, only one binary complex (MCR·methyl-SCoM) is productive whereas the other (MCR·CoB7SH) is inhibitory. Moreover, the kinetic data demonstrate that binding of methyl-SCoM to the inhibitory MCR·CoB7SH complex is highly disfavored (Kd = 56 mm). However, binding of CoB7SH to the productive MCR·methyl-SCoM complex to form the active ternary complex (CoB7SH·MCR(NiI)·CH3SCoM) is highly favored (Kd = 79 μm). Only then can the chemical reaction occur (kobs = 20 s−1 at 25 °C), leading to rapid formation and dissociation of CH4 leaving the binary product complex (MCR(NiII)·CoB7S−·SCoM), which undergoes electron transfer to regenerate Ni(I) and the final product CoBS-SCoM. This first rapid kinetics study of MCR with its natural substrates describes how an enzyme can enforce a strictly ordered ternary complex mechanism and serves as a template for identification of the reaction intermediates. PMID:25691570

  6. The Effect of Protein Mass Modulation on Human Dihydrofolate Reductase

    PubMed Central

    Francis, Kevin; Sapienza, Paul J.; Lee, Andrew L.; Kohen, Amnon

    2016-01-01

    Dihydrofolate reductase (DHFR) from Escherichia coli has long served as a model enzyme with which to elucidate possible links between protein dynamics and the catalyzed reaction. Such physical properties of its human counterpart have not been rigorously studied so far, but recent computer-based simulations suggest that these two DHFRs differ significantly in how closely coupled the protein dynamics and the catalyzed C-H→C hydride transfer step are. To test this prediction, two contemporary probes for studying the effect of protein dynamics on catalysis were combined here: temperature dependence of intrinsic kinetic isotope effects (KIEs) that are sensitive to the physical nature of the chemical step, and protein mass-modulation that slows down fast dynamics (femto- to picosecond timescale) throughout the protein. The intrinsic H/T KIEs of human DHFR, like those of E. coli DHFR, are shown to be temperature-independent in the range from 5–45 °C, indicating fast sampling of donor and acceptor distances (DADs) at the reaction’s transition state (or tunneling ready state – TRS). Mass modulation of these enzymes through isotopic labeling with 13C, 15N, and 2H at nonexchangeable hydrogens yield an 11% heavier enzyme. The additional mass has no effect on the intrinsic KIEs of the human enzyme. This finding indicates that the mass-modulation of the human DHFR affects neither DAD distribution nor the DAD’s conformational sampling dynamics. Furthermore, reduction in the enzymatic turnover number and the dissociation rate constant for the product indicate that the isotopic substitution affects kinetic steps that are not the catalyzed C-H→C hydride transfer. The findings are discussed in terms of fast dynamics and their role in catalysis, the comparison of calculations and experiments, and the interpretation of isotopically-modulated heavy enzymes in general. PMID:26813442

  7. Environmental Adaptation of Dihydrofolate Reductase from Deep-Sea Bacteria.

    PubMed

    Ohmae, Eiji; Gekko, Kunihiko; Kato, Chiaki

    2015-01-01

    In order to elucidate the molecular adaptation mechanisms of enzymes to the high hydrostatic pressure of the deep sea, we cloned, purified, and characterized more than ten dihydrofolate reductases (DHFRs) from bacteria living in deep-sea and ambient atmospheric pressure environments. The nucleotide and amino acid sequences of these DHFRs indicate the deep-sea bacteria are adapted to their environments after the differentiation of their genus from ancestors inhabiting atmospheric pressure environments. In particular, the backbone structure of the deep-sea DHFR from Moritella profunda (mpDHFR) almost overlapped with the normal homolog from Escherichia coli (ecDHFR). Thus, those of other DHFRs would also overlap on the basis of their sequence similarities. However, the structural stability of both DHFRs was quite different: compared to ecDHFR, mpDHFR was more thermally stable but less stable against urea and pressure unfolding. The smaller volume changes due to unfolding suggest that the native structure of mpDHFR has a smaller cavity and/or enhanced hydration compared to ecDHFR. High hydrostatic pressure reduced the enzymatic activity of many DHFRs, but three deep-sea DHFRs and the D27E mutant of ecDHFR exhibited pressure-dependent activation. The inverted activation volumes from positive to negative values indicate the modification of their structural dynamics, conversion of the rate-determining step of the enzymatic reaction, and different contributions of the cavity and hydration to the transition-state structure. Since the cavity and hydration depend on amino acid side chains, DHFRs would adapt to the deep-sea environment by regulating the cavity and hydration by substituting their amino acid side chains without altering their backbone structure. The results of this study clearly indicate that the cavity and hydration play important roles in the adaptation of enzymes to the deep-sea environment.

  8. Pyranopterin Coordination Controls Molybdenum Electrochemistry in Escherichia coli Nitrate Reductase.

    PubMed

    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.

  9. Short-chain dehydrogenases/reductases in cyanobacteria.

    PubMed

    Kramm, Anneke; Kisiela, Michael; Schulz, Rüdiger; Maser, Edmund

    2012-03-01

    The short-chain dehydrogenases/reductases (SDRs) represent a large superfamily of enzymes, most of which are NAD(H)-dependent or NADP(H)-dependent oxidoreductases. They display a wide substrate spectrum, including steroids, alcohols, sugars, aromatic compounds, and xenobiotics. On the basis of characteristic sequence motifs, the SDRs are subdivided into two main (classical and extended) and three smaller (divergent, intermediate, and complex) families. Despite low residue identities in pairwise comparisons, the three-dimensional structure among the SDRs is conserved and shows a typical Rossmann fold. Here, we used a bioinformatics approach to determine whether and which SDRs are present in cyanobacteria, microorganisms that played an important role in our ecosystem as the first oxygen producers. Cyanobacterial SDRs could indeed be identified, and were clustered according to the SDR classification system. Furthermore, because of the early availability of its genome sequence and the easy application of transformation methods, Synechocystis sp. PCC 6803, one of the most important cyanobacterial strains, was chosen as the model organism for this phylum. Synechocystis sp. SDRs were further analysed with bioinformatics tools, such as hidden Markov models (HMMs). It became evident that several cyanobacterial SDRs show remarkable sequence identities with SDRs in other organisms. These so-called 'homologous' proteins exist in plants, model organisms such as Drosophila melanogaster and Caenorhabditis  elegans, and even in humans. As sequence identities of up to 60% were found between Synechocystis and humans, it was concluded that SDRs seemed to have been well conserved during evolution, even after dramatic terrestrial changes such as the conversion of the early reducing atmosphere to an oxidizing one by cyanobacteria.

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

    PubMed

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

    2014-01-01

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

  11. Characterization of thioredoxin glutathione reductase in Schiotosoma japonicum.

    PubMed

    Han, Yanhui; Zhang, Min; Hong, Yang; Zhu, Zhu; Li, Dong; Li, Xiangrui; Fu, Zhiqiang; Lin, Jiaojiao

    2012-09-01

    Schistosomiasis is one of the most prevalent and serious parasitic diseases in the world and remains an important public health problem in China. Screening and discovery of an effective vaccine candidate or new drug target is crucial for the control of this disease. In this study, we cloned a cDNA encoding Schistosoma japonicum (S. japonicum) thioredoxin glutathione reductase (SjTGR) from the cDNA of 42-day-old adult worms. The open reading frame (ORF) of the gene was 1791 base pairs (bp) encoding a protein of 596 amino acids. SjTGR was subcloned into pET-32a (+) and expressed in Escherichia coli (E. coli) BL21 (DE3). The recombinant protein rSjTGR exhibited enzymatic activity of 5.13U/mg with DTNB as the substrate, and showed strong immunogenecity. Real-time PCR results indicated that SjTGR was expressed at a higher level in 35-day-old schistosome worms in transcript. We vaccinated BALB/c mice with rSjTGR in combination with MONTANIDE™ ISA 206 VG (ISA 206) and observed a 33.50% to 36.51% (P<0.01) decrease in the adult worm burden and a 33.73%to 43.44% (P<0.01) decrease in the number of eggs counted compared to the ISA 206 or blank control groups in two independent vaccination tests. ELISA analysis demonstrated that rSjTGR induced a high level of SjTGR-specific IgG, IgG1, and IgG 2a antibodies and induced elevated production of IFN-γ. This study provides the basis for further investigations into the biological function of SjTGR and further evaluation of the potential use of this molecule as a vaccine candidate or new drug target is warranted.

  12. Human carbonyl reductase catalyzes reduction of 4-oxonon-2-enal.

    PubMed

    Doorn, Jonathan A; Maser, Edmund; Blum, Andreas; Claffey, David J; Petersen, Dennis R

    2004-10-19

    4-Oxonon-2-enal (4ONE) was demonstrated to be a product of lipid peroxidation, and previous studies found that it was highly reactive toward DNA and protein. The present study sought to determine whether carbonyl reductase (CR) catalyzes reduction of 4ONE, representing a potential pathway for metabolism of the lipid peroxidation product. Recombinant CR was cloned from a human liver cDNA library, expressed in Escherichia coli, and purified by metal chelate chromatography. Both 4ONE and its glutathione conjugate were found to be substrates for CR, and kinetic parameters were calculated. TLC analysis of reaction products revealed the presence of three compounds, two of which were identified as 4-hydroxynon-2-enal (4HNE) and 1-hydroxynon-2-en-4-one (1HNO). GC/MS analysis confirmed 4HNE and 1HNO and identified the unknown reaction product as 4-oxononanal (4ONA). Analysis of oxime derivatives of the reaction products via LC/MS confirmed the unknown as 4ONA. The time course for CR-mediated, NADPH-dependent 4ONE reduction and appearance of 4HNE and 1HNO was determined using HPLC, demonstrating 4HNE to be a major product and 1HNO and 4ONA to be minor products. Simulated structures of 4ONE in the active site of CR/NADPH calculated via docking experiments predict the ketone positioned as primary hydride acceptor. Results of the present study demonstrate that 4ONE is a substrate for CR/NADPH and the enzyme may represent a pathway for biotransformation of the lipid. Furthermore, these findings reveal that CR catalyzes hydride transfer selectively to the ketone but also to the aldehyde and C=C of 4ONE, resulting in 4HNE, 1HNO, and 4ONA, respectively.

  13. Tales of Dihydrofolate Binding to R67 Dihydrofolate Reductase

    PubMed Central

    2015-01-01

    Homotetrameric R67 dihydrofolate reductase possesses 222 symmetry and a single active site pore. This situation results in a promiscuous binding site that accommodates either the substrate, dihydrofolate (DHF), or the cofactor, NADPH. NADPH interacts more directly with the protein as it is larger than the substrate. In contrast, the p-aminobenzoyl-glutamate tail of DHF, as monitored by nuclear magnetic resonance and crystallography, is disordered when bound. To explore whether smaller active site volumes (which should decrease the level of tail disorder by confinement effects) alter steady state rates, asymmetric mutations that decreased the half-pore volume by ∼35% were constructed. Only minor effects on kcat were observed. To continue exploring the role of tail disorder in catalysis, 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide-mediated cross-linking between R67 DHFR and folate was performed. A two-folate, one-tetramer complex results in the loss of enzyme activity where two symmetry-related K32 residues in the protein are cross-linked to the carboxylates of two bound folates. The tethered folate could be reduced, although with a ≤30-fold decreased rate, suggesting decreased dynamics and/or suboptimal positioning of the cross-linked folate for catalysis. Computer simulations that restrain the dihydrofolate tail near K32 indicate that cross-linking still allows movement of the p-aminobenzoyl ring, which allows the reaction to occur. Finally, a bis-ethylene-diamine-α,γ-amide folate adduct was synthesized; both negatively charged carboxylates in the glutamate tail were replaced with positively charged amines. The Ki for this adduct was ∼9-fold higher than for folate. These various results indicate a balance between folate tail disorder, which helps the enzyme bind substrate while dynamics facilitates catalysis. PMID:26637016

  14. Hydride transfer during catalysis by dihydrofolate reductase from Thermotoga maritima.

    PubMed Central

    Maglia, Giovanni; Javed, Masood H; Allemann, Rudolf K

    2003-01-01

    DHFR (dihydrofolate reductase) catalyses the metabolically important reduction of 7,8-dihydrofolate by NADPH. DHFR from the hyperthermophilic bacterium Thermotoga maritima (TmDHFR), which shares similarity with DHFR from Escherichia coli, has previously been characterized structurally. Its tertiary structure is similar to that of DHFR from E. coli but it is the only DHFR characterized so far that relies on dimerization for stability. The midpoint of the thermal unfolding of TmDHFR was at approx. 83 degrees C, which was 30 degrees C higher than the melting temperature of DHFR from E. coli. The turnover and the hydride-transfer rates in the kinetic scheme of TmDHFR were derived from measurements of the steady-state and pre-steady-state kinetics using absorbance and stopped-flow fluorescence spectroscopy. The rate constant for hydride transfer was found to depend strongly on the temperature and the pH of the solution. Hydride transfer was slow (0.14 s(-1) at 25 degrees C) and at least partially rate limiting at low temperatures but increased dramatically with temperature. At 80 degrees C the hydride-transfer rate of TmDHFR was 20 times lower than that observed for the E. coli enzyme at its physiological temperature. Hydride transfer depended on ionization of a single group in the active site with a p K(a) of 6.0. While at 30 degrees C, turnover of substrate by TmDHFR was almost two orders of magnitude slower than by DHFR from E. coli; the steady-state rates of the two enzymes differed only 8-fold at their respective working temperatures. PMID:12765545

  15. Structural and biochemical properties of cloned and expressed human and rat steroid 5. alpha. -reductases

    SciTech Connect

    Andersson, S.; Russell, D.W. )

    1990-05-01

    The microsomal enzyme steroid 5{alpha}-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 5{alpha}-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 5{alpha}-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 5{alpha}-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 5{alpha}-reductases.

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

    PubMed

    Montalvetti, A; Peña-Díaz, J; Hurtado, R; Ruiz-Pérez, L M; González-Pacanowska, D

    2000-07-01

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

  17. Daio-Orengedokuto inhibits HMG-CoA reductase and pancreatic lipase.

    PubMed

    Kim, Young-Suk; Jung, Eun-Ah; Shin, Ji-Eun; Chang, Jong-Chul; Yang, Hyung-Kil; Kim, Nam-Jae; Cho, Ki-Ho; Bae, Hyung-Sup; Moon, Sang-Kwan; Kim, Dong-Hyun

    2002-11-01

    To evaluate the antihyperlipidemic activities of Orengedokuto (OT) and Daio-Orengedokuto (DOT), the inhibitory effects of these polyprescriptions on HMG-CoA reductase and pancreatic lipase and on the rat hyperlipidemic model induced by Triton WR-1339 were measured. OT potently inhibited HMG-CoA reductase but did not inhibit lipase. Among their ingredients, Coptidis Rhizoma was the most potent inhibitor, followed by Rhei Rhizoma. The HMG-CoA reductase-inhibitory activity of 80% EtOH extract was superior to that of water extract. However, DOT potently inhibited HMG CoA-reductase as well as pancreatic lipase. In the rat hyperlipidemic model induced by Triton WR-1339, OT and DOT decreased serum total cholesterol and low-density lipoprotein cholesterol levels. DOT also decreased serum triglyceride levels, but OT did not decrease it. These results suggest that the antihyperlipidemic activity of DOT may originate from the inhibition of pancreatic lipase as well as HMG-CoA reductase.

  18. Organization of dimethyl sulfoxide reductase in the plasma membrane of Escherichia coli.

    PubMed Central

    Sambasivarao, D; Scraba, D G; Trieber, C; Weiner, J H

    1990-01-01

    Dimethyl sulfoxide reductase is a trimeric, membrane-bound, iron-sulfur molybdoenzyme induced in Escherichia coli under anaerobic growth conditions. The enzyme catalyzes the reduction of dimethyl sulfoxide, trimethylamine N-oxide, and a variety of S- and N-oxide compounds. The topology of dimethyl sulfoxide reductase subunits was probed by a combination of techniques. Immunoblot analysis of the periplasmic proteins from the osmotic shock and chloroform wash fluids indicated that the subunits were not free in the periplasm. The reductase was susceptible to proteases in everted membrane vesicles, but the enzyme in outer membrane-permeabilized cells became protease sensitive only after detergent solubilization of the E. coli plasma membrane. Lactoperoxidase catalyzed the iodination of each of the three subunits in an everted membrane vesicle preparation. Antibodies to dimethyl sulfoxide reductase and fumarate reductase specifically agglutinated the everted membrane vesicles. No TnphoA fusions could be found in the dmsA or -B genes, indicating that these subunits were not translocated to the periplasm. Immunogold electron microscopy of everted membrane vesicles and thin sections by using antibodies to the DmsABC, DmsA, DmsB subunits resulted in specific labeling of the cytoplasmic surface of the inner membrane. These results show that the DmsA (catalytic subunit) and DmsB (electron transfer subunit) are membrane-extrinsic subunits facing the cytoplasmic side of the plasma membrane. Images PMID:2170332

  19. α-Glucosidase and aldose reductase inhibitory activities from the fruiting body of Phellinus merrillii.

    PubMed

    Huang, Guan-Jhong; Hsieh, Wen-Tsong; Chang, Heng-Yuan; Huang, Shyh-Shyun; Lin, Ying-Chih; Kuo, Yueh-Hsiung

    2011-05-25

    The inhibitory activity from the isolated component of the fruiting body Phellinus merrillii (PM) was evaluated against α-glucosidase and lens aldose reductase from Sprague-Dawley male rats and compared to the quercetin as an aldose reductase inhibitor and acarbose as an α-glucosidase inhibitor. The ethanol extracts of PM (EPM) showed the strong α-glucosidase and aldose reductase activities. α-Glucosidase and aldose reductase inhibitors were identified as hispidin (A), hispolon (B), and inotilone (C), which were isolated from EtOAc-soluble fractions of EPM. The above structures were elucidated by their spectra and comparison with the literatures. Among them, hispidin, hispolon, and inotilone exhibited potent against α-glucosidase inhibitor activity with IC(50) values of 297.06 ± 2.06, 12.38 ± 0.13, and 18.62 ± 0.23 μg/mL, respectively, and aldose reductase inhibitor activity with IC(50) values of 48.26 ± 2.48, 9.47 ± 0.52, and 15.37 ± 0.32 μg/mL, respectively. These findings demonstrated that PM may be a good source for lead compounds as alternatives for antidiabetic agents currently used. The importance of finding effective antidiabetic therapeutics led us to further investigate natural compounds.

  20. Structural basis for cyclopropanation by a unique enoyl-acyl carrier protein reductase

    PubMed Central

    Khare, Dheeraj; Hale, Wendi A.; Tripathi, Ashootosh; Gu, Liangcai; Sherman, David H.; Gerwick, William H.; Håkansson, Kristina; Smith, Janet L.

    2015-01-01

    The natural product curacin A, a potent anticancer agent, contains a rare cyclopropane group. The five enzymes for cyclopropane biosynthesis are highly similar to enzymes that generate a vinyl chloride moiety in the jamaicamide natural product. The structural biology of this remarkable catalytic adaptability is probed with high-resolution crystal structures of the curacin cyclopropanase (CurF ER), an in vitro enoyl reductase (JamJ ER), and a canonical curacin enoyl reductase (CurK ER). The JamJ and CurK ERs catalyze NADPH-dependent double bond reductions typical of enoyl reductases (ERs) of the medium chain dehydrogenase reductase (MDR) superfamily. Cyclopropane formation by CurF ER is specified by a short loop which, when transplanted to JamJ ER, confers cyclopropanase activity on the chimeric enzyme. Detection of an adduct of NADPH with the model substrate crotonyl-CoA provides indirect support for a recent proposal of a C2-ene intermediate on the reaction pathway of MDR enoyl-thioester reductases. PMID:26526850

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

    PubMed Central

    Montalvetti, A; Peña-Díaz, J; Hurtado, R; Ruiz-Pérez, L M; González-Pacanowska, D

    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 Leishmania lacks the membrane domain characteristic of eukaryotic cells but exhibits sequence similarity with eukaryotic reductases. Highly purified protein was achieved by ammonium sulphate precipitation followed by chromatography on hydroxyapatite. Kinetic parameters were determined for the protozoan reductase, obtaining K(m) values for the overall reaction of 40.3+/-5.8 microM for (R,S)-HMG-CoA and 81.4+/-5.3 microM for NADPH; V(max) was 33.55+/-1.8 units x mg(-1). Gel-filtration experiments suggested an apparent molecular mass of 184 kDa with subunits of 46 kDa. Finally, in order to achieve a better understanding of the role of this enzyme in trypanosomatids, the effect of possible regulators of isoprenoid biosynthesis in cultured promastigote cells was studied. Neither mevalonic acid nor serum sterols appear to modulate enzyme activity whereas incubation with lovastatin results in significant increases in the amount of reductase protein. Western- and Northern-blot analyses indicate that this activation is apparently performed via post-transcriptional control. PMID:10861207

  2. Enzymatic removal of diacetyl from beer. II. Further studies on the use of diacetyl reductase.

    PubMed

    Tolls, T N; Shovers, J; Sandine, W E; Elliker, P R

    1970-04-01

    Diacetyl removal from beer was studied with whole cells and crude enzyme extracts of yeasts and bacteria. Cells of Streptococcus diacetilactis 18-16 destroyed diacetyl in solutions at a rate almost equal to that achieved by the addition of whole yeast cells. Yeast cells impregnated in a diatomaceous earth filter bed removed all diacetyl from solutions percolated through the bed. Undialyzed crude enzyme extracts from yeast cells removed diacetyl very slowly from beer at its normal pH (4.1); at a pH of 5.0 or higher, rapid diacetyl removal was achieved. Dialyzed crude enzyme extracts from yeast cells were found to destroy diacetyl in a manner quite similar to that of diacetyl reductase from Aerobacter aerogenes, and both the bacterial and the yeast extracts were stimulated significantly by the addition of reduced nicotinamide adenine dinucleotide (NADH). Diacetyl reductase activity of four strains of A. aerogenes was compared; three of the strains produced enzyme with approximately twice the specific activity of the other strain (8724). Gel electrophoresis results indicated that at least three different NADH-oxidizing enzymes were present in crude extracts of diacetyl reductase. Sephadex-gel chromotography separated NADH oxidase from diacetyl reductase. It was also noted that ethyl alcohol concentrations approximately equivalent to those found in beer were quite inhibitory to diacetyl reductase.

  3. Regulation of 5alpha-reductase isoforms by oxytocin in the rat ventral prostate.

    PubMed

    Assinder, S J; Johnson, C; King, K; Nicholson, H D

    2004-12-01

    Oxytocin (OT) is present in the male reproductive tract, where it is known to modulate contractility, cell growth, and steroidogenesis. Little is known about how OT regulates these processes. This study describes the localization of OT receptor in the rat ventral prostate and investigates if OT regulates gene expression and/or activity of 5alpha-reductase isoforms I and II. The ventral prostates of adult male Wistar rats were collected following daily sc administration of saline (control), OT, a specific OT antagonist or both OT plus antagonist for 3 d. Expression of the OT receptor was identified in the ventral prostate by RT-PCR and Western blot, and confirmed to be a single active binding site by radioreceptor assay. Immunohistochemistry localized the receptor to the epithelium of prostatic acini and to the stromal tissue. Real-time RT-PCR determined that OT treatment significantly reduced expression of 5alpha-reductase I but significantly increased 5alpha-reductase II expression in the ventral prostate. Activity of both isoforms of 5alpha-reductase was significantly increased by OT, resulting in increased concentration of prostatic dihydrotestosterone. In conclusion, OT is involved in regulating conversion of testosterone to the biologically active dihydrotestosterone in the rat ventral prostate. It does so by differential regulation of 5alpha-reductase isoforms I and II.

  4. Aldose reductase expression as a risk factor for cataract.

    PubMed

    Snow, Anson; Shieh, Biehuoy; Chang, Kun-Che; Pal, Arttatrana; Lenhart, Patricia; Ammar, David; Ruzycki, Philip; Palla, Suryanarayana; Reddy, G Bhanuprakesh; Petrash, J Mark

    2015-06-05

    Aldose reductase (AR) is thought to play a role in the pathogenesis of diabetic eye diseases, including cataract and retinopathy. However, not all diabetics develop ocular complications. Paradoxically, some diabetics with poor metabolic control appear to be protected against retinopathy, while others with a history of excellent metabolic control develop severe complications. These observations indicate that one or more risk factors may influence the likelihood that an individual with diabetes will develop cataracts and/or retinopathy. We hypothesize that an elevated level of AR gene expression could confer higher risk for development of diabetic eye disease. To investigate this hypothesis, we examined the onset and severity of diabetes-induced cataract in transgenic mice, designated AR-TG, that were either heterozygous or homozygous for the human AR (AKR1B1) transgene construct. AR-TG mice homozygous for the transgene demonstrated a conditional cataract phenotype, whereby they developed lens vacuoles and cataract-associated structural changes only after induction of experimental diabetes; no such changes were observed in AR-TG heterozygotes or nontransgenic mice with or without experimental diabetes induction. We observed that nondiabetic AR-TG mice did not show lens structural changes even though they had lenticular sorbitol levels almost as high as the diabetic AR-TG lenses that showed early signs of cataract. Over-expression of AR led to increases in the ratio of activated to total levels of extracellular signal-regulated kinase (ERK1/2) and c-Jun N-terminal (JNK1/2), which are known to be involved in cell growth and apoptosis, respectively. After diabetes induction, AR-TG but not WT controls had decreased levels of phosphorylated as well as total ERK1/2 and JNK1/2 compared to their nondiabetic counterparts. These results indicate that high AR expression in the context of hyperglycemia and insulin deficiency may constitute a risk factor that could predispose the

  5. Endothelial human dihydrofolate reductase low activity limits vascular tetrahydrobiopterin recycling

    PubMed Central

    Whitsett, Jennifer; Filho, Artur Rangel; Sethumadhavan, Savitha; Celinska, Joanna; Widlansky, Michael; Vásquez-Vivar, Jeannette

    2013-01-01

    Tetrahydrobiopterin (BH4) is required for NO synthesis and inhibition of superoxide release from eNOS. Clinical trials using BH4 to treat endothelial dysfunction have produced mixed results. Poor outcomes may be explained by the rapid systemic and cellular oxidation of BH4. One of the oxidation products of BH4, 7,8-dihydrobiopterin (7,8-BH2), is recycled back to BH4 by dihydrofolate reductase (DHFR). This enzyme is ubiquitously distributed and shows a wide range of activity depending on species-specific factors and cell type. Information about the kinetics and efficiency of BH4 recycling in human endothelial cells receiving BH4 treatment is lacking. To characterize this reaction, we applied a novel multi-electrode coulometric HPLC method that enabled the direct quantification of 7,8-BH2 and BH4 which is not possible with fluorescent-based methodologies. We found that basal untreated BH4 and 7,8-BH2 concentrations in human ECs is lower than bovine and murine endothelioma cells. Treatment of human ECs with BH4 transiently increased intracellular BH4 while accumulating the more stable 7,8-BH2. This was different from bovine or murine ECs that resulted in preferential BH4 increase. Using BH4 diastereomers, 6S-BH4 and 6R-BH4, the narrow contribution of enzymatic DHFR recycling to total intracellular BH4 was demonstrated. Reduction of 7,8-BH2 to BH4 occurs at very slow rates in cells and needs supra-physiological levels of 7,8-BH2, indicating this reaction is kinetically limited. Activity assays verified that hDHFR has very low affinity for 7,8-BH2 (DHF7,8-BH2) and folic acid inhibits 7,8-BH2 recycling. We conclude that low activity of endothelial DHFR is an important factor limiting the benefits of BH4 therapies which may be further aggravated by folate supplements. PMID:23707606

  6. Expression and purification of spinach nitrite reductase in E. coli

    SciTech Connect

    Bellissimo, D.; Privalle, L. )

    1991-03-11

    The study of structure-function relationships in nitrite reductase (NiR) by site-directed mutagenesis requires an expression system from which suitable quantities of active enzyme can be purified. Spinach NiR cDNA was cloned into pUC18 and expressed in E.coli JM109 as a beta-galactosidase fusion protein. The IPTG-induced fusion protein contains five additional amino acids at the N-terminus. The expressed NiR in aerobic cultures was mostly insoluble and inactive indicating the presence of inclusion bodies. By altering growth conditions, active NiR could represent 0.5-1.0% of the total E.coli protein, Effects of the addition of delta-aminolevulinic acid, a heme precursor, and anaerobic growth were also examined. Spinach NiR was purified approximately 200 fold to homogeneity. When subjected to electrophoresis on SDS polyacrylamide gels, the NiR migrated as a single band with similar mobility to pure spinach enzyme. The expressed enzyme also reacted with rabbit anti-spinach NiR antibody as visualized by Western blot analysis. The absorption spectrum of the E.coli-expressed enzyme was identical to spinach enzyme with a Soret and alpha band a 386 and 573 nm, respectively, and an A{sub 278}/A{sub 386} = 1.9. The addition of nitrite produced the characteristic shifts in the spectrum. The E. coli-expressed NiR catalyzed the methylviologen-dependent reduction of nitrite. The specific activity was 100 U/mg. The K{sub m} determined for nitrite was 0.3 mM which is in agreement with values reported for the enzyme. These results indicate that the E.coli-expressed NiR is fully comparable to spinach NiR in purity, catalytic activity and physical state. Site-directed mutants have been made using PCR to examine structure-function relationships in this enzyme.

  7. Characterization of two dissimilatory sulfite reductases from sulfate-reducing bacteria

    NASA Astrophysics Data System (ADS)

    Huynh, B. H.; Moura, I.; Lino, A. R.; Moura, J. J. G.; Legall, J.

    1988-02-01

    Mössbauer, EPR, and biochemical techniques were used to characterize two dissimilatory sulfite reductases: desulforubidin from Desulfovibrio baculatus strain DSM 1743 and desulfoviridin from Desulfovibrio gigas. For each molecule of desulforubidin, there are two sirohemes and four [4Fe-4S] clusters. The [4Fe-4S] clusters are in the diamagnetic 2+ oxidation state. The sirohemes are high-spin ferric (S=5/2) and each siroheme is exchanged-coupled to a [4Fe-4S]2+ cluster. Such an exchange-coupled siroheme-[4Fe-4S] unit has also been found in the assimilatory sulfite reductase from Escherichia coli/1/ and in a low-molecular weight sulfite reductase from Desulfovibrio vulgaris/2/. For each molecule of defulfoviridin, there are two tetrahydroporphyrin groups and four [4Fe-4S]2+ clusters. To our surprise, we discovered that about 80% of the tetrahydroporphyrin groups, however, do not bind iron.

  8. Partial Purification and Characterization of d-Ribose-5-phosphate Reductase from Adonis vernalis L. Leaves

    PubMed Central

    Negm, Fayek B.; Marlow, Gary C.

    1985-01-01

    This study presents evidence for a new enzyme, d-ribose-5-P reductase, which catalyzes the reaction: d-ribose-5-P + NADPH + H+ → d-ribitol-5-P + NADP+. The enzyme was isolated from Adonis vernalis L. leaves in 38% yield and was purified 71-fold. The reductase was NADPH specific and had a pH optimum in the range of 5.5 to 6.0. The Michaelis constant value for d-ribose-5-P reduction was 1.35 millimolar. The enzyme also reduced d-erythrose-4-P, d-erythrose, dl-glyceraldehyde, and the aromatic aldehyde 3-pyridinecarboxaldehyde. Hexoses, hexose phosphates, pentoses, and dihydroxyacetone did not serve as substrates. d-Ribose-5-P reductase is distinct from the other known ribitol synthesizing enzymes detected in bacteria and yeast, and may be responsible for ribitol synthesis in Adonis vernalis. PMID:16664320

  9. Denitrification by plant roots? New aspects of plant plasma membrane-bound nitrate reductase.

    PubMed

    Eick, Manuela; Stöhr, Christine

    2012-10-01

    A specific form of plasma membrane-bound nitrate reductase in plants is restricted to roots. Two peptides originated from plasma membrane integral proteins isolated from Hordeum vulgare have been assigned as homologues to the subunit NarH of respiratory nitrate reductase of Escherichia coli. Corresponding sequences have been detected for predicted proteins of Populus trichocarpa with high degree of identities for the subunits NarH (75%) and NarG (65%), however, with less accordance for the subunit NarI. These findings coincide with biochemical properties, particularly in regard to the electron donors menadione and succinate. Together with the root-specific and plasma membrane-bound nitrite/NO reductase, nitric oxide is produced under hypoxic conditions in the presence of nitrate. In this context, a possible function in nitrate respiration of plant roots and an involvement of plants in denitrification processes are discussed.

  10. Synthetic and Crystallographic Studies of a New Inhibitor Series Targeting Bacillus anthracis Dihydrofolate Reductase

    PubMed Central

    Beierlein, Jennifer M.; Frey, Kathleen M.; Bolstad, David B.; Pelphrey, Phillip M.; Joska, Tammy M.; Smith, Adrienne E.; Priestley, Nigel D.; Wright, Dennis L.; Anderson, Amy C.

    2008-01-01

    Bacillus anthracis, the causative agent of anthrax, poses a significant biodefense danger. Serious limitations in approved therapeutics and the generation of resistance have produced a compelling need for new therapeutic agents against this organism. Bacillus anthracis is known to be insensitive to the clinically used antifolate, trimethoprim, because of a lack of potency against the dihydrofolate reductase enzyme. Herein, we describe a novel lead series of B. anthracis dihydrofolate reductase inhibitors characterized by an extended trimethoprim-like scaffold. The best lead compound adds only 22 Da to the molecular weight and is 82-fold more potent than trimethoprim. An X-ray crystal structure of this lead compound bound to B. anthracis dihydrofolate reductase in the presence of NADPH was determined to 2.25 Å resolution. The structure reveals several features that can be exploited for further development of this lead series. PMID:19007108

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

    PubMed

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

    2016-11-01

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

  12. Synthetic and Crystallographic Studies of a New Inhibitor Series Targeting Bacillus anthracis Dihydrofolate Reductase

    SciTech Connect

    Beierlein, J.; Frey, K; Bolstad, D; Pelphrey, P; Joska, T; Smith, A; Priestley, N; Wright, D; Anderson, A

    2008-01-01

    Bacillus anthracis, the causative agent of anthrax, poses a significant biodefense danger. Serious limitations in approved therapeutics and the generation of resistance have produced a compelling need for new therapeutic agents against this organism. Bacillus anthracis is known to be insensitive to the clinically used antifolate, trimethoprim, because of a lack of potency against the dihydrofolate reductase enzyme. Herein, we describe a novel lead series of B. anthracis dihydrofolate reductase inhibitors characterized by an extended trimethoprim-like scaffold. The best lead compound adds only 22 Da to the molecular weight and is 82-fold more potent than trimethoprim. An X-ray crystal structure of this lead compound bound to B. anthracis dihydrofolate reductase in the presence of NADPH was determined to 2.25 A resolution. The structure reveals several features that can be exploited for further development of this lead series.

  13. Crystallization and preliminary X-ray crystallographic studies of pig heart carbonyl reductase

    SciTech Connect

    Aoki, Ken-ichi; Tanaka, Nobutada; Ishikura, Shuhei; Araki, Naoko; Imamura, Yorishige; Hara, Akira; Nakamura, Kazuo T.

    2006-10-01

    Pig heart carbonyl reductase has been crystallized in the presence of NADPH. Diffraction data have been collected using synchrotron radiation. Pig heart carbonyl reductase (PHCR), which belongs to the short-chain dehydrogenase/reductase (SDR) family, has been crystallized by the hanging-drop vapour-diffusion method. Two crystal forms (I and II) have been obtained in the presence of NADPH. Form I crystals belong to the tetragonal space group P4{sub 2}, with unit-cell parameters a = b = 109.61, c = 94.31 Å, and diffract to 1.5 Å resolution. Form II crystals belong to the tetragonal space group P4{sub 1}2{sub 1}2, with unit-cell parameters a = b = 120.10, c = 147.00 Å, and diffract to 2.2 Å resolution. Both crystal forms are suitable for X-ray structure analysis at high resolution.

  14. Circadian variation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in swine liver and ileum.

    PubMed

    Rogers, D H; Kim, D N; Lee, K T; Reiner, J M; Thomas, W A

    1981-07-01

    The temporal variation of HMG-CoA reductase activity in the liver and intestine of swine was investigated. The thin-layer chromatographic method widely used in the assay of the reductase was successfully applied to the porcine enzymes. Parallel circadian rhythms were demonstrated in both hepatic and ileal reductases from mash-fed animals. Peak activity occurred approximately 6 hr after feeding, 2.7-fold over the basal level in the liver, and 1.6-fold in the ileum. A milk-cholesterol diet caused a marked depression of both rhythms (90% in liver, 50% in ileum); however, the hourly variation in activity persisted in both organs. Cholestyramine was found to elevate hepatic activity (2.7-fold throughout the rhythm) without affecting that of the intestine. Clofibrate had no effect on either enzyme at any time during the cycle despite a 34% reduction in serum cholesterol concentrations.

  15. Molecular docking analysis of UniProtKB nitrate reductase enzyme with known natural flavonoids

    PubMed Central

    Shaik, Ayub; Thumma, Vishnu; Kotha, Aruna Kumari; Kramadhati, Sandhya; Pochampally, Jalapathy; Bandi, Seshagiri

    2016-01-01

    The functional inference of UniProtKB nitrate reductase enzyme (UniProtKB - P0AF33) through structural modeling is of interest in plant biology. Therefore, a homology model for UniProtKB variant of the enzyme was constructed using available data with the MODELER software tool. The model was further docked with five natural flavonoid structures such as hesperetin, naringenin, leucocyanidin, quercetin and hesperetin triacetate using the AUTODOCK (version 4.2) software tool. The structure aided molecular interactions of these flavonoids with nitrate reductase is documented in this study. The binding features (binding energy (ΔG) value, H bonds and docking score) hesperetin to the enzyme model is relatively high, satisfactory and notable. This data provides valuable insights to the relative binding of several naturally occurring flavonoids to nitrate reductase enzyme and its relevance in plant biology.

  16. Purification and properties of a dissimilatory nitrate reductase from Haloferax denitrificans

    NASA Technical Reports Server (NTRS)

    Hochstein, L. I.; Lang, F.

    1991-01-01

    A membrane-bound nitrate reductase (nitrite:(acceptor) oxidoreductase, EC 1.7.99.4) from the extremely halophilic bacterium Haloferax denitrificans was solubilized by incubating membranes in buffer lacking NaCl and purified by DEAE, hydroxylapatite, and Sepharose 6B gel filtration chromatography. The purified nitrate reductase reduced chlorate and was inhibited by azide and cyanide. Preincubating the enzyme with cyanide increased the extent of inhibition which in turn was intensified when dithionite was present. Although cyanide was a noncompetitive inhibitor with respect to nitrate, nitrate protected against inhibition. The enzyme, as isolated, was composed of two subunits (Mr 116,000 and 60,000) and behaved as a dimer during gel filtration (Mr 380,000). Unlike other halobacterial enzymes, this nitrate reductase was most active, as well as stable, in the absence of salt.

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

    PubMed Central

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

    2017-01-01

    Biliverdin reductase catalyses the last step in haem degradation and produces the major lipophilic antioxidant bilirubin via reduction of biliverdin, using NAD(P)H as a cofactor. Despite the importance of biliverdin reductase in maintaining the redox balance, the molecular details of the reaction it catalyses remain unknown. Here we present the crystal structure of biliverdin reductase in complex with biliverdin and NADP+. Unexpectedly, two biliverdin molecules, which we designated the proximal and distal biliverdins, bind with stacked geometry in the active site. The nicotinamide ring of the NADP+ is located close to the reaction site on the proximal biliverdin, supporting that the hydride directly attacks this position of the proximal biliverdin. The results of mutagenesis studies suggest that a conserved Arg185 is essential for the catalysis. The distal biliverdin probably acts as a conduit to deliver the proton from Arg185 to the proximal biliverdin, thus yielding bilirubin. PMID:28169272

  18. Rnr4p, a novel ribonucleotide reductase small-subunit protein.

    PubMed Central

    Wang, P J; Chabes, A; Casagrande, R; Tian, X C; Thelander, L; Huffaker, T C

    1997-01-01

    Ribonucleotide reductases catalyze the formation of deoxyribonucleotides by the reduction of the corresponding ribonucleotides. Eukaryotic ribonucleotide reductases are alpha2beta2 tetramers; each of the larger, alpha subunits possesses binding sites for substrate and allosteric effectors, and each of the smaller, beta subunits contains a binuclear iron complex. The iron complex interacts with a specific tyrosine residue to form a tyrosyl free radical which is essential for activity. Previous work has identified two genes in the yeast Saccharomyces cerevisiae, RNR1 and RNR3, that encode alpha subunits and one gene, RNR2, that encodes a beta subunit. Here we report the identification of a second gene from this yeast, RNR4, that encodes a protein with significant similarity to the beta-subunit proteins. The phenotype of rnr4 mutants is consistent with that expected for a defect in ribonucleotide reductase; rnr4 mutants are supersensitive to the ribonucleotide reductase inhibitor hydroxyurea and display an S-phase arrest at their restrictive temperature. rnr4 mutant extracts are deficient in ribonucleotide reductase activity, and this deficiency can be remedied by the addition of exogenous Rnr4p. As is the case for the other RNR genes, RNR4 is induced by agents that damage DNA. However, Rnr4p lacks a number of sequence elements thought to be essential for iron binding, and mutation of the critical tyrosine residue does not affect Rnr4p function. These results suggest that Rnr4p is catalytically inactive but, nonetheless, does play a role in the ribonucleotide reductase complex. PMID:9315671

  19. Biocatalytic Asymmetric Alkene Reduction: Crystal Structure and Characterization of a Double Bond Reductase from Nicotiana tabacum

    PubMed Central

    2013-01-01

    The application of biocatalysis for the asymmetric reduction of activated C=C is a powerful tool for the manufacture of high-value chemical commodities. The biocatalytic potential of “-ene” reductases from the Old Yellow Enzyme (OYE) family of oxidoreductases is well-known; however, the specificity of these enzymes toward mainly small molecule substrates has highlighted the need to discover “-ene” reductases from different enzymatic classes to broaden industrial applicability. Here, we describe the characterization of a flavin-free double bond reductase from Nicotiana tabacum (NtDBR), which belongs to the leukotriene B4 dehydrogenase (LTD) subfamily of the zinc-independent, medium chain dehydrogenase/reductase superfamily of enzymes. Using steady-state kinetics and biotransformation reactions, we have demonstrated the regio- and stereospecificity of NtDBR against a variety of α,β-unsaturated activated alkenes. In addition to catalyzing the reduction of typical LTD substrates and several classical OYE-like substrates, NtDBR also exhibited complementary activity by reducing non-OYE substrates (i.e., reducing the exocyclic C=C double bond of (R)-pulegone) and in some cases showing an opposite stereopreference in comparison with the OYE family member pentaerythritol tetranitrate (PETN) reductase. This serves to augment classical OYE “-ene” reductase activity and, coupled with its aerobic stability, emphasizes the potential industrial value of NtDBR. Furthermore, we also report the X-ray crystal structures of the holo-, binary NADP(H)-bound, and ternary [NADP+ and 4-hydroxy-3-methoxycinnamaldehyde (9a)-bound] NtDBR complexes. These will underpin structure-driven site-saturated mutagenesis studies aimed at enhancing the reactivity, stereochemistry, and specificity of this enzyme. PMID:27547488

  20. Adverse Effects and Safety of 5-alpha Reductase Inhibitors (Finasteride, Dutasteride): A Systematic Review

    PubMed Central

    Hirshburg, Jason M.; Kelsey, Petra A.; Therrien, Chelsea A.; Gavino, A. Carlo; Reichenberg, Jason S.

    2016-01-01

    Finasteride and dutasteride, both 5-alpha reductase inhibitors, are considered first-line treatment for androgenetic hair loss in men and used increasingly in women. In each case, patients are expected to take the medications indefinitely despite the lack of research regarding long-term adverse effects. Concerns regarding the adverse effects of these medications has led the United States National Institutes of Health to add a link for post-finasteride syndrome to its Genetic and Rare Disease Information Center. Herein, the authors report the results of a literature search reviewing adverse events of 5-alpha reductase inhibitors as they relate to prostate cancer, psychological effects, sexual health, and use in women. Several large studies found no increase in incidence of prostate cancer, a possible increase of high-grade cancer when detected, and no change in survival rate with 5-alpha reductase inhibitor use. Currently, there is no direct link between 5-alpha reductase inhibitor use and depression; however, several small studies have led to depression being listed as a side effect on the medication packaging. Sexual effects including erectile dysfunction and decreased libido and ejaculate were reported in as many as 3.4 to 15.8 percent of men. To date, there are very few studies evaluating 5-alpha reductase inhibitor use in women. Risks include birth defects in male fetuses if used in pregnancy, decreased libido, headache, gastrointestinal discomfort, and isolated reports of changes in menstruation, acne, and dizziness. Overall, 5-alpha reductase inhibitors were well-tolerated in both men and women, but not without risk, highlighting the importance of patient education prior to treatment. PMID:27672412

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

    PubMed

    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

  2. HMG-CoA reductase activity in human liver microsomes: comparative inhibition by statins.

    PubMed

    Dansette, P M; Jaoen, M; Pons, C

    2000-05-01

    The aim of this study was to compare a number of vastatins, HMG-CoA reductase inhibitors, in human liver microsomes. HMG-CoA reductase activity was four times lower than the activity in untreated rat liver microsomes. Vastatins could be classified in this in vitro assay in three classes both in human and rat microsomes: the first one including cerivastatin with an IC50 of 6 nM, the second one with atorvastatin and fluvastatin (IC50) between 40 and 100 nM) and the third one containing pravastatin, simvastatin and lovastatin (IC50 between 100 and 300 nM).

  3. Affinity purifications of aldose reductase and xylitol dehydrogenase from the xylose-fermenting yeast Pachysolen tannophilus

    SciTech Connect

    Bolen, P.L.; Roth, K.A.; Freer, S.N.

    1986-10-01

    Although xylose is a major product of hydrolysis of lignocellulosic materials, few yeasts are able to convert it to ethanol. In Pachysolen tannophilus, one of the few xylose-fermenting yeasts found, aldose reductase and xylitol dehydrogenase were found to be key enzymes in the metabolic pathway for xylose fermentation. This paper presents a method for the rapid and simultaneous purification of both aldose reductase and xylitol dehydrogenase from P. tannophilus. Preliminary studies indicate that this method may be easily adapted to purify similar enzymes from other xylose-fermenting yeasts.

  4. Isolation of xylose reductase gene of Pichia stipitis and its expression in Saccharomyces cerevisiae

    SciTech Connect

    Takuma, Shinya; Nakashima, Noriyuki; Tantirungkij, Manee

    1991-12-31

    A NADPH/NADH-dependent xylose reductase gene was isolated from the xylose-assimilating yeast, Pichia stipitis. DNA sequence analysis showed that the gene consists of 951 bp. The gene introduced in Saccharomyces cerevisiae was transcribed to mRNA, and a considerable amount of enzyme activity was observed constitutively, whereas transcription and translation in P steps were inducible. S. cerevisiae carrying the xylose reductase gene could not, however, grow on xylose medium, and could not produce ethanol from xylose. Since xylose uptake and accumulation of xylitol by S. cerevisiae were observed, the conversion of xylitol to xylulose seemed to be limited.

  5. Synthesis and degradation of nitrate reductase during the cell cycle of Chlorella sorokiniana

    NASA Technical Reports Server (NTRS)

    Velasco, P. J.; Tischner, R.; Huffaker, R. C.; Whitaker, J. R.

    1989-01-01

    Studies on the diurnal variations of nitrate reductase (NR) activity during the life cycle of synchronized Chlorella sorokiniana cells grown with a 7:5 light-dark cycle showed that the NADH:NR activity, as well as the NR partial activities NADH:cytochrome c reductase and reduced methyl viologen:NR, closely paralleled the appearance and disappearance of NR protein as shown by sodium dodecyl sulfate gel electrophoresis and immunoblots. Results of pulse-labeling experiments with [35S]methionine further confirmed that diurnal variations of the enzyme activities can be entirely accounted for by the concomitant synthesis and degradation of the NR protein.

  6. Interaction of Product Analogues With the Active Site of Rhodobacter Sphaeroides Dimethyl Sulfoxide Reductase

    SciTech Connect

    George, G.N.; Nelson, K.J.; Harris, H.H.; Doonan, C.J.; Rajagopalan, K.V.; /Saskatchewan U. /Duke U. /Sydney U.

    2007-07-09

    We report a structural characterization using X-ray absorption spectroscopy of Rhodobacter sphaeroides dimethylsulfoxide (DMSO) reductase reduced with trimethylarsine, and show that this is structurally analogous to the physiologically relevant dimethylsulfide-reduced DMSO reductase. Our data unambiguously indicate that these species should be regarded as formal MoIV species, and indicate a classical coordination complex of trimethylarsine oxide, with no special structural distortions. The similarity of the trimethylarsine and dimethylsulfide complexes suggests in turn that the dimethylsulfide reduced enzyme possesses a classical coordination of DMSO with no special elongation of the S-O bond, as previously suggested.

  7. Production of a recombinant hybrid hemoflavoprotein: engineering a functional NADH:cytochrome c reductase.

    PubMed

    Barber, M J; Quinn, G B

    2001-11-01

    A gene has been constructed coding for a unique fusion protein, NADH:cytochrome c reductase, that comprises the soluble heme-containing domain of rat hepatic cytochrome b(5) as the amino-terminal portion of the protein and the soluble flavin-containing domain of rat hepatic cytochrome b(5) reductase as the carboxyl terminus. The gene has been expressed in Escherichia coli resulting in the highly efficient production of a functional hybrid hemoflavoprotein which has been purified to homogeneity by a combination of ammonium sulfate precipitation, affinity chromatography on 5'-ADP agarose, and size-exclusion chromatography. The purified protein exhibited a molecular mass of approximately 46 kDa by polyacrylamide gel electrophoresis and 40,875 Da, for the apoprotein, using mass spectrometry which also confirmed the presence of both heme and FAD prosthetic groups. The fusion protein showed immunological cross-reactivity with both anti-rat cytochrome b(5) and anti-rat cytochrome b(5) reductase antibodies indicating the conservation of antigenic determinants from both native domains. Spectroscopic analysis indicated the fusion protein contained both a b-type cytochrome and flavin chromophors with properties identical to those of the native proteins. Amino-terminal and internal amino acid sequencing confirmed the identity of peptides derived from both the heme- and flavin-binding domains with sequences identical to the deduced amino acid sequence. The isolated fusion protein retained NADH:ferricyanide reductase activity (k(cat) = 8.00 x 10(2) s(-1), K(NADH)(m) = 4 microM, K(FeCN(6))(m) = 11 microM) comparable to that of that of native NADH:cytochrome b(5) reductase and also exhibited both NADH:cytochrome c reductase activity (k(cat) = 2.17 x 10(2) s(-1), K(NADH)(m) = 2 microM, K(FeCN(6))(m) = 11 microM, K(Cyt.c)(m) = 1 microM) and NADH:methemoglobin reductase activity (k(cat) = 4.40 x 10(-1) s(-1), K(NADH)(m) = 3 microM, K(mHb)(m) = 47 microM), the latter two activities

  8. The haem-copper oxygen reductase of Desulfovibrio vulgaris contains a dihaem cytochrome c in subunit II.

    PubMed

    Lobo, Susana A L; Almeida, Claúdia C; Carita, João N; Teixeira, Miguel; Saraiva, Lígia M

    2008-12-01

    The genome of the sulphate reducing bacterium Desulfovibrio vulgaris Hildenborough, still considered a strict anaerobe, encodes two oxygen reductases of the bd and haem-copper types. The haem-copper oxygen reductase deduced amino acid sequence reveals that it is a Type A2 enzyme, which in its subunit II contains two c-type haem binding motifs. We have characterized the cytochrome c domain of subunit II and confirmed the binding of two haem groups, both with Met-His iron coordination. Hence, this enzyme constitutes the first example of a ccaa3 haem-copper oxygen reductase. The expression of D. vulgaris haem-copper oxygen reductase was found to be independent of the electron donor and acceptor source and is not altered by stress factors such as oxygen exposure, nitrite, nitrate, and iron; therefore the haem-copper oxygen reductase seems to be constitutive. The KCN sensitive oxygen reduction by D. vulgaris membranes demonstrated in this work indicates the presence of an active haem-copper oxygen reductase. D. vulgaris membranes perform oxygen reduction when accepting electrons from the monohaem cytochrome c553, thus revealing the first possible electron donor to the terminal oxygen reductase of D. vulgaris. The physiological implication of the presence of the oxygen reductase in this organism is discussed.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 ...

  10. Cysteine-286 as the site of acylation of the Lux-specific fatty acyl-CoA reductase.

    PubMed

    Lee, C Y; Meighen, E A

    1997-04-04

    The channelling of fatty acids into the fatty aldehyde substrate for the bacterial bioluminescence reaction is catalyzed by a fatty acid reductase multienzyme complex, which channels fatty acids through the thioesterase (LuxD), synthetase (LuxE) and reductase (LuxC) components. Although all three components can be readily acylated in extracts of different luminescent bacteria, this complex has been successfully purified only from Photobacterium phosphoreum and the sites of acylation identified on LuxD and LuxE. To identify the acylation site on LuxC, the nucleotide sequence of P. phosphoreum luxC has been determined and the gene expressed in a mutant Escherichia coli strain. Even in crude extracts, the acylated reductase intermediate as well as acyl-CoA reductase activity could be readily detected, providing the basis for analysis of mutant reductases. Comparison of the amino-acid sequences of LuxC from P. phosphoreum, P. leiognathi and other luminescent bacteria, showed that only three cysteine residues (C171, C279, and C286) were conserved. As a cysteine residue on LuxC has been implicated in fatty acyl transfer, each of the conserved cysteine residues of the P. phosphoreum and P. leiognathi reductases was converted to a serine residue, and the properties of the mutant proteins examined. Only mutation of C286-blocked reductase activity and prevented formation of the acylated reductase intermediate, showing that C286 is the site of acylation on LuxC.

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

    PubMed Central

    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

  12. Multiple types of 8-vinyl reductases for (bacterio)chlorophyll biosynthesis occur in many green sulfur bacteria.

    PubMed

    Liu, Zhenfeng; Bryant, Donald A

    2011-09-01

    Two 8-vinyl reductases, BciA and BciB, have been identified in chlorophototrophs. The bciA gene of Chlorobaculum tepidum was replaced with genes similar to bciB from other green sulfur bacteria. Pigment analyses of the complemented strains showed that the bciB homologs encode 8-vinyl reductases similar to those of cyanobacteria.

  13. Induction of aldose reductase gene expression in LEC rats during the development of the hereditary hepatitis and hepatoma.

    PubMed

    Takahashi, M; Hoshi, A; Fujii, J; Miyoshi, E; Kasahara, T; Suzuki, K; Aozasa, K; Taniguchi, N

    1996-04-01

    We examined age-related changes in the protein and the mRNA expression of aldose reductase in livers of Long-Evans with a cinnamon-like color (LEC) rats, which develop hereditary hepatitis and hepatoma with aging, using Long-Evans with an agouti color rats as controls. The levels of the protein and mRNA of aldose reductase increased after 20 weeks, at the stage of acute hepatitis, and were maintained at 60 weeks of age, while those of aldehyde reductase seemed to be constant at all ages. The expression of aldose reductase was marked in cancerous lesions in hepatoma-bearing LEC rat liver compared to uninvolved surrounding tissues. These results indicated that elevation of aldose reductase accompanied hepatocarcinogenesis and may be related to the acquisition of immortality of the cancer cells through detoxifying cytotoxic aldehyde compounds.

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

    PubMed Central

    Giachetti, Daniela; Biagi, Marco; Manetti, Fabrizio; De Vico, Luca

    2016-01-01

    Benign prostatic hyperplasia is a common disease in men aged over 50 years old, with an incidence increasing to more than 80% over the age of 70, that is increasingly going to attract pharmaceutical interest. Within conventional therapies, such as α-adrenoreceptor antagonists and 5α-reductase inhibitor, there is a large requirement for treatments with less adverse events on, e.g., blood pressure and sexual function: phytotherapy may be the right way to fill this need. Serenoa repens standardized extract has been widely studied and its ability to reduce lower urinary tract symptoms related to benign prostatic hyperplasia is comprehensively described in literature. An innovative investigation on the mechanism of inhibition of 5α-reductase by Serenoa repens extract active principles is proposed in this work through computational methods, performing molecular docking simulations on the crystal structure of human liver 5β-reductase. The results confirm that both sterols and fatty acids can play a role in the inhibition of the enzyme, thus, suggesting a competitive mechanism of inhibition. This work proposes a further confirmation for the rational use of herbal products in the management of benign prostatic hyperplasia, and suggests computational methods as an innovative, low cost, and non-invasive process for the study of phytocomplex activity toward proteic targets. PMID:27904805

  15. Three spinach leaf nitrate reductase-3-hydroxy-3-methylglutaryl-CoA reductase kinases that are required by reversible phosphorylation and/or Ca2+ ions.

    PubMed Central

    Douglas, P; Pigaglio, E; Ferrer, A; Halfords, N G; MacKintosh, C

    1997-01-01

    In spinach (Spinacea oleracea L.) leaf extracts, three protein kinases (PKI, PKII and PKIII) were identified each of which phosphorylated spinach nitrate reductase on serine-543, and inactivated the enzyme in the presence of nitrate reductase inhibitor, 14-3-3. PKIII was also very active in phosphorylating and inactivating Arabidopsis (Landsberg erecta) 3-hydroxy-3-methylglutaryl-coenzyme A reductase 1 (HMGR1). PKI and PKII phosphorylated HMGR1 more slowly than PKIII, compared with their relative rates of phosphorylation of nitrate reductase. HMGR1 identical with those that are seen after phosphorylation of serine-577 by the sucrose non-fermenting (SNF1)-like PK, 3-hydroxy-3-methylglutaryl-Co A reductase kinase A (HRK-A), from cauliflower [Dale, Arró, Becerra, Morrice, Boronat, Hardie and Ferrer (1995) Eur. J. Biochem. 233, 506-513]. PKI was Ca2+-dependent when prepared in the absence of protein phosphatase (PP) inhibitors, and largely Ca2+-dependent when prepared in the presence of PP inhibitors (NaF and EGTA). The Ca2+-independent portion of PKI was inactivated by either PP2A or PP2C, while the Ca2+-dependent portion of PKI became increasingly activated during storage, which we presume was mimicking the effect of an unidentified PP. These findings indicate that PK1 is regulated by two functionally distinct phosphorylations. PKI had a molecular mass of 45 kDa on gel filtration and was active towards substrate peptides that terminated at the +2 residue from the phosphorylation site, whereas PKIII was inactive towards these peptides. PKII was Ca2+-stimulated under all conditions tested. PKIII was Ca2+-indepdented, inactivated by PP2A or PP2C, had a requirement for a hydrophobic residue in the +4 position of peptide substrates, had a molecular mass by gel filtration of approximately 140 kDa, and an antibody against the rye SNF1-related PK (RKIN1) recognized a 58 kDa subunit in fractions containing PKIII. These properties of PKIII are identical with those reported

  16. Structural and docking studies of Leucaena leucocephala Cinnamoyl CoA reductase.

    PubMed

    Prasad, Nirmal K; Vindal, Vaibhav; Kumar, Vikash; Kabra, Ashish; Phogat, Navneet; Kumar, Manoj

    2011-03-01

    Lignin, a major constituent of plant call wall, is a phenolic heteropolymer. It plays a major role in the development of plants and their defense mechanism against pathogens. Therefore Lignin biosynthesis is one of the critical metabolic pathways. In lignin biosynthesis, the Cinnamoyl CoA reductase is a key enzyme which catalyzes the first step in the pathway. Cinnamoyl CoA reductase provides the substrates which represent the main transitional molecules of lignin biosynthesis pathway, exhibits a high in vitro kinetic preference for feruloyl CoA. In present study, the three-dimensional model of cinnamoyl CoA reductase was constructed based on the crystal structure of Grape Dihydroflavonol 4-Reductase. Furthermore, the docking studies were performed to understand the substrate interactions to the active site of CCR. It showed that residues ARG51, ASN52, ASP54 and ASN58 were involved in substrate binding. We also suggest that residue ARG51 in CCR is the determinant residue in competitive inhibition of other substrates. This structural and docking information have prospective implications to understand the mechanism of CCR enzymatic reaction with feruloyl CoA, however the approach will be applicable in prediction of substrates and engineering 3D structures of other enzymes as well.

  17. Type I and type II fatty acid biosynthesis in Eimeria tenella: enoyl reductase activity and structure.

    PubMed

    Lu, J Z; Muench, S P; Allary, M; Campbell, S; Roberts, C W; Mui, E; McLeod, R L; Rice, D W; Prigge, S T

    2007-12-01

    Apicomplexan parasites of the genus Eimeria are the major causative agent of avian coccidiosis, leading to high economic losses in the poultry industry. Recent results show that Eimeria tenella harbours an apicoplast organelle, and that a key biosynthetic enzyme, enoyl reductase, is located in this organelle. In related parasites, enoyl reductase is one component of a type II fatty acid synthase (FAS) and has proven to be an attractive target for antimicrobial compounds. We cloned and expressed the mature form of E. tenella enoyl reductase (EtENR) for biochemical and structural studies. Recombinant EtENR exhibits NADH-dependent enoyl reductase activity and is inhibited by triclosan with an IC50 value of 60 nm. The crystal structure of EtENR reveals overall similarity with other ENR enzymes; however, the active site of EtENR is unoccupied, a state rarely observed in other ENR structures. Furthermore, the position of the central beta-sheet appears to block NADH binding and would require significant movement to allow NADH binding, a feature not previously seen in the ENR family. We analysed the E. tenella genomic database for orthologues of well-characterized bacterial and apicomplexan FAS enzymes and identified 6 additional genes, suggesting that E. tenella contains a type II FAS capable of synthesizing saturated, but not unsaturated, fatty acids. Interestingly, we also identified sequences that appear to encode multifunctional type I FAS enzymes, a feature also observed in Toxoplasma gondii, highlighting the similarity between these apicomplexan parasites.

  18. Feedback regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase in Saccharomyces cerevisiae.

    PubMed Central

    Dimster-Denk, D; Thorsness, M K; Rine, J

    1994-01-01

    In eukaryotic cells all isoprenoids are synthesized from a common precursor, mevalonate. The formation of mevalonate from 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) is catalyzed by HMG-CoA reductase and is the first committed step in isoprenoid biosynthesis. In mammalian cells, synthesis of HMG-CoA reductase is subject to feedback regulation at multiple molecular levels. We examined the state of feedback regulation of the synthesis of the HMG-CoA reductase isozyme encoded by the yeast gene HMG1 to examine the generality of this regulatory pattern. In yeast, synthesis of Hmg1p was subject to feedback regulation. This regulation of HMG-CoA reductase synthesis was independent of any change in the level of HMG1 mRNA. Furthermore, regulation of Hmg1p synthesis was keyed to the level of a nonsterol product of the mevalonate pathway. Manipulations of endogenous levels of several isoprenoid intermediates, either pharmacologically or genetically, suggested that mevalonate levels may control the synthesis of Hmg1p through effects on translation. Images PMID:7949422

  19. The role of enoyl reductase genes in phloridzin biosynthesis in apple.

    PubMed

    Dare, Andrew P; Tomes, Sumathi; Cooney, Janine M; Greenwood, David R; Hellens, Roger P

    2013-11-01

    Phloridzin is the predominant polyphenol in apple (Malus × domestica Borkh.) where it accumulates to high concentrations in many tissues including the leaves, bark, roots and fruit. Despite its relative abundance in apple the biosynthesis of phloridzin and other related dihydrochalcones remains only partially understood. The key unidentified enzyme in phloridzin biosynthesis is a putative carbon double bond reductase which is thought to act on p-coumaroyl-CoA to produce the dihydro-p-coumaroyl-CoA precursor. A functional screen of six apple enoyl reductase-like (ENRL) genes was carried out using transient infiltration into tobacco and gene silencing by RNA interference (RNAi) in order to determine carbon double bond reductase activity and contribution to foliar phloridzin concentrations. The ENRL-3 gene caused a significant increase in phloridzin concentration when infiltrated into tobacco leaves whilst a second protein ENRL-5, with over 98% amino acid sequence similarity to ENRL-3, showed p-coumaroyl-CoA reductase activity in enzyme assays. Finally, an RNAi study showed that reducing the transcript levels of ENRL-3 in transgenic 'Royal Gala' led to a 66% decrease in the concentration of dihydrochalcones in the leaves in the one available silenced line. Overall these results suggest that ENRL-3, and its close homolog ENRL-5, may contribute to the biosynthesis of phloridzin in apple.

  20. The anaerobic ribonucleoside triphosphate reductase from Escherichia coli requires S-adenosylmethionine as a cofactor.

    PubMed Central

    Eliasson, R; Fontecave, M; Jörnvall, H; Krook, M; Pontis, E; Reichard, P

    1990-01-01

    Extracts from anaerobically grown Escherichia coli contain an oxygen-sensitive activity that reduces CTP to dCTP in the presence of NADPH, dithiothreitol, Mg2+ ions, and ATP, different from the aerobic ribonucleoside diphosphate reductase (2'-deoxyribonucleoside-diphosphate: oxidized-thioredoxin 2'-oxidoreductase, EC 1.17.4.1) present in aerobically grown E. coli. After fractionation, the activity required at least five components, two heat-labile protein fractions and several low molecular weight fractions. One protein fraction, suggested to represent the actual ribonucleoside triphosphate reductase was purified extensively and on denaturing gel electrophoresis gave rise to several defined protein bands, all of which were stained by a polyclonal antibody against one of the two subunits (protein B1) of the aerobic reductase but not by monoclonal anti-B1 antibodies. Peptide mapping and sequence analyses revealed partly common structures between two types of protein bands but also suggested the presence of an additional component. Obviously, the preparations are heterogeneous and the structure of the reductase is not yet established. The second, crude protein fraction is believed to contain several ancillary enzymes required for the reaction. One of the low molecular weight components is S-adenosylmethionine; a second component is a loosely bound metal. We propose that S-adenosylmethionine together with a metal participates in the generation of the radical required for the reduction of carbon 2' of the ribosyl moiety of CTP. Images PMID:2185465

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

    SciTech Connect

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

    1989-10-01

    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.

  2. Rhythms of glutathione peroxidase and glutathione reductase in brain of chick and their inhibition by light.

    PubMed

    Pablos, M I; Reiter, R J; Ortiz, G G; Guerrero, J M; Agapito, M T; Chuang, J I; Sewerynek, E

    1998-01-01

    Melatonin was recently shown to be a component of the antioxidative defense system of organisms due to its free radical scavenging and antioxidant activities. Pharmacologically, melatonin stimulates the activity of the peroxide detoxifying enzyme glutathione peroxidase in rat brain and in several tissues of chicks. In this report, we studied the endogenous rhythm of two antioxidant enzymes, glutathione peroxidase and glutathione reductase, in five regions (hippocampus, hypothalamus, striatum, cortex and cerebellum) of chick brain and correlated them with physiological blood melatonin concentrations. Glutathione peroxidase exhibited a marked 24 h rhythm with peak activity in each brain region which had acrophases about 8 h after lights off and about 4 h after the serum melatonin peak was detected. Glutathione reductase activity exhibited similar robust rhythms with the peaks occurring roughly 2 h after those of glutathione peroxidase. We suggest that neural glutathione peroxidase increases due to the rise of nocturnal melatonin levels while glutathione reductase activity rises slightly later possibly due to an increase of its substrate, oxidized glutathione. The exposure of chicks to constant light for 6 days eliminated the melatonin rhythm as well as the peaks in both glutathione peroxidase and glutathione reductase activities. These findings suggest that the melatonin rhythm may be related to the nighttime increases in the enzyme activities, although other explanations cannot be excluded.

  3. Fumarate-Mediated Inhibition of Erythrose Reductase, a Key Enzyme for Erythritol Production by Torula corallina

    PubMed Central

    Lee, Jung-Kul; Koo, Bong-Seong; Kim, Sang-Yong

    2002-01-01

    Torula corallina, a strain presently being used for the industrial production of erythritol, has the highest erythritol yield ever reported for an erythritol-producing microorganism. The increased production of erythritol by Torula corallina with trace elements such as Cu2+ has been thoroughly reported, but the mechanism by which Cu2+ increases the production of erythritol has not been studied. This study demonstrated that supplemental Cu2+ enhanced the production of erythritol, while it significantly decreased the production of a major by-product that accumulates during erythritol fermentation, which was identified as fumarate by instrumental analyses. Erythrose reductase, a key enzyme that converts erythrose to erythritol in T. corallina, was purified to homogeneity by chromatographic methods, including ion-exchange and affinity chromatography. In vitro, purified erythrose reductase was significantly inhibited noncompetitively by increasing the fumarate concentration. In contrast, the enzyme activity remained almost constant regardless of Cu2+ concentration. This suggests that supplemental Cu2+ reduced the production of fumarate, a strong inhibitor of erythrose reductase, which led to less inhibition of erythrose reductase and a high yield of erythritol. This is the first report that suggests catabolite repression by a tricarboxylic acid cycle intermediate in T. corallina. PMID:12200310

  4. Glutathione reductase-mediated synthesis of tellurium-containing nanostructures exhibiting antibacterial properties.

    PubMed

    Pugin, Benoit; Cornejo, Fabián A; Muñoz-Díaz, Pablo; Muñoz-Villagrán, Claudia M; Vargas-Pérez, Joaquín I; Arenas, Felipe A; Vásquez, Claudio C

    2014-11-01

    Tellurium, a metalloid belonging to group 16 of the periodic table, displays very interesting physical and chemical properties and lately has attracted significant attention for its use in nanotechnology. In this context, the use of microorganisms for synthesizing nanostructures emerges as an eco-friendly and exciting approach compared to their chemical synthesis. To generate Te-containing nanostructures, bacteria enzymatically reduce tellurite to elemental tellurium. In this work, using a classic biochemical approach, we looked for a novel tellurite reductase from the Antarctic bacterium Pseudomonas sp. strain BNF22 and used it to generate tellurium-containing nanostructures. A new tellurite reductase was identified as glutathione reductase, which was subsequently overproduced in Escherichia coli. The characterization of this enzyme showed that it is an NADPH-dependent tellurite reductase, with optimum reducing activity at 30°C and pH 9.0. Finally, the enzyme was able to generate Te-containing nanostructures, about 68 nm in size, which exhibit interesting antibacterial properties against E. coli, with no apparent cytotoxicity against eukaryotic cells.

  5. Part of respiratory nitrate reductase of Klebsiella aerogenes is intimately associated with the peptidoglycan.

    PubMed

    Abraham, P R; Wientjes, F B; Nanninga, N; Van't Riet, J

    1987-02-01

    Lysozyme digestion and sonication of sodium dodecyl sulfate (SDS)-purified Klebsiella aerogenes murein sacculi resulted in the quantitative release of both subunits of nitrate reductase, as well as a number of other cytoplasmic membrane polypeptides (5.2%, by weight, of the total membrane proteins). Similar results were obtained after lysozyme digestion of SDS-prepared peptidoglycan fragments, which excluded the phenomenon of simple trapping of the polypeptides by the surrounding peptidoglycan matrix. About 28% of membrane-bound nitrate reductase appears to be tightly associated with the peptidoglycan. Additional evidence for this association was demonstrated by positive immunogold labeling of SDS-murein sacculi and thin sections of plasmolyzed bacteria. Qualitative amino acid analysis of trypsin-treated sacculi, a tryptic product of holo-nitrate reductase, and amino- and carboxypeptidase digests of both nitrate reductase subunits indicated the possible existence of a terminal anchoring peptide containing the following amino acids: (Gly)n, Trp, Ser, Pro, Ile, Leu, Phe, Cys, Tyr, Asp, and Lys.

  6. Electron microscopy of nickel-containing methanogenic enzymes: methyl reductase and F420-reducing hydrogenase.

    PubMed Central

    Wackett, L P; Hartwieg, E A; King, J A; Orme-Johnson, W H; Walsh, C T

    1987-01-01

    Methanogens catalyze the hydrogen-dependent eight-electron reduction of carbon dioxide to methane. Two of the key catalysts in the eight-electron reduction pathway are the nickel-containing enzymes F420-reducing hydrogenase and methyl reductase. In the present study, the structures of these archaebacterial enzymes from Methanobacterium thermoautotrophicum delta H have been determined by electron microscopy. By negative stain techniques, F420 hydrogenase was found to be a ring structure with a diameter of 15.7 nm and an inner channel 4 nm in diameter. Shadow-casting experiments demonstrated that the rings were 8.5 nm deep, indicating a holoenzyme molecular weight of 8.0 X 10(5). Methyl reductase appeared to be an oligomeric complex of dimensions 8.5 by 9 by 11 nm, with a central stain-penetrating region. The morphology and known subunit composition suggest a model in which the subunits are arranged as an eclipsed pair of open trimers. Methyl reductase was also found in the form of larger aggregates and in paracrystalline arrays derived from highly concentrated solutions. The extremely large size of F420 hydrogenase and the methyl reductase supramolecular assemblies may have relevance in vivo in the construction of multiprotein arrays that function in methane biogenesis. Images PMID:3804976

  7. Identification of the Arabidopsis CHL3 gene as the nitrate reductase structural gene NIA2.

    PubMed Central

    Wilkinson, J Q; Crawford, N M

    1991-01-01

    Chlorate, the chlorine analog of nitrate, is a herbicide that has been used to select mutants impaired in the process of nitrate assimilation. In Arabidopsis thaliana, mutations at any one of eight distinct loci confer resistance to chlorate. The molecular identities of the genes at these loci are not known; however, one of these loci--chl3--maps very near the nitrate reductase structural gene NIA2. Through the isolation, characterization, and genetic analysis of new chlorate-resistant mutants generated by gamma irradiation, we have been able to demonstrate that the CHL3 gene and the NIA2 gene are identical. Three new chlorate-resistant mutants were identified that had deletions of the entire NIA2 gene. These nia2 null mutants were viable and still retained 10% of wild-type nitrate reductase activity in the leaves of the plants. All three deletion mutations were found to be new alleles of chl3. Introduction of the NIA2 gene back into these chl3 mutants by Agrobacterium-mediated transformation partially complemented their mutant phenotype. From these data, we conclude that Arabidopsis has at least two functional nitrate reductase genes and that the NIA2 gene product accounts for the majority of the leaf nitrate reductase activity and chlorate sensitivity of Arabidopsis plants. PMID:1840922

  8. A rational approach to identify inhibitors of Mycobacterium tuberculosis enoyl acyl carrier protein reductase.

    PubMed

    Chhabria, Mahesh T; Parmar, Kailash B; Brahmkshatriya, Pathik S

    2013-01-01

    Mycobacterial enoyl acyl carrier protein (ACP) reductase is an attractive target for focused design of novel antitubercular agents. Structural information available on enoyl-ACP reductase in complex with different ligands was used to generate receptor-based pharmacophore model in Discovery Studio (DS). In parallel, pharmacophore models were also generated using ligand-based approach (HypoGen module in DS). Statistically significant models were generated (r(2) = 0.85) which were found to be predictive as indicated from internal and external cross-validations. The model was used as a query tool to search Zinc and Maybridge databases to identify lead compounds and predict their activity in silico. Database searching retrieved many potential lead compounds having better estimated IC50 values than the training set compounds. These compounds were then evaluated for their drug-likeliness and pharmacokinetic properties using DS. Few selected compounds were then docked into the crystal structure of enoyl-ACP reductase using Dock 6.5. Most compounds were found to have high score values, which was found to be consistent with the results from pharmacophore mapping. Additionally, molecular docking provided useful insights into the nature of binding of the identified hit molecules. In summary, we show a useful strategy employing ligand- and structure-based approaches (pharmacophore modeling coupled with molecular docking) to identify new enoyl- ACP reductase inhibitors for antimycobacterial chemotherapy.

  9. Biochemical characterization of an L-Xylulose reductase from Neurospora crassa.

    PubMed

    Nair, Nikhil; Zhao, Huimin

    2007-03-01

    An l-xylulose reductase identified from the genome sequence of the filamentous fungus Neurospora crassa was heterologously expressed in Escherichia coli as a His(6) tag fusion protein, purified, and characterized. The enzyme may be used in the production of xylitol from the major pentose components of hemicellulosic waste, d-xylose and l-arabinose.

  10. Copper-dependent inhibition and oxidative inactivation with affinity cleavage of yeast glutathione reductase.

    PubMed

    Murakami, Keiko; Tsubouchi, Ryoko; Fukayama, Minoru; Yoshino, Masataka

    2014-06-01

    Effects of copper on the activity and oxidative inactivation of yeast glutathione reductase were analyzed. Glutathione reductase from yeast was inhibited by cupric ion and more potently by cuprous ion. Copper ion inhibited the enzyme noncompetitively with respect to the substrate GSSG and NADPH. The Ki values of the enzyme for Cu(2+) and Cu(+) ion were determined to be 1 and 0.35 μM, respectively. Copper-dependent inactivation of glutathione reductase was also analyzed. Hydrogen peroxide and copper/ascorbate also caused an inactivation with the cleavage of peptide bond of the enzyme. The inactivation/fragmentation of the enzyme was prevented by addition of catalase, suggesting that hydroxyl radical produced through the cuprous ion-dependent reduction of oxygen is responsible for the inactivation/fragmentation of the enzyme. SDS-PAGE and TOF-MS analysis confirmed eight fragments, which were further determined to result from the cleavage of the Met17-Ser18, Asn20-Thr21, Glu251-Gly252, Ser420-Pro421, Pro421-Thr422 bonds of the enzyme by amino-terminal sequencing analysis. Based on the kinetic analysis and no protective effect of the substrates, GSSG and NADPH on the copper-mediated inactivation/fragmentation of the enzyme, copper binds to the sites apart from the substrate-sites, causing the peptide cleavage by hydroxyl radical. Copper-dependent oxidative inactivation/fragmentation of glutathione reductase can explain the prooxidant properties of copper under the in vivo conditions.

  11. Structural characterization and functional validation of aldose reductase from the resurrection plant Xerophyta viscosa.

    PubMed

    Singh, Preeti; Sarin, Neera Bhalla

    2014-11-01

    Aldose reductases are key enzymes in the detoxification of reactive aldehyde compounds like methylglyoxal (MG) and malondialdehyde. The present study describes for first time the preliminary biochemical and structural characterization of the aldose reductase (ALDRXV4) enzyme from the resurrection plant Xerophyta viscosa. The ALDRXV4 cDNA was expressed in E. coli using pET28a expression vector, and the protein was purified using affinity chromatography. The recombinant protein showed a molecular mass of ~36 kDa. The K M (1.2 mM) and k cat (14.5 s(-1)) of the protein determined using MG as substrate was found to be comparable with other reported homologs. Three-dimensional structure prediction based on homology modeling suggested several similarities with the other aldose reductases reported from plants. Circular dichroism spectroscopy results supported the bioinformatic prediction of alpha-beta helix nature of aldose reductase proteins. Subcellular localization studies revealed that the ALDRXV4-GFP fusion protein was localized both in the nucleus and the cytoplasm. The E. coli cells overexpressing ALDRXV4 exhibited improved growth and showed tolerance against diverse abiotic stresses induced by high salt (500 mM NaCl), osmoticum (10 % PEG 6000), heavy metal (20 mM CdCl2), and MG (5 mM). Based on these results, we propose that ALDRXV4 gene from X. viscosa could be a potential candidate for developing stress-tolerant crop plants.

  12. Glutathione Reductase-Mediated Synthesis of Tellurium-Containing Nanostructures Exhibiting Antibacterial Properties

    PubMed Central

    Pugin, Benoit; Cornejo, Fabián A.; Muñoz-Díaz, Pablo; Muñoz-Villagrán, Claudia M.; Vargas-Pérez, Joaquín I.; Arenas, Felipe A.

    2014-01-01

    Tellurium, a metalloid belonging to group 16 of the periodic table, displays very interesting physical and chemical properties and lately has attracted significant attention for its use in nanotechnology. In this context, the use of microorganisms for synthesizing nanostructures emerges as an eco-friendly and exciting approach compared to their chemical synthesis. To generate Te-containing nanostructures, bacteria enzymatically reduce tellurite to elemental tellurium. In this work, using a classic biochemical approach, we looked for a novel tellurite reductase from the Antarctic bacterium Pseudomonas sp. strain BNF22 and used it to generate tellurium-containing nanostructures. A new tellurite reductase was identified as glutathione reductase, which was subsequently overproduced in Escherichia coli. The characterization of this enzyme showed that it is an NADPH-dependent tellurite reductase, with optimum reducing activity at 30°C and pH 9.0. Finally, the enzyme was able to generate Te-containing nanostructures, about 68 nm in size, which exhibit interesting antibacterial properties against E. coli, with no apparent cytotoxicity against eukaryotic cells. PMID:25193000

  13. The crystal structure of dihydrofolate reductase from Thermotoga maritima: molecular features of thermostability.

    PubMed

    Dams, T; Auerbach, G; Bader, G; Jacob, U; Ploom, T; Huber, R; Jaenicke, R

    2000-03-31

    Two high-resolution structures have been obtained for dihydrofolate reductase from the hyperthermophilic bacterium Thermotoga maritima in its unliganded state, and in its ternary complex with the cofactor NADPH and the inhibitor, methotrexate. While the overall fold of the hyperthermophilic enzyme is closely similar to monomeric mesophilic dihydrofolate reductase molecules, its quaternary structure is exceptional, in that T. maritima dihydrofolate reductase forms a highly stable homodimer. Here, the molecular reasons for the high intrinsic stability of the enzyme are elaborated and put in context with the available data on the physical parameters governing the folding reaction. The molecule is extremely rigid, even with respect to structural changes during substrate binding and turnover. Subunit cooperativity can be excluded from structural and biochemical data. Major contributions to the high intrinsic stability of the enzyme result from the formation of the dimer. Within the monomer, only subtle stabilizing interactions are detectable, without clear evidence for any of the typical increments of thermal stabilization commonly reported for hyperthermophilic proteins. The docking of the subunits is optimized with respect to high packing density in the dimer interface, additional salt-bridges and beta-sheets. The enzyme does not show significant structural changes upon binding its coenzyme, NADPH, and the inhibitor, methotrexate. The active-site loop, which is known to play an important role in catalysis in mesophilic dihydrofolate reductase molecules, is rearranged, participating in the association of the subunits; it no longer participates in catalysis.

  14. Aldose reductase induced by hyperosmotic stress mediates cardiomyocyte apoptosis: differential effects of sorbitol and mannitol.

    PubMed

    Galvez, Anita S; Ulloa, Juan Alberto; Chiong, Mario; Criollo, Alfredo; Eisner, Verónica; Barros, Luis Felipe; Lavandero, Sergio

    2003-10-03

    Cells adapt to hyperosmotic conditions by several mechanisms, including accumulation of sorbitol via induction of the polyol pathway. Failure to adapt to osmotic stress can result in apoptotic cell death. In the present study, we assessed the role of aldose reductase, the key enzyme of the polyol pathway, in cardiac myocyte apoptosis. Hyperosmotic stress, elicited by exposure of cultured rat cardiac myocytes to the nonpermeant solutes sorbitol and mannitol, caused identical cell shrinkage and adaptive hexose uptake stimulation. In contrast, only sorbitol induced the polyol pathway and triggered stress pathways as well as apoptosis-related signaling events. Sorbitol resulted in activation of the extracellular signal-regulated kinase (ERK), p54 c-Jun N-terminal kinase (JNK), and protein kinase B. Furthermore, sorbitol treatment resulting in induction and activation of aldose reductase, decreased expression of the antiapoptotic protein Bcl-xL, increased DNA fragmentation, and glutathione depletion. Apoptosis was attenuated by aldose reductase inhibition with zopolrestat and also by glutathione replenishment with N-acetylcysteine. In conclusion, our data show that hypertonic shrinkage of cardiac myocytes alone is not sufficient to induce cardiac myocyte apoptosis. Hyperosmolarity-induced cell death is sensitive to the nature of the osmolyte and requires induction of aldose reductase as well as a decrease in intracellular glutathione levels.

  15. Biochemical and crystallographic characterization of ferredoxin-NADP(+) reductase from nonphotosynthetic tissues.

    PubMed

    Aliverti, A; Faber, R; Finnerty, C M; Ferioli, C; Pandini, V; Negri, A; Karplus, P A; Zanetti, G

    2001-12-04

    Distinct forms of ferredoxin-NADP(+) reductase are expressed in photosynthetic and nonphotosynthetic plant tissues. Both enzymes catalyze electron transfer between NADP(H) and ferredoxin; whereas in leaves the enzyme transfers reducing equivalents from photoreduced ferredoxin to NADP(+) in photosynthesis, in roots it has the opposite physiological role, reducing ferredoxin at the expense of NADPH mainly for use in nitrate assimilation. Here, structural and kinetic properties of a nonphotosynthetic isoform were analyzed to define characteristics that may be related to tissue-specific function. Compared with spinach leaf ferredoxin-NADP(+) reductase, the recombinant corn root isoform showed a slightly altered absorption spectrum, a higher pI, a >30-fold higher affinity for NADP(+), greater susceptibility to limited proteolysis, and an approximately 20 mV more positive redox potential. The 1.7 A resolution crystal structure is very similar to the structures of ferredoxin-NADP(+) reductases from photosynthetic tissues. Four distinct structural features of this root ferredoxin-NADP(+) reductases are an alternate conformation of the bound FAD molecule, an alternate path for the amino-terminal extension, a disulfide bond in the FAD-binding domain, and changes in the surface that binds ferredoxin.

  16. Properties of some reductase enzymes in the nitrifying bacteria and their relationship to the oxidase systems

    PubMed Central

    Wallace, W.; Nicholas, D. J. D.

    1968-01-01

    The reductase enzymes in Nitrosomonas and Nitrobacter were studied under anaerobic conditions when the oxidase enzymes were inactive. The most effective electron-donor systems for nitrate reductase in Nitrobacter were reduced benzyl viologen alone, phenazine methosulphate with either NADH or NADPH, and FMN or FAD with NADH. Nitrite and hydroxylamine reductases were found in both nitrifying bacteria, and optimum activity for each enzyme was obtained with NADH or NADPH with either FMN or FAD. The product of both these enzymes was identified as ammonia. In extracts of Nitrosomonas the ammonia was further utilized by an NADPH-specific glutamate dehydrogenase. 15N-labelled nitrite, hydroxylamine and ammonia were rapidly incorporated into cell protein by Nitrosomonas, and Nitrobacter in addition incorporated [15N]nitrate. Relatively gentle methods of cell disruption were compared with ultrasonic treatment, to enable a more exact study to be undertaken of the intracellular distribution of the oxidase and reductase enzymes. The functional relationship of these opposing enzyme systems in the nitrifying bacteria is considered. PMID:4386932

  17. Novel anti-Prelog stereospecific carbonyl reductases from Candida parapsilosis for asymmetric reduction of prochiral ketones

    PubMed Central

    Nie, Yao; Xiao, Rong; Xu, Yan; Montelione, Gaetano T.

    2014-01-01

    Application of biocatalysis in the synthesis of chiral molecules is one of the greenest technologies for the replacement of chemical routes due to its environmentally benign reaction conditions and unparalleled chemo-, regio-and stereoselectivities. We have been interested in searching for carbonyl reductase enzymes and assessing their substrate specificity and stereoselectivity. We now report a gene cluster identified in Candida parapsilosis that consists of four open reading frames including three putative stereospecific carbonyl reductases (scr1, scr2, and scr3) and an alcohol dehydrogenase (cpadh). These newly identified three stereospecific carbonyl reductases (SCRs) showed high catalytic activities for producing (S)-1-phenyl-1,2-ethanediol from 2-hydroxyacetophenone with NADPH as the coenzyme. Together with CPADH, all four enzymes from this cluster are carbonyl reductases with novel anti-Prelog stereoselectivity. SCR1 and SCR3 exhibited distinct specificities to acetophenone derivatives and chloro-substituted 2-hydroxyacetophenones, and especially very high activities to ethyl 4-chloro-3-oxobutyrate, a β-ketoester with important pharmaceutical potentials. Our study also showed that genomic mining is a powerful tool for the discovery of new enzymes. PMID:21505708

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  19. Differential antioxidant and quinone reductase inducing activity of American, Asian, and Siberian ginseng

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The antioxidant and quinone reductase (QR) inducing activities of American, Asian, and Siberian ginseng have been reported using various plant materials, solvents, and assays. To directly establish their comparative bioactivity, the effects of extracts obtained from acidified methanol (MeOH), a gas...

  20. Cloning and sequencing of the genes from Salmonella typhimurium encoding a new bacterial ribonucleotide reductase.

    PubMed Central

    Jordan, A; Gibert, I; Barbé, J

    1994-01-01

    A plasmid library of Salmonella typhimurium was used to complement a temperature-sensitive nrdA mutant of Escherichia coli. Complementation was obtained with two different classes of plasmids, one carrying the E. coli nrdAB-like genes and the second containing an operon encoding a new bacterial ribonucleotide reductase. Plasmids harboring these new reductase genes also enable obligately anaerobic nrdB::Mud1 E. coli mutants to grow in the presence of oxygen. This operon consists of two open reading frames, which have been designated nrdE (2,145 bp) and nrdF (969 bp). The deduced amino acid sequences of the nrdE and nrdF products include the catalytically important residues conserved in ribonucleotide reductase enzymes of class I and show 25 and 28% overall identity with the R1 and R2 protein, respectively, of the aerobic ribonucleoside diphosphate reductase of E. coli. The 3' end of the sequenced 4.9-kb fragment corresponds to the upstream region of the previously published proU operon of both S. typhimurium and E. coli, indicating that the nrdEF genes are at 57 min on the chromosomal maps of these two bacterial species. Analysis of the nrdEF and proU sequences demonstrates that transcription of the nrdEF genes is in the clockwise direction on the S. typhimurium and E. coli maps. Images PMID:8195103

  1. Novel anti-Prelog stereospecific carbonyl reductases from Candida parapsilosis for asymmetric reduction of prochiral ketones.

    PubMed

    Nie, Yao; Xiao, Rong; Xu, Yan; Montelione, Gaetano T

    2011-06-07

    The application of biocatalysis to the synthesis of chiral molecules is one of the greenest technologies for the replacement of chemical routes due to its environmentally benign reaction conditions and unparalleled chemo-, regio- and stereoselectivities. We have been interested in searching for carbonyl reductase enzymes and assessing their substrate specificity and stereoselectivity. We now report a gene cluster identified in Candida parapsilosis that consists of four open reading frames including three putative stereospecific carbonyl reductases (scr1, scr2, and scr3) and an alcohol dehydrogenase (cpadh). These newly identified three stereospecific carbonyl reductases (SCRs) showed high catalytic activities for producing (S)-1-phenyl-1,2-ethanediol from 2-hydroxyacetophenone with NADPH as the coenzyme. Together with CPADH, all four enzymes from this cluster are carbonyl reductases with novel anti-Prelog stereoselectivity. SCR1 and SCR3 exhibited distinct specificities to acetophenone derivatives and chloro-substituted 2-hydroxyacetophenones, and especially very high activities towards ethyl 4-chloro-3-oxobutyrate, a β-ketoester with important pharmaceutical potential. Our study also showed that genomic mining is a powerful tool for the discovery of new enzymes.

  2. Loss and stabilization of amplified dihydrofolate reductase genes in mouse sarcoma S-180 cell lines

    SciTech Connect

    Kaufman, R.J.; Brown, P.C.; Schimke, R.T.

    1981-12-01

    The authors studied the loss and stabilization of dihydrofolate reductase genes in clones of a methotrexate-resistant murine S-180 cell line. These cells contained multiple copies of the dihydrofolate reductase gene which were associated with double minute chromosomes. The growth rate of these cells in the absence of methotrexate was inversely related to the degree of gene amplification (number of double minute chromosomes). Cells could both gain and lose genes as a result of an unequal distribution of double minute chromosomes into daughter cells at mitosis. The loss of amplified dihydrofolate reductase genes during growth in the absence of methotrexate resulted from the continual generation of cells containing lower numbers of double minute chromosomes. Because of the growth advantage of these cells, they became dominant in the population. They also studied an unstably resistant S-180 cell line (clone) that, after 3 years of continuous growth in methotrexate, generated cells containing stably amplified dihydrofolate reductase genes. These genes were present on one or more chromosomes, and they were retained in a stable state.

  3. Use of a Simple, Colorimetric Assay to Demonstrate Conditions for Induction of Nitrate Reductase in Plants.

    ERIC Educational Resources Information Center

    Harley, Suzanne M.

    1993-01-01

    Nitrate assimilation by plants provides an excellent system for demonstrating control of gene expression in a eukaryotic organism. Describes an assay method that allows students to complete experiments designed around the measurement of nitrate reductase within a three-hour laboratory experiment. (PR)

  4. Nitrate reduction in Haloferax alexandrinus: the case of assimilatory nitrate reductase.

    PubMed

    Kilic, Volkan; Kilic, Gözde Aydoğan; Kutlu, Hatice Mehtap; Martínez-Espinosa, Rosa María

    2017-03-21

    Haloferax alexandrinus Strain TM JCM 10717(T) = IFO 16590(T) is an extreme halophilic archaeon able to produce significant amounts of canthaxanthin. Its genome sequence has been analysed in this work using bioinformatics tools available at Expasy in order to look for genes encoding nitrate reductase-like proteins: respiratory nitrate reductase (Nar) and/or assimilatory nitrate reductase (Nas). The ability of the cells to reduce nitrate under aerobic conditions was tested. The enzyme in charge of nitrate reduction under aerobic conditions (Nas) has been purified and characterised. It is a monomeric enzyme (72 ± 1.8 kDa) that requires high salt concentration for stability and activity. The optimum pH value for activity was 9.5. Effectiveness of different substrates, electron donors, cofactors and inhibitors was also reported. High nitrite concentrations were detected within the culture media during aerobic/microaerobic cells growth. The main conclusion from the results is that this haloarchaeon reduces nitrate aerobically thanks to Nas and may induce denitrification under anaerobic/microaerobic conditions using nitrate as electron acceptor. The study sheds light on the role played by haloarchaea in the biogeochemical cycle of nitrogen, paying special attention to nitrate reduction processes. Besides, it provides useful information for future attempts on microecological and biotechnological implications of haloarchaeal nitrate reductases.

  5. Inhibition of human anthracycline reductases by emodin - A possible remedy for anthracycline resistance.

    PubMed

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

    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 IC50- and Ki-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.

  6. X-Ray Absorption Spectroscopic Characterization of the Molybdenum Site of 'Escherichia Coli' Dimethyl Sulfoxide Reductase

    SciTech Connect

    George, G.N.; Doonan, C.J.; Rothery, R.A.; Boroumand, N.; Weiner, J.H.; /Saskatchewan U. /Alberta U.

    2007-07-09

    Structural studies of dimethyl sulfoxide (DMSO) reductases were hampered by modification of the active site during purification. We report an X-ray absorption spectroscopic analysis of the molybdenum active site of Escherichia coli DMSO reductase contained within its native membranes. The enzyme in these preparations is expected to be very close to the form found in vivo. The oxidized active site was found to have four Mo-S ligands at 2.43 angstroms, one Mo=O at 1.71 angstroms, and a longer Mo-O at 1.90 angstroms. We conclude that the oxidized enzyme is a monooxomolybdenum(VI) species coordinated by two molybdopterin dithiolenes and a serine. The bond lengths determined for E. coli DMSO reductase are very similar to those determined for the well-characterized Rhodobacter sphaeroides DMSO reductase, suggesting similar active site structures for the two enzymes. Furthermore, our results suggest that the form found in vivo is the monooxobis(molybdopterin) species.

  7. Structure of Physarum polycephalum cytochrome b{sub 5} reductase at 1.56 Å resolution

    SciTech Connect

    Kim, Sangwoo; Suga, Michihiro; Ogasahara, Kyoko; Ikegami, Terumi; Minami, Yoshiko; Yubisui, Toshitsugu; Tsukihara, Tomitake

    2007-04-01

    The structure of P. polycephalum cytochrome b{sub 5} reductase, an enzyme which catalyzes the reduction of cytochrome b{sub 5} by NADH, was determined at a resolution of 1.56 Å. Physarum polycephalum cytochrome b{sub 5} reductase catalyzes the reduction of cytochrome b{sub 5} by NADH. The structure of P. polycephalum cytochrome b{sub 5} reductase was determined at a resolution of 1.56 Å. The molecular structure was compared with that of human cytochrome b{sub 5} reductase, which had previously been determined at 1.75 Å resolution [Bando et al. (2004 ▶), Acta Cryst. D60, 1929–1934]. The high-resolution structure revealed conformational differences between the two enzymes in the adenosine moiety of the FAD, the lid region and the linker region. The structural properties of both proteins were inspected in terms of hydrogen bonding, ion pairs, accessible surface area and cavity volume. The differences in these structural properties between the two proteins were consistent with estimates of their thermostabilities obtained from differential scanning calorimetry data.

  8. 5 Alpha-reductase inhibitory and antiandrogenic activities of novel steroids in hamster seminal vesicles.

    PubMed

    Cabeza, Marisa; Bratoeff, Eugene; Flores, Eugenio; Ramírez, Elena; Calleros, Jorge; Montes, Diana; Quiroz, Alexandra; Heuze, Ivonne

    2002-11-01

    The pharmacological activity of several 16-bromosubstituted trienediones 4 and 5, 16-methyl substituted dienediones 6 and 7 and the 16-methyl substituted trienedione 8 was determined on gonadectomized hamster seminal vesicles by measuring the in vitro conversion of testosterone (T) to dihydrotestosterone (DHT) as 5alpha-reductase inhibitors and also the ability of these steroids to bind to the androgen receptor. Steroids 6 and 7 when injected together with T decreased the weight of the seminal vesicles thus showing an antiandrogenic effect. Compounds 5 and 6 reduced substantially the conversion of T to DHT and therefore can be considered good inhibitors for the enzyme 5alpha-reductase; however both steroids failed to form a complex with the androgen receptor. On the other hand compound 7 which showed a very small inhibitory activity for the enzyme 5alpha-reductase, exhibited a very high affinity for the androgen receptor and thus can be considered an effective antiandrogen. This compound also reduced substantially the weight of the seminal vesicles. Steroids 4 and 8 did not reduce the weight of the seminal vesicles and exhibited a low affinity for the androgen receptor; 8 showed a weak 5alpha-reductase inhibitory activity, whereas 4 exhibited a weak androgenic effect.

  9. [Dutasteride (Avodart): a novel 5-alpha reductase inhibitor for treatment of benign prostate hypertrophy].

    PubMed

    Vanden Bossche, M; Sternon, J

    2005-01-01

    Dutasteride (Avodart), a novel dual 5-alpha reductase inhibitor is effective for the treatment of benign prostate hypertrophy, of more than 30 cc because the reduction of the level of dihydrotestosterone. By reducing prostatic volume, dutasteride improves moderate to severe symptoms and flow rate. It allows a reduction of disease progression by reducing the rate of acute urinary retention and need for surgery.

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

    PubMed

    Windahl, Sara H; Andersson, Niklas; Börjesson, Anna E; Swanson, Charlotte; Svensson, Johan; Movérare-Skrtic, Sofia; Sjögren, Klara; Shao, Ruijin; Lagerquist, Marie K; Ohlsson, Claes

    2011-01-01

    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.

  11. Identification of structural domains within the large subunit of herpes simplex virus ribonucleotide reductase.

    PubMed

    Conner, J; Cross, A; Murray, J; Marsden, H

    1994-12-01

    The large subunit (R1) of herpes simplex virus (HSV) ribonucleotide reductase is a bifunctional protein consisting of a unique N-terminal protein kinase domain and a ribonucleotide reductase domain. Previous studies showed that the two functional domains are linked by a protease sensitive site. Here we provide evidence for two subdomains, of 30K and 53K, within the reductase domain. The two fragments, which were produced by limited proteolysis and were resistant to further degradation, remained tightly associated in a complex containing two molecules of each. They were capable of binding the R2 subunit of HSV ribonucleotide reductase with approximately the same affinity as the intact protein but the complex did not complement the small subunit (R2) to give an active enzyme. At low concentrations (0.4 micrograms/ml) of trypsin or V8 protease, cleavage between the subdomains was prevented by the presence of the N-terminal protein kinase domain. At higher protease concentrations (1 micrograms/ml) the N-terminal domain is extensively proteolysed and the 30K and 53K domains were generated. Identical results were obtained using purified R1 isolated from infected cell extracts or following expression in Escherichia coli. The origin of the two domains was investigated by N-terminal sequencing of the 53K fragment and by examining their reactivity with a panel of R1-specific monoclonal antibodies which we isolated and epitope mapped for that purpose. The trypsin cleavage site was found to lie between arginine 575 and asparagine 576, and proteolysis in this region was not prevented by the presence of R2 or the nonapeptide YAGAVVNDL. We propose that the ribonucleotide reductase region of HSV R1 exists in a two domain structure, and that the interdomain linking region is protected by the unique N terminus.

  12. Expression of a fully functional cd1 nitrite reductase from Pseudomonas aeruginosa in Pseudomonas stutzeri.

    PubMed

    Arese, Marzia; Zumft, Walter G; Cutruzzolà, Francesca

    2003-01-01

    Nitrite reductases are redox enzymes catalysing the one electron reduction of nitrite to nitrogen monoxide (NO) within the bacterial denitrification process. We have cloned the gene for cd(1) nitrite reductase (Pa-nirS) from Pseudomonas aeruginosa into the NiRS(-) strain MK202 of Pseudomonas stutzeri and expressed the enzyme under denitrifying conditions. In the MK202 strain, denitrification is abolished by the disruption of the endogenous nitrite reductase gene; thus, cells can be grown only in the presence of oxygen. After complementation with Pa-nirS gene, cells supplemented with nitrate can be grown in the absence of oxygen. The presence of nitrite reductase was proven in vivo by the demonstration of NO production, showing that the enzyme was expressed in the active form, containing both heme c and d(1). A purification procedure for the recombinant PaNir has been developed, based on the P. aeruginosa purification protocol; spectroscopic analysis of the purified protein fully confirms the presence of the d(1) heme cofactor. Moreover, the functional characterisation of the recombinant NiR has been carried out by monitoring the production of NO by the purified NiR enzyme in the presence of nitrite by an NO electrode. The full recovery of the denitrification properties in the P. stutzeri MK202 strain by genetic complementation with Pa-NiR underlines the high homology between enzymes of nitrogen oxianion respiration. Our work provides an expression system for cd(1) nitrite reductase and its site-directed mutants in a non-pathogenic strain and is a starting point for the in vivo study of recombinant enzyme variants.

  13. Purification, Characterization, and Overexpression of Flavin Reductase Involved in Dibenzothiophene Desulfurization by Rhodococcus erythropolis D-1

    PubMed Central

    Matsubara, Toshiyuki; Ohshiro, Takashi; Nishina, Yoshihiro; Izumi, Yoshikazu

    2001-01-01

    The dibenzothiophene (DBT)-desulfurizing bacterium, Rhodococcus erythropolis D-1, removes sulfur from DBT to form 2-hydroxybiphenyl using four enzymes, DszC, DszA, DszB, and flavin reductase. In this study, we purified and characterized the flavin reductase from R. erythropolis D-1 grown in a medium containing DBT as the sole source of sulfur. It is conceivable that the enzyme is essential for two monooxygenase (DszC and DszA) reactions in vivo. The purified flavin reductase contains no chromogenic cofactors and was found to have a molecular mass of 86 kDa and four identical 22-kDa subunits. The enzyme catalyzed NADH-dependent reduction of flavin mononucleotide (FMN), and the Km values for NADH and FMN were 208 and 10.8 μM, respectively. Flavin adenine dinucleotide was a poor substrate, and NADPH was inert. The enzyme did not catalyze reduction of any nitroaromatic compound. The optimal temperature and optimal pH for enzyme activity were 35°C and 6.0, respectively, and the enzyme retained 30% of its activity after heat treatment at 80°C for 30 min. The N-terminal amino acid sequence of the purified flavin reductase was identical to that of DszD of R. erythropolis IGTS8 (K. A. Gray, O. S. Pogrebinsky, G. T. Mrachko, L. Xi, D. J. Monticello, and C. H. Squires, Nat. Biotechnol. 14:1705–1709, 1996). The flavin reductase gene was amplified with primers designed by using dszD of R. erythropolis IGTS8, and the enzyme was overexpressed in Escherichia coli. The specific activity in crude extracts of the overexpressed strain was about 275-fold that of the wild-type strain. PMID:11229908

  14. Sequence diversity and enzyme activity of ferric-chelate reductase LeFRO1 in tomato.

    PubMed

    Kong, Danyu; Chen, Chunlin; Wu, Huilan; Li, Ye; Li, Junming; Ling, Hong-Qing

    2013-11-20

    Ferric-chelate reductase which functions in the reduction of ferric to ferrous iron on root surface is a critical protein for iron homeostasis in strategy I plants. LeFRO1 is a major ferric-chelate reductase involved in iron uptake in tomato. To identify the natural variations of LeFRO1 and to assess their effect on the ferric-chelate reductase activity, we cloned the coding sequences of LeFRO1 from 16 tomato varieties collected from different regions, and detected three types of LeFRO1 (LeFRO1(MM), LeFRO1(Ailsa) and LeFRO1(Monita)) with five amino acid variations at the positions 21, 24, 112, 195 and 582. Enzyme activity assay revealed that the three types of LeFRO1 possessed different ferric-chelate reductase activity (LeFRO1(Ailsa) > LeFRO1(MM) > LeFRO1(Monita)). The 112th amino acid residue Ala of LeFRO1 is critical for maintaining the high activity of ferric-chelate reductase, because modification of this amino acid resulted in a significant reduction of enzyme activity. Further, we showed that the combination of the amino acid residue Ile at the site 24 with Lys at the site 582 played a positive role in the enzyme activity of LeFRO1. In conclusion, the findings are helpful to understand the natural adaptation mechanisms of plants to iron-limiting stress, and may provide new knowledge to select and manipulate LeFRO1 for improving the iron deficiency tolerance in tomato.

  15. Biotransformation of arsenic by bacterial strains mediated by oxido-reductase enzyme system.

    PubMed

    Vishnoi, N; Singh, D P

    2014-12-24

    The present study deals with the enzyme mediated biotransformation of arsenic in five arsenic tolerant strains (Bacillus subtilis, Bacillus megaterium, Bacillus pumilus, Paenibacillus macerans and Escherichia coli). Biotransformation ability of these isolates was evaluated by monitoring arsenite oxidase and arsenate reductase activity. Results showed that arsenic oxidase activity was exclusively present in P. macerans and B. pumilus while B. subtilis, B. megaterium and E. coli strains showed presence of Arsenic oxido-reductase enzyme. The reversible nature of arsenic oxido- reductase suggested that same enzyme can carry out oxidation and reduction of arsenic depending upon the relative concentration of arsenic species. Lineweaver-Burk plot of the arsenite oxidase activity in P. macerans showed highest Km value (Km- 200 μM) and lower Vmax (0.012 μmol mg-1 protein min-1) indicating lowest affinity of the enzyme for arsenite. On the contrary, E. coli showed the lower Km value ( Km- 38.46 μM) and higher Vmax (0.044 μmol mg-1 protein min-1) suggesting for higher affinity for the arsenite. Lineweaver-Burk plot of arsenate reductase activity showed the presence of this enzyme in B. subtilis, B. megaterium and E. coli which were in the range of 200-360 μM Km and Vmax value between 0.256- 0.129 mmol mg-1 protein min-1. These results suggested that affinity of the as reductase enzyme is lowest for arsenate than that for the arsenite. Thus, arsenite oxidase system appears to be a predominant mechanism of cellular defense in these bacterial strains.

  16. Purification and characterization of an NADPH-cytochrome P450 (cytochrome c) reductase from spearmint (Mentha spicata) glandular trichomes.

    PubMed

    Ponnamperuma, K; Croteau, R

    1996-05-01

    Solubilized NADPH-cytochrome c (P450) reductase was purified to homogeneity from an extract of spearmint (Mentha spicata) glandular trichomes by dye-ligand interaction chromatography on Matrex-Gel Red A and affinity chromatography on 2', 5'-adenosine diphosphate agarose. SDS-PAGE of the purified enzyme preparation revealed the presence of two similar proteins with masses of 82 kDa (major) and 77 kDa (minor) that crossreacted on immunoblot analysis with polyclonal antibodies directed against NADPH-cytochrome P450 reductase from Jerusalem artichoke and from mung bean. Complete immunoinhibition of reductase activity was observed with both types of polyclonal antibodies, while only partial inhibition of activity resulted using a family of monoclonal antibodies directed against the Jerusalem artichoke cytochrome P450 reductase. Inhibition of the spearmint oil gland cytochrome c reductase was also observed with the diphenyliodonium ion. The K(m) values for the cosubstrates NADPH and cytochrome c were 6.2 and 3.7 microM, respectively, and the pH optimum for activity was at 8.5. The NADPH-cytochrome c reductase reconstituted NADPH-dependent (-)-4S-limonene-6-hydroxylase activity in the presence of cytochrome P450, purified from the microsomal fraction of spearmint oil gland cells and dilauroyl phosphatidyl choline. These characteristics establish the identity of the purified enzyme as a NADPH-cytochrome P450 reductase.

  17. Biochemical Characterization of Inducible 'Reductase' Component of Benzoate Dioxygenase and Phthalate Isomer Dioxygenases from Pseudomonas aeruginosa strain PP4.

    PubMed

    Karandikar, Rohini; Badri, Abinaya; Phale, Prashant S

    2015-09-01

    The first step involved in the degradation of phthalate isomers (phthalate, isophthalate and terephthalate) is the double hydroxylation by respective aromatic-ring hydroxylating dioxygenases. These are two component enzymes consisting of 'oxygenase' and 'reductase' components. Soil isolate Pseudomonas aeruginosa strain PP4 degrades phthalate isomers via protocatechuate and benzoate via catechol 'ortho' ring cleavage pathway. Metabolic studies suggest that strain PP4 has carbon source-specific inducible phthalate isomer dioxygenase and benzoate dioxygenase. Thus, it was of interest to study the properties of reductase components of these enzymes. Reductase activity from phthalate isomer-grown cells was 3-5-folds higher than benzoate grown cells. In-gel activity staining profile showed a reductase activity band of R f 0.56 for phthalate isomer-grown cells as compared to R f 0.73 from benzoate-grown cells. Partially purified reductase components from phthalate isomer grown cells showed K m in the range of 30-40 μM and V max = 34-48 μmol min(-1) mg(-1). However, reductase from benzoate grown cells showed K m = 49 μM and V max = 10 μmol min(-1) mg(-1). Strikingly similar molecular and kinetic properties of reductase component from phthalate isomer-grown cells suggest that probably the same reductase component is employed in three phthalate isomer dioxygenases. However, reductase component is different, with respect to kinetic properties and zymogram analysis, from benzoate-grown cells when compared to that from phthalate isomer grown cells of PP4.

  18. Assignment of the human dihydrofolate reductase gene to the q11. -->. q22 region of chromosome 5

    SciTech Connect

    Funanage, V.L.; Myoda, T.T.; Moses, P.A.; Cowell, H.R.

    1984-10-01

    Cells from a dihydrofolate reductase-deficit Chinese hamster ovary cell line were hybridized to human fetal skin fibroblast cells. Nineteen dihydrofolate reductase-positive hybrid clones were isolated and characterized. Cytogenetic and biochemical analyses of these clones have shown that the human dihydrofolate reductase (DHFR) gene is located on chromosome 5. Three of these hybrid cell lines contained different terminal deletions of chromosome 5. An analysis of the breakpoints of these deletions has demonstrated that the DHFR gene resides in the q11..-->..q22 region.

  19. Alkyl nitrate (C1-C3) depth profiles in the tropical Pacific Ocean

    NASA Astrophysics Data System (ADS)

    Dahl, E. E.; Yvon-Lewis, S. A.; Saltzman, E. S.

    2007-01-01

    This paper reports the first depth profile measurements of methyl, ethyl, isopropyl and n-propyl nitrates in the tropical Pacific Ocean. Depth profile measurements were made at 22 stations during the Project Halocarbon Air Sea Exchange cruise, in warm pool, equatorial, subequatorial, and gyre waters. The highest concentrations, up to several hundred pM of methyl nitrate, were observed in the central Pacific within 8 degrees of the equator. In general, alkyl nitrate levels were highest in the surface mixed layer, and decreased with depth below the mixed layer. The spatial distribution of the alkyl nitrates suggests that there is a strong source associated with biologically productive ocean regions, that is characterized by high ratios of methyl:ethyl nitrate. However, the data do not allow discrimination between direct biological emissions and photochemistry as production mechanisms. Alkyl nitrates were consistently detectable at several hundred meters depth. On the basis of the estimated chemical loss rate of these compounds, we conclude that deep water alkyl nitrates must be produced in situ. Possible sources include free radical processes initiated by radioactive decay or cosmic rays, enzymatically mediated reactions involving bacteria, or unidentified chemical mechanisms involving dissolved organic matter.

  20. Nucleotide sequence of the luxC gene encoding fatty acid reductase of the lux operon from Photobacterium leiognathi.

    PubMed

    Lin, J W; Chao, Y F; Weng, S F

    1993-02-26

    The nucleotide sequence of the luxC gene (EMBL Accession No. 65156) encoding fatty acid reductase (FAR) of the lux operon from Photobacterium leiognathi PL741 was determined and the encoded amino acid sequence deduced. The fatty acid reductase is a component of the fatty acid reductase complex. The complex is responsible for converting fatty acid to aldehyde which serves as the substrate in the luciferase-catalyzed bioluminescent reaction. The protein comprises 478 amino acid residues and has a calculated M(r) of 53,858. Alignment and comparison of the fatty acid reductase of P. leiognathi with that of Vibrio harveyi B392 and Vibrio fischeri ATCC 7744 shows that there is 70% and 59% amino acid residues identity, respectively.

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

    PubMed

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

    2014-08-01

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

  2. Molecular characterization of nosRZDFYLX genes coding for denitrifying nitrous oxide reductase of Bradyrhizobium japonicum.

    PubMed

    Velasco, Leonardo; Mesa, Socorro; Xu, Chang-Ai; Delgado, María J; Bedmar, Eulogio J

    2004-04-01

    The nosRZDFYLX gene cluster for the respiratory nitrous oxide reductase from Bradyrhizobium japonicum strain USDA110 has been cloned and sequenced. Seven protein coding regions corresponding to nosR, nosZ, the structural gene, nosD, nosF, nosY, nosL, and nosX were detected. The deduced amino acid sequence exhibited a high degree of similarity to other nitrous oxide reductases from various sources. The NosZ protein included a signal peptide for protein export. Mutant strains carrying either a nosZ or a nosR mutation accumulated nitrous oxide when cultured microaerobically in the presence of nitrate. Maximal expression of a P nosZ-lacZ fusion in strain USDA110 required simultaneously both low level oxygen conditions and the presence of nitrate. Microaerobic activation of the fusion required FixLJ and FixK(2).

  3. Cloning, sequencing, and expression of the adenosylcobalamin-dependent ribonucleotide reductase from Lactobacillus leichmannii.

    PubMed Central

    Booker, S; Stubbe, J

    1993-01-01

    Ribonucleoside-triphosphate reductase (RTPR, EC 1.17.4.2) from Lactobacillus leichmannii, a monomeric adenosylcobalamin-requiring enzyme, catalyzes the conversion of nucleoside triphosphates to deoxynucleoside triphosphates. The gene for this enzyme has been cloned and sequenced. In contrast to expectations based on mechanistic considerations, there is no statistically significant sequence homology with the Escherichia coli reductase that requires a dinuclear-iron center and tyrosyl radical cofactor. The RTPR has been overexpressed and purified to homogeneity, yielding 90 mg of protein from 2.5 g of bacteria. Initial characterization of the recombinant RTPR indicates that its properties are identical to those of the RTPR isolated from L. leichmannii. PMID:8397403

  4. Crystal Structure of ChrR -- A Quinone Reductase with the Capacity to Reduce Chromate

    SciTech Connect

    Eswaramoorthy S.; Poulain, S.; Hienerwadel, R.; Bremond, N.; Sylvester, M. D.; Zhang, Y.-B.; Berthomieu, C.; van der Lelie, D.; Matin, A.

    2012-04-01

    The Escherichia coli ChrR enzyme is an obligatory two-electron quinone reductase that has many applications, such as in chromate bioremediation. Its crystal structure, solved at 2.2 {angstrom} resolution, shows that it belongs to the flavodoxin superfamily in which flavin mononucleotide (FMN) is firmly anchored to the protein. ChrR crystallized as a tetramer, and size exclusion chromatography showed that this is the oligomeric form that catalyzes chromate reduction. Within the tetramer, the dimers interact by a pair of two hydrogen bond networks, each involving Tyr128 and Glu146 of one dimer and Arg125 and Tyr85 of the other; the latter extends to one of the redox FMN cofactors. Changes in each of these amino acids enhanced chromate reductase activity of the enzyme, showing that this network is centrally involved in chromate reduction.

  5. Biphasic Kinetic Behavior of Nitrate Reductase from Heterocystous, Nitrogen-Fixing Cyanobacteria 1

    PubMed Central

    Martin-Nieto, José; Flores, Enrique; Herrero, Antonia

    1992-01-01

    Nitrate reductase activity from filamentous, heterocyst-forming cyanobacteria showed a biphasic kinetic behavior with respect to nitrate as the variable substrate. Two kinetic components were detected, the first showing a higher affinity for nitrate (Km, 0.05-0.25 mm) and a lower catalytic activity and the second showing a lower affinity for nitrate (Km, 5-25 mm) and a higher (3- to 5-fold) catalytic activity. In contrast, among unicellular cyanobacteria, most representatives studied exhibited a monophasic, Michaelis-Menten kinetic pattern for nitrate reductase activity. Biphasic kinetics remained unchanged with the use of different assay conditions (i.e. cell disruption or permeabilization, two different electron donors) or throughout partial purification of the enzyme. PMID:16652939

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

    PubMed

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

    2013-09-01

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

  7. A qualitative and quantitative cytochemical assay of dihydrofolate reductase in erythroid cells.

    PubMed

    Nano, R; Gerzeli, G; Invernizzi, R; Supino, R

    1989-01-01

    The distribution and intensity of dihydrofolate reductase (DHFR) cytochemically demonstrable was studied in erythroid cells. Cells of normal human bone marrow, of human erythroleukaemia (M6), and cells of the Friend (MEL) clone 745A murine erythroleukaemia (also after differentiation with dimethylsulphoxide, DMSO) were stained according to Gerzeli and de Piceis Polver (1969) technique; quantification of the reaction product was made using a Vickers M86 microdensitometer. The enzyme activity progressively decreased during the normal differentiation of the erythropoietic series while persisted at high levels in erythroleukaemia cells. It can be suggested that in the 1st case, the cytochemical pattern of dihydrofolate reductase may be a useful added tool for studying the erythroid differentiation. In the 2nd case, the increased level of this enzyme may be related to an amplification of the gene of DHFR in the malignant transformation.

  8. Structure-based design of pteridine reductase inhibitors targeting African sleeping sickness and the leishmaniases.

    PubMed

    Tulloch, Lindsay B; Martini, Viviane P; Iulek, Jorge; Huggan, Judith K; Lee, Jeong Hwan; Gibson, Colin L; Smith, Terry K; Suckling, Colin J; Hunter, William N

    2010-01-14

    Pteridine reductase (PTR1) is a target for drug development against Trypanosoma and Leishmania species, parasites that cause serious tropical diseases and for which therapies are inadequate. We adopted a structure-based approach to the design of novel PTR1 inhibitors based on three molecular scaffolds. A series of compounds, most newly synthesized, were identified as inhibitors with PTR1-species specific properties explained by structural differences between the T. brucei and L. major enzymes. The most potent inhibitors target T. brucei PTR1, and two compounds displayed antiparasite activity against the bloodstream form of the parasite. PTR1 contributes to antifolate drug resistance by providing a molecular bypass of dihydrofolate reductase (DHFR) inhibition. Therefore, combining PTR1 and DHFR inhibitors might improve therapeutic efficacy. We tested two new compounds with known DHFR inhibitors. A synergistic effect was observed for one particular combination highlighting the potential of such an approach for treatment of African sleeping sickness.

  9. Crystal Structure of Saccharomyces Cerevisiae 3'-Phosphoadenosine-5'-Phosphosulfate Reductase Complexed With Adenosine 3',5'-Bisphosphate

    SciTech Connect

    Yu, Z.; Lemongello, D.; Segel, I.H.; Fisher, A.J.

    2009-05-28

    Most assimilatory bacteria, fungi, and plants species reduce sulfate (in the activated form of APS or PAPS) to produce reduced sulfur. In yeast, PAPS reductase reduces PAPS to sulfite and PAP. Despite the difference in substrate specificity and catalytic cofactor, PAPS reductase is homologous to APS reductase in both sequence and structure, and they are suggested to share the same catalytic mechanism. Metazoans do not possess the sulfate reduction pathway, which makes APS/PAPS reductases potential drug targets for human pathogens. Here, we present the 2.05 A resolution crystal structure of the yeast PAPS reductase binary complex with product PAP bound. The N-terminal region mediates dimeric interactions resulting in a unique homodimer assembly not seen in previous APS/PAPS reductase structures. The 'pyrophosphate-binding' sequence (47)TTAFGLTG(54) defines the substrate 3'-phosphate binding pocket. In yeast, Gly54 replaces a conserved aspartate found in APS reductases vacating space and charge to accommodate the 3'-phosphate of PAPS, thus regulating substrate specificity. Also, for the first time, the complete C-terminal catalytic motif (244)ECGIH(248) is revealed in the active site. The catalytic residue Cys245 is ideally positioned for an in-line attack on the beta-sulfate of PAPS. In addition, the side chain of His248 is only 4.2 A from the Sgamma of Cys245 and may serve as a catalytic base to deprotonate the active site cysteine. A hydrophobic sequence (252)RFAQFL(257) at the end of the C-terminus may provide anchoring interactions preventing the tail from swinging away from the active site as seen in other APS/PAPS reductases.

  10. A conservative region of the mercuric reductase gene (mera) as a molecular marker of bacterial mercury resistance

    PubMed Central

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

    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. PMID:24031221

  11. Aldose reductases influence prostaglandin F2α levels and adipocyte differentiation in male mouse and human species.

    PubMed

    Pastel, Emilie; Pointud, Jean-Christophe; Loubeau, Gaëlle; Dani, Christian; Slim, Karem; Martin, Gwenaëlle; Volat, Fanny; Sahut-Barnola, Isabelle; Val, Pierre; Martinez, Antoine; Lefrançois-Martinez, Anne-Marie

    2015-05-01

    Aldose reductases (AKR1B) are widely expressed oxidoreductases whose physiological function remains elusive. Some isoforms are genuine prostaglandin F2α (PGF2α) synthases, suggesting they might influence adipose homeostasis because PGF2α inhibits adipogenesis. This was shown by Akr1b7 gene ablation in the mouse, which resulted in increased adiposity related to a lower PGF2α content in fat. Yet humans have no ortholog gene for Akr1b7, so the role of aldose reductases in human adipose homeostasis remains to be explored. We analyzed expression of genes encoding human and mouse aldose reductase isoforms in adipose tissues and differentiating adipocytes to assess conserved mechanisms regulating PGF2α synthesis and adipogenesis. The Akr1b3 gene encoded the most abundant isoform in mouse adipose tissue, whereas Akr1b7 encoded the only isoform enriched in the stromal vascular fraction. Most mouse aldose reductase gene expression peaked in early adipogenesis of 3T3-L1 cells and diminished with differentiation. In contrast with its mouse ortholog Akr1b3, AKR1B1 expression increased throughout differentiation of human multipotent adipose-derived stem cells, paralleling PGF2α release, whereas PGF2α receptor (FP) levels collapsed in early differentiation. Pharmacological inhibition of aldose reductase using Statil altered PGF2α production and enhanced human multipotent adipose-derived stem adipocyte differentiation. As expected, the adipogenic effects of Statil were counteracted by an FP agonist (cloprostenol). Thus, in both species aldose reductase-dependent PGF2α production could be important in early differentiation to restrict adipogenesis. PGF2α antiadipogenic signaling could then be toned down through the FP receptor or aldose reductases down-regulation in human and mouse cells, respectively. Our data suggest that aldose reductase inhibitors could have obesogenic potential.

  12. Isolation and characterization of genes encoding leucoanthocyanidin reductase (FeLAR) and anthocyanidin reductase (FeANR) in buckwheat (Fagopyrum esculentum).

    PubMed

    Matsui, Katsuhiro; Hisano, Tomomi; Yasui, Yasuo; Mori, Masashi; Walker, Amanda R; Morishita, Toshikazu; Katsu, Kenjiro

    2016-10-20

    Proanthocyanidins (PAs) are a major group of flavonoids synthesized via the phenylpropanoid biosynthesis pathway, however the pathway has not been fully characterized in buckwheat. Anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR) are involved in the last steps of PA biosynthesis. To isolate the genes for these enzymes from buckwheat we performed PCR using degenerate primers and obtained cDNAs of ANR and LAR, which we designated FeANR and FeLAR1. A search for homologs in a buckwheat genome database with both sequences returned two more LAR sequences, designated FeLAR2 and FeLAR3. Linkage analysis with an F2 segregating population indicated that the three LAR loci were not genetically linked. We detected high levels of PAs in roots and cotyledons of buckwheat seedlings and in buds and flowers of mature plants. FeANR and FeLAR1-3 were expressed in most organs but had different expression patterns. Our findings would be useful for breeding and further analysis of PA synthesis and its regulation in buckwheat.

  13. Separation and distribution of thiosulfate-oxidizing enzyme, tetrathionate reductase, and thiosulfate reductase in extracts of marine heterotroph strain 16B.

    PubMed

    Whited, G M; Tuttle, J H

    1983-11-01

    Thiosulfate-oxidizing enzyme (TSO), tetrathionate reductase (TTR), and thiosulfate reductase (TSR) were demonstrated in cell-free extracts of the marine heterotrophic thiosulfate-oxidizing bacterium strain 16B. Extracts prepared from cells cultured aerobically in the absence of thiosulfate or tetrathionate exhibited constitutive TSO and TTR activity which resided in the soluble fraction of ultracentrifuged crude extracts. Constitutive TSO and TTR cochromatographed on DEAE-Sephadex A-50, Cellex D, Sephadex G-150, and orange A dye-ligand affinity gels. Extracts prepared from cells cultured anaerobically with tetrathionate or aerobically with thiosulfate followed by oxygen deprivation showed an 11- to 30-fold increase in TTR activity, with no increase in TSO activity. The inducible TTR resided in both the ultracentrifuge pellet and supernatant fractions and was readily separated from constitutive TSO and TTR in the latter by DEAE-Sephadex chromatography. Inducible TTR exhibited TSR activity, which was also located in both membrane and soluble extract fractions and which cochromatographed with inducible TTR. The results indicate that constitutive TSO and TTR in marine heterotroph 16B represent reverse activities of the same enzyme whose major physiological function is thiosulfate oxidation. Evidence is also presented which suggests a possible association of inducible TTR and TSR in strain 16B.

  14. Gene expression of monodehydroascorbate reductase and dehydroascorbate reductase during fruit ripening and in response to environmental stresses in acerola (Malpighia glabra).

    PubMed

    Eltelib, Hani A; Badejo, Adebanjo A; Fujikawa, Yukichi; Esaka, Muneharu

    2011-04-15

    Acerola (Malpighia glabra) is an exotic fruit cultivated primarily for its abundant ascorbic acid (AsA) content. The molecular mechanisms that regulate the metabolism of AsA in acerola have yet to be defined. Monodehydroascorbate reductase (MDHAR) and dehydroascorbate reductase (DHAR) are key enzymes of the ascorbate-glutathione cycle that maintain reduced pools of ascorbic acid and serve as important antioxidants. cDNAs encoding MDHAR and DHAR were isolated from acerola using RT-PCR and RACE. Phylogenetic trees associated acerola MDHAR and DHAR with other plant cytosolic MDHARs and DHARs. Expressions of the two genes correlated with their enzymatic activities and were differentially regulated during fruit ripening. Interestingly, MDHAR expression was only detected in overripe fruits, whereas the transcript level of DHAR was highest at the intermediate stage of fruit ripening. Under dark conditions, there was a sharp and significant decline in the total and reduced ascorbate contents, accompanied by a decrease in the level of transcripts and enzyme activities of the two genes in acerola leaves. MDHAR and DHAR transcripts and enzyme activities were significantly up-regulated in the leaves of acerola under cold and salt stress conditions, indicating that expression of both genes are transcriptionally regulated under these stresses.

  15. Transgenic Tobacco Overexpressing Tea cDNA Encoding Dihydroflavonol 4-Reductase and Anthocyanidin Reductase Induces Early Flowering and Provides Biotic Stress Tolerance

    PubMed Central

    Kumar, Vinay; Nadda, Gireesh; Kumar, Sanjay; Yadav, Sudesh Kumar

    2013-01-01

    Flavan-3-ols contribute significantly to flavonoid content of tea (Camellia sinensis L.). Dihydroflavonol 4-reductase (DFR) and anthocyanidin reductase (ANR) are known to be key regulatory enzymes of flavan-3-ols biosynthesis. In this study, we have generated the transgenic tobacco overexpressing individually tea cDNA CsDFR and CsANR encoding for DFR and ANR to evaluate their influ