Sample records for mtrc terminal reductases

  1. Targeted Protein Degradation of Outer Membrane Decaheme Cytochrome MtrC Metal Reductase in Shewanella oneidensis MR-1 Measured Using Biarsenical Probe CrAsH-EDT2

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Xiong, Yijia; Chen, Baowei; Shi, Liang

    2011-10-14

    Development of efficient microbial biofuel cells requires an ability to exploit interfacial electron transfer reactions to external electron acceptors, such as metal oxides; such reactions occur in the facultative anaerobic gram-negative bacterium Shewanella oneidensis MR-1 through the catalytic activity of the outer membrane decaheme c-type cytochrome MtrC. Central to the utility of this pathway to synthetic biology is an understanding of cellular mechanisms that maintain optimal MtrC function, cellular localization, and renewal by degradation and resynthesis. In order to monitor trafficking to the outer membrane, and the environmental sensitivity of MtrC, we have engineered a tetracysteine tag (i.e., CCPGCC) atmore » its C-terminus that permits labeling by the cell impermeable biarsenical fluorophore, carboxy-FlAsH (CrAsH) of MtrC at the surface of living Shewanella oneidensis MR-1 cells. In comparison, the cell permeable reagent FlAsH permits labeling of the entire population of MtrC, including proteolytic fragments resulting from incorrect maturation. We demonstrate specific labeling by CrAsH of engineered MtrC which is dependent on the presence of a functional type-2 secretion system (T2S), as evidenced by T2S system gspD or gspG deletion mutants which are incapable of CrAsH labeling. Under these latter conditions, MtrC undergoes proteolytic degradation to form a large 35-38 kDa fragment; this degradation product is also resolved during normal turnover of the CrAsH-labeled MtrC protein. No MtrC protein is released into the medium during turnover, suggesting the presence of cellular turnover systems involving MtrC reuptake and degradation. The mature MtrC localized on the outer membrane is a long-lived protein, with a turnover rate of 0.043 hr-1 that is insensitive to O2 concentration. Maturation of MtrC is relatively inefficient, with substantial rates of turnover of the immature protein prior to export to the outer membrane (i.e., 0.028 hr-1) that are

  2. Flavin binding to the deca-heme cytochrome MtrC: Insights from computational molecular simulation

    DOE PAGES

    Breuer, Marian; Rosso, Kevin  M.; Blumberger, Jochen

    2015-12-15

    Here, certain dissimilatory bacteria have the remarkable ability to use extracellular metal oxide minerals instead of oxygen as terminal electron sinks, using a process known as “extracellular respiration”. Specialized multiheme cytochromes located on the outer membrane of the microbe were shown to be crucial for electron transfer from the cell surface to the mineral. This process is facilitated by soluble, biogenic flavins secreted by the organism for the purpose of acting as an electron shuttle. However, their interactions with the outer-membrane cytochromes are not established on a molecular scale. Here, we study the interaction between the outer-membrane deca-heme cytochrome MtrCmore » from Shewanella oneidensis and flavin mononucleotide (FMN in fully oxidized quinone form) using computational docking. We find that interaction of FMN with MtrC is significantly weaker than with known FMN-binding proteins, but identify a mildly preferred interaction site close to heme 2 with a dissociation constant (K d) = 490 μM, in good agreement with recent experimental estimates, K d = 255 μM. The weak interaction with MtrC can be qualitatively explained by the smaller number of hydrogen bonds that the planar headgroup of FMN can form with this protein compared to FMN-binding proteins. Molecular dynamics simulation gives indications for a possible conformational switch upon cleavage of the disulphide bond of MtrC, but without concomitant increase in binding affinities according to this docking study. Overall, our results suggest that binding of FMN to MtrC is reversible and not highly specific, which may be consistent with a role as redox shuttle that facilitates extracellular respiration.« less

  3. Analysis of Structural MtrC Models Based on Homology with the Crystal Structure of MtrF

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Edwards, Marcus; Fredrickson, Jim K.; Zachara, John M.

    2012-12-01

    The outer-membrane decahaem cytochrome MtrC is part of the transmembrane MtrCAB complex required for mineral respiration by Shewanella oneidensis. MtrC has significant sequence similarity to the paralogous decahaem cytochrome MtrF, which has been structurally solved through X-ray crystallography. This now allows for homology-based models of MtrC to be generated. The structure of these MtrC homology models contain ten bis-histidine-co-ordinated c-type haems arranged in a staggered cross through a four-domain structure. This model is consistent with current spectroscopic data and shows that the areas around haem 5 and haem 10, at the termini of an octahaem chain, are likely to havemore » functions similar to those of the corresponding haems in MtrF. The electrostatic surfaces around haem 7, close to the β-barrels, are different in MtrF and MtrC, indicating that these haems may have different potentials and interact with substrates differently.« less

  4. Role of Outer Membrane C-Type Cytochromes MtrC and OmcA in Shewanella Oneidensis MR-1 Cell Production, Accumulation, and Detachment During Respiration on Hematite

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Mitchell, Andrew C.; Peterson, L.; Reardon, Catherine L.

    2012-07-01

    Solid phase iron oxides are considered to be important terminal electron acceptors for microbial respiration in many anoxic environments. Besides the knowledge that cells attach to and reduce these substrates, other aspects of surface-associated cell behavior and the related cell surface components that influence cell-mineral interactions are not well understood. In the present study, wild-type cells of the dissimilatory iron-reducing bacterium Shewanella oneidensis MR-1 formed thin biofilms one-to-two cell layers in thickness when respiring on natural specular hematite under flow conditions similar to those which exist in aquatic sediments and subsurface environments. The distribution of cells within the biofilm indicatedmore » that direct contact was not required for electron transfer from cells to the mineral surface. Detached biomass in the form of single cells represented >99% of the surface-associated wild-type cell production from respiration on hematite over the biofilm life cycle. A mutant deficient in the outer membrane c35 type cytochrome OmcA, while still able to respire and replicate on hematite, established a lower steady-state cell density on the mineral surface than that of the wild-type strain. A mutant deficient in MtrC, another outer membrane c-type cytochrome, and a mutant deficient in both cytochromes were unable to reduce sufficient amounts of hematite to support detectable growth on the mineral surface. When considered in the context of previous work, the results support a growing body of evidence that the relative importance of OmcA and MtrC to cell respiration and replication depends on the form of iron oxide available as terminal electron acceptor.« less

  5. Single-Cell Imaging and Spectroscopic Analyses of Cr(VI) Reduction on the Surface of Bacterial Cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wang, Yuanmin; Sevinc, Papatya C.; Belchik, Sara M.

    2013-01-22

    We investigate single-cell reduction of toxic Cr(VI) by the dissimilatory metal-reducing bacterium Shewanella oneidensis MR-1 (MR-1), an important bioremediation process, using Raman spectroscopy and scanning electron microscopy (SEM) combined with energy-dispersive X-ray spectroscopy (EDX). Our experiments indicate that the toxic and highly soluble Cr(VI) can be efficiently reduced to the less toxic and non-soluble Cr2O3 nanoparticles by MR-1. Cr2O3 is observed to emerge as nanoparticles adsorbed on the cell surface and its chemical nature is identified by EDX imaging and Raman spectroscopy. Co-localization of Cr2O3 and cytochromes by EDX imaging and Raman spectroscopy suggests a terminal reductase role for MR-1more » surface-exposed cytochromes MtrC and OmcA. Our experiments revealed that the cooperation of surface proteins OmcA and MtrC makes the reduction reaction most efficient, and the sequence of the reducing reactivity of the MR-1 is: wild type > single mutant @mtrC or mutant @omcA > double mutant (@omcA-@mtrC). Moreover, our results also suggest that the direct microbial Cr(VI) reduction and Fe(II) (hematite)-mediated Cr(VI) reduction mechanisms may co-exist in the reduction processes.« less

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

    PubMed

    Son, Minky; Bang, Woo Young; Park, Chanin; Lee, Yuno; Kwon, Seul Gi; Kim, Sam Woong; Kim, Chul Wook; Lee, Keun Woo

    2014-01-01

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

  7. Functional Mechanism of C-Terminal Tail in the Enzymatic Role of Porcine Testicular Carbonyl Reductase: A Combined Experiment and Molecular Dynamics Simulation Study of the C-Terminal Tail in the Enzymatic Role of PTCR

    PubMed Central

    Park, Chanin; Lee, Yuno; Kwon, Seul Gi; Kim, Sam Woong; Kim, Chul Wook; Lee, Keun Woo

    2014-01-01

    Porcine testicular carbonyl reductase, PTCR which is one of the short chain dehydrogenases/reductases (SDR) superfamily catalyzes the NADPH-dependent reduction of carbonyl compounds including steroids and prostaglandins. Previously we reported C- terminal tail of PTCR was deleted due to a nonsynonymous single nucleotide variation (nsSNV). Here we identified from kinetic studies that the enzymatic properties for 5α-dihydrotestosterone (5α-DHT) were different between wild-type and C-terminal-deleted PTCRs. Compared to wild-type PTCR, C-terminal-deleted PTCR has much higher reduction rate. To investigate structural difference between wild-type and C-terminal-deleted PTCRs upon 5α-DHT binding, we performed molecular dynamics simulations for two complexes. Using trajectories, molecular interactions including hydrogen bonding patterns, distance between 5α-DHT and catalytic Tyr193, and interaction energies are analyzed and compared. During the MD simulation time, the dynamic behavior of C-terminal tail in wild-type PTCR is also examined using essential dynamics analysis. The results of our simulations reveal that the binding conformation of 5α-DHT in C-terminal-deleted PTCR is more favorable for reduction reaction in PTCR, which shows strong agreement with kinetic data. These structural findings provide valuable information to understand substrate specificity of PTCR and further kinetic properties of enzymes belonging to the SDR superfamily. PMID:24646606

  8. N-terminal domain of the dual-targeted pea glutathione reductase signal peptide controls organellar targeting efficiency.

    PubMed

    Rudhe, Charlotta; Clifton, Rachel; Whelan, James; Glaser, Elzbieta

    2002-12-06

    Import of nuclear-encoded proteins into mitochondria and chloroplasts is generally organelle specific and its specificity depends on the N-terminal signal peptide. Yet, a group of proteins known as dual-targeted proteins have a targeting peptide capable of leading the mature protein to both organelles. We have investigated the domain structure of the dual-targeted pea glutathione reductase (GR) signal peptide by using N-terminal truncations. A mutant of the GR precursor (pGR) starting with the second methionine residue of the targeting peptide, pGRdelta2-4, directed import into both organelles, negating the possibility that dual import was controlled by the nature of the N terminus. The deletion of the 30 N-terminal residues (pGRdelta2-30) inhibited import efficiency into chloroplasts substantially and almost completely into mitochondria, whereas the removal of only 16 N-terminal amino acid residues (pGRdelta2-16) resulted in the strongly stimulated mitochondrial import without significantly affecting chloroplast import. Furthermore, N-terminal truncations of the signal peptide (pGRdelta2-16 and pGRdelta2-30) greatly stimulated the mitochondrial processing activity measured with the isolated processing peptidase. These results suggest a domain structure for the dual-targeting peptide of pGR and the existence of domains controlling organellar import efficiency therein.

  9. Role of outer membrane c-type cytochromes MtrC and OmcA in Shewanella oneidensis MR-1 cell production, accumulation and detachment during respiration on hematite

    USDA-ARS?s Scientific Manuscript database

    The iron-reducing bacterium Shewanella oneidensis MR-1 has the capacity to contribute to iron cycling over the long term by respiring on crystalline iron oxides such as hematite when poorly crystalline phases are depleted. The ability of outer membrane cytochromes OmcA and MtrC of MR-1 to bind to an...

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

    PubMed

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

    2017-04-01

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

  11. Quinone Reductase 2 Is a Catechol Quinone Reductase

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    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 betweenmore » 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.« less

  12. Isolation and characterization of a cDNA from Cuphea lanceolata encoding a beta-ketoacyl-ACP reductase.

    PubMed

    Klein, B; Pawlowski, K; Höricke-Grandpierre, C; Schell, J; Töpfer, R

    1992-05-01

    A cDNA encoding beta-ketoacyl-ACP reductase (EC 1.1.1.100), an integral part of the fatty acid synthase type II, was cloned from Cuphea lanceolata. This cDNA of 1276 bp codes for a polypeptide of 320 amino acids with 63 N-terminal residues presumably representing a transit peptide and 257 residues corresponding to the mature protein of 27 kDa. The encoded protein shows strong homology with the amino-terminal sequence and two tryptic peptides from avocado mesocarp beta-ketoacyl-ACP reductase, and its total amino acid composition is highly similar to those of the beta-ketoacyl-ACP reductases of avocado and spinach. Amino acid sequence homologies to polyketide synthase, beta-ketoreductases and short-chain alcohol dehydrogenases are discussed. An engineered fusion protein lacking most of the transit peptide, which was produced in Escherichia coli, was isolated and proved to possess beta-ketoacyl-ACP reductase activity. Hybridization studies revealed that in C. lanceolata beta-ketoacyl-ACP reductase is encoded by a small family of at least two genes and that members of this family are expressed in roots, leaves, flowers and seeds.

  13. Differential cytochrome content and reductase activity in Geospirillum barnesii strain SeS3

    USGS Publications Warehouse

    Stolz, J.F.; Gugliuzza, T.; Switzer, Blum J.; Oremland, R.; Martinez, Murillo F.

    1997-01-01

    The protein composition, cytochrome content, and reductase activity in the dissimilatory selenate-reducing bacterium Geospirillum barnesii strain SeS3, grown with thiosulfate, nitrate, selenate, or fumarate as the terminal electron acceptor, was investigated. Comparison of seven high-molecular-mass membrane proteins (105.3, 90.3, 82.6, 70.2, 67.4, 61.1, and 57.3 kDa) by SDS-PAGE showed that their detection was dependent on the terminal electron acceptor used. Membrane fractions from cells grown on thiosulfate contained a 70.2-kDa c-type cytochrome with absorbance maxima at 552, 522, and 421 nm. A 61.1-kDa c-type cytochrome with absorption maxima at 552, 523, and 423 nm was seen in membrane fractions from cells grown on nitrate. No c-type cytochromes were detected in membrane fractions of either selenate- or fumarate-grown cells. Difference spectra, however, revealed the presence of a cytochrome b554 (absorption maxima at 554, 523, and 422 nm) in membrane fractions from selenate-grown cells and a cytochrome b556 (absorption maxima at 556, 520, and 416 nm) in membrane fractions from fumarate-grown cells. Analysis of reductase activity in the different membrane fractions showed variability in substrate specificity. However, enzyme activity was greatest for the substrate on which the cells had been grown (e.g., membranes from nitrate-grown cells exhibited the greatest activity with nitrate). These results show that protein composition, cytochrome content, and reductase activity are dependent on the terminal electron acceptor used for growth.

  14. Ketopantoyl lactone reductase is a conjugated polyketone reductase.

    PubMed

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

    1989-03-01

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

  15. Molecular dissection of a putative iron reductase from Desulfotomaculum reducens MI-1

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Li, Zhi; Kim, David D.; Nelson, Ornella D.

    2015-10-08

    Desulfotomaculum reducens MI-1 is a Firmicute strain capable of reducing a variety of heavy metal ions and has a great potential in heavy metal bioremediation.We recently identified Dred_2421 as a potential iron reductase through proteomic study of D. reducens. The current study examines its iron-reduction mechanism. Dred_2421, like its close homolog from Escherichia coli (2, 4-dienoyl-CoA reductase), has an FMN-binding N-terminal domain (NTD), an FAD-binding C-terminal domain (CTD), and a 4Fee4S cluster between the two domains. To understand the mechanism of the iron-reduction activity and the role of each domain, we generated a series of variants for each domain andmore » investigated their iron reduction activity. Our results suggest that CTD is the main contributor of the iron-reduction activity, and that NTD and the 4Fee4S cluster are not directly involved in such activity. This study provides a mechanistic understanding of the ironereductase activity of Dred_2421 and may also help to elucidate other physiological activities this enzyme may have.« less

  16. Dimethyl sulfoxide reductase activity by anaerobically grown Escherichia coli HB101.

    PubMed Central

    Bilous, P T; Weiner, J H

    1985-01-01

    Escherichia coli grew anaerobically on a minimal medium with glycerol as the carbon and energy source and dimethyl sulfoxide (DMSO) as the terminal electron acceptor. DMSO reductase activity, measured with an artificial electron donor (reduced benzyl viologen), was preferentially associated with the membrane fraction (77 +/- 10% total cellular activity). A Km for DMSO reduction of 170 +/- 60 microM was determined for the membrane-bound activity. Methyl viologen, reduced flavin mononucleotide, and reduced flavin adenine dinucleotide also served as electron donors for DMSO reduction. Methionine sulfoxide, a DMSO analog, could substitute for DMSO in both the growth medium and in the benzyl viologen assay. DMSO reductase activity was present in cells grown anaerobically on DMSO but was repressed by the presence of nitrate or by aerobic growth. Anaerobic growth on DMSO coinduced nitrate, fumarate, and and trimethylamine-N-oxide reductase activities. The requirement of a molybdenum cofactor for DMSO reduction was suggested by the inhibition of growth and a 60% reduction in DMSO reductase activity in the presence of 10 mM sodium tungstate. Furthermore, chlorate-resistant mutants chlA, chlB, chlE, and chlG were unable to grow anaerobically on DMSO. DMSO reduction appears to be under the control of the fnr gene. PMID:3888958

  17. The crystal structure of NADPH:ferredoxin reductase from Azotobacter vinelandii.

    PubMed Central

    Sridhar Prasad, G.; Kresge, N.; Muhlberg, A. B.; Shaw, A.; Jung, Y. S.; Burgess, B. K.; Stout, C. D.

    1998-01-01

    NADPH:ferredoxin reductase (AvFPR) is involved in the response to oxidative stress in Azotobacter vinelandii. The crystal structure of AvFPR has been determined at 2.0 A resolution. The polypeptide fold is homologous with six other oxidoreductases whose structures have been solved including Escherichia coli flavodoxin reductase (EcFldR) and spinach, and Anabaena ferredoxin:NADP+ reductases (FNR). AvFPR is overall most homologous to EcFldR. The structure is comprised of a N-terminal six-stranded antiparallel beta-barrel domain, which binds FAD, and a C-terminal five-stranded parallel beta-sheet domain, which binds NADPH/NADP+ and has a classical nucleotide binding fold. The two domains associate to form a deep cleft where the NADPH and FAD binding sites are juxtaposed. The structure displays sequence conserved motifs in the region surrounding the two dinucleotide binding sites, which are characteristic of the homologous enzymes. The folded over conformation of FAD in AvFPR is similar to that in EcFldR due to stacking of Phe255 on the adenine ring of FAD, but it differs from that in the FNR enzymes, which lack a homologous aromatic residue. The structure of AvFPR displays three unique features in the environment of the bound FAD. Two features may affect the rate of reduction of FAD: the absence of an aromatic residue stacked on the isoalloxazine ring in the NADPH binding site; and the interaction of a carbonyl group with N10 of the flavin. Both of these features are due to the substitution of a conserved C-terminal tyrosine residue with alanine (Ala254) in AvFPR. An additional unique feature may affect the interaction of AvFPR with its redox partner ferredoxin I (FdI). This is the extension of the C-terminus by three residues relative to EcFldR and by four residues relative to FNR. The C-terminal residue, Lys258, interacts with the AMP phosphate of FAD. Consequently, both phosphate groups are paired with a basic group due to the simultaneous interaction of the FMN

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

    PubMed

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

    2017-08-15

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

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

    PubMed

    Wang, Jinling; de Montellano, Paul R Ortiz

    2003-05-30

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

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

    PubMed

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

    2007-10-12

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

  1. Iodate Reduction by Shewanella oneidensis Does Not Involve Nitrate Reductase

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Mok, Jung Kee; Toporek, Yael J.; Shin, Hyun-Dong

    Microbial iodate (IO 3 -) reduction is a major component of the iodine biogeochemical reaction network and is the basis of alternative strategies for remediation of iodine-contaminated environments. The molecular mechanism of microbial IO 3 - reduction, however, is not well understood. In microorganisms displaying IO 3 - and nitrate (NO 3 -) reduction activities, NO 3 - reductase is postulated to reduce IO 3 - as alternate electron acceptor. In the present study, whole genome analyses of 25 NO 3 --reducing Shewanella strains identified various combinations of genes encoding one assimilatory (cytoplasmic Nas) and three dissimilatory (membrane-associated Nar andmore » periplasmic Napα and Napβ) NO 3 - reductases. S. oneidensis was the only Shewanella strain whose genome encoded a single NO 3 - reductase (Napβ). Terminal electron acceptor competition experiments in S. oneidensis batch cultures amended with both NO 3 - and IO 3 - demonstrated that neither NO 3 - nor IO 3 - reduction activities were competitively inhibited by the presence of the competing electron acceptor. The lack of involvement of S. oneidensis Napβ in IO 3 - reduction was confirmed via phenotypic analysis of an in-frame gene deletion mutant lacking napβΑ (encoding the NO 3 --reducing NapβA catalytic subunit). S. oneidensis ΔnapβA was unable to reduce NO 3 -, yet reduced IO 3 - at rates higher than the wild-type strain. Thus, NapβA is required for dissimilatory NO 3 - reduction by S. oneidensis, while neither the assimilatory (Nas) nor dissimilatory (Napα, Napβ, and Nar) NO 3 - reductases are required for IO 3 - reduction. These findings oppose the traditional view that NO 3 - reductase reduces IO 3 - as alternate electron acceptor and indicate that S. oneidensis reduces IO 3 - via an as yet undiscovered enzymatic mechanism.« less

  2. Functional evidence for the critical amino-terminal conserved domain and key amino acids of Arabidopsis 4-HYDROXY-3-METHYLBUT-2-ENYL DIPHOSPHATE REDUCTASE.

    PubMed

    Hsieh, Wei-Yu; Sung, Tzu-Ying; Wang, Hsin-Tzu; Hsieh, Ming-Hsiun

    2014-09-01

    The plant 4-HYDROXY-3-METHYLBUT-2-ENYL DIPHOSPHATE REDUCTASE (HDR) catalyzes the last step of the methylerythritol phosphate pathway to synthesize isopentenyl diphosphate and its allyl isomer dimethylallyl diphosphate, which are common precursors for the synthesis of plastid isoprenoids. The Arabidopsis (Arabidopsis thaliana) genomic HDR transgene-induced gene-silencing lines are albino, variegated, or pale green, confirming that HDR is essential for plants. We used Escherichia coli isoprenoid synthesis H (Protein Data Bank code 3F7T) as a template for homology modeling to identify key amino acids of Arabidopsis HDR. The predicted model reveals that cysteine (Cys)-122, Cys-213, and Cys-350 are involved in iron-sulfur cluster formation and that histidine (His)-152, His-241, glutamate (Glu)-242, Glu-243, threonine (Thr)-244, Thr-312, serine-379, and asparagine-381 are related to substrate binding or catalysis. Glu-242 and Thr-244 are conserved only in cyanobacteria, green algae, and land plants, whereas the other key amino acids are absolutely conserved from bacteria to plants. We used site-directed mutagenesis and complementation assay to confirm that these amino acids, except His-152 and His-241, were critical for Arabidopsis HDR function. Furthermore, the Arabidopsis HDR contains an extra amino-terminal domain following the transit peptide that is highly conserved from cyanobacteria, and green algae to land plants but not existing in the other bacteria. We demonstrated that the amino-terminal conserved domain was essential for Arabidopsis and cyanobacterial HDR function. Further analysis of conserved amino acids in the amino-terminal conserved domain revealed that the tyrosine-72 residue was critical for Arabidopsis HDR. These results suggest that the structure and reaction mechanism of HDR evolution have become specific for oxygen-evolving photosynthesis organisms and that HDR probably evolved independently in cyanobacteria versus other prokaryotes. © 2014

  3. Lamin B receptor regulates the growth and maturation of myeloid progenitors via its sterol reductase domain: implications for cholesterol biosynthesis in regulating myelopoiesis.

    PubMed

    Subramanian, Gayathri; Chaudhury, Pulkit; Malu, Krishnakumar; Fowler, Samantha; Manmode, Rahul; Gotur, Deepali; Zwerger, Monika; Ryan, David; Roberti, Rita; Gaines, Peter

    2012-01-01

    Lamin B receptor (LBR) is a bifunctional nuclear membrane protein with N-terminal lamin B and chromatin-binding domains plus a C-terminal sterol Δ(14) reductase domain. LBR expression increases during neutrophil differentiation, and deficient expression disrupts neutrophil nuclear lobulation characteristic of Pelger-Huët anomaly. Thus, LBR plays a critical role in regulating myeloid differentiation, but how the two functional domains of LBR support this role is currently unclear. We previously identified abnormal proliferation and deficient functional maturation of promyelocytes (erythroid, myeloid, and lymphoid [EML]-derived promyelocytes) derived from EML-ic/ic cells, a myeloid model of ichthyosis (ic) bone marrow that lacks Lbr expression. In this study, we provide new evidence that cholesterol biosynthesis is important to myeloid cell growth and is supported by the sterol reductase domain of Lbr. Cholesterol biosynthesis inhibitors caused growth inhibition of EML cells that increased in EML-derived promyelocytes, whereas cells lacking Lbr exhibited complete growth arrest at both stages. Lipid production increased during wild-type neutrophil maturation, but ic/ic cells exhibited deficient levels of lipid and cholesterol production. Ectopic expression of a full-length Lbr in EML-ic/ic cells rescued both nuclear lobulation and growth arrest in cholesterol starvation conditions. Lipid production also was rescued, and a deficient respiratory burst was corrected. Expression of just the C-terminal sterol reductase domain of Lbr in ic/ic cells also improved each of these phenotypes. Our data support the conclusion that the sterol Δ(14) reductase domain of LBR plays a critical role in cholesterol biosynthesis and that this process is essential to both myeloid cell growth and functional maturation.

  4. 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

  5. Lamin B receptor (LBR) regulates the growth and maturation of myeloid progenitors via its sterol reductase domain: Implications for cholesterol biosynthesis in regulating myelopoiesis

    PubMed Central

    Subramanian, Gayathri; Chaudhury, Pulkit; Malu, Krishnakumar; Fowler, Samantha; Manmode, Rahul; Gotur, Deepali; Zwerger, Monika; Ryan, David; Roberti, Rita; Gaines, Peter

    2011-01-01

    Lamin B receptor (LBR) is a bifunctional nuclear membrane protein with N-terminal lamin B and chromatin binding domains plus a C-terminal sterol Δ14 reductase domain. LBR expression increases during neutrophil differentiation and deficient expression disrupts neutrophil nuclear lobulation characteristic of Pelger-Huët anomaly. Thus LBR plays a critical role in regulating myeloid differentiation, but how the two functional domains of LBR support this role is currently unclear. We previously identified abnormal proliferation and deficient functional maturation of promyelocytes (EPRO cells) derived from EML-ic/ic cells, a myeloid model of ichthyosis (ic) bone marrow that lacks Lbr expression. Here we provide new evidence that cholesterol biosynthesis is important to myeloid cell growth and is supported by the sterol reductase domain of Lbr. Cholesterol biosynthesis inhibitors caused growth inhibition of EML cells that increased in EPRO cells, whereas cells lacking Lbr exhibited complete growth arrest at both stages. Lipid production increased during wild-type neutrophil maturation, but ic/ic cells exhibited deficient levels of lipid and cholesterol production. Ectopic expression of a full length Lbr in EML-ic/ic cells rescued both nuclear lobulation and growth arrest in cholesterol starvation conditions. Lipid production also was rescued, and a deficient respiratory burst was corrected. Expression of just the C-terminal sterol reductase domain of Lbr in ic/ic cells also improved each of these phenotypes. Our data support the conclusion that the sterol Δ14 reductase domain of LBR plays a critical role in cholesterol biosynthesis, and that this process is essential to both myeloid cell growth and functional maturation. PMID:22140257

  6. Production of a highly active, soluble form of the cytochrome P450 reductase (CPR A) from Candida tropicalis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Donnelly, Mark

    2006-08-01

    The present invention provides soluble cytochrome p450 reductase (CPR) proteins from Candida sp. having an altered N-terminal region which results in reduced hydrophobicity of the N-terminal region. Also provided are host cells comprising the subject soluble CPR proteins. In addition, the present invention provides nucleotide and corresponding amino acid sequences for soluble CPR proteins and vectors comprising the nucleotide sequences. Methods for producing a soluble CPR, for increasing production of a dicarboxylic acid, and for detecting a cytochrome P450 are also provided.

  7. A Conserved Acidic Motif in the N-Terminal Domain of Nitrate Reductase Is Necessary for the Inactivation of the Enzyme in the Dark by Phosphorylation and 14-3-3 Binding1

    PubMed Central

    Pigaglio, Emmanuelle; Durand, Nathalie; Meyer, Christian

    1999-01-01

    It has previously been shown that the N-terminal domain of tobacco (Nicotiana tabacum) nitrate reductase (NR) is involved in the inactivation of the enzyme by phosphorylation, which occurs in the dark (L. Nussaume, M. Vincentz, C. Meyer, J.P. Boutin, and M. Caboche [1995] Plant Cell 7: 611–621). The activity of a mutant NR protein lacking this N-terminal domain was no longer regulated by light-dark transitions. In this study smaller deletions were performed in the N-terminal domain of tobacco NR that removed protein motifs conserved among higher plant NRs. The resulting truncated NR-coding sequences were then fused to the cauliflower mosaic virus 35S RNA promoter and introduced in NR-deficient mutants of the closely related species Nicotiana plumbaginifolia. We found that the deletion of a conserved stretch of acidic residues led to an active NR protein that was more thermosensitive than the wild-type enzyme, but it was relatively insensitive to the inactivation by phosphorylation in the dark. Therefore, the removal of this acidic stretch seems to have the same effects on NR activation state as the deletion of the N-terminal domain. A hypothetical explanation for these observations is that a specific factor that impedes inactivation remains bound to the truncated enzyme. A synthetic peptide derived from this acidic protein motif was also found to be a good substrate for casein kinase II. PMID:9880364

  8. Pseudomonas stutzeri N2O reductase contains CuA-type sites.

    PubMed Central

    Scott, R A; Zumft, W G; Coyle, C L; Dooley, D M

    1989-01-01

    N2O reductase (N2O----N2) is the terminal enzyme in the energy-conserving denitrification pathway of soil and marine denitrifying bacteria. The protein is composed of two identical subunits and contains eight copper ions per enzyme molecule. The magnetic circular dichroism spectrum of resting (oxidized) N2O reductase is strikingly similar to the magnetic circular dichroism spectrum of the CuA site in mammalian cytochrome c oxidase [Greenwood, C., Hull, B. C., Barber, D., Eglinton, D. G. & Thomson, A. J. (1983) Biochem. J. 215, 303-316] and is unlike the magnetic circular dichroism spectra of all other biological copper chromophores obtained to date. Sulfur (or chlorine) scatterers are required to fit the copper extended x-ray absorption fine structure data of both the oxidized and reduced forms of N2O reductase. Satisfactory fits require a Cu-N or Cu-O [denoted Cu-(N, O)] interaction at 2.0 A, a Cu-(S, Cl) interaction at 2.3 A and an additional Cu(S, Cl) interaction at approximately 2.6 A (oxidized) or approximately 2.7 A (reduced). Approximately eight sulfur ions (per eight copper ions) at approximately 2.3 A are required to fit the extended x-ray absorption fine structure data for both the oxidized and reduced N2O reductase. The 2.3-A Cu-(S, Cl) distance is nearly identical to that previously determined for the CuA site in cytochrome c oxidase. A 2.6-2.7 A Cu-(S, Cl) interaction is also present in resting and fully reduced cytochrome c oxidase. Comparison of the N2O reductase sequence, determined by translating the structural NosZ gene, with cytochrome c oxidase subunit II sequences from several sources indicates that a Gly-Xaa-Xaa-Xaa-Xaa-Xaa-Cys-Ser-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-His stretch is highly conserved. This sequence contains three of the probable ligands (two cysteines and one histidine) in a CuA-type site. Collectively these data establish that Pseudomonas stutzeri N2O reductase contains CuA-type sites. PMID:2542963

  9. Selenium as an electron acceptor during the catalytic mechanism of thioredoxin reductase.

    PubMed

    Lothrop, Adam P; Snider, Gregg W; Ruggles, Erik L; Patel, Amar S; Lees, Watson J; Hondal, Robert J

    2014-02-04

    Mammalian thioredoxin reductase (TR) is a pyridine nucleotide disulfide oxidoreductase that uses the rare amino acid selenocysteine (Sec) in place of the more commonly used amino acid cysteine (Cys) in the redox-active tetrapeptide Gly-Cys-Sec-Gly motif to catalyze thiol/disulfide exchange reactions. Sec can accelerate the rate of these exchange reactions (i) by being a better nucleophile than Cys, (ii) by being a better electrophile than Cys, (iii) by being a better leaving group than Cys, or (iv) by using a combination of all three of these factors, being more chemically reactive than Cys. The role of the selenolate as a nucleophile in the reaction mechanism was recently demonstrated by creating a mutant of human thioredoxin reductase-1 in which the Cys497-Sec498 dyad of the C-terminal redox center was mutated to either a Ser497-Cys498 dyad or a Cys497-Ser498 dyad. Both mutant enzymes were incubated with human thioredoxin (Trx) to determine which mutant formed a mixed disulfide bond complex. Only the mutant containing the Ser497-Cys498 dyad formed a complex, and this structure has been determined by X-ray crystallography [Fritz-Wolf, K., Kehr, S., Stumpf, M., Rahlfs, S., and Becker, K. (2011) Crystal structure of the human thioredoxin reductase-thioredoxin complex. Nat. Commun. 2, 383]. This experimental observation most likely means that the selenolate is the nucleophile initially attacking the disulfide bond of Trx because a complex resulted only when Cys was present in the second position of the dyad. As a nucleophile, the selenolate of Sec helps to accelerate the rate of this exchange reaction relative to Cys in the Sec → Cys mutant enzyme. Another thiol/disulfide exchange reaction that occurs in the enzymatic cycle of the enzyme is the transfer of electrons from the thiolate of the interchange Cys residue of the N-terminal redox center to the eight-membered selenosulfide ring of the C-terminal redox center. The selenium atom of the selenosulfide could

  10. Nitrate, nitrite and nitric oxide reductases: from the last universal common ancestor to modern bacterial pathogens

    PubMed Central

    Vázquez-Torres, Andrés; Bäumler, Andreas

    2016-01-01

    The electrochemical gradient that ensues from the enzymatic activity of cytochromes such as nitrate reductase, nitric oxide reductase, and quinol oxidase contributes to the bioenergetics of the bacterial cell. Reduction of nitrogen oxides by bacterial pathogens can, however, be uncoupled from proton translocation and biosynthesis of ATP or NH4+, but still linked to quinol and NADH oxidation. Ancestral nitric oxide reductases, as well as cytochrome coxidases and quinol bo oxidases evolved from the former, are capable of binding and detoxifying nitric oxide to nitrous oxide. The NO-metabolizing activity associated with these cytochromes can be a sizable source of antinitrosative defense in bacteria during their associations with host cells. Nitrosylation of terminal cytochromes arrests respiration, reprograms bacterial metabolism, stimulates antioxidant defenses and alters antibiotic cytotoxicity. Collectively, the bioenergetics and regulation of redox homeostasis that accompanies the utilization of nitrogen oxides and detoxification of nitric oxide by cytochromes of the electron transport chain increases fitness of many Gram-positive and –negative pathogens during their associations with invertebrate and vertebrate hosts. PMID:26426528

  11. Insights on the mechanism of thioredoxin reductase inhibition by gold N-heterocyclic carbene compounds using the synthetic linear selenocysteine containing C-terminal peptide hTrxR(488-499): an ESI-MS investigation.

    PubMed

    Pratesi, Alessandro; Gabbiani, Chiara; Michelucci, Elena; Ginanneschi, Mauro; Papini, Anna Maria; Rubbiani, Riccardo; Ott, Ingo; Messori, Luigi

    2014-07-01

    Gold-based drugs typically behave as strong inhibitors of the enzyme thioredoxin reductase (hTrxR), possibly as the consequence of direct Gold(I) coordination to its active site selenocysteine. To gain a deeper insight into the molecular basis of enzyme inhibition and prove gold-selenocysteine coordination, the reactions of three parent Gold(I) NHC compounds with the synthetic C-terminal dodecapeptide of hTrxR containing Selenocysteine at position 498, were investigated by electrospray ionization mass spectrometry (ESI-MS). Formation of 1:1 Gold-peptide adducts, though in highly different amounts, was demonstrated in all cases. In these adducts the same [Au-NHC](+) moiety is always associated to the intact peptide. Afterward, tandem MS experiments, conducted on a specific Gold-peptide complex, pointed out that Gold is coordinated to the selenolate group. The relatively large strength of the Gold-selenolate coordinative bond well accounts for potent enzyme inhibition typically afforded by these Gold(I) compounds. In a selected case, the time course of enzyme inhibition was explored. Interestingly, enzyme inhibition turned out to show up very quickly and reached its maximum just few minutes after mixing. Overall, the present results offer some clear insight into the process of thioredoxin reductase inhibition by Gold-based compounds. Copyright © 2014 Elsevier Inc. All rights reserved.

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

    PubMed

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

    1989-02-24

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

  13. S-nitrosoglutathione reductase in human lung cancer.

    PubMed

    Marozkina, Nadzeya V; Wei, Christina; Yemen, Sean; Wallrabe, Horst; Nagji, Alykhan S; Liu, Lei; Morozkina, Tatiana; Jones, David R; Gaston, Benjamin

    2012-01-01

    S-Nitrosoglutathione (GSNO) reductase regulates cell signaling pathways relevant to asthma and protects cells from nitrosative stress. Recent evidence suggests that this enzyme may prevent human hepatocellular carcinoma arising in the setting of chronic hepatitis. We hypothesized that GSNO reductase may also protect the lung against potentially carcinogenic reactions associated with nitrosative stress. We report that wild-type Ras is S-nitrosylated and activated by nitrosative stress and that it is denitrosylated by GSNO reductase. In human lung cancer, the activity and expression of GSNO reductase are decreased. Further, the distribution of the enzyme (including its colocalization with wild-type Ras) is abnormal. We conclude that decreased activity of GSNO reductase could leave the human lung vulnerable to the oncogenic effects of nitrosative stress, as is the case in the liver. This potential should be considered when developing therapies that inhibit pulmonary GSNO reductase to treat asthma and other conditions.

  14. Purification and Characterization of (Per)Chlorate Reductase from the Chlorate-Respiring Strain GR-1

    PubMed Central

    Kengen, Servé W. M.; Rikken, Geoffrey B.; Hagen, Wilfred R.; van Ginkel, Cees G.; Stams, Alfons J. M.

    1999-01-01

    Strain GR-1 is one of several recently isolated bacterial species that are able to respire by using chlorate or perchlorate as the terminal electron acceptor. The organism performs a complete reduction of chlorate or perchlorate to chloride and oxygen, with the intermediate formation of chlorite. This study describes the purification and characterization of the key enzyme of the reductive pathway, the chlorate and perchlorate reductase. A single enzyme was found to catalyze both the chlorate- and perchlorate-reducing activity. The oxygen-sensitive enzyme was located in the periplasm and had an apparent molecular mass of 420 kDa, with subunits of 95 and 40 kDa in an α3β3 composition. Metal analysis showed the presence of 11 mol of iron, 1 mol of molybdenum, and 1 mol of selenium per mol of heterodimer. In accordance, quantitative electron paramagnetic resonance spectroscopy showed the presence of one [3Fe-4S] cluster and two [4Fe-4S] clusters. Furthermore, two different signals were ascribed to Mo(V). The Kmvalues for perchlorate and chlorate were 27 and <5 μM, respectively. Besides perchlorate and chlorate, nitrate, iodate, and bromate were also reduced at considerable rates. The resemblance of the enzyme to nitrate reductases, formate dehydrogenases, and selenate reductase is discussed. PMID:10542172

  15. A Decaheme Cytochrome as a Molecular Electron Conduit in Dye-Sensitized Photoanodes

    PubMed Central

    Hwang, Ee Taek; Sheikh, Khizar; Orchard, Katherine L; Hojo, Daisuke; Radu, Valentin; Lee, Chong-Yong; Ainsworth, Emma; Lockwood, Colin; Gross, Manuela A; Adschiri, Tadafumi; Reisner, Erwin; Butt, Julea N; Jeuken, Lars J C

    2015-01-01

    In nature, charge recombination in light-harvesting reaction centers is minimized by efficient charge separation. Here, it is aimed to mimic this by coupling dye-sensitized TiO2 nanocrystals to a decaheme protein, MtrC from Shewanella oneidensis MR-1, where the 10 hemes of MtrC form a ≈7-nm-long molecular wire between the TiO2 and the underlying electrode. The system is assembled by forming a densely packed MtrC film on an ultra-flat gold electrode, followed by the adsorption of approximately 7 nm TiO2 nanocrystals that are modified with a phosphonated bipyridine Ru(II) dye (RuP). The step-by-step construction of the MtrC/TiO2 system is monitored with (photo)electrochemistry, quartz-crystal microbalance with dissipation (QCM-D), and atomic force microscopy (AFM). Photocurrents are dependent on the redox state of the MtrC, confirming that electrons are transferred from the TiO2 nanocrystals to the surface via the MtrC conduit. In other words, in these TiO2/MtrC hybrid photodiodes, MtrC traps the conduction-band electrons from TiO2 before transferring them to the electrode, creating a photobioelectrochemical system in which a redox protein is used to mimic the efficient charge separation found in biological photosystems. PMID:26180522

  16. 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.

  17. The diheme cytochrome c4 from Vibrio cholerae is a natural electron donor to the respiratory cbb3 oxygen reductase

    PubMed Central

    Chang, Hsin-Yang; Ahn, Young; Pace, Laura A.; Lin, Myat T.; Lin, Yun-Hui; Gennis, Robert B.

    2010-01-01

    The respiratory chain of Vibrio cholerae contains three bd-type quinol oxygen reductases as well as one cbb3 oxygen reductase. The cbb3 oxygen reductase has been previously isolated and characterized, however the natural mobile electron donor(s) which shuttles electrons between the bc1 complex and the cbb3 oxygen reductase is not known. The most likely candidates are the diheme cytochrome c4 and mono-heme cytochrome c5, which have been previously shown to be present in the periplasm of aerobically grown cultures of V. cholerae. Both cytochromes c4 and c5 from V. cholerae have been cloned and expressed heterologously in E. coli. It is shown that reduced cytochrome c4 is a substrate for the purified cbb3 oxygen reductase and can support steady state oxygen reductase activity of at least 300 e−1/s. In contrast, reduced cytochrome c5 is not a good substrate for the cbb3 oxygen reductase. Surprisingly, the dependence of the oxygen reductase activity on the concentration of cytochrome c4 does not exhibit saturation. Global spectroscopic analysis of the time course of the oxidation of cytochrome c4 indicates that the apparent lack of saturation is due to the strong dependence of KM and Vmax on the concentration of oxidized cytochrome c4. Whether this is an artifact of the in vitro assay or has physiological significance remains unknown. Cyclic voltammetry was used to determine that the midpoint potentials of the two hemes in cytochrome c4 are 240 mV and 340 mV (vs SHE), similar to the electrochemical properties of other c4-type cytochromes. Genomic analysis shows a strong correlation between the presence of a c4-type cytochrome and a cbb3 oxygen reductase within the β- and γ- proteobacterial clades, suggesting that cytochrome c4 is the likely natural electron donor to the cbb3 oxygen reductases within these organisms. These would include the β-proteobacteria Neisseria meningitidis and Neisseria gonnorhoeae, in which the cbb3 oxygen reductases are the only terminal

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

    DOE PAGES

    Wei, Yifeng; Li, Bin; Prakash, Divya; ...

    2015-11-04

    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 RNRsmore » reflects the diversity of electron carriers used in anaerobic metabolism« less

  19. 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.

  20. 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

  1. Structure and function of NADPH-cytochrome P450 reductase and nitric oxide synthase reductase domain

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Iyanagi, Takashi

    2005-12-09

    NADPH-cytochrome P450 reductase (CPR) and the nitric oxide synthase (NOS) reductase domains are members of the FAD-FMN family of proteins. The FAD accepts two reducing equivalents from NADPH (dehydrogenase flavin) and FMN acts as a one-electron carrier (flavodoxin-type flavin) for the transfer from NADPH to the heme protein, in which the FMNH {sup {center_dot}}/FMNH{sub 2} couple donates electrons to cytochrome P450 at constant oxidation-reduction potential. Although the interflavin electron transfer between FAD and FMN is not strictly regulated in CPR, electron transfer is activated in neuronal NOS reductase domain upon binding calmodulin (CaM), in which the CaM-bound activated form canmore » function by a similar mechanism to that of CPR. The oxygenated form and spin state of substrate-bound cytochrome P450 in perfused rat liver are also discussed in terms of stepwise one-electron transfer from CPR. This review provides a historical perspective of the microsomal mixed-function oxidases including CPR and P450. In addition, a new model for the redox-linked conformational changes during the catalytic cycle for both CPR and NOS reductase domain is also discussed.« less

  2. Identification and Characterization of the Missing Pyrimidine Reductase in the Plant Riboflavin Biosynthesis Pathway1[W][OA

    PubMed Central

    Hasnain, Ghulam; Frelin, Océane; Roje, Sanja; Ellens, Kenneth W.; Ali, Kashif; Guan, Jiahn-Chou; Garrett, Timothy J.; de Crécy-Lagard, Valérie; Gregory, Jesse F.; McCarty, Donald R.; Hanson, Andrew D.

    2013-01-01

    Riboflavin (vitamin B2) is the precursor of the flavin coenzymes flavin mononucleotide and flavin adenine dinucleotide. In Escherichia coli and other bacteria, sequential deamination and reduction steps in riboflavin biosynthesis are catalyzed by RibD, a bifunctional protein with distinct pyrimidine deaminase and reductase domains. Plants have two diverged RibD homologs, PyrD and PyrR; PyrR proteins have an extra carboxyl-terminal domain (COG3236) of unknown function. Arabidopsis (Arabidopsis thaliana) PyrD (encoded by At4g20960) is known to be a monofunctional pyrimidine deaminase, but no pyrimidine reductase has been identified. Bioinformatic analyses indicated that plant PyrR proteins have a catalytically competent reductase domain but lack essential zinc-binding residues in the deaminase domain, and that the Arabidopsis PyrR gene (At3g47390) is coexpressed with riboflavin synthesis genes. These observations imply that PyrR is a pyrimidine reductase without deaminase activity. Consistent with this inference, Arabidopsis or maize (Zea mays) PyrR (At3g47390 or GRMZM2G090068) restored riboflavin prototrophy to an E. coli ribD deletant strain when coexpressed with the corresponding PyrD protein (At4g20960 or GRMZM2G320099) but not when expressed alone; the COG3236 domain was unnecessary for complementing activity. Furthermore, recombinant maize PyrR mediated NAD(P)H-dependent pyrimidine reduction in vitro. Import assays with pea (Pisum sativum) chloroplasts showed that PyrR and PyrD are taken up and proteolytically processed. Ablation of the maize PyrR gene caused early seed lethality. These data argue that PyrR is the missing plant pyrimidine reductase, that it is plastid localized, and that it is essential. The role of the COG3236 domain remains mysterious; no evidence was obtained for the possibility that it catalyzes the dephosphorylation that follows pyrimidine reduction. PMID:23150645

  3. Isolation and Characterization of a Soluble NADPH-Dependent Fe(III) Reductase from Geobacter sulfurreducens

    PubMed Central

    Kaufmann, Franz; Lovley, Derek R.

    2001-01-01

    NADPH is an intermediate in the oxidation of organic compounds coupled to Fe(III) reduction in Geobacter species, but Fe(III) reduction with NADPH as the electron donor has not been studied in these organisms. Crude extracts of Geobacter sulfurreducens catalyzed the NADPH-dependent reduction of Fe(III)-nitrilotriacetic acid (NTA). The responsible enzyme, which was recovered in the soluble protein fraction, was purified to apparent homogeneity in a four-step procedure. Its specific activity for Fe(III) reduction was 65 μmol · min−1 · mg−1. The soluble Fe(III) reductase was specific for NADPH and did not utilize NADH as an electron donor. Although the enzyme reduced several forms of Fe(III), Fe(III)-NTA was the preferred electron acceptor. The protein possessed methyl viologen:NADP+ oxidoreductase activity and catalyzed the reduction of NADP+ with reduced methyl viologen as electron donor at a rate of 385 U/mg. The enzyme consisted of two subunits with molecular masses of 87 and 78 kDa and had a native molecular mass of 320 kDa, as determined by gel filtration. The purified enzyme contained 28.9 mol of Fe, 17.4 mol of acid-labile sulfur, and 0.7 mol of flavin adenine dinucleotide per mol of protein. The genes encoding the two subunits were identified in the complete sequence of the G. sulfurreducens genome from the N-terminal amino acid sequences derived from the subunits of the purified protein. The sequences of the two subunits had about 30% amino acid identity to the respective subunits of the formate dehydrogenase from Moorella thermoacetica, but the soluble Fe(III) reductase did not possess formate dehydrogenase activity. This soluble Fe(III) reductase differs significantly from previously characterized dissimilatory and assimilatory Fe(III) reductases in its molecular composition and cofactor content. PMID:11443080

  4. Cloning, sequence determination, and regulation of the ribonucleotide reductase subunits from Plasmodium falciparum: a target for antimalarial therapy.

    PubMed Central

    Rubin, H; Salem, J S; Li, L S; Yang, F D; Mama, S; Wang, Z M; Fisher, A; Hamann, C S; Cooperman, B S

    1993-01-01

    Malaria remains a leading cause of morbidity and mortality worldwide, accounting for more than one million deaths annually. We have focused on the reduction of ribonucleotides to 2'-deoxyribonucleotides, catalyzed by ribonucleotide reductase, which represents the rate-determining step in DNA replication as a target for antimalarial agents. We report the full-length DNA sequence corresponding to the large (PfR1) and small (PfR2) subunits of Plasmodium falciparum ribonucleotide reductase. The small subunit (PfR2) contains the major catalytic motif consisting of a tyrosyl radical and a dinuclear Fe site. Whereas PfR2 shares 59% amino acid identity with human R2, a striking sequence divergence between human R2 and PfR2 at the C terminus may provide a selective target for inhibition of the malarial enzyme. A synthetic oligopeptide corresponding to the C-terminal 7 residues of PfR2 inhibits mammalian ribonucleotide reductase at concentrations approximately 10-fold higher than that predicted to inhibit malarial R2. The gene encoding the large subunit (PfR1) contains a single intron. The cysteines thought to be involved in the reduction mechanism are conserved. In contrast to mammalian ribonucleotide reductase, the genes for PfR1 and PfR2 are located on the same chromosome and the accumulation of mRNAs for the two subunits follow different temporal patterns during the cell cycle. Images Fig. 2 Fig. 4 Fig. 5 PMID:8415692

  5. Evidence for a Ustilago maydis steroid 5alpha-reductase by functional expression in Arabidopsis det2-1 mutants.

    PubMed

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

    2002-06-01

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

  6. 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.

  7. The enzymes with benzil reductase activity conserved from bacteria to mammals.

    PubMed

    Maruyama, Reiji; Nishizawa, Mikio; Itoi, Yasushi; Ito, Seiji; Inoue, Masami

    2002-03-28

    The diketone compound, benzil is reduced to (S)-benzoin with living Bacillus cereus cells. Recently, we isolated a gene responsible for benzil reduction, and Escherichia coli cells in which this gene was overexpressed transformed benzil to (S)-benzoin. Although this benzil reductase showed high identity to the short-chain dehydrogenase/reductase (SDR) family, enzymological features were unknown. Here, we demonstrated that many B. cereus strains had benzil reductase activity in vivo, and that the benzil reductases shared 94-100% amino acid identities. Recombinant B. cereus benzil reductase produced optically pure (S)-benzoin with NADPH in vitro, and the ketone group distal to a benzene ring was asymmetrically reduced. B. cereus benzil reductase showed 31% amino acid identity to the yeast open reading frame YIR036C protein and 28-30% to mammalian sepiapterin reductases, sharing the seven residues consensus for the SDR family. We isolated the genes encoding yeast YIR036C protein and gerbil sepiapterin reductase, and both recombinant proteins also reduced benzil to (S)-benzoin in vitro. Green fluorescent protein-tagged B. cereus benzil reductase distributed in the bipolar cytoplasm in B. cereus cells. Asymmetric reduction with B. cereus benzil reductase, yeast YIR036C protein and gerbil sepiapterin reductase will be utilized to produce important chiral compounds.

  8. Modeled structure of trypanothione reductase of Leishmania infantum.

    PubMed

    Singh, Bishal K; Sarkar, Nandini; Jagannadham, M V; Dubey, Vikash K

    2008-06-30

    Trypanothione reductase is an important target enzyme for structure-based drug design against Leishmania. We used homology modeling to construct a three-dimensional structure of the trypanothione reductase (TR) of Leishmania infantum. The structure shows acceptable Ramachandran statistics and a remarkably different active site from glutathione reductase(GR). Thus, a specific inhibitor against TR can be designed without interfering with host (human) GR activity.

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

    PubMed Central

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

    2002-01-01

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

  10. 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.

  11. Canopy and seasonal profiles of nitrate reductase in soybeans

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Harper, J.E.; Hageman, R.H.

    1972-01-01

    Nitrate reductase activity of soybeans (Glycine max L. Merr.) was evaluated in soil plots and outdoor hydroponic gravel culture systems throughout the growing season. Nitrate reductase profiles within the plant canopy were also established. Mean activity per gram fresh weight per hour of the entire plant canopy was highest in the seedling stage while total activity (activity per gram fresh weight per hour times the total leaf weight) reached a maximum when plants were in the full bloom to midpod fill stage. Nitrate reductase activity per gram fresh weight per hour was highest in the uppermost leaf just prior tomore » full expansion and declined with leaf positions lower in the canopy. Total nitrate reductase activity per leaf was also highest in the uppermost fully expanded leaf during early growth stages. Maximum total activity shifted to leaf positions lower in the plant canopy with later growth stages. Nitrate reductase activity of soybeans grown in hydroponic systems was significantly higher than activity of adjacent soil grown plants at later growth stages, which suggested that under normal field conditions the potential for nitrate utilization may not be realized. Nitrate reductase activity per gram fresh weight per hour and nitrate content were positively correlated over the growing season with plants grown in either soil or solution culture. Computations based upon the nitrate reductase assay of plants grown in hydroponics indicated that from 1.7 to 1.8 grams N could have been supplied to the plant via the nitrate reductase process. 11 references, 9 figures, 3 tables.« less

  12. Purification and characterization of protein PC, a component of glycine reductase from Eubacterium acidaminophilum.

    PubMed

    Schräder, T; Andreesen, J R

    1992-05-15

    Protein PC of the glycine reductase from Eubacterium acidaminophilum was purified to homogeneity by chromatography on phenyl-Sepharose and Sepharose S. The apparent molecular mass of the native protein, which showed an associating/dissociating behaviour, was about 420 kDa. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of protein PC revealed two protein bands corresponding to 48 and 57 kDa, indicating an alpha 4 beta 4 composition. The smaller subunit was identified as an acetyl-group-transferring protein, the 57-kDa protein was hydrophobic. N-terminal amino acid sequences were determined for both subunits. Antibodies raised against the 48-kDa subunit showed cross-reactions with extracts of E. acidaminophilum grown on different substrates and with extracts from other glycine-utilizing anaerobic bacteria such as Clostridium purinolyticum, C. sticklandii, and C. sporogenes. The respective protein from the former two organisms corresponded in molecular mass. When protein PA was chemically carboxymethylated by iodo[2-14C]acetate and incubated with protein PC, acetyl phosphate was a reaction product, thus establishing it as the product of the glycine reductase reaction by using homogeneous preparations of these two proteins from E. acidaminophilum.

  13. Haloarcula marismortui cytochrome b-561 is encoded by the narC gene in the dissimilatory nitrate reductase operon.

    PubMed

    Yoshimatsu, Katsuhiko; Araya, Osamu; Fujiwara, Taketomo

    2007-01-01

    The composition of membrane-bound electron-transferring proteins from denitrifying cells of Haloarcula marismortui was compared with that from the aerobic cells. Accompanying nitrate reductase catalytic NarGH subcomplex, cytochrome b-561, cytochrome b-552, and halocyanin-like blue copper protein were induced under denitrifying conditions. Cytochrome b-561 was purified to homogeneity and was shown to be composed of a polypeptide with a molecular mass of 40 kDa. The cytochrome was autooxidizable and its redox potential was -27 mV. The N-terminal sequence of the cytochrome was identical to the deduced amino acid sequence of the narC gene product encoded in the third ORF of the nitrate reductase operon with a unique arrangement of ORFs. The sequence of the cytochrome was homologous with that of the cytochrome b subunit of respiratory cytochrome bc. A possibility that the cytochrome bc and the NarGH constructed a supercomplex was discussed.

  14. Sortase A-mediated crosslinked short-chain dehydrogenases/reductases as novel biocatalysts with improved thermostability and catalytic efficiency.

    PubMed

    Li, Kunpeng; Zhang, Rongzhen; Xu, Yan; Wu, Zhimeng; Li, Jing; Zhou, Xiaotian; Jiang, Jiawei; Liu, Haiyan; Xiao, Rong

    2017-06-08

    (S)-carbonyl reductase II (SCRII) from Candida parapsilosis is a short-chain alcohol dehydrogenase/reductase. It catalyses the conversion of 2-hydroxyacetophenone to (S)-1-phenyl-1,2-ethanediol with low efficiency. Sortase was reported as a molecular "stapler" for site-specific protein conjugation to strengthen or add protein functionality. Here, we describe Staphylococcus aureus sortase A-mediated crosslinking of SCRII to produce stable catalysts for efficient biotransformation. Via a native N-terminal glycine and an added GGGGSLPETGG peptide at C-terminus of SCRII, SCRII subunits were conjugated by sortase A to form crosslinked SCRII, mainly dimers and trimers. The crosslinked SCRII showed over 6-fold and 4-fold increases, respectively, in activity and k cat /K m values toward 2-hydroxyacetophenone compared with wild-type SCRII. Moreover, crosslinked SCRII was much more thermostable with its denaturation temperature (T m ) increased to 60 °C. Biotransformation result showed that crosslinked SCRII gave a product optical purity of 100% and a yield of >99.9% within 3 h, a 16-fold decrease in transformation duration with respect to Escherichia coli/pET-SCRII. Sortase A-catalysed ligation also obviously improved T m s and product yields of eight other short-chain alcohol dehydrogenases/reductases. This work demonstrates a generic technology to improve enzyme function and thermostability through sortase A-mediated crosslinking of oxidoreductases.

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

    NASA Astrophysics Data System (ADS)

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

    2018-03-01

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

  16. 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.

  17. High Performance Reduction of H2O2 with an Electron Transport Decaheme Cytochrome on a Porous ITO Electrode

    PubMed Central

    2017-01-01

    The decaheme cytochrome MtrC from Shewanella oneidensis MR-1 immobilized on an ITO electrode displays unprecedented H2O2 reduction activity. Although MtrC showed lower peroxidase activity in solution compared to horseradish peroxidase, the ten heme cofactors enable excellent electronic communication and a superior activity on the electrode surface. A hierarchical ITO electrode enabled optimal immobilization of MtrC and a high current density of 1 mA cm–2 at 0.4 V vs SHE could be obtained at pH 6.5 (Eonset = 0.72 V). UV–visible and Resonance Raman spectroelectrochemical studies suggest the formation of a high valent iron-oxo species as the catalytic intermediate. Our findings demonstrate the potential of multiheme cytochromes to catalyze technologically relevant reactions and establish MtrC as a new benchmark in biotechnological H2O2 reduction with scope for applications in fuel cells and biosensors. PMID:28221032

  18. Changes in glutathione redox cycle during diapause determination and termination in the bivoltine silkworm, Bombyx mori.

    PubMed

    Zhao, Lin-Chuan; Hou, Yi-Sheng; Sima, Yang-Hu

    2014-02-01

    To explore whether glutathione regulates diapause determination and termination in the bivoltine silkworm Bombyx mori, we monitored the changes in glutathione redox cycle in the ovary of both diapause- and nondiapause-egg producers, as well as those in diapause eggs incubated at different temperatures. The activity of thioredoxin reductase (TrxR) was detected in ovaries but not in eggs, while neither ovaries nor eggs showed activity of glutathione peroxidase. A lower reduced glutathione/oxidized glutathione (GSH/GSSG) ratio was observed in the ovary of diapause-egg producers, due to weaker reduction of oxidized glutathione (GSSG) to the reduced glutathione (GSH) catalyzed by glutathione reductase (GR) and TrxR. This indicates an oxidative shift in the glutathione redox cycle during diapause determination. Compared with the 25°C-treated diapause eggs, the 5°C-treated diapause eggs showed lower GSH/GSSG ratio, a result of stronger oxidation of GSH catalyzed by thioredoxin peroxidase and weaker reduction of GSSG catalyzed by GR. Our study demonstrated the important regulatory role of glutathione in diapause determination and termination of the bivoltine silkworm. © 2013 Institute of Zoology, Chinese Academy of Sciences.

  19. Structural and biochemical characterization of cinnamoyl-coa reductases

    USDA-ARS?s Scientific Manuscript database

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

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

    PubMed

    Skubatz, Hanna; Howald, William N

    2013-03-01

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

  1. Structure of the detoxification catalyst mercuric ion reductase from Bacillus sp. strain RC607

    NASA Astrophysics Data System (ADS)

    Schiering, N.; Kabsch, W.; Moore, M. J.; Distefano, M. D.; Walsh, C. T.; Pai, E. F.

    1991-07-01

    SEVERAL hundred million tons of toxic mercurials are dispersed in the biosphere1. Microbes can detoxify organo-mercurials and mercury salts through sequential action of two enzymes, organomercury lyase2 and mercuric ion reductase (MerA) 3-5. The latter, a homodimer with homology to the FAD-dependent disulphide oxidoreductases6, catalyses the reaction NADPH + Hg(II) --> NADP+ + H+Hg(0), one of the very rare enzymic reactions with metal substrates. Human glutathione reductase7,8 serves as a reference molecule for FAD-dependent disulphide reductases and between its primary structure9 and that of MerA from Tn501 (Pseudomonas), Tn21 (Shigella), pI258 (Staphylococcus) and Bacillus, 25-30% of the residues have been conserved10,11. All MerAs have a C-terminal extension about 15 residues long but have very varied N termini. Although the enzyme from Streptomyces lividans has no addition, from Pseudomonas aeruginosa Tn5Ol and Bacillus sp. strain RC607 it has one and two copies respectively of a domain of 80-85 residues, highly homologous to MerP, the periplasmic component of proteins encoded by the mer operon11. These domains can be proteolytically cleaved off without changing the catalytic efficiency3. We report here the crystal structure of MerA from the Gram-positive bacterium Bacillus sp. strain RC607. Analysis of its complexes with nicotinamide dinucleotide substrates and the inhibitor Cd(II) reveals how limited structural changes enable an enzyme to accept as substrate what used to be a dangerous inhibitor. Knowledge of the mode of mercury ligation is a prerequisite for understanding this unique detoxification mechanism.

  2. 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.; hide

    2003-01-01

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

  3. Essential role of the flexible linker on the conformational equilibrium of bacterial peroxiredoxin reductase for effective regeneration of peroxiredoxin

    PubMed Central

    Kamariah, Neelagandan; Eisenhaber, Birgit; Eisenhaber, Frank; Grüber, Gerhard

    2017-01-01

    Reactive oxygen species (ROS) can damage DNA, proteins, and lipids, so cells have antioxidant systems that regulate ROS. In many bacteria, a dedicated peroxiredoxin reductase, alkyl hydroperoxide reductase subunit F (AhpF), catalyzes the rapid reduction of the redox-active disulfide center of the antioxidant protein peroxiredoxin (AhpC) to detoxify ROS such as hydrogen peroxide, organic hydroperoxide, and peroxynitrite. AhpF is a flexible multidomain protein that enables a series of electron transfers among the redox centers by accepting reducing equivalents from NADH. A flexible linker connecting the N-terminal domain (NTD) and C-terminal domain (CTD) of AhpF suggests that the enzyme adopts a large-scale domain motion that alternates between the closed and open states to shuttle electrons from the CTD via the NTD to AhpC. Here, we conducted comprehensive mutational, biochemical, and biophysical analyses to gain insights into the role of the flexible linker and the residues critical for the domain motions of Escherichia coli AhpF (EcAhpF) during electron transfer. Small-angle X-ray scattering studies of linker mutants revealed that a group of charged residues, 200EKR202, is crucial for the swiveling motion of the NTD. Moreover, NADH binding significantly affected EcAhpF flexibility and the movement of the NTD relative to the CTD. The mutants also exhibited a decrease in H2O2 reduction by the AhpF-AhpC ensemble. We propose that a concerted movement involving the NTD, C-terminal NADH, and FAD domains, and the flexible linker between them is essential for optimal intra-domain cross-talk and for efficient electron transfer to the redox partner AhpC required for peroxidation. PMID:28270505

  4. Resolution and partial characterization of two aldehyde reductases of mammalian liver.

    PubMed

    Tulsiani, D R; Touster

    1977-04-25

    Investigation of NADP-dependent aldehyde reductase activity in mouse liver led to the finding that two distinct reductases are separable by DE52 ion exchange chromatography. Aldehyde reductase I (AR I) appears in the effluent, while aldehyde reductase II (AR II) is eluted with a salt gradient. By several procedures AR II was purified over 1100-fold from liver supernatant fraction, but AR I could be pruified only 107-fold because of its instability. The two enzymes are different in regard to pH optimum, substrate specificity, response to inhibitors, and reactivity with antibody to AR II. While both enzymes utilize aromatic aldehydes well, only AR II ACTS ON D-glucuronate, indicating that it is the aldyhyde reductase recently reported to be identical to NADP-L-gulonate dehydrogenase. The presence of two NADP-linked aldehyde reductases in liver has apparently not heretofore been reported.

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

    PubMed

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

    2017-07-01

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

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

    PubMed

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

    2008-01-01

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

  7. Chlorate reductase is cotranscribed with cytochrome c and other downstream genes in the gene cluster for chlorate respiration of Ideonella dechloratans.

    PubMed

    Hellberg Lindqvist, Miriam; Nilsson, Thomas; Sundin, Pontus; Rova, Maria

    2015-03-01

    The chlorate-respiring bacterium Ideonella dechloratans is a facultative anaerobe that can use both oxygen and chlorate as terminal electron acceptors. The genes for the enzymes chlorate reductase (clrABDC) and chlorite dismutase, necessary for chlorate metabolism and probably acquired by lateral gene transfer, are located in a gene cluster that also includes other genes potentially important for chlorate metabolism. Among those are a gene for cytochrome c (cyc) whose gene product may serve as an electron carrier during chlorate reduction, a cofactor biosynthesis gene (mobB) and a predicted transcriptional regulator (arsR). Only chlorate reductase and chlorite dismutase have been shown to be expressed in vivo. Here, we report the in vivo production of a single polycistronic transcript covering eight open reading frames including clrABDC, cyc, mobB and arsR. Transcription levels of the cyc and clrA genes were compared to each other by the use of qRT-PCR in RNA preparations from cells grown under aerobic or chlorate reducing anaerobic conditions. The two genes showed the same mRNA levels under both growth regimes, indicating that no transcription termination occurs between them. Higher transcription levels were observed at growth without external oxygen supply. Implications for electron pathway integration following lateral gene transfer are discussed. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  8. Physiological and transcriptional approaches reveal connection between nitrogen and manganese cycles in Shewanella algae C6G3.

    PubMed

    Aigle, Axel; Bonin, Patricia; Iobbi-Nivol, Chantal; Méjean, Vincent; Michotey, Valérie

    2017-03-20

    To explain anaerobic nitrite/nitrate production at the expense of ammonium mediated by manganese oxide (Mn(IV)) in sediment, nitrate and manganese respirations were investigated in a strain (Shewanella algae C6G3) presenting these features. In contrast to S. oneidensis MR-1, a biotic transitory nitrite accumulation at the expense of ammonium was observed in S. algae during anaerobic growth with Mn(IV) under condition of limiting electron acceptor, concomitantly, with a higher electron donor stoichiometry than expected. This low and reproducible transitory accumulation is the result of production and consumption since the strain is able to dissimilative reduce nitrate into ammonium. Nitrite production in Mn(IV) condition is strengthened by comparative expression of the nitrate/nitrite reductase genes (napA, nrfA, nrfA-2), and rates of the nitrate/nitrite reductase activities under Mn(IV), nitrate or fumarate conditions. Compared with S. oneidensis MR-1, S. algae contains additional genes that encode nitrate and nitrite reductases (napA-α and nrfA-2) and an Outer Membrane Cytochrome (OMC)(mtrH). Different patterns of expression of the OMC genes (omcA, mtrF, mtrH and mtrC) were observed depending on the electron acceptor and growth phase. Only gene mtrF-2 (SO1659 homolog) was specifically expressed under the Mn(IV) condition. Nitrate and Mn(IV) respirations seem connected at the physiological and transcriptional levels.

  9. Physiological and transcriptional approaches reveal connection between nitrogen and manganese cycles in Shewanella algae C6G3

    NASA Astrophysics Data System (ADS)

    Aigle, Axel; Bonin, Patricia; Iobbi-Nivol, Chantal; Méjean, Vincent; Michotey, Valérie

    2017-03-01

    To explain anaerobic nitrite/nitrate production at the expense of ammonium mediated by manganese oxide (Mn(IV)) in sediment, nitrate and manganese respirations were investigated in a strain (Shewanella algae C6G3) presenting these features. In contrast to S. oneidensis MR-1, a biotic transitory nitrite accumulation at the expense of ammonium was observed in S. algae during anaerobic growth with Mn(IV) under condition of limiting electron acceptor, concomitantly, with a higher electron donor stoichiometry than expected. This low and reproducible transitory accumulation is the result of production and consumption since the strain is able to dissimilative reduce nitrate into ammonium. Nitrite production in Mn(IV) condition is strengthened by comparative expression of the nitrate/nitrite reductase genes (napA, nrfA, nrfA-2), and rates of the nitrate/nitrite reductase activities under Mn(IV), nitrate or fumarate conditions. Compared with S. oneidensis MR-1, S. algae contains additional genes that encode nitrate and nitrite reductases (napA-α and nrfA-2) and an Outer Membrane Cytochrome (OMC)(mtrH). Different patterns of expression of the OMC genes (omcA, mtrF, mtrH and mtrC) were observed depending on the electron acceptor and growth phase. Only gene mtrF-2 (SO1659 homolog) was specifically expressed under the Mn(IV) condition. Nitrate and Mn(IV) respirations seem connected at the physiological and transcriptional levels.

  10. Heme inhibition of ferrisiderophore reductase in Bacillus subtilis.

    PubMed

    Lodge, J S; Gaines, C G; Arceneaux, J E; Byers, B R

    1982-11-01

    Heme was a noncompetitive inhibitor (apparent K(i) and K'(i) = 0.043 mM) of a ferrisiderophore reductase purified from Bacillus subtilis; protoporphyrin IX had no effect. The cellular level of heme may partly regulate the function of this reductase to yield a controlled flow of iron into metabolism.

  11. Domain Evolution and Functional Diversification of Sulfite Reductases

    NASA Astrophysics Data System (ADS)

    Dhillon, Ashita; Goswami, Sulip; Riley, Monica; Teske, Andreas; Sogin, Mitchell

    2005-02-01

    Sulfite reductases are key enzymes of assimilatory and dissimilatory sulfur metabolism, which occur in diverse bacterial and archaeal lineages. They share a highly conserved domain "C-X5-C-n-C-X3-C" for binding siroheme and iron-sulfur clusters that facilitate electron transfer to the substrate. For each sulfite reductase cluster, the siroheme-binding domain is positioned slightly differently at the N-terminus of dsrA and dsrB, while in the assimilatory proteins the siroheme domain is located at the C-terminus. Our sequence and phylogenetic analysis of the siroheme-binding domain shows that sulfite reductase sequences diverged from a common ancestor into four separate clusters (aSir, alSir, dsr, and asrC) that are biochemically distinct; each serves a different assimilatory or dissimilatory role in sulfur metabolism. The phylogenetic distribution and functional grouping in sulfite reductase clusters (dsrA and dsrB vs. aSiR, asrC, and alSir) suggest that their functional diversification during evolution may have preceded the bacterial/archaeal divergence.

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

    PubMed

    Sarangi, Abhipsa; Krishnan, Chandraraj

    2016-02-01

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

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

    PubMed

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

    2002-03-01

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

  14. Isolation of Assimilatory- and Dissimilatory-Type Sulfite Reductases from Desulfovibrio vulgaris

    PubMed Central

    Lee, Jin-Po; LeGall, Jean; Peck, Harry D.

    1973-01-01

    Bisulfite reductase (desulfoviridin) and an assimilatory sulfite reductase have been purified from extracts of Desulfovibrio vulgaris. The bisulfite reductase has absorption maxima at 628, 580, 408, 390, and 279 nm, and a molecular weight of 226,000 by sedimentation equilibrium, and was judged to be free of other proteins by disk electrophoresis and ultracentrifugation. On gels, purified bisulfite reductase exhibited two green bands which coincided with activity and protein. The enzyme appears to be a tetramer but was shown to have two different types of subunits having molecular weights of 42,000 and 50,000. The chromophore did not form an alkaline ferrohemochromogen, was not reduced with dithionite or borohydride, and did not form a spectrally visible complex with CO. The assimilatory sulfite reductase has absorption maxima at 590, 545, 405 and 275 nm and a molecular weight of 26,800, and appears to consist of a single polypeptide chain as it is not dissociated into subunits by sodium dodecyl sulfate. By disk electrophoresis, purified sulfite reductase exhibited a single greenish-brown band which coincided with activity and protein. The sole product of the reduction was sulfide, and the chromophore was reduced by borohydride in the presence of sulfite. Carbon monoxide reacted with the reduced chromophore but it did not form a typical pyridine ferrohemochromogen. Thiosulfate, trithionate, and tetrathionate were not reduced by either enzyme preparation. In the presence of 8 M urea, the spectrum of bisulfite reductase resembles that of the sulfite reductase, thus suggesting a chemical relationship between the two chromophores. Images PMID:4725615

  15. Terminal Supraparticle Assemblies from Similarly Charged Protein Molecules and Nanoparticles

    PubMed Central

    Park, Jai Il; Nguyen, Trung Dac; de Queirós Silveira, Gleiciani; Bahng, Joong Hwan; Srivastava, Sudhanshu; Sun, Kai; Zhao, Gongpu; Zhang, Peijun; Glotzer, Sharon C.; Kotov, Nicholas A.

    2015-01-01

    Self-assembly of proteins and inorganic nanoparticles into terminal assemblies makes possible a large family of uniformly sized hybrid colloids. These particles can be compared in terms of utility, versatility and multifunctionality to other known types of terminal assemblies. They are simple to make and offer theoretical tools for designing their structure and function. To demonstrate such assemblies, we combine cadmium telluride nanoparticles with cytochrome C protein and observe spontaneous formation of spherical supraparticles with a narrow size distribution. Such self-limiting behaviour originates from the competition between electrostatic repulsion and non-covalent attractive interactions. Experimental variation of supraparticle diameters for several assembly conditions matches predictions obtained in simulations. Similar to micelles, supraparticles can incorporate other biological components as exemplified by incorporation of nitrate reductase. Tight packing of nanoscale components enables effective charge and exciton transport in supraparticles as demonstrated by enzymatic nitrate reduction initiated by light absorption in the nanoparticle. PMID:24845400

  16. Exploiting the synthetic lethality between terminal respiratory oxidases to kill Mycobacterium tuberculosis and clear host infection

    PubMed Central

    Kalia, Nitin P.; Hasenoehrl, Erik J.; Ab Rahman, Nurlilah B.; Koh, Vanessa H.; Ang, Michelle L. T.; Sajorda, Dannah R.; Hards, Kiel; Grüber, Gerhard; Alonso, Sylvie; Cook, Gregory M.; Berney, Michael; Pethe, Kevin

    2017-01-01

    The recent discovery of small molecules targeting the cytochrome bc1:aa3 in Mycobacterium tuberculosis triggered interest in the terminal respiratory oxidases for antituberculosis drug development. The mycobacterial cytochrome bc1:aa3 consists of a menaquinone:cytochrome c reductase (bc1) and a cytochrome aa3-type oxidase. The clinical-stage drug candidate Q203 interferes with the function of the subunit b of the menaquinone:cytochrome c reductase. Despite the affinity of Q203 for the bc1:aa3 complex, the drug is only bacteriostatic and does not kill drug-tolerant persisters. This raises the possibility that the alternate terminal bd-type oxidase (cytochrome bd oxidase) is capable of maintaining a membrane potential and menaquinol oxidation in the presence of Q203. Here, we show that the electron flow through the cytochrome bd oxidase is sufficient to maintain respiration and ATP synthesis at a level high enough to protect M. tuberculosis from Q203-induced bacterial death. Upon genetic deletion of the cytochrome bd oxidase-encoding genes cydAB, Q203 inhibited mycobacterial respiration completely, became bactericidal, killed drug-tolerant mycobacterial persisters, and rapidly cleared M. tuberculosis infection in vivo. These results indicate a synthetic lethal interaction between the two terminal respiratory oxidases that can be exploited for anti-TB drug development. Our findings should be considered in the clinical development of drugs targeting the cytochrome bc1:aa3, as well as for the development of a drug combination targeting oxidative phosphorylation in M. tuberculosis. PMID:28652330

  17. Photoreduction of Shewanella oneidensis Extracellular Cytochromes by Organic Chromophores and Dye‐Sensitized TiO2

    PubMed Central

    Ainsworth, Emma V.; Lockwood, Colin W. J.; White, Gaye F.; Hwang, Ee Taek; Sakai, Tsubasa; Gross, Manuela A.; Richardson, David J.; Clarke, Thomas A.

    2016-01-01

    Abstract The transfer of photoenergized electrons from extracellular photosensitizers across a bacterial cell envelope to drive intracellular chemical transformations represents an attractive way to harness nature's catalytic machinery for solar‐assisted chemical synthesis. In Shewanella oneidensis MR‐1 (MR‐1), trans‐outer‐membrane electron transfer is performed by the extracellular cytochromes MtrC and OmcA acting together with the outer‐membrane‐spanning porin⋅cytochrome complex (MtrAB). Here we demonstrate photoreduction of solutions of MtrC, OmcA, and the MtrCAB complex by soluble photosensitizers: namely, eosin Y, fluorescein, proflavine, flavin, and adenine dinucleotide, as well as by riboflavin and flavin mononucleotide, two compounds secreted by MR‐1. We show photoreduction of MtrC and OmcA adsorbed on RuII‐dye‐sensitized TiO2 nanoparticles and that these protein‐coated particles perform photocatalytic reduction of solutions of MtrC, OmcA, and MtrCAB. These findings provide a framework for informed development of strategies for using the outer‐membrane‐associated cytochromes of MR‐1 for solar‐driven microbial synthesis in natural and engineered bacteria. PMID:27685371

  18. 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.

  19. 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.

  20. A high-throughput assay format for determination of nitrate reductase and nitrite reductase enzyme activities

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    McNally, N.; Liu, Xiang Yang; Choudary, P.V.

    1997-01-01

    The authors describe a microplate-based high-throughput procedure for rapid assay of the enzyme activities of nitrate reductase and nitrite reductase, using extremely small volumes of reagents. The new procedure offers the advantages of rapidity, small sample size-nanoliter volumes, low cost, and a dramatic increase in the throughput sample number that can be analyzed simultaneously. Additional advantages can be accessed by using microplate reader application software packages that permit assigning a group type to the wells, recording of the data on exportable data files and exercising the option of using the kinetic or endpoint reading modes. The assay can also bemore » used independently for detecting nitrite residues/contamination in environmental/food samples. 10 refs., 2 figs.« less

  1. Identifying the role of cytochromes upon the attachment, growth and detachment of Shewanella oneidensis MR-1 on hematite during dissimilatory iron reduction under natural- flow conditions

    NASA Astrophysics Data System (ADS)

    Mitchell, A. C.; Geesey, G. G.

    2006-12-01

    Current understanding of bacterial respiration by dissimilatory iron (Fe) reduction is based primarily on studies of closed systems using soluble Fe(III). However, natural environments likely to support Fe reduction are typically open systems and contain Fe(III) primarily in the form of crystalline (hydr)oxides. Mechanisms by which electrons are transported between bacteria and mineral terminal electron acceptors (TEAs) under open system conditions are still poorly understood. However, a number of cytochromes have been identified as potentially playing a critical role in the electron transport system of some Fe reducing bacteria. Experiments were performed using (i) omcA, (ii) mtrC, or (iii) omcA and mtrC cytochrome deficient mutants of the Fe-reducing bacteria, Shewanella oneidensis MR-1, in transparent-window flow- reactors containing hematite as the only TEA. These were operated under defined hydrodynamic and anaerobic conditions. Cells expressed green fluorescent protein (gfp), allowing real time measurement of cells at the mineral surface by epifluorescence microscopy. Cytochromes which play a critical role in the anaerobic growth of S. Oneidensis by Fe reduction under open system natural-flow conditions could then be identified. Differences in the accumulation, maximum density, detachment and total production of surface-associated cells growing on hematite surfaces were apparent between the mutants, and between the mutants and the wild-type. Mutants deficient in cytochromes grew to a lower max density by up to 2 orders of magnitude than the wild-type, and exhibited no reduced Fe in the reactor effluent or at the surface of the hematite at the conclusion of the experiment, as revealed by X-Ray photoelectron spectroscopy (XPS). Therefore omcA and / or mtrC cytochromes appear critical for electron shuttling and anaerobic growth of S. Oneidensis on hematite under natural-flow conditions.

  2. Respiratory arsenate reductase as a bidirectional enzyme

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Richey, Christine; Chovanec, Peter; Department of Chemistry and Biochemistry, Duquesne University, Pittsburgh, PA 15282

    2009-05-01

    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 asmore » 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.« less

  3. Partial vinylphenol reductase purification and characterization from Brettanomyces bruxellensis.

    PubMed

    Tchobanov, Iavor; Gal, Laurent; Guilloux-Benatier, Michèle; Remize, Fabienne; Nardi, Tiziana; Guzzo, Jean; Serpaggi, Virginie; Alexandre, Hervé

    2008-07-01

    Brettanomyces is the major microbial cause for wine spoilage worldwide and causes significant economic losses. The reasons are the production of ethylphenols that lead to an unpleasant taint described as 'phenolic odour'. Despite its economic importance, Brettanomyces has remained poorly studied at the metabolic level. The origin of the ethylphenol results from the conversion of vinylphenols in ethylphenol by Brettanomyces hydroxycinnamate decarboxylase. However, no information is available on the vinylphenol reductase responsible for the conversion of vinylphenols in ethylphenols. In this study, a vinylphenol reductase was partially purified from Brettanomyces bruxellensis that was active towards 4-vinylguaiacol and 4-vinylphenol only among the substrates tested. First, a vinylphenol reductase activity assay was designed that allowed us to show that the enzyme was NADH dependent. The vinylphenol reductase was purified 152-fold with a recovery yield of 1.77%. The apparent K(m) and V(max) values for the hydrolysis of 4-vinylguaiacol were, respectively, 0.14 mM and 1900 U mg(-1). The optimal pH and temperature for vinylphenol reductase were pH 5-6 and 30 degrees C, respectively. The molecular weight of the enzyme was 26 kDa. Trypsic digest of the protein was performed and the peptides were sequenced, which allowed us to identify in Brettanomyces genome an ORF coding for a 210 amino acid protein.

  4. Inhibitory effect of chalcone derivatives on recombinant human aldose reductase.

    PubMed

    Iwata, S; Nagata, N; Omae, A; Yamaguchi, S; Okada, Y; Shibata, S; Okuyama, T

    1999-03-01

    More than fifty chalcone derivatives were synthesized to examine structure-activity relationships against human aldose reductase. Certain 2',4'-dihydroxychalcone derivatives inhibited human aldose reductase activities, and 2',4',2, 4-tetrahydroxychalcone and 2',4',2-trihydroxychalcone showed potent inhibitory activity with IC50 values of 7.4x10(-9) M and 1.6x10(-7) M, respectively. On the other hand, cis-form chalcones, which were isomerized from the original trans-forms by irradiation of daylight in methanol solution, promoted the activity of human aldose reductase.

  5. A Novel Arsenate Reductase from the Arsenic Hyperaccumulating Fern Pteris vittata1

    PubMed Central

    Ellis, Danielle R.; Gumaelius, Luke; Indriolo, Emily; Pickering, Ingrid J.; Banks, Jo Ann; Salt, David E.

    2006-01-01

    Pteris vittata sporophytes hyperaccumulate arsenic to 1% to 2% of their dry weight. Like the sporophyte, the gametophyte was found to reduce arsenate [As(V)] to arsenite [As(III)] and store arsenic as free As(III). Here, we report the isolation of an arsenate reductase gene (PvACR2) from gametophytes that can suppress the arsenate sensitivity and arsenic hyperaccumulation phenotypes of yeast (Saccharomyces cerevisiae) lacking the arsenate reductase gene ScACR2. Recombinant PvACR2 protein has in vitro arsenate reductase activity similar to ScACR2. While PvACR2 and ScACR2 have sequence similarities to the CDC25 protein tyrosine phosphatases, they lack phosphatase activity. In contrast, Arath;CDC25, an Arabidopsis (Arabidopsis thaliana) homolog of PvACR2 was found to have both arsenate reductase and phosphatase activities. To our knowledge, PvACR2 is the first reported plant arsenate reductase that lacks phosphatase activity. CDC25 protein tyrosine phosphatases and arsenate reductases have a conserved HCX5R motif that defines the active site. PvACR2 is unique in that the arginine of this motif, previously shown to be essential for phosphatase and reductase activity, is replaced with a serine. Steady-state levels of PvACR2 expression in gametophytes were found to be similar in the absence and presence of arsenate, while total arsenate reductase activity in P. vittata gametophytes was found to be constitutive and unaffected by arsenate, consistent with other known metal hyperaccumulation mechanisms in plants. The unusual active site of PvACR2 and the arsenate reductase activities of cell-free extracts correlate with the ability of P. vittata to hyperaccumulate arsenite, suggesting that PvACR2 may play an important role in this process. PMID:16766666

  6. Three-dimensional structure of NADPH–cytochrome P450 reductase: Prototype for FMN- and FAD-containing enzymes

    PubMed Central

    Wang, Ming; Roberts, David L.; Paschke, Rosemary; Shea, Thomas M.; Masters, Bettie Sue Siler; Kim, Jung-Ja P.

    1997-01-01

    Microsomal NADPH–cytochrome P450 reductase (CPR) is one of only two mammalian enzymes known to contain both FAD and FMN, the other being nitric-oxide synthase. CPR is a membrane-bound protein and catalyzes electron transfer from NADPH to all known microsomal cytochromes P450. The structure of rat liver CPR, expressed in Escherichia coli and solubilized by limited trypsinolysis, has been determined by x-ray crystallography at 2.6 Å resolution. The molecule is composed of four structural domains: (from the N- to C- termini) the FMN-binding domain, the connecting domain, and the FAD- and NADPH-binding domains. The FMN-binding domain is similar to the structure of flavodoxin, whereas the two C-terminal dinucleotide-binding domains are similar to those of ferredoxin–NADP+ reductase (FNR). The connecting domain, situated between the FMN-binding and FNR-like domains, is responsible for the relative orientation of the other domains, ensuring the proper alignment of the two flavins necessary for efficient electron transfer. The two flavin isoalloxazine rings are juxtaposed, with the closest distance between them being about 4 Å. The bowl-shaped surface near the FMN-binding site is likely the docking site of cytochrome c and the physiological redox partners, including cytochromes P450 and b5 and heme oxygenase. PMID:9237990

  7. Male Sterile2 Encodes a Plastid-Localized Fatty Acyl Carrier Protein Reductase Required for Pollen Exine Development in Arabidopsis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Chen, W.; Shanklin, J.; Yu, X.-H.

    Male Sterile2 (MS2) is predicted to encode a fatty acid reductase required for pollen wall development in Arabidopsis (Arabidopsis thaliana). Transient expression of MS2 in tobacco (Nicotiana benthamiana) leaves resulted in the accumulation of significant levels of C16 and C18 fatty alcohols. Expression of MS2 fused with green fluorescent protein revealed that an amino-terminal transit peptide targets the MS2 to plastids. The plastidial localization of MS2 is biologically important because genetic complementation of MS2 in ms2 homozygous plants was dependent on the presence of its amino-terminal transit peptide or that of the Rubisco small subunit protein amino-terminal transit peptide. Inmore » addition, two domains, NAD(P)H-binding domain and sterile domain, conserved in MS2 and its homologs were also shown to be essential for MS2 function in pollen exine development by genetic complementation testing. Direct biochemical analysis revealed that purified recombinant MS2 enzyme is able to convert palmitoyl-Acyl Carrier Protein to the corresponding C16:0 alcohol with NAD(P)H as the preferred electron donor. Using optimized reaction conditions (i.e. at pH 6.0 and 30 C), MS2 exhibits a K{sub m} for 16:0-Acyl Carrier Protein of 23.3 {+-} 4.0 {mu}m, a V{sub max} of 38.3 {+-} 4.5 nmol mg{sup -1} min{sup -1}, and a catalytic efficiency/K{sub m} of 1,873 m{sup -1} s{sup -1}. Based on the high homology of MS2 to other characterized fatty acid reductases, it was surprising that MS2 showed no activity against palmitoyl- or other acyl-coenzyme A; however, this is consistent with its plastidial localization. In summary, genetic and biochemical evidence demonstrate an MS2-mediated conserved plastidial pathway for the production of fatty alcohols that are essential for pollen wall biosynthesis in Arabidopsis.« less

  8. COP9 signalosome subunit 7 from Arabidopsis interacts with and regulates the small subunit of ribonucleotide reductase (RNR2).

    PubMed

    Halimi, Yair; Dessau, Moshe; Pollak, Shaul; Ast, Tslil; Erez, Tamir; Livnat-Levanon, Nurit; Karniol, Baruch; Hirsch, Joel A; Chamovitz, Daniel A

    2011-09-01

    The COP9 Signalosome protein complex (CSN) is a pleiotropic regulator of plant development and contains eight-subunits. Six of these subunits contain the PCI motif which mediates specific protein interactions necessary for the integrity of the complex. COP9 complex subunit 7 (CSN7) contains an N-terminal PCI motif followed by a C-terminal extension which is also necessary for CSN function. A yeast-interaction trap assay identified the small subunit of ribonucelotide reductase (RNR2) from Arabidopsis as interacting with the C-terminal section of CSN7. This interaction was confirmed in planta by both bimolecular fluorescence complementation and immuoprecipitation assays with endogenous proteins. The subcellular localization of RNR2 was primarily nuclear in meristematic regions, and cytoplasmic in adult cells. RNR2 was constitutively nuclear in csn7 mutant seedlings, and was also primarily nuclear in wild type seedlings following exposure to UV-C. These two results correlate with constitutive expression of several DNA-damage response genes in csn7 mutants, and to increased tolerance of csn7 seedlings to UV-C treatment. We propose that the CSN is a negative regulator of RNR activity in Arabidopsis.

  9. Isolation and expression of a Bacillus cereus gene encoding benzil reductase.

    PubMed

    Maruyama, R; Nishizawa, M; Itoi, Y; Ito, S; Inoue, M

    2001-12-20

    Benzil was reduced stereospecifically to (S)-benzoin by Bacillus cereus strain Tim-r01. To isolate the gene responsible for asymmetric reduction, we constructed a library consisting of Escherichia coli clones that harbored plasmids expressing Bacillus cereus genes. The library was screened using the halo formation assay, and one clone showed benzil reduction to (S)-benzoin. Thus, this clone seemed to carry a plasmid encoding a Bacillus cereus benzil reductase. The deduced amino acid sequence had marked homologies to the Bacillus subtilis yueD protein (41% identity), the yeast open reading frame YIR036C protein (31%), and the mammalian sepiapterin reductases (28% to 30%), suggesting that benzil reductase is a novel short-chain de-hydrogenases/ reductase. Copyright 2001 John Wiley & Sons, Inc.

  10. The C-type Arabidopsis thioredoxin reductase ANTR-C acts as an electron donor to 2-Cys peroxiredoxins in chloroplasts

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Moon, Jeong Chan; Jang, Ho Hee; Chae, Ho Byoung

    2006-09-22

    2-Cys peroxiredoxins (Prxs) play important roles in the antioxidative defense systems of plant chloroplasts. In order to determine the interaction partner for these proteins in Arabidopsis, we used a yeast two-hybrid screening procedure with a C175S-mutant of Arabidopsis 2-Cys Prx-A as bait. A cDNA encoding an NADPH-dependent thioredoxin reductase (NTR) isotype C was identified and designated ANTR-C. We demonstrated that this protein effected efficient transfer of electrons from NADPH to the 2-Cys Prxs of chloroplasts. Interaction between 2-Cys Prx-A and ANTR-C was confirmed by a pull-down experiment. ANTR-C contained N-terminal TR and C-terminal Trx domains. It exhibited both TR andmore » Trx activities and co-localized with 2-Cys Prx-A in chloroplasts. These results suggest that ANTR-C functions as an electron donor for plastidial 2-Cys Prxs and represents the NADPH-dependent TR/Trx system in chloroplasts.« less

  11. Physiological and transcriptional approaches reveal connection between nitrogen and manganese cycles in Shewanella algae C6G3

    PubMed Central

    Aigle, Axel; Bonin, Patricia; Iobbi-Nivol, Chantal; Méjean, Vincent; Michotey, Valérie

    2017-01-01

    To explain anaerobic nitrite/nitrate production at the expense of ammonium mediated by manganese oxide (Mn(IV)) in sediment, nitrate and manganese respirations were investigated in a strain (Shewanella algae C6G3) presenting these features. In contrast to S. oneidensis MR-1, a biotic transitory nitrite accumulation at the expense of ammonium was observed in S. algae during anaerobic growth with Mn(IV) under condition of limiting electron acceptor, concomitantly, with a higher electron donor stoichiometry than expected. This low and reproducible transitory accumulation is the result of production and consumption since the strain is able to dissimilative reduce nitrate into ammonium. Nitrite production in Mn(IV) condition is strengthened by comparative expression of the nitrate/nitrite reductase genes (napA, nrfA, nrfA-2), and rates of the nitrate/nitrite reductase activities under Mn(IV), nitrate or fumarate conditions. Compared with S. oneidensis MR-1, S. algae contains additional genes that encode nitrate and nitrite reductases (napA-α and nrfA-2) and an Outer Membrane Cytochrome (OMC)(mtrH). Different patterns of expression of the OMC genes (omcA, mtrF, mtrH and mtrC) were observed depending on the electron acceptor and growth phase. Only gene mtrF-2 (SO1659 homolog) was specifically expressed under the Mn(IV) condition. Nitrate and Mn(IV) respirations seem connected at the physiological and transcriptional levels. PMID:28317859

  12. [Membrane lipids and electron transfer. Effects of four detergents on NADH-ferricyanide reductase and NADH-cytochrome c reductase activities of potato tuber microsomes].

    PubMed

    Jolliot, A; Mazliak, P

    1977-10-17

    The NADH-ferricyanure reductase activity of Potato microsomes is stimulated by non ionic detergents (Triton X100 and Tween80) and is partially inhibited by ionic detergents (sodium-cholate and deoxycholate). All these four detergents progressively decreased the NADH-cytochrome c reductase in the following order: sodium deoxycholate greater than Triton X100 greater than sodium cholate greater than Tween80.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

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

    1987-11-03

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

  14. 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.

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

    PubMed Central

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

    2011-01-01

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

  16. Relative adrenal insufficiency in mice deficient in 5α-reductase 1

    PubMed Central

    Livingstone, Dawn E W; Di Rollo, Emma M; Yang, Chenjing; Codrington, Lucy E; Mathews, John A; Kara, Madina; Hughes, Katherine A; Kenyon, Christopher J; Walker, Brian R; Andrew, Ruth

    2014-01-01

    Patients with critical illness or hepatic failure exhibit impaired cortisol responses to ACTH, a phenomenon known as ‘relative adrenal insufficiency’. A putative mechanism is that elevated bile acids inhibit inactivation of cortisol in liver by 5α-reductases type 1 and type 2 and 5β-reductase, resulting in compensatory downregulation of the hypothalamic–pituitary–adrenal axis and adrenocortical atrophy. To test the hypothesis that impaired glucocorticoid clearance can cause relative adrenal insufficiency, we investigated the consequences of 5α-reductase type 1 deficiency in mice. In adrenalectomised male mice with targeted disruption of 5α-reductase type 1, clearance of corticosterone was lower after acute or chronic (eightfold, P<0.05) administration, compared with WT control mice. In intact 5α-reductase-deficient male mice, although resting plasma corticosterone levels were maintained, corticosterone responses were impaired after ACTH administration (26% lower, P<0.05), handling stress (2.5-fold lower, P<0.05) and restraint stress (43% lower, P<0.05) compared with WT mice. mRNA levels of Nr3c1 (glucocorticoid receptor), Crh and Avp in pituitary or hypothalamus were altered, consistent with enhanced negative feedback. These findings confirm that impaired peripheral clearance of glucocorticoids can cause ‘relative adrenal insufficiency’ in mice, an observation with important implications for patients with critical illness or hepatic failure, and for patients receiving 5α-reductase inhibitors for prostatic disease. PMID:24872577

  17. The 3-hydroxy-3-methylglutaryl coenzyme-A (HMG-CoA) reductases

    PubMed Central

    Friesen, Jon A; Rodwell, Victor W

    2004-01-01

    The enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase catalyzes the conversion of HMG-CoA to mevalonate, a four-electron oxidoreduction that is the rate-limiting step in the synthesis of cholesterol and other isoprenoids. The enzyme is found in eukaryotes and prokaryotes; and phylogenetic analysis has revealed two classes of HMG-CoA reductase, the Class I enzymes of eukaryotes and some archaea and the Class II enzymes of eubacteria and certain other archaea. Three-dimensional structures of the catalytic domain of HMG-CoA reductases from humans and from the bacterium Pseudomonas mevalonii, in conjunction with site-directed mutagenesis studies, have revealed details of the mechanism of catalysis. The reaction catalyzed by human HMG-CoA reductase is a target for anti-hypercholesterolemic drugs (statins), which are intended to lower cholesterol levels in serum. Eukaryotic forms of the enzyme are anchored to the endoplasmic reticulum, whereas the prokaryotic enzymes are soluble. Probably because of its critical role in cellular cholesterol homeostasis, mammalian HMG-CoA reductase is extensively regulated at the transcriptional, translational, and post-translational levels. PMID:15535874

  18. 1-Ene-steroid reductase of Mycobacterium sp. NRRL B-3805.

    PubMed

    Goren, T; Harnik, M; Rimon, S; Aharonowitz, Y

    1983-12-01

    The microbial enzymatic reduction of 1,4-androstadiene-3,17-dione (ADD) to 4-androstene-3,17-dione (AD), testosterone and 1-dehydrotestosterone (DHT) is described. Two reducing activities observed in washed cell suspensions and cell free extracts of Mycobacterium sp. NRRL B-3805 were found to account for these bioconversions. One was a 1-ene-steroid reductase and the other a 17-keto steroid reductase. The first reducing activity was found to appear in the soluble cell fraction whereas the latter could be precipitated by centrifugation. Maximum 1-ene-steroid reductase specific activity was achieved during the exponential growth phase of the organism and significantly increased upon induction with ADD. The 1-ene-steroid reductase was partially purified (30-fold) by ammonium sulfate fractionation, gel-filtration and ion-exchange chromatography, and was eluted from a Sephacryl S-300 column with an Mr = 115,000. The 1-ene-steroid reductase activity was NADPH-dependent and had specificity towards steroid compounds containing C-1,2 double bond with an apparent Km for ADD of 2.2 X 10(-5) M. The reverse reaction catalyzing C-1,2 dehydrogenation could not be detected in our preparations. The results suggest that in Mycobacterium sp NRRL B-3805 and B-3683 the steroid C-1,2 dehydrogenation and 1-ene reduction are two separable activities.

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

    NASA Astrophysics Data System (ADS)

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

    2018-03-01

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

  20. 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

  1. Purification and Characterization of Ferredoxin-Nicotinamide Adenine Dinucleotide Phosphate Reductase from a Nitrogen-Fixing Bacterium

    PubMed Central

    Yoch, Duane C.

    1973-01-01

    Evidence suggesting that Bacillus polymyxa has an active ferredoxin-NADP+ reductase (EC 1.6.99.4) was obtained when NADPH was found to provide reducing power for the nitrogenase of this organism; direct evidence was provided when it was shown that B. polymyxa extracts could substitute for the native ferredoxin-NADP+ reductase in the photochemical reduction of NADP+ by blue-green algal particles. The ferredoxin-NADP+ reductase was purified about 80-fold by a combination of high-speed centrifugation, ammonium sulfate fractionation, and chromatography on Sephadex G-100 and diethylaminoethyl-cellulose. The molecular weight was estimated by gel filtration to be 60,000. A small amount of the enzyme was further purified by polyacrylamide gel electrophoresis and shown to be a flavoprotein. The reductase was specific for NADPH in the ferredoxin-dependent reduction of cytochrome c and methyl viologen diaphorase reactions; furthermore, NADP+ was the acceptor of preference when the electron donor was photoreduced ferredoxin. The reductase also has an irreversible NADPH-NAD+ transhydrogenase (reduced-NADP:NAD oxidoreductase, EC 1.6.1.1) activity, the rate of which was proportional to the concentration of NAD (Km = 5.0 × 10−3M). The reductase catalyzed electron transfer from NADPH not only to B. polymyxa ferredoxin but also to the ferredoxins of Clostridium pasteurianum, Azotobacter vinelandii, and spinach chloroplasts, although less effectively. Rubredoxin from Clostridium acidi-urici and azotoflavin from A. vinelandii also accept electrons from the B. polymyxa reductase. The pH optima for the various reactions catalyzed by the B. polymyxa ferredoxin-NADP reductase are similar to those of the chloroplast reductase. NAD and acetyl-coenzyme A, which obligatorily activate NADPH- and NADH-ferredoxin reductases, respectively, in Clostridium kluyveri, have no effect on B. polymyxa reductase. PMID:4147648

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

    NASA Astrophysics Data System (ADS)

    Basyuni, M.; Wati, R.

    2018-03-01

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

  3. 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.

  4. Characterization of 5α-reductase activity and isoenzymes in human abdominal adipose tissues.

    PubMed

    Fouad Mansour, Mohamed; Pelletier, Mélissa; Tchernof, André

    2016-07-01

    The substrate for the generation of 5α-dihydrotestosterone (DHT) is either androstenedione (4-dione) which is first converted to androstanedione and then to DHT through 17-oxoreductase activity, or testosterone, which is directly converted to DHT. Three 5α-reductase isoenzymes have been characterized and designated as types 1, 2 and 3 (SRD5A1, 2 and 3). To define the predominant source of local DHT production in human adipose tissues, identify 5α-reductase isoenzymes and test their impact on preadipocyte differentiation. Cultures of omental (OM) and subcutaneous (SC) preadipocytes were treated for 0, 6 or 24h with 30nM (14)C-4-dione or (14)C-testosterone, with and without 500nM 5α-reductase inhibitors 17-N,N-diethylcarbamoyl-4-methyl-4-aza-5-androstan-3-one (4-MA) or finasteride. Protein level and mRNA abundance of 5α-reductase isoenzymes/transcripts were examined in whole SC and OM adipose tissue. HEK-293 cells stably transfected with 5α-reductase type 1, 2 or 3 were used to test 5α-reductase inhibitors. We also assessed the impact of 5α-reductase inhibitors on preadipocyte differentiation. Over 24h, DHT formation from 4-dione increased gradually (p<0.05) and was significantly higher compared to that generated from testosterone (p<0.001). DHT formation from both 4-dione and testosterone was blocked by both 5α-reductase inhibitors. In whole adipose tissue from both fat compartments, SRD5A3 was the most highly expressed isoenzyme followed by SRD5A1 (p<0.001). SRD5A2 was not expressed. In HEK-293 cells, 4-MA and finasteride inhibited activity of 5α-reductases types 2 and 3 but not type 1. In preadipocyte cultures where differentiation was inhibited by 4-dione (p<0.05, n=7) or testosterone (p<0.05, n=5), the inhibitors 4-MA and finasteride abolished these effects. Although 4-dione is the main source of DHT in human preadipocytes, production of this steroid by 5α-reductase isoenzymes mediates the inhibitory effect of both 4-dione and testosterone on

  5. Connection between nitrogen and manganese cycles revealed by transcriptomic analysis in Shewanella algae C6G3

    NASA Astrophysics Data System (ADS)

    Michotey, V.; Aigle, A.; Armougom, F.; Mejean, V.; Guasco, S.; Bonin, P.

    2016-02-01

    In sedimentary systems, the repartition of terminal electron-accepting molecules is often stratified on a permanent or seasonal basis. Just below to oxic zone, the suboxic one is characterized by high concentrations of oxidized inorganic compounds such as nitrate, manganese oxides (MnIII/IV) and iron oxides that are in close vicinity. Several studies have reported unexpected anaerobic nitrite/nitrate production at the expense of ammonium mediated by MnIII/IV, however this transient processes is difficult to discern and poorly understood. In the frame of this study, genes organization of nitrate and MnIII/IV respiration was investigated in S.algae. Additional genes were identified in S. algae compare to S. oneidensis: genes coding for nitrate and nitrite reductase (napA-a and nrfA-2) and an OMC protein (mtrH). In contrast to S. oneidensis, an anaerobic transitory nitrite accumulation at the expense of ammonium was observed in S. algae during growth with MnIII/IV, concomitantly with expression of nitrate/nitrite reductase genes (napA, nrfA, nrfA-2). Among the hypothesis explaining this data, the potential putative expression of unidentified gene able to perform ammonium oxidation was not observed on the global transcriptional level, however several signs of oxidative stress were detected and the existence of a secondary reaction generated by a putative oxidative s could not be excluded. Another option could be the action of reverse reaction by an enzyme such as NrfA or NrfA-2 due to the electron flow equilibrium. Whatever the electron acceptor (Nitrate/ MnIII/IV), the unexpected expression level of of omcA, mtrF, mtrH, mtrC was observed and peaked at the end of the exponential phase. Different expression patterns of the omc genes were observed depending on electron acceptor and growth phase. Only mtrF-2 gene was specifically expressed in Mn(III/IV) condition. Nitrate and Mn(III/IV) respirations seem connected at physiological as well as at transcriptional level

  6. 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.

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

    PubMed

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

    2004-04-01

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

  8. Medium-chain dehydrogenase/reductase and aldo-keto reductase scavenge reactive carbonyls in Synechocystis sp. PCC 6803.

    PubMed

    Shimakawa, Ginga; Kohara, Ayaka; Miyake, Chikahiro

    2018-03-01

    Reactive carbonyls (RCs), which are inevitably produced during respiratory and photosynthetic metabolism, have the potential to cause oxidative damage to photosynthetic organisms. Previously, we proposed a scavenging model for RCs in the cyanobacterium Synechocystis sp. PCC 6803 (S. 6803). In the current study, we constructed mutants deficient in the enzymes medium-chain dehydrogenase/reductase (ΔMDR) and aldo-keto reductase (ΔAKR) to investigate their contributions to RC scavenging in vivo. We found that treatment with the lipid-derived RC acrolein causes growth inhibition and promotes greater protein carbonylation in ΔMDR, compared with the wild-type and ΔAKR. In both ΔMDR and ΔAKR, photosynthesis is severely inhibited in the presence of acrolein. These results suggest that these enzymes function as part of the scavenging systems for RCs in S. 6803 in vivo. © 2018 Federation of European Biochemical Societies.

  9. 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

  10. 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.

  11. X-ray Structure of a Hg 2+ Complex of Mercuric Reductase (MerA) and Quantum Mechanical/Molecular Mechanical Study of Hg 2+ Transfer between the C-Terminal and Buried Catalytic Site Cysteine Pairs

    DOE PAGES

    Lian, Peng; Guo, Hao-Bo; Riccardi, Demian; ...

    2014-10-24

    Here we report that mercuric reductase, MerA, is a key enzyme in bacterial mercury resistance. This homodimeric enzyme captures and reduces toxic Hg 2+ to Hg 0, which is relatively unreactive and can exit the cell passively. Prior to reduction, the Hg 2+ is transferred from a pair of cysteines (C558' and C559' using Tn501 numbering) at the C-terminus of one monomer to another pair of cysteines (C136 and C141) in the catalytic site of the other monomer. Here, we present the X-ray structure of the C-terminal Hg 2+ complex of the C136A/C141A double mutant of the Tn501 MerA catalyticmore » core and explore the molecular mechanism of this Hg transfer with quantum mechanical/molecular mechanical (QM/MM) calculations. The transfer is found to be nearly thermoneutral and to pass through a stable tricoordinated intermediate that is marginally less stable than the two end states. For the overall process, Hg 2+ is always paired with at least two thiolates and thus is present at both the C-terminal and catalytic binding sites as a neutral complex. Prior to Hg 2+ transfer, C141 is negatively charged. As Hg 2+ is transferred into the catalytic site, a proton is transferred from C136 to C559' while C558' becomes negatively charged, resulting in the net transfer of a negative charge over a distance of ~7.5 Å. Thus, the transport of this soft divalent cation is made energetically feasible by pairing a competition between multiple Cys thiols and/or thiolates for Hg 2+ with a competition between the Hg 2+ and protons for the thiolates.« less

  12. Pyrroline-5-Carboxylate Reductase in Chlorella autotrophica and Chlorella saccharophila in Relation to Osmoregulation 1

    PubMed Central

    Laliberté, Gilles; Hellebust, Johan A.

    1989-01-01

    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 Km 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. PMID:16667157

  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. 49 CFR 1242.27 - Coal marine terminals, ore marine terminals, TOFC/COFC terminals, other marine terminals, motor...

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 49 Transportation 9 2014-10-01 2014-10-01 false Coal marine terminals, ore marine terminals, TOFC/COFC terminals, other marine terminals, motor vehicle loading and distribution facilities, and... Structures § 1242.27 Coal marine terminals, ore marine terminals, TOFC/COFC terminals, other marine terminals...

  15. 49 CFR 1242.27 - Coal marine terminals, ore marine terminals, TOFC/COFC terminals, other marine terminals, motor...

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 49 Transportation 9 2013-10-01 2013-10-01 false Coal marine terminals, ore marine terminals, TOFC/COFC terminals, other marine terminals, motor vehicle loading and distribution facilities, and... Structures § 1242.27 Coal marine terminals, ore marine terminals, TOFC/COFC terminals, other marine terminals...

  16. 49 CFR 1242.27 - Coal marine terminals, ore marine terminals, TOFC/COFC terminals, other marine terminals, motor...

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 49 Transportation 9 2012-10-01 2012-10-01 false Coal marine terminals, ore marine terminals, TOFC/COFC terminals, other marine terminals, motor vehicle loading and distribution facilities, and... Structures § 1242.27 Coal marine terminals, ore marine terminals, TOFC/COFC terminals, other marine terminals...

  17. 49 CFR 1242.27 - Coal marine terminals, ore marine terminals, TOFC/COFC terminals, other marine terminals, motor...

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 49 Transportation 9 2011-10-01 2011-10-01 false Coal marine terminals, ore marine terminals, TOFC/COFC terminals, other marine terminals, motor vehicle loading and distribution facilities, and... Structures § 1242.27 Coal marine terminals, ore marine terminals, TOFC/COFC terminals, other marine terminals...

  18. 49 CFR 1242.27 - Coal marine terminals, ore marine terminals, TOFC/COFC terminals, other marine terminals, motor...

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 9 2010-10-01 2010-10-01 false Coal marine terminals, ore marine terminals, TOFC/COFC terminals, other marine terminals, motor vehicle loading and distribution facilities, and... Structures § 1242.27 Coal marine terminals, ore marine terminals, TOFC/COFC terminals, other marine terminals...

  19. 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. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  20. 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

  1. Tropinone reductases, enzymes at the branch point of tropane alkaloid metabolism.

    PubMed

    Dräger, Birgit

    2006-02-01

    Two stereospecific oxidoreductases constitute a branch point in tropane alkaloid metabolism. Products of tropane metabolism are the alkaloids hyoscyamine, scopolamine, cocaine, and polyhydroxylated nortropane alkaloids, the calystegines. Both tropinone reductases reduce the precursor tropinone to yield either tropine or pseudotropine. In Solanaceae, tropine is incorporated into hyoscyamine and scopolamine; pseudotropine is the first specific metabolite on the way to the calystegines. Isolation, cloning and heterologous expression of both tropinone reductases enabled kinetic characterisation, protein crystallisation, and structure elucidation. Stereospecificity of reduction is achieved by binding tropinone in the respective enzyme active centre in opposite orientation. Immunolocalisation of both enzyme proteins in cultured roots revealed a tissue-specific protein accumulation. Metabolite flux through both arms of the tropane alkaloid pathway appears to be regulated by the activity of both enzymes and by their access to the precursor tropinone. Both tropinone reductases are NADPH-dependent short-chain dehydrogenases with amino acid sequence similarity of more than 50% suggesting their descent from a common ancestor. Putative tropinone reductase sequences annotated in plant genomes other that Solanaceae await functional characterisation.

  2. Nitrate Transport Is Independent of NADH and NAD(P)H Nitrate Reductases in Barley Seedlings 1

    PubMed Central

    Warner, Robert L.; Huffaker, Ray 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. PMID:11537465

  3. Enzyme phylogenies as markers for the oxidation state of the environment: the case of respiratory arsenate reductase and related enzymes.

    PubMed

    Duval, Simon; Ducluzeau, Anne-Lise; Nitschke, Wolfgang; Schoepp-Cothenet, Barbara

    2008-07-16

    Phylogenies of certain bioenergetic enzymes have proved to be useful tools for deducing evolutionary ancestry of bioenergetic pathways and their relationship to geochemical parameters of the environment. Our previous phylogenetic analysis of arsenite oxidase, the molybdopterin enzyme responsible for the biological oxidation of arsenite to arsenate, indicated its probable emergence prior to the Archaea/Bacteria split more than 3 billion years ago, in line with the geochemical fact that arsenite was present in biological habitats on the early Earth. Respiratory arsenate reductase (Arr), another molybdopterin enzyme involved in microbial arsenic metabolism, serves as terminal oxidase, and is thus situated at the opposite end of bioenergetic electron transfer chains as compared to arsenite oxidase. The evolutionary history of the Arr-enzyme has not been studied in detail so far. We performed a genomic search of genes related to arrA coding for the molybdopterin subunit. The multiple alignment of the retrieved sequences served to reconstruct a neighbor-joining phylogeny of Arr and closely related enzymes. Our analysis confirmed the previously proposed proximity of Arr to the cluster of polysulfide/thiosulfate reductases but also unravels a hitherto unrecognized clade even more closely related to Arr. The obtained phylogeny strongly suggests that Arr originated after the Bacteria/Archaea divergence in the domain Bacteria, and was subsequently laterally distributed within this domain. It further more indicates that, as a result of accumulation of arsenate in the environment, an enzyme related to polysulfide reductase and not to arsenite oxidase has evolved into Arr. These findings are paleogeochemically rationalized by the fact that the accumulation of arsenate over arsenite required the increase in oxidation state of the environment brought about by oxygenic photosynthesis.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  5. 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

  6. The prenyltransferase UBIAD1 is the target of geranylgeraniol in degradation of HMG CoA reductase.

    PubMed

    Schumacher, Marc M; Elsabrouty, Rania; Seemann, Joachim; Jo, Youngah; DeBose-Boyd, Russell A

    2015-03-05

    Schnyder corneal dystrophy (SCD) is an autosomal dominant disorder in humans characterized by abnormal accumulation of cholesterol in the cornea. SCD-associated mutations have been identified in the gene encoding UBIAD1, a prenyltransferase that synthesizes vitamin K2. Here, we show that sterols stimulate binding of UBIAD1 to the cholesterol biosynthetic enzyme HMG CoA reductase, which is subject to sterol-accelerated, endoplasmic reticulum (ER)-associated degradation augmented by the nonsterol isoprenoid geranylgeraniol through an unknown mechanism. Geranylgeraniol inhibits binding of UBIAD1 to reductase, allowing its degradation and promoting transport of UBIAD1 from the ER to the Golgi. CRISPR-CAS9-mediated knockout of UBIAD1 relieves the geranylgeraniol requirement for reductase degradation. SCD-associated mutations in UBIAD1 block its displacement from reductase in the presence of geranylgeraniol, thereby preventing degradation of reductase. The current results identify UBIAD1 as the elusive target of geranylgeraniol in reductase degradation, the inhibition of which may contribute to accumulation of cholesterol in SCD.

  7. Manumycin A Is a Potent Inhibitor of Mammalian Thioredoxin Reductase-1 (TrxR-1).

    PubMed

    Tuladhar, Anupama; Rein, Kathleen S

    2018-04-12

    The anticancer effect of manumycin A (Man A) has been attributed to the inhibition of farnesyl transferase (FTase), an enzyme that is responsible for post-translational modification of Ras proteins. However, we have discovered that Man A inhibits mammalian cytosolic thioredoxin reductase 1 (TrxR-1) in a time-dependent manner, with an IC 50 of 272 nM with preincubation and 1586 nM without preincubation. The inhibition of TrxR-1 by Man A is irreversible and is the result of a covalent interaction between Man A and TrxR-1. Evidence presented herein demonstrates that Man A forms a Michael adduct with the selenocysteine residue, which is located in the C-terminal redox center of TrxR-1. Inhibitors of TrxR-1, which act through this mechanism, convert TrxR-1 into a SecTRAP, which utilizes NADPH to reduce oxygen to superoxide radical anion (O 2 -• ).

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

    PubMed Central

    Vincentz, M; Caboche, M

    1991-01-01

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

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

    PubMed

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

    2017-12-01

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

  10. Thyroid hormone stimulation of NADPH P450 reductase expression in liver and extrahepatic tissues. Regulation by multiple mechanisms.

    PubMed

    Ram, P A; Waxman, D J

    1992-02-15

    The role of thyroid hormone in regulating the expression of the flavoprotein NADPH cytochrome P450 reductase was studied in adult rats. Depletion of circulating thyroid hormone by hypophysectomy, or more selectively, by treatment with the anti-thyroid drug methimazole led to a 75-85% depletion of hepatic microsomal P450 reductase activity and protein in both male and female rats. Thyroxine substantially restored P450 reductase activity at a dose that rendered the thyroid-depleted rats euthyroid. Microsomal P450 reductase activity in several extrahepatic tissues was also dependent on thyroid hormone, but to a lesser extent than in liver (30-50% decrease in kidney, adrenal, lung, and heart but not in testis from hypothyroid rats). Hepatic P450 reductase mRNA levels were also decreased in the hypothyroid state, indicating that the loss of P450 reductase activity is not a consequence of the associated decreased availability of the FMN and FAD cofactors of P450 reductase. Parallel analysis of S14 mRNA, which has been studied extensively as a model thyroid-regulated liver gene product, indicated that P450 reductase and S14 mRNA respond similarly to these changes in thyroid state. In contrast, while the expression of S14 and several other thyroid hormone-dependent hepatic mRNAs is stimulated by feeding a high carbohydrate, fat-free diet, hepatic P450 reductase expression was not increased by this lipogenic diet. Injection of hypothyroid rats with T3 at a supraphysiologic, receptor-saturating dose stimulated a major induction of hepatic P450 reductase mRNA that was detectable 4 h after the T3 injection, and peaked at approximately 650% of euthyroid levels by 12 h. However, this same treatment stimulated a biphasic increase in P450 reductase protein and activity that required 3 days to reach normal euthyroid levels. T3 treatment of euthyroid rats also stimulated a major induction of P450 reductase mRNA that was maximal (12-fold increase) by 12 h, but in this case no major

  11. 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.

  12. Overexpression of tropinone reductases alters alkaloid composition in Atropa belladonna root cultures.

    PubMed

    Richter, Ute; Rothe, Grit; Fabian, Anne-Katrin; Rahfeld, Bettina; Dräger, Birgit

    2005-02-01

    The medicinally applied tropane alkaloids hyoscyamine and scopolamine are produced in Atropa belladonna L. and in a small number of other Solanaceae. Calystegines are nortropane alkaloids that derive from a branching point in the tropane alkaloid biosynthetic pathway. In A. belladonna root cultures, calystegine molar concentration is 2-fold higher than that of hyoscyamine and scopolamine. In this study, two tropinone reductases forming a branching point in the tropane alkaloid biosynthesis were overexpressed in A. belladonna. Root culture lines with strong overexpression of the transcripts contained more enzyme activity of the respective reductase and enhanced enzyme products, tropine or pseudotropine. High pseudotropine led to an increased accumulation of calystegines in the roots. Strong expression of the tropine-forming reductase was accompanied by 3-fold more hyoscyamine and 5-fold more scopolamine compared with control roots, and calystegine levels were decreased by 30-90% of control. In some of the transformed root cultures, an increase of total tropane alkaloids was observed. Thus, transformation with cDNA of tropinone reductases successfully altered the ratio of tropine-derived alkaloids versus pseudotropine-derived alkaloids.

  13. An orally effective dihydropyrimidone (DHPM) analogue induces apoptosis-like cell death in clinical isolates of Leishmania donovani overexpressing pteridine reductase 1.

    PubMed

    Singh, Neeloo; Kaur, Jaspreet; Kumar, Pranav; Gupta, Swati; Singh, Nasib; Ghosal, Angana; Dutta, Avijit; Kumar, Ashutosh; Tripathi, Ramapati; Siddiqi, Mohammad Imran; Mandal, Chitra; Dube, Anuradha

    2009-10-01

    The protozoan parasite Leishmania donovani is the causative agent of visceral leishmaniasis. The enzyme pteridine reductase 1 (PTR1) of L. donovani acts as a metabolic bypass for drugs targeting dihydrofolate reductase (DHFR); therefore, for successful antifolate chemotherapy to be developed against Leishmania, it must target both enzyme activities. Leishmania cells overexpressing PTR1 tagged at the N-terminal with green fluorescent protein were established to screen for proprietary dihydropyrimidone (DHPM) derivatives of DHFR specificity synthesised in our laboratory. A cell-permeable molecule with impressive antileishmanial in vitro and in vivo oral activity was identified. Structure activity relationship based on homology model drawn on our recombinant enzyme established the highly selective inhibition of the enzyme by this analogue. It was seen that the leishmanicidal effect of this analogue is triggered by programmed cell death mediated by the loss of plasma membrane integrity as detected by binding of annexin V and propidium iodide (PI), loss of mitochondrial membrane potential culminating in cell cycle arrest at the sub-G0/G1 phase and oligonucleosomal DNA fragmentation. Hence, this DHPM analogue [(4-fluoro-phenyl)-6-methyl-2-thioxo-1, 2, 3, 4-tetrahydropyrimidine-5-carboxylic acid ethyl ester] is a potent antileishmanial agent that merits further pharmacological investigation.

  14. 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

  15. The prenyltransferase UBIAD1 is the target of geranylgeraniol in degradation of HMG CoA reductase

    PubMed Central

    Schumacher, Marc M; Elsabrouty, Rania; Seemann, Joachim; Jo, Youngah; DeBose-Boyd, Russell A

    2015-01-01

    Schnyder corneal dystrophy (SCD) is an autosomal dominant disorder in humans characterized by abnormal accumulation of cholesterol in the cornea. SCD-associated mutations have been identified in the gene encoding UBIAD1, a prenyltransferase that synthesizes vitamin K2. Here, we show that sterols stimulate binding of UBIAD1 to the cholesterol biosynthetic enzyme HMG CoA reductase, which is subject to sterol-accelerated, endoplasmic reticulum (ER)-associated degradation augmented by the nonsterol isoprenoid geranylgeraniol through an unknown mechanism. Geranylgeraniol inhibits binding of UBIAD1 to reductase, allowing its degradation and promoting transport of UBIAD1 from the ER to the Golgi. CRISPR-CAS9-mediated knockout of UBIAD1 relieves the geranylgeraniol requirement for reductase degradation. SCD-associated mutations in UBIAD1 block its displacement from reductase in the presence of geranylgeraniol, thereby preventing degradation of reductase. The current results identify UBIAD1 as the elusive target of geranylgeraniol in reductase degradation, the inhibition of which may contribute to accumulation of cholesterol in SCD. DOI: http://dx.doi.org/10.7554/eLife.05560.001 PMID:25742604

  16. Enzyme phylogenies as markers for the oxidation state of the environment: The case of respiratory arsenate reductase and related enzymes

    PubMed Central

    2008-01-01

    Background Phylogenies of certain bioenergetic enzymes have proved to be useful tools for deducing evolutionary ancestry of bioenergetic pathways and their relationship to geochemical parameters of the environment. Our previous phylogenetic analysis of arsenite oxidase, the molybdopterin enzyme responsible for the biological oxidation of arsenite to arsenate, indicated its probable emergence prior to the Archaea/Bacteria split more than 3 billion years ago, in line with the geochemical fact that arsenite was present in biological habitats on the early Earth. Respiratory arsenate reductase (Arr), another molybdopterin enzyme involved in microbial arsenic metabolism, serves as terminal oxidase, and is thus situated at the opposite end of bioenergetic electron transfer chains as compared to arsenite oxidase. The evolutionary history of the Arr-enzyme has not been studied in detail so far. Results We performed a genomic search of genes related to arrA coding for the molybdopterin subunit. The multiple alignment of the retrieved sequences served to reconstruct a neighbor-joining phylogeny of Arr and closely related enzymes. Our analysis confirmed the previously proposed proximity of Arr to the cluster of polysulfide/thiosulfate reductases but also unravels a hitherto unrecognized clade even more closely related to Arr. The obtained phylogeny strongly suggests that Arr originated after the Bacteria/Archaea divergence in the domain Bacteria, and was subsequently laterally distributed within this domain. It further more indicates that, as a result of accumulation of arsenate in the environment, an enzyme related to polysulfide reductase and not to arsenite oxidase has evolved into Arr. Conclusion These findings are paleogeochemically rationalized by the fact that the accumulation of arsenate over arsenite required the increase in oxidation state of the environment brought about by oxygenic photosynthesis. PMID:18631373

  17. Ribonucleotide reductase activity is regulated by proliferating cell nuclear antigen (PCNA)

    PubMed Central

    Salguero, Israel; Guarino, Estrella; Shepherd, Marianne; Deegan, Tom; Havens, Courtney G.; MacNeill, Stuart A.; Walter, Johannes C.; Kearsey, Stephen E.

    2014-01-01

    Summary Synthesis of dNTPs is required for both DNA replication and DNA repair and is catalyzed by ribonucleotide reductases (RNR), which convert ribonucleotides to their deoxy forms [1, 2]. Maintaining the correct levels of dNTPs for DNA synthesis is important for minimising the mutation rate [3-7], and this is achieved by tight regulation of ribonucleotide reductase [2, 8, 9]. In fission yeast, ribonucleotide reductase is regulated in part by a small protein inhibitor, Spd1, which is degraded in S phase and after DNA damage to allow up-regulation of dNTP supply [10-12]. Spd1 degradation is mediated by the activity of the CRL4Cdt2 ubiquitin ligase complex [5, 13, 14]. This has been reported to be dependent on modulation of Cdt2 levels which are cell cycle regulated, peaking in S phase, and which also increase after DNA damage in a checkpoint-dependent manner [7, 13]. We show here that Cdt2 levels fluctuations are not sufficient to regulate Spd1 proteolysis and that the key step in this event is the interaction of Spd1 with the polymerase processivity factor PCNA, complexed onto DNA. This mechanism thus provides a direct link between DNA synthesis and ribonucleotide reductase regulation. PMID:22464192

  18. NADPH-dependent coenzyme Q reductase is the main enzyme responsible for the reduction of non-mitochondrial CoQ in cells.

    PubMed

    Takahashi, Takayuki; Okuno, Masaaki; Okamoto, Tadashi; Kishi, Takeo

    2008-01-01

    We purified an NADPH-dependent coenzyme Q reductase (NADPH-CoQ reductase) in rat liver cytosol and compared its enzymatic properties with those of the other CoQ10 reductases such as NADPH: quinone acceptor oxidoreductase 1 (NQO1), lipoamide dehydrogenase, thioredoxine reductase and glutathione reductase. NADPH-CoQ reductase was the only enzyme that preferred NADPH to NADH as an electron donor and was also different from the other CoQ10 reductases in the sensitivities to its inhibitors and stimulators. Especially, Zn2+ was the most powerful inhibitor for NADPH-CoQ reductase, but CoQ10 reduction by the other CoQ10 reductases could not be inhibited by Zn2+. Furthermore, the reduction of the CoQ9 incorporated into HeLa cells was also inhibited by Zn2+ in the presence of pyrithione, a zinc ionophore. Moreover, NQO1 gene silencing in HeLa cells by transfection of a small interfering RNA resulted in lowering of both the NQO1 protein level and the NQO1 activity by about 75%. However, this transfection did not affect the NADPH-CoQ reductase activity and the reduction of CoQ9 incorporated into the cells. These results suggest that the NADPH-CoQ reductase located in cytosol may be the main enzyme responsible for the reduction of non-mitochondrial CoQ in cells.

  19. Aerobic degradation of 2,4,6-trinitrotoluene by Enterobacter cloacae PB2 and by pentaerythritol tetranitrate reductase

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    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 colimore » 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.« less

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

    PubMed

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

    1989-02-15

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

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

    PubMed

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

    1997-04-01

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

  2. Nitrogen fixation in transposon mutants from Bradyrhizobium japonicum USDA 110 impaired in nitrate reductase.

    PubMed

    Camacho, María; Burgos, Araceli; Chamber-Pérez, Manuel A

    2003-04-01

    Tn5 transposon mutagenesis was carried out in Bradyrhizobium japonicum strain USDA 110 to produce defective mutants. From over one thousand clones expressing low levels of nitrate reductase activity as free-living bacteria, approximately five percent had significantly different ratios of nodulation, N2 fixation or nitrate reductase activity compared to the wild strain when determined in bacteroids from soybean nodules. Tn5 insertions were checked previously and mutants were arranged into four different groups. Only one of these groups, designated AN, was less effective at N2 fixation than the wild strain, suggesting a mutation in a domain shared by nitrogenase and NR. The remaining groups of insertions successfully nodulated and were as effective at N2 fixation as the wild strain, but showed diminished ability to reduce nitrate both in nodules and in the isolated bacteroids when assayed in vitro with NADH or methyl viologen as electron donors. PCR amplification demonstrated that Tn5 insertions took place in different genes on each mutant group and the type of mutant (CC) expressing almost no nitrate reductase activity under all treatments seemed to possess transposable elements in two genes. Induction of nitrate reductase activity by nitrate was observed only in those clones expressing a low constitutive activity (AN and AE). Nitrate reductase activity in bacteroids along nodule growth decreased in all groups including the ineffective AN group, whose nodulation was highly inhibited by nitrate at 5 mmol/L N. Host-cultivar interaction seemed to influence the regulation of nitrate reductase activity in bacteroids. Total or partial repression of nitrate reductase activity in bacteroids unaffected by N2 fixation (CC, AJ and AE groups) improved nodule resistance to nitrate and N yields of shoots over those of the wild strain. These observations may suggest that some of the energy supplied to bacteroids was wasted by its constitutive NRA.

  3. The pH Requirement for in Vivo Activity of the Iron-Deficiency-Induced "Turbo" Ferric Chelate Reductase (A Comparison of the Iron-Deficiency-Induced Iron Reductase Activities of Intact Plants and Isolated Plasma Membrane Fractions in Sugar Beet).

    PubMed Central

    Susin, S.; Abadia, A.; Gonzalez-Reyes, J. A.; Lucena, J. J.; Abadia, J.

    1996-01-01

    The characteristics of the Fe reduction mechanisms induced by Fe deficiency have been studied in intact plants of Beta vulgaris and in purified plasma membrane vesicles from the same plants. In Fe-deficient plants the in vivo Fe(III)-ethylenediaminetetraacetic complex [Fe(III)-EDTA] reductase activity increased over the control values 10 to 20 times when assayed at a pH of 6.0 or below ("turbo" reductase) but increased only 2 to 4 times when assayed at a pH of 6.5 or above. The Fe(III)-EDTA reductase activity of root plasma membrane preparations increased 2 and 3.5 times over the controls, irrespective of the assay pH. The Km for Fe(III)-EDTA of the in vivo ferric chelate reductase in Fe-deficient plants was approximately 510 and 240 [mu]M in the pH ranges 4.5 to 6.0 and 6.5 to 8.0, respectively. The Km for Fe(III)-EDTA of the ferric chelate reductase in intact control plants and in plasma membrane preparations isolated from Fe-deficient and control plants was approximately 200 to 240 [mu]M. Therefore, the turbo ferric chelate reductase activity of Fe-deficient plants at low pH appears to be different from the constitutive ferric chelate reductase. PMID:12226175

  4. The pH Requirement for in Vivo Activity of the Iron-Deficiency-Induced "Turbo" Ferric Chelate Reductase (A Comparison of the Iron-Deficiency-Induced Iron Reductase Activities of Intact Plants and Isolated Plasma Membrane Fractions in Sugar Beet).

    PubMed

    Susin, S.; Abadia, A.; Gonzalez-Reyes, J. A.; Lucena, J. J.; Abadia, J.

    1996-01-01

    The characteristics of the Fe reduction mechanisms induced by Fe deficiency have been studied in intact plants of Beta vulgaris and in purified plasma membrane vesicles from the same plants. In Fe-deficient plants the in vivo Fe(III)-ethylenediaminetetraacetic complex [Fe(III)-EDTA] reductase activity increased over the control values 10 to 20 times when assayed at a pH of 6.0 or below ("turbo" reductase) but increased only 2 to 4 times when assayed at a pH of 6.5 or above. The Fe(III)-EDTA reductase activity of root plasma membrane preparations increased 2 and 3.5 times over the controls, irrespective of the assay pH. The Km for Fe(III)-EDTA of the in vivo ferric chelate reductase in Fe-deficient plants was approximately 510 and 240 [mu]M in the pH ranges 4.5 to 6.0 and 6.5 to 8.0, respectively. The Km for Fe(III)-EDTA of the ferric chelate reductase in intact control plants and in plasma membrane preparations isolated from Fe-deficient and control plants was approximately 200 to 240 [mu]M. Therefore, the turbo ferric chelate reductase activity of Fe-deficient plants at low pH appears to be different from the constitutive ferric chelate reductase.

  5. Cytidine 5'-diphosphate reductase activity in phytohemagglutinin stimulated human lymphocytes.

    PubMed Central

    Tyrsted, G; Gamulin, V

    1979-01-01

    The optimal conditions and the effect of deoxyribonucleoside triphosphates were determined for CDP reductase activity in PHA-stimulated lymphocytes. The enzymatic reaction showed an absolute requirement for ATP. In the absence of ATP, only dATP showed a minor stimulation of the reduction of CDP to dCDP. During transformation the CDP reductase activity reached a maximum at the same time as the four deoxyribonucleoside triphosphate pools, corresponding to mid S-phase at about 50 h after PHA addition. The DNA polymerase activity reached a maximum at 57 h. PMID:424294

  6. Peach MYB7 activates transcription of the proanthocyanidin pathway gene encoding leucoanthocyanidin reductase, but not anthocyanidin reductase

    PubMed Central

    Zhou, Hui; Lin-Wang, Kui; Liao, Liao; Gu, Chao; Lu, Ziqi; Allan, Andrew C.; Han, Yuepeng

    2015-01-01

    Proanthocyanidins (PAs) are a group of natural phenolic compounds that have a great effect on both flavor and nutritious value of fruit. It has been shown that PA synthesis is regulated by R2R3-MYB transcription factors (TFs) via activation of PA-specific pathway genes encoding leucoanthocyanidin reductase and anthocyanidin reductase. Here, we report the isolation and characterization of a MYB gene designated PpMYB7 in peach. The peach PpMYB7 represents a new group of R2R3-MYB genes regulating PA synthesis in plants. It is able to activate transcription of PpLAR1 but not PpANR, and has a broader selection of potential bHLH partners compared with PpMYBPA1. Transcription of PpMYB7 can be activated by the peach basic leucine-zipper 5 TF (PpbZIP5) via response to ABA. Our study suggests a transcriptional network regulating PA synthesis in peach, with the results aiding the understanding of the functional divergence between R2R3-MYB TFs in plants. PMID:26579158

  7. 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.

  8. The location of dissimilatory nitrite reductase and the control of dissimilatory nitrate reductase by oxygen in Paracoccus denitrificans.

    PubMed Central

    Alefounder, P R; Ferguson, S J

    1980-01-01

    1. A method is described for preparing spheroplasts from Paracoccus denitrificans that are substantially depleted of dissimilatory nitrate reductase (cytochrome cd) activity. Treatment of cells with lysozyme + EDTA together with a mild osmotic shock, followed by centrifugation, yielded a pellet of spheroplasts and a supernatant that contained d-type cytochrome. The spheroplasts were judged to have retained an intact plasma membrane on the basis that less than 1% of the activity of a cytoplasmic marker protein, malate dehydrogenase, was released from the spheroplasts. In addition to a low activity towards added nitrite, the suspension of spheroplasts accumulated the nitrite that was produced by respiratory chain-linked reduction of nitrate. It is concluded that nitrate reduction occurs at the periplasmic side of the plasma membrane irrespective of whether nitrite is generated by nitrate reduction or is added exogenously. 2. Further evidence for the integrity of the spheroplasts was that nitrate reduction was inhibited by O2, and that chlorate was reduced at a markedly lower rate than nitrate. These data are taken as evidence for an intact plasma membrane because it was shown that cells acquire the capability to reduce nitrate under aerobic conditions after addition of low amounts of Triton X-100 which, with the same titre, also overcame the permeability barrier to chlorate reduction by intact cells. The close relationship between the appearance of chlorate reduction and the loss of the inhibitory effect of O2 on nitrate reduction also suggests that the later feature of nitrate respiration is due to a control on the accessibility of nitrate to its reductase rather than on the flow of electrons to nitrate reductase. PMID:7197918

  9. Purification, cloning, functional expression and characterization of perakine reductase: the first example from the AKR enzyme family, extending the alkaloidal network of the plant Rauvolfia.

    PubMed

    Sun, Lianli; Ruppert, Martin; Sheludko, Yuri; Warzecha, Heribert; Zhao, Yu; Stöckigt, Joachim

    2008-07-01

    Perakine reductase (PR) catalyzes an NADPH-dependent step in a side-branch of the 10-step biosynthetic pathway of the alkaloid ajmaline. The enzyme was cloned by a "reverse-genetic" approach from cell suspension cultures of the plant Rauvolfia serpentina (Apocynaceae) and functionally expressed in Escherichia coli as the N-terminal His(6)-tagged protein. PR displays a broad substrate acceptance, converting 16 out of 28 tested compounds with reducible carbonyl function which belong to three substrate groups: benzaldehyde, cinnamic aldehyde derivatives and monoterpenoid indole alkaloids. The enzyme has an extraordinary selectivity in the group of alkaloids. Sequence alignments define PR as a new member of the aldo-keto reductase (AKR) super family, exhibiting the conserved catalytic tetrad Asp52, Tyr57, Lys84, His126. Site-directed mutagenesis of each of these functional residues to an alanine residue results in >97.8% loss of enzyme activity, in compounds of each substrate group. PR represents the first example of the large AKR-family which is involved in the biosynthesis of plant monoterpenoid indole alkaloids. In addition to a new esterase, PR significantly extends the Rauvolfia alkaloid network to the novel group of peraksine alkaloids.

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

    PubMed

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

    2009-03-01

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

  11. Regulation of succinate-ubiquinone reductase and fumarate reductase activities in human complex II by phosphorylation of its flavoprotein subunit.

    PubMed

    Tomitsuka, Eriko; Kita, Kiyoshi; Esumi, Hiroyasu

    2009-01-01

    Complex II (succinate-ubiquinone reductase; SQR) is a mitochondrial respiratory chain enzyme that is directly involved in the TCA cycle. Complex II exerts a reverse reaction, fumarate reductase (FRD) activity, in various species such as bacteria, parasitic helminths and shellfish, but the existence of FRD activity in humans has not been previously reported. Here, we describe the detection of FRD activity in human cancer cells. The activity level was low, but distinct, and it increased significantly when the cells were cultured under hypoxic and glucose-deprived conditions. Treatment with phosphatase caused the dephosphorylation of flavoprotein subunit (Fp) with a concomitant increase in SQR activity, whereas FRD activity decreased. On the other hand, treatment with protein kinase caused an increase in FRD activity and a decrease in SQR activity. These data suggest that modification of the Fp subunit regulates both the SQR and FRD activities of complex II and that the phosphorylation of Fp might be important for maintaining mitochondrial energy metabolism within the tumor microenvironment.

  12. 21 CFR 864.7375 - Glutathione reductase assay.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

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

  13. 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 Glutathione...

  14. 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 Glutathione...

  15. 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 Glutathione...

  16. 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 Glutathione...

  17. Direct cloning of the trxB gene that encodes thioredoxin reductase.

    PubMed Central

    Russel, M; Model, P

    1985-01-01

    A strain was constructed which contains mutations in the genes encoding thioredoxin (trxA) and thioredoxin reductase (trxB) such that filamentous phage f1 cannot grow. The complementation of either mutation with its wild-type allele permits phage growth. We used this strain to select f1 phage which contain a cloned trxB gene. The location of the gene on the cloned fragment was determined, and its protein product was identified. Plasmid subclones that contain this gene overproduce thioredoxin reductase. Images PMID:2989245

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

    PubMed Central

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

    2011-01-01

    Most species of invertebrate animals cannot synthesise sterols de novo and many that feed on plants dealkylate phytosterols (mostly C29 and C28) yielding cholesterol (C27). The final step of this dealkylation pathway involves desmosterol reductase (DHCR24)-catalysed reduction of desmosterol to cholesterol. We now report the molecular characterisation in the silkworm, Bombyx mori, of such a desmosterol reductase involved in production of cholesterol from phytosterol, rather than in de novo synthesis of cholesterol. Phylogenomic analysis of putative desmosterol reductases revealed the occurrence of various clades that allowed for the identification of a strong reductase candidate gene in Bombyx mori (BGIBMGA 005735). Following PCR-based cloning of the cDNA (1.6 kb) and its heterologous expression in Saccharomyces cerevisae, the recombinant protein catalysed reduction of desmosterol to cholesterol in an NADH- and FAD- dependent reaction. Conceptual translation of the cDNA, that encodes a 58.9 kDa protein, and database searching, revealed that the enzyme belongs to an FAD-dependent oxidoreductase family. Western blotting revealed reductase protein expression exclusively in the microsomal subcellular fraction and primarily in the gut. The protein is peripherally associated with microsomal membranes. 2D-native gel and PAGE analysis revealed that the reductase is part of a large complex with molecular weight approximately 250kDa. The protein occurs in midgut microsomes at a fairly constant level throughout development in the last two instars, but is drastically reduced during the wandering stage in preparation for metamorphosis. Putative Broad Complex transcription factor-binding sites detectable upstream of the DHCR24 gene may play a role in this down-regulation. PMID:21738635

  19. 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

  20. Regulation of schistosome egg production by HMG CoA reductase

    DOE Office of Scientific and Technical Information (OSTI.GOV)

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

    1986-03-05

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

  1. Nitrate Reductase Activity and Polyribosomal Content of Corn (Zea mays L.) Having Low Leaf Water Potentials 1

    PubMed Central

    Morilla, Camila A.; Boyer, J. S.; Hageman, R. H.

    1973-01-01

    Desiccation of 8- to 13-day-old seedlings, achieved by withholding nutrient solution from the vermiculite root medium, caused a reduction in nitrate reductase activity of the leaf tissue. Activity declined when leaf water potentials decreased below −2 bars and was 25% of the control at a leaf water potential of −13 bars. Experiments were conducted to determine whether the decrease in nitrate reductase activity was due to reduced levels of nitrate in the tissue, direct inactivation of the enzyme by low leaf water potentials, or to changes in rates of synthesis or decay of the enzyme. Although tissue nitrate content decreased with the onset of desiccation, it did not continue to decline with tissue desiccation and loss of enzyme activity. Nitrate reductase activity recovered when the plants were rewatered with nitrate-free medium, suggesting that the nitrate in the plant was adequate for high nitrate reductase activity. The rate of decay of nitrate reductase activity from desiccated tissue was essentially identical to that of the control, in vivo or in vitro, regardless of the rapidity of desiccation of the tissue. Direct inactivation of the enzyme by the low water potentials was not detected. Polyribosomal content of the tissue declined with the decrease in water potential, prior to the decline in nitrate reductase activity. Changes in ribosomal profiles occurred during desiccation, regardless of whether the tissue had been excised or not and whether desiccation was rapid or slow. Reduction in polyribosomal content did not appear to be associated with changes in ribonuclease activity. Nitrate reductase activity and the polyribosomal content of the tissue recovered upon rewatering, following the recovery in water potential. The increase in polyribosomal content preceded the increase in nitrate reductase activity. Recovery of enzyme activity was prevented by cycloheximide. Based on these results, it appears that nitrate reductase activity was affected primarily by

  2. A Bioinformatics Classifier and Database for Heme-Copper Oxygen Reductases

    PubMed Central

    Sousa, Filipa L.; Alves, Renato J.; Pereira-Leal, José B.; Teixeira, Miguel; Pereira, Manuela M.

    2011-01-01

    Background Heme-copper oxygen reductases (HCOs) are the last enzymatic complexes of most aerobic respiratory chains, reducing dioxygen to water and translocating up to four protons across the inner mitochondrial membrane (eukaryotes) or cytoplasmatic membrane (prokaryotes). The number of completely sequenced genomes is expanding exponentially, and concomitantly, the number and taxonomic distribution of HCO sequences. These enzymes were initially classified into three different types being this classification recently challenged. Methodology We reanalyzed the classification scheme and developed a new bioinformatics classifier for the HCO and Nitric oxide reductases (NOR), which we benchmark against a manually derived gold standard sequence set. It is able to classify any given sequence of subunit I from HCO and NOR with a global recall and precision both of 99.8%. We use this tool to classify this protein family in 552 completely sequenced genomes. Conclusions We concluded that the new and broader data set supports three functional and evolutionary groups of HCOs. Homology between NORs and HCOs is shown and NORs closest relationship with C Type HCOs demonstrated. We established and made available a classification web tool and an integrated Heme-Copper Oxygen reductase and NOR protein database (www.evocell.org/hco). PMID:21559461

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

    PubMed

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

    2018-02-07

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

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

    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

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

    PubMed

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

    2012-05-01

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

  6. Purification and Thermal Dependence of Glutathione Reductase from Two Forage Legume Species 1

    PubMed Central

    Kidambi, Saranga P.; Mahan, James R.; Matches, Arthur G.

    1990-01-01

    Alfalfa (Medicago sativa L.) and sainfoin (Onobrychis viciifolia Scop.) are forage legumes that differ in their responses to high and low temperature stresses. Thermal limitations on the function of glutathione reductase (EC 1.6.4.2) could adversely affect the ability of the plant to cope with adverse temperatures. Our objectives were to (a) purify glutathione reductase from `Cimarron' alfalfa and `PI 212241' sainfoin and (b) investigate the intraspecies variation in the thermal dependency of glutathione reductase from each of three cultivars of alfalfa and two cultivars and an introduction of sainfoin. Glutathione reductase was purified 1222-and 1948-fold to a specific activity of 281 and 273 units per milligram of protein, from one species each of alfalfa and sainfoin, respectively. The relative molecular mass of the protein was approximately 140 kilodaltons with subunits of 57 and 37 kilodaltons under denaturing conditions. The activation energies were approximately 50 kilojoules per mole for both species. Over a 5 to 45°C temperature gradient, large variation among species and genotypes within species was found for: (a) the minimum apparent Michaelis constant (0.6-2.1 micromoles of NADPH), (b) the temperature at which the minimum apparent Michaelis constant was observed (10-25°C), and (c) the thermal kinetic windows (6-19°C width). Future studies will focus on relating the thermal dependence of the Michaelis constant of the glutathione reductases and plant growth rates and forage quality of these species throughout the growing season. PMID:16667283

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

    PubMed

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

    2013-11-01

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

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Hintzpeter, Jan, E-mail: hintzpeter@toxi.uni-kiel.de; Seliger, Jan Moritz; Hofman, Jakub

    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,more » in particular emodin and anthraflavic acid. Intense inhibition kinetic data for the most effective daunorubicin reductases, including IC{sub 50}- and K{sub i}-values, the mode of inhibition, as well as molecular docking, were compiled. Subsequently, a cytotoxicity profile and the ability of emodin to reverse daunorubicin resistance were determined using multiresistant A549 lung cancer and HepG2 liver cancer cells. Emodin potently inhibited the four main human daunorubicin reductases in vitro. Further, we could demonstrate that emodin is able to synergistically sensitize human cancer cells towards daunorubicin at clinically relevant concentrations. Therefore, emodin may yield the potential to enhance the therapeutic effectiveness of anthracyclines by preventing anthracycline resistance via inhibition of the anthracycline reductases. In symphony with its known pharmacological properties, emodin might be a compound of particular interest in the management of anthracycline chemotherapy efficacy and their adverse effects. - Highlights: • Natural and synthetic compounds were identified as inhibitors for human daunorubicin reductases. • Emodin is a potent inhibitor for human daunorubicin

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

    PubMed Central

    Kim, Tae Hyeon; Kim, Jin Kyu; Kang, Young-Hee; Lee, Jae-Yong; Kang, Il Jun; Lim, Soon Sung

    2013-01-01

    Aldose reductase (AR) inhibitors have a considerable therapeutic potential against diabetes complications and do not increase the risk of hypoglycemia. Through bioassay-guided fractionation of an EtOH extract of the kernel from purple corn (Zea mays L.), 7 nonanthocyanin phenolic compounds (compound 1–7) and 5 anthocyanins (compound 8–12) were isolated. These compounds were investigated by rat lens aldose reductase (RLAR) inhibitory assays. Kinetic analyses of recombinant human aldose reductase (rhAR) were performed, and intracellular galactitol levels were measured. Hirsutrin, one of 12 isolated compounds, showed the most potent RLAR inhibitory activity (IC50, 4.78 μM). In the kinetic analyses using Lineweaver-Burk plots of 1/velocity and 1/substrate concentration, hirsutrin showed competitive inhibition against rhAR. Furthermore, hirsutrin inhibited galactitol formation in rat lens and erythrocytes sample incubated with a high concentration of galactose; this finding indicates that hirsutrin may effectively prevent osmotic stress in hyperglycemia. Therefore, hirsutrin derived from Zea mays L. may be a potential therapeutic agent against diabetes complications. PMID:23586057

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

    PubMed

    Waddell, D; Ullman, B

    1983-04-10

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

  11. Membrane-bound oxygen reductases of the anaerobic sulfate-reducing Desulfovibrio vulgaris Hildenborough: roles in oxygen defence and electron link with periplasmic hydrogen oxidation.

    PubMed

    Ramel, F; Amrani, A; Pieulle, L; Lamrabet, O; Voordouw, G; Seddiki, N; Brèthes, D; Company, M; Dolla, A; Brasseur, G

    2013-12-01

    Cytoplasmic membranes of the strictly anaerobic sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough contain two terminal oxygen reductases, a bd quinol oxidase and a cc(b/o)o3 cytochrome oxidase (Cox). Viability assays pointed out that single Δbd, Δcox and double ΔbdΔcox deletion mutant strains were more sensitive to oxygen exposure than the WT strain, showing the involvement of these oxygen reductases in the detoxification of oxygen. The Δcox strain was slightly more sensitive than the Δbd strain, pointing to the importance of the cc(b/o)o3 cytochrome oxidase in oxygen protection. Decreased O2 reduction rates were measured in mutant cells and membranes using lactate, NADH, ubiquinol and menadiol as substrates. The affinity for oxygen measured with the bd quinol oxidase (Km, 300 nM) was higher than that of the cc(b/o)o3 cytochrome oxidase (Km, 620 nM). The total membrane activity of the bd quinol oxidase was higher than that of the cytochrome oxidase activity in line with the higher expression of the bd oxidase genes. In addition, analysis of the ΔbdΔcox mutant strain indicated the presence of at least one O2-scavenging membrane-bound system able to reduce O2 with menaquinol as electron donor with an O2 affinity that was two orders of magnitude lower than that of the bd quinol oxidase. The lower O2 reductase activity in mutant cells with hydrogen as electron donor and the use of specific inhibitors indicated an electron transfer link between periplasmic H2 oxidation and membrane-bound oxygen reduction via the menaquinol pool. This linkage is crucial in defence of the strictly anaerobic bacterium Desulfovibrio against oxygen stress.

  12. Reduction of Brain β-Amyloid (Aβ) by Fluvastatin, a Hydroxymethylglutaryl-CoA Reductase Inhibitor, through Increase in Degradation of Amyloid Precursor Protein C-terminal Fragments (APP-CTFs) and Aβ Clearance*

    PubMed Central

    Shinohara, Mitsuru; Sato, Naoyuki; Kurinami, Hitomi; Takeuchi, Daisuke; Takeda, Shuko; Shimamura, Munehisa; Yamashita, Toshihide; Uchiyama, Yasuo; Rakugi, Hiromi; Morishita, Ryuichi

    2010-01-01

    Epidemiological studies suggest that statins (hydroxymethylglutaryl-CoA reductase inhibitors) could reduce the risk of Alzheimer disease. Although one possible explanation is through an effect on β-amyloid (Aβ) metabolism, its effect remains to be elucidated. Here, we explored the molecular mechanisms of how statins influence Aβ metabolism. Fluvastatin at clinical doses significantly reduced Aβ and amyloid precursor protein C-terminal fragment (APP-CTF) levels among APP metabolites in the brain of C57BL/6 mice. Chronic intracerebroventricular infusion of lysosomal inhibitors blocked these effects, indicating that up-regulation of the lysosomal degradation of endogenous APP-CTFs is involved in reduced Aβ production. Biochemical analysis suggested that this was mediated by enhanced trafficking of APP-CTFs from endosomes to lysosomes, associated with marked changes of Rab proteins, which regulate endosomal function. In primary neurons, fluvastatin enhanced the degradation of APP-CTFs through an isoprenoid-dependent mechanism. Because our previous study suggests additive effects of fluvastatin on Aβ metabolism, we examined Aβ clearance rates by using the brain efflux index method and found its increased rates at high Aβ levels from brain. As LRP1 in brain microvessels was increased, up-regulation of LRP1-mediated Aβ clearance at the blood-brain barrier might be involved. In cultured brain microvessel endothelial cells, fluvastatin increased LRP1 and the uptake of Aβ, which was blocked by LRP1 antagonists, through an isoprenoid-dependent mechanism. Overall, the present study demonstrated that fluvastatin reduced Aβ level by an isoprenoid-dependent mechanism. These results have important implications for the development of disease-modifying therapy for Alzheimer disease as well as understanding of Aβ metabolism. PMID:20472556

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

    PubMed

    Ono, S; Hirano, H

    1984-04-01

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

  14. Studies on Marek's Disease Virus Encoded Ribonucleotide Reductase

    USDA-ARS?s Scientific Manuscript database

    Ribonucleotide reductase (RR) is an essential enzyme for the conversion of ribonucleotides to deoxyribonucleotides in prokaryotic and eukaryotic cells. The enzyme consists of two subunits namely RR1 and RR2, both of which associate to form an active holoenzyme. Herpesviruses express a functional R...

  15. Enzymatic reduction of disulfide bonds in lysosomes: Characterization of a Gamma-interferon-inducible lysosomal thiol reductase (GILT)

    NASA Astrophysics Data System (ADS)

    Arunachalam, Balasubramanian; Phan, Uyen T.; Geuze, Hans J.; Cresswell, Peter

    2000-01-01

    Proteins internalized into the endocytic pathway are usually degraded. Efficient proteolysis requires denaturation, induced by acidic conditions within lysosomes, and reduction of inter- and intrachain disulfide bonds. Cytosolic reduction is mediated enzymatically by thioredoxin, but the mechanism of lysosomal reduction is unknown. We describe here a lysosomal thiol reductase optimally active at low pH and capable of catalyzing disulfide bond reduction both in vivo and in vitro. The active site, determined by mutagenesis, consists of a pair of cysteine residues separated by two amino acids, similar to other enzymes of the thioredoxin family. The enzyme is a soluble glycoprotein that is synthesized as a precursor. After delivery into the endosomal/lysosomal system by the mannose 6-phosphate receptor, N- and C-terminal prosequences are removed. The enzyme is expressed constitutively in antigen-presenting cells and induced by IFN-γ in other cell types, suggesting a potentially important role in antigen processing.

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

    PubMed

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

    2017-12-01

    Recent efforts to develop cure for chronic diabetic complications have led to the discovery of potent inhibitors against aldose reductase (AKR1B1, EC 1.1.1.21) whose role in diabetes is well-evident. In the present work, two new natural products were isolated from the ariel part of Ocimum basilicum; 7-(3-hydroxypropyl)-3-methyl-8-β-O-d-glucoside-2H-chromen-2-one (1) and E-4-(6'-hydroxyhex-3'-en-1-yl)phenyl propionate (2) and confirmed their structures with different spectroscopic techniques including NMR spectroscopy etc. The isolated compounds (1, 2) were evaluated for in vitro inhibitory activity against aldose reductase (AKR1B1) and aldehyde reductase (AKR1A1). The natural product (1) showed better inhibitory activity for AKR1B1 with IC 50 value of 2.095±0.77µM compare to standard sorbinil (IC 50 =3.14±0.02µM). Moreover, the compound (1) also showed multifolds higher activity (IC 50 =0.783±0.07µM) against AKR1A1 as compared to standard valproic acid (IC 50 =57.4±0.89µM). However, the natural product (2) showed slightly lower activity for AKR1B1 (IC 50 =4.324±1.25µM). Moreover, the molecular docking studies of the potent inhibitors were also performed to identify the putative binding modes within the active site of aldose/aldehyde reductases. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Deep-Sea Bacterium Shewanella piezotolerans WP3 Has Two Dimethyl Sulfoxide Reductases in Distinct Subcellular Locations

    PubMed Central

    Xiong, Lei; Jian, Huahua

    2017-01-01

    ABSTRACT Dimethyl sulfoxide (DMSO) acts as a substantial sink for dimethyl sulfide (DMS) in deep waters and is therefore considered a potential electron acceptor supporting abyssal ecosystems. Shewanella piezotolerans WP3 was isolated from west Pacific deep-sea sediments, and two functional DMSO respiratory subsystems are essential for maximum growth of WP3 under in situ conditions (4°C/20 MPa). However, the relationship between these two subsystems and the electron transport pathway underlying DMSO reduction by WP3 remain unknown. In this study, both DMSO reductases (type I and type VI) in WP3 were found to be functionally independent despite their close evolutionary relationship. Moreover, immunogold labeling of DMSO reductase subunits revealed that the type I DMSO reductase was localized on the outer leaflet of the outer membrane, whereas the type VI DMSO reductase was located within the periplasmic space. CymA, a cytoplasmic membrane-bound tetraheme c-type cytochrome, served as a preferential electron transport protein for the type I and type VI DMSO reductases, in which type VI accepted electrons from CymA in a DmsE- and DmsF-independent manner. Based on these results, we proposed a core electron transport model of DMSO reduction in the deep-sea bacterium S. piezotolerans WP3. These results collectively suggest that the possession of two sets of DMSO reductases with distinct subcellular localizations may be an adaptive strategy for WP3 to achieve maximum DMSO utilization in deep-sea environments. IMPORTANCE As the dominant methylated sulfur compound in deep oceanic water, dimethyl sulfoxide (DMSO) has been suggested to play an important role in the marine biogeochemical cycle of the volatile anti-greenhouse gas dimethyl sulfide (DMS). Two sets of DMSO respiratory systems in the deep-sea bacterium Shewanella piezotolerans WP3 have previously been identified to mediate DMSO reduction under in situ conditions (4°C/20 MPa). Here, we report that the two DMSO

  18. 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.

  19. Reduced Bone Mass and Muscle Strength in Male 5α-Reductase Type 1 Inactivated Mice

    PubMed Central

    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. PMID:21731732

  20. Expression, purification, crystallization and preliminary X-ray crystallographic analysis of a novel plant-type ferredoxin/thioredoxin reductase-like protein from Methanosarcina acetivorans

    PubMed Central

    Kumar, Adepu K.; Yennawar, Neela H.; Yennawar, Hemant P.; Ferry, James G.

    2011-01-01

    The genome of Methanosarcina acetivorans contains a gene (ma1659) that is predicted to encode an uncharacterized chimeric protein containing a plant-type ferredoxin/thioredoxin reductase-like catalytic domain in the N-terminal region and a bacterial-like rubredoxin domain in the C-terminal region. To understand the structural and functional properties of the protein, the ma1659 gene was cloned and overexpressed in Escherichia coli. Crystals of the MA1659 protein were grown by the sitting-drop method using 2 M ammonium sulfate, 0.1 M HEPES buffer pH 7.5 and 0.1 M urea. Diffraction data were collected to 2.8 Å resolution using the remote data-collection feature of the Advanced Light Source, Lawrence Berkeley National Laboratory. The crystal belonged to the primitive cubic space group P23 or P213, with unit-cell parameters a = b = c = 92.72 Å. Assuming the presence of one molecule in the asymmetric unit gave a Matthews coefficient (V M) of 3.55 Å3 Da−1, corresponding to a solvent content of 65%. PMID:21795791

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

    PubMed

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

    2013-12-05

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

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

    DTIC Science & Technology

    2013-10-01

    Novel Bifunctional Aldo -Keto Reductase 1C3 Inhibitor and Androgen Receptor Antagonist” PRINCIPAL INVESTIGATOR: ADEGOKE ADENIJI, Ph.D...therapeutic benefit relative to targeting either mechanism alone. Aldo -keto reductase 1C3 (AKR1C3) is highly upregulated in APC and is localized within...therapy of Abi with MDV3100 has been proposed as a way to reduce resistance. 14, 15 Aldo -keto reductase IC3 (AKR1C3, type 5 17β hydroxysteroid

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

    PubMed

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

    2016-11-01

    In many organisms, hydroxyurea (HU) inhibits class I ribonucleotide reductase, leading to lowered cellular pools of deoxyribonucleoside triphosphates. The reduced levels for DNA precursors is believed to cause replication fork stalling. Upon treatment of the hyperthermophilic archaeon Sulfolobus solfataricus with HU, we observe dose-dependent cell cycle arrest, accumulation of DNA double-strand breaks, stalled replication forks, and elevated levels of recombination structures. However, Sulfolobus has a HU-insensitive class II ribonucleotide reductase, and we reveal that HU treatment does not significantly impact cellular DNA precursor pools. Profiling of protein and transcript levels reveals modulation of a specific subset of replication initiation and cell division genes. Notably, the selective loss of the regulatory subunit of the primase correlates with cessation of replication initiation and stalling of replication forks. Furthermore, we find evidence for a detoxification response induced by HU treatment. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

  4. 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.

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

    PubMed

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

    2006-07-31

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

  6. Isolation and Characterization of cDNAs Encoding Leucoanthocyanidin Reductase and Anthocyanidin Reductase from Populus trichocarpa

    PubMed Central

    Lu, Wanxiang; Yang, Li; Karim, Abdul; Luo, Keming

    2013-01-01

    Proanthocyanidins (PAs) contribute to poplar defense mechanisms against biotic and abiotic stresses. Transcripts of PA biosynthetic genes accumulated rapidly in response to infection by the fungus Marssonina brunnea f.sp. multigermtubi, treatments of salicylic acid (SA) and wounding, resulting in PA accumulation in poplar leaves. Anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR) are two key enzymes of the PA biosynthesis that produce the main subunits: (+)-catechin and (−)-epicatechin required for formation of PA polymers. In Populus, ANR and LAR are encoded by at least two and three highly related genes, respectively. In this study, we isolated and functionally characterized genes PtrANR1 and PtrLAR1 from P. trichocarpa. Phylogenetic analysis shows that Populus ANR1 and LAR1 occurr in two distinct phylogenetic lineages, but both genes have little difference in their tissue distribution, preferentially expressed in roots. Overexpression of PtrANR1 in poplar resulted in a significant increase in PA levels but no impact on catechin levels. Antisense down-regulation of PtrANR1 showed reduced PA accumulation in transgenic lines, but increased levels of anthocyanin content. Ectopic expression of PtrLAR1 in poplar positively regulated the biosynthesis of PAs, whereas the accumulation of anthocyanin and flavonol was significantly reduced (P<0.05) in all transgenic plants compared to the control plants. These results suggest that both PtrANR1 and PtrLAR1 contribute to PA biosynthesis in Populus. PMID:23741362

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

    PubMed

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

    2001-12-01

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

  8. 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

  9. [Establishment of an in vitro screening model for steroid 5 alpha-reductase inhibitors with the microplate reader].

    PubMed

    Wu, Jian-Hui; Sun, Zu-Yue

    2013-06-01

    To establish an in vitro screening model for steroid 5 alpha-reductase inhibitors using the microplate reader. Steroid 5 alpha-reductase was obtained from the liver of female rats, an in vitro screening model for steroid 5 alpha-reductase inhibitors established using the 96-well plate and microplate reader after determination of the enzymatic activity, and the reliability of the model verified with the known 5 alpha-reductase inhibitors epristeride and finasteride. Added to the 96-well plate were the final concentrations of testosterone (0-40 micromol/L), NADPH (22 micromol/L), epristeride (0-60 nmol/L) or finasteride (0-60 nmol/ L) and steroid 5 alpha-reductase (20 microl), the total volume of each well adjusted to 200 microl with Tris-Hcl buffer. The 96-well plate was placed in the microplate reader, mixed and incubated at 37 degrees C, followed by detection of the A340nm value at 0 and 10 min and analysis of the data. The Km value of steroid 5 alpha-reductase was 3.794 micromol/L, with a Vmax of 0.271 micromol/(L. min). The Ki of epristeride was 148.2 nmol/L, with an IC50 of 31.5 nmol/L, and the enzymatic reaction kinetic curve suggested that epristeride was an uncompetitive enzyme inhibitor. The Ki of finasteride was 158. 8 nmol/L, with an IC50 of 13.6 nmol/L. The enzymatic reaction kinetic curve showed that both epristeride and finasteride were competitive enzyme inhibitors, similar to those reported in the published literature. A screening model was successfully established, which could rapidly and effectively screen steroid 5 alpha-reductase inhibitors in vitro.

  10. Evidence for an Inactivating System of Nitrate Reductase in Hordeum vulgare L. during Darkness That Requires Protein Synthesis 1

    PubMed Central

    Travis, R. L.; Jordan, W. R.; Huffaker, R. C.

    1969-01-01

    The disappearance of nitrate reductase activity in leaves of Hordeum vulgare L. during darkness was inhibited by cycloheximide, actinomycin D, and low temperature. Thus, protein synthesis was probably required for the disappearance of nitrate reductase in the dark. Since chloramphenicol did not affect the rate of loss of activity, the degradation or inactivation apparently required protein synthesis by the cytoplasmic ribosomal system. Consistent with this observation, nitrate reductase is also reportedly located in the cytoplasm. Thus, the amount of nitrate reductase activity present in leaves of barley may be controlled by a balance between activating and inactivating systems. PMID:16657182

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

    PubMed

    Pais, Pilar

    2010-08-01

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

  12. Aldo-Keto Reductases 1B in Adrenal Cortex Physiology

    PubMed Central

    Pastel, Emilie; Pointud, Jean-Christophe; Martinez, Antoine; Lefrançois-Martinez, A. Marie

    2016-01-01

    Aldose reductase (AKR1B) proteins are monomeric enzymes, belonging to the aldo-keto reductase (AKR) superfamily. They perform oxidoreduction of carbonyl groups from a wide variety of substrates, such as aliphatic and aromatic aldehydes or ketones. Due to the involvement of human aldose reductases in pathologies, such as diabetic complications and cancer, AKR1B subgroup enzymatic properties have been extensively characterized. However, the issue of AKR1B function in non-pathologic conditions remains poorly resolved. Adrenal activities generated large amount of harmful aldehydes from lipid peroxidation and steroidogenesis, including 4-hydroxynonenal (4-HNE) and isocaproaldehyde (4-methylpentanal), which can both be reduced by AKR1B proteins. More recently, some AKR1B isoforms have been shown to be endowed with prostaglandin F synthase (PGFS) activity, suggesting that, in addition to possible scavenger function, they could instigate paracrine signals. Interestingly, the adrenal gland is one of the major sites for human and murine AKR1B expression, suggesting that their detoxifying/signaling activity could be specifically required for the correct handling of adrenal function. Moreover, chronic effects of ACTH result in a coordinated regulation of genes encoding the steroidogenic enzymes and some AKR1B isoforms. This review presents the molecular mechanisms accounting for the adrenal-specific expression of some AKR1B genes. Using data from recent mouse genetic models, we will try to connect their enzymatic properties and regulation with adrenal functions. PMID:27499746

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

    PubMed

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

    2006-10-01

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

  14. Laue Crystal Structure of Shewanella oneidensis Cytochrome c Nitrite Reductase from a High-yield Expression System

    PubMed Central

    Youngblut, Matthew; Judd, Evan T.; Srajer, Vukica; Sayyed, Bilal; Goelzer, Tyler; Elliott, Sean J.; Schmidt, Marius; Pacheco, A. Andrew

    2012-01-01

    The high-yield expression and purification of Shewanella oneidensis cytochrome c nitrite reductase (ccNiR), and its characterization by a variety of methods, notably Laue crystallography, is reported. A key component of the expression system is an artificial ccNiR gene in which the N-terminal signal peptide from the highly expressed S. oneidensis protein “Small Tetra-heme c” replaces the wild-type signal peptide. This gene, inserted into the plasmid pHSG298 and expressed in S. oneidensis TSP-1 strain, generated ~20 mg crude ccNiR/L culture, compared with 0.5–1 mg/L for untransformed cells. Purified ccNiR has nitrite and hydroxylamine reductase activities comparable to those previously reported for E. coli ccNiR, and is stable for over two weeks in pH 7 solution at 4° C. UV/Vis spectropotentiometric titrations and protein film voltammetry identified 5 independent 1-electron reduction processes. Global analysis of the spectropotentiometric data also allowed determination of the extinction coefficient spectra for the 5 reduced ccNiR species. The characteristics of the individual extinction coefficient spectra suggest that, within each reduced species, the electrons are distributed amongst the various hemes, rather than being localized on specific heme centers. The purified ccNiR yielded good quality crystals, with which the 2.59 Å resolution structure was solved at room temperature using the Laue diffraction method. The structure is similar to that of E. coli ccNiR, except in the region where the enzyme interacts with its physiological electron donor (CymA in the case of S. oneidensis ccNiR, NrfB in the case of the E. coli protein). PMID:22382353

  15. Characterization and localization of progesterone 5 alpha-reductase from cell cultures of foxglove (Digitalis lanata EHRH).

    PubMed Central

    Wendroth, S; Seitz, H U

    1990-01-01

    Progesterone 5 alpha-reductase, which catalyses the reduction of progesterone to 5 alpha-pregnane-3,20-dione, was isolated and characterized from cell cultures of Digitalis lanata (foxglove). Optimum enzyme activity was observed at pH 7.0, and the enzyme had an apparent Km value of 30 microM for its substrate progesterone. The enzyme needs NADPH as reductant, which could not be replaced by NADH. For NADPH, the apparent Km value is 130 microM. The optimum temperature was 40 degrees C; at temperatures below 45 degrees C, the product 5 alpha-pregnane-3,20-dione was reduced by a second reaction to 5 alpha-pregnan-3 beta-ol-20-one. Progesterone 5 alpha-reductase activity was not dependent on bivalent cations. In the presence of EDTA, 0.1 mM-Mn2+ had no influence on enzyme activity, whereas 0.1 mM-Ca2+, -Co2+ and -Zn2+ decreased progesterone 5 alpha-reductase activity. Only 0.1 mM-Mg2+ was slightly stimulatory. EDTA and thiol reagents such as dithiothreitol stimulate progesterone 5 alpha-reductase activity. By means of linear sucrose gradient fractionation of the cellular membranes, progesterone 5 alpha-reductase was found to be located in the endoplasmic reticulum. PMID:2106876

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

    PubMed Central

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

    2006-01-01

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

  17. Evolution of plant δ 1-pyrroline-5-carboxylate reductases from phylogenetic and structural perspectives

    DOE PAGES

    Forlani, Giuseppe; Makarova, Kira S.; Ruszkowski, Milosz; ...

    2015-08-03

    Proline plays a crucial role in cell growth and stress responses, and its accumulation is essential for the tolerance of adverse environmental conditions in plants. Two routes are used to biosynthesize proline in plants. The main route uses glutamate as a precursor, while in the other route proline is derived from ornithine. The terminal step of both pathways, the conversion of δ 1-pyrroline-5-carboxylate (P5C) to L-proline, is catalyzed by P5C reductase (P5CR) using NADH or NADPH as a cofactor. Since P5CRs are important housekeeping enzymes, they are conserved across all domains of life and appear to be relatively unaffected throughoutmore » evolution. However, global analysis of these enzymes unveiled significant functional diversity in the preference for cofactors (NADPH vs. NADH), variation in metal dependence and the differences in the oligomeric state. In our study we investigated evolutionary patterns through phylogenetic and structural analysis of P5CR representatives from all kingdoms of life, with emphasis on the plant species. We attempted to correlate local sequence/structure variation among the functionally and structurally characterized members of the family.« less

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

    PubMed Central

    2013-01-01

    Background The proanthocyanidins (PAs), a subgroup of flavonoids, accumulate to levels of approximately 10% total dry weight of cacao seeds. PAs have been associated with human health benefits and also play important roles in pest and disease defense throughout the plant. Results To dissect the genetic basis of PA biosynthetic pathway in cacao (Theobroma cacao), we have isolated three genes encoding key PA synthesis enzymes, anthocyanidin synthase (ANS), anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR). We measured the expression levels of TcANR, TcANS and TcLAR and PA content in cacao leaves, flowers, pod exocarp and seeds. In all tissues examined, all three genes were abundantly expressed and well correlated with PA accumulation levels, suggesting their active roles in PA synthesis. Overexpression of TcANR in an Arabidopsis ban mutant complemented the PA deficient phenotype in seeds and resulted in reduced anthocyanidin levels in hypocotyls. Overexpression of TcANS in tobacco resulted in increased content of both anthocyanidins and PAs in flower petals. Overexpression of TcANS in an Arabidopsis ldox mutant complemented its PA deficient phenotype in seeds. Recombinant TcLAR protein converted leucoanthocyanidin to catechin in vitro. Transgenic tobacco overexpressing TcLAR had decreased amounts of anthocyanidins and increased PAs. Overexpressing TcLAR in Arabidopsis ldox mutant also resulted in elevated synthesis of not only catechin but also epicatechin. Conclusion Our results confirm the in vivo function of cacao ANS and ANR predicted based on sequence homology to previously characterized enzymes from other species. In addition, our results provide a clear functional analysis of a LAR gene in vivo. PMID:24308601

  19. ESR studies on reactivity of protein-derived tyrosyl radicals formed by prostaglandin H synthase and ribonucleotide reductase.

    PubMed

    Lassmann, G; Curtis, J; Liermann, B; Mason, R P; Eling, T E

    1993-01-01

    Using ESR spectroscopy, the ability of enzyme inhibitors to quench protein-derived tyrosyl radicals was studied in two different enzymes, prostaglandin H synthase and ribonucleotide reductase. The prostaglandin H synthase inhibitors indomethacin, eugenol, and MK-410 effectively prevent the formation of tyrosyl radicals during the oxidation of arachidonic acid by prostaglandin H synthase from ram seminal vesicles. A direct reaction with preformed tyrosyl radicals was observed only with eugenol. The other prostaglandin H synthase inhibitors were ineffective. The ribonucleotide reductase inhibitors hydroxyurea and 4-hydroxyanisole, which effectively inactivate the tyrosyl radical in the active site of ribonucleotide reductase present in tumor cells, exhibit a different reactivity with tyrosyl radicals formed by prostaglandin H synthase. Hydroxyurea quenches preformed tyrosyl radicals in prostaglandin H synthase weakly, whereas 4-hydroxyanisole does not quench tyrosyl radicals in prostaglandin H synthase at all. Eugenol, which quenches preformed prostaglandin H synthase-derived tyrosyl radicals, also quenches the tyrosyl radical in ribonucleotide reductase. The results suggest that the reactivity of protein-linked tyrosyl radicals in ribonucleotide reductase and those formed during prostaglandin H synthase catalysis are very different and have unrelated roles in enzyme catalysis.

  20. Sepiapterin Reductase Mediates Chemical Redox Cycling in Lung Epithelial Cells*

    PubMed Central

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

    2013-01-01

    In the lung, chemical redox cycling generates highly toxic reactive oxygen species that can cause alveolar inflammation and damage to the epithelium, as well as fibrosis. In this study, we identified a cytosolic NADPH-dependent redox cycling activity in mouse lung epithelial cells as sepiapterin reductase (SPR), an enzyme important for the biosynthesis of tetrahydrobiopterin. Human SPR was cloned and characterized. In addition to reducing sepiapterin, SPR mediated chemical redox cycling of bipyridinium herbicides and various quinones; this activity was greatest for 1,2-naphthoquinone followed by 9,10-phenanthrenequinone, 1,4-naphthoquinone, menadione, and 2,3-dimethyl-1,4-naphthoquinone. Whereas redox cycling chemicals inhibited sepiapterin reduction, sepiapterin had no effect on redox cycling. Additionally, inhibitors such as dicoumarol, N-acetylserotonin, and indomethacin blocked sepiapterin reduction, with no effect on redox cycling. Non-redox cycling quinones, including benzoquinone and phenylquinone, were competitive inhibitors of sepiapterin reduction but noncompetitive redox cycling inhibitors. Site-directed mutagenesis of the SPR C-terminal substrate-binding site (D257H) completely inhibited sepiapterin reduction but had minimal effects on redox cycling. These data indicate that SPR-mediated reduction of sepiapterin and redox cycling occur by distinct mechanisms. The identification of SPR as a key enzyme mediating chemical redox cycling suggests that it may be important in generating cytotoxic reactive oxygen species in the lung. This activity, together with inhibition of sepiapterin reduction by redox-active chemicals and consequent deficiencies in tetrahydrobiopterin, may contribute to tissue injury. PMID:23640889

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

    PubMed

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

    2010-09-01

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

  2. DNA from uncultured organisms as a source of 2,5-diketo-L-gluconic acid reductases.

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Eschenfeldt, W. H.; Stols, L.; Rosenbaum, H.

    2001-09-01

    Total DNA of a population of uncultured organisms was extracted from soil samples, and by using PCR methods, the genes encoding two different 2,5-diketo-D-gluconic acid reductases (DKGRs) were recovered. Degenerate PCR primers based on published sequence information gave internal gene fragments homologous to known DKGRs. Nested primers specific for the internal fragments were combined with random primers to amplify flanking gene fragments from the environmental DNA, and two hypothetical full-length genes were predicted from the combined sequences. Based on these predictions, specific primers were used to amplify the two complete genes in single PCRs. These genes were cloned and expressedmore » in Escherichia coli. The purified gene products catalyzed the reduction of 2,5-diketo-D-gluconic acid to 2-keto-L-gulonic acid. Compared to previously described DKGRs isolated from Corynebacterium spp., these environmental reductases possessed some valuable properties. Both exhibited greater than 20-fold-higher k{sub cat}/K{sub m} values than those previously determined, primarily as a result of better binding of substrate. The K{sub m} values for the two new reductases were 57 and 67 {mu}M, versus 2 and 13 mM for the Corynebacterium enzymes. Both environmental DKGRs accepted NADH as well as NADPH as a cosubstrate; other DKGRs and most related aldo-keto reductases use only NADPH. In addition, one of the new reductases was more thermostable than known DKGRs.« less

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

    PubMed

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

    2011-05-30

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

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

    PubMed

    Thapliyal, Charu; Jain, Neha; Chaudhuri, Pratima

    2015-01-01

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

  5. 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

  6. Role of Aldo-Keto Reductase Family 1 (AKR1) Enzymes in Human Steroid Metabolism

    PubMed Central

    Rižner, Tea Lanišnik; Penning, Trevor M.

    2013-01-01

    Human aldo-keto reductases AKR1C1-AKR1C4 and AKR1D1 play essential roles in the metabolism of all steroid hormones, the biosynthesis of neurosteroids and bile acids, the metabolism of conjugated steroids, and synthetic therapeutic steroids. These enzymes catalyze NADPH dependent reductions at the C3, C5, C17 and C20 positions on the steroid nucleus and side-chain. AKR1C1-AKR1C4 act as 3-keto, 17-keto and 20-ketosteroid reductases to varying extents, while AKR1D1 acts as the sole Δ4-3-ketosteroid-5β-reductase (steroid 5β-reductase) in humans. AKR1 enzymes control the concentrations of active ligands for nuclear receptors and control their ligand occupancy and trans-activation, they also regulate the amount of neurosteroids that can modulate the activity of GABAA and NMDA receptors. As such they are involved in the pre-receptor regulation of nuclear and membrane bound receptors. Altered expression of individual AKR1C genes is related to development of prostate, breast, and endometrial cancer. Mutations in AKR1C1 and AKR1C4 are responsible for sexual development dysgenesis and mutations in AKR1D1 are causative in bile-acid deficiency. PMID:24189185

  7. 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.

  8. 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)

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

    PubMed

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

    2000-08-01

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

  10. Resolving the cofactor-binding site in the proline biosynthetic enzyme human pyrroline-5-carboxylate reductase 1

    PubMed Central

    Christensen, Emily M.; Patel, Sagar M.; Korasick, David A.; Campbell, Ashley C.; Krause, Kurt L.; Becker, Donald F.; Tanner, John J.

    2017-01-01

    Pyrroline-5-carboxylate reductase (PYCR) is the final enzyme in proline biosynthesis, catalyzing the NAD(P)H-dependent reduction of Δ1-pyrroline-5-carboxylate (P5C) to proline. Mutations in the PYCR1 gene alter mitochondrial function and cause the connective tissue disorder cutis laxa. Furthermore, PYCR1 is overexpressed in multiple cancers, and the PYCR1 knock-out suppresses tumorigenic growth, suggesting that PYCR1 is a potential cancer target. However, inhibitor development has been stymied by limited mechanistic details for the enzyme, particularly in light of a previous crystallographic study that placed the cofactor-binding site in the C-terminal domain rather than the anticipated Rossmann fold of the N-terminal domain. To fill this gap, we report crystallographic, sedimentation-velocity, and kinetics data for human PYCR1. Structures of binary complexes of PYCR1 with NADPH or proline determined at 1.9 Å resolution provide insight into cofactor and substrate recognition. We see NADPH bound to the Rossmann fold, over 25 Å from the previously proposed site. The 1.85 Å resolution structure of a ternary complex containing NADPH and a P5C/proline analog provides a model of the Michaelis complex formed during hydride transfer. Sedimentation velocity shows that PYCR1 forms a concentration-dependent decamer in solution, consistent with the pentamer-of-dimers assembly seen crystallographically. Kinetic and mutational analysis confirmed several features seen in the crystal structure, including the importance of a hydrogen bond between Thr-238 and the substrate as well as limited cofactor discrimination. PMID:28258219

  11. Exploring the molecular mechanisms of electron shuttling across the microbe/metal space

    PubMed Central

    Paquete, Catarina M.; Fonseca, Bruno M.; Cruz, Davide R.; Pereira, Tiago M.; Pacheco, Isabel; Soares, Cláudio M.; Louro, Ricardo O.

    2014-01-01

    Dissimilatory metal reducing organisms play key roles in the biogeochemical cycle of metals as well as in the durability of submerged and buried metallic structures. The molecular mechanisms that support electron transfer across the microbe-metal interface in these organisms remain poorly explored. It is known that outer membrane proteins, in particular multiheme cytochromes, are essential for this type of metabolism, being responsible for direct and indirect, via electron shuttles, interaction with the insoluble electron acceptors. Soluble electron shuttles such as flavins, phenazines, and humic acids are known to enhance extracellular electron transfer. In this work, this phenomenon was explored. All known outer membrane decaheme cytochromes from Shewanella oneidensis MR-1 with known metal terminal reductase activity and a undecaheme cytochrome from Shewanella sp. HRCR-6 were expressed and purified. Their interactions with soluble electron shuttles were studied using stopped-flow kinetics, NMR spectroscopy, and molecular simulations. The results show that despite the structural similarities, expected from the available structural data and sequence homology, the detailed characteristics of their interactions with soluble electron shuttles are different. MtrC and OmcA appear to interact with a variety of different electron shuttles in the close vicinity of some of their hemes, and with affinities that are biologically relevant for the concentrations typical found in the medium for this type of compounds. All data support a view of a distant interaction between the hemes of MtrF and the electron shuttles. For UndA a clear structural characterization was achieved for the interaction with AQDS a humic acid analog. These results provide guidance for future work of the manipulation of these proteins toward modulation of their role in metal attachment and reduction. PMID:25018753

  12. Trichomonas vaginalis Flavin Reductase 1 and its Role in Metronidazole Resistance

    PubMed Central

    Leitsch, David; Janssen, Brian D.; Kolarich, Daniel; Johnson, Patricia J.; Duchêne, Michael

    2015-01-01

    Summary The enzyme flavin reductase 1 (FR1) from Trichomonas vaginalis, formerly known as NADPH oxidase, was isolated and identified. Flavin reductase is part of the antioxidative defense in T. vaginalis and indirectly reduces molecular oxygen to hydrogen peroxide via free flavins. Importantly, a reduced or absent flavin reductase activity has been reported in metronidazole-resistant T. vaginalis, resulting in elevated intracellular oxygen levels and futile cycling of metronidazole. Interestingly, FR1 has no close homologue in any other sequenced genome, but seven full-length and three truncated isoforms exist in the T. vaginalis genome. However, out of these, only FR1 has an affinity for flavins, i.e. FMN, FAD, and riboflavin, which is high enough to be of physiological relevance. Although there are no relevant changes in the gene sequence or any alterations of the predicted FR1-mRNA structure in any of the strains studied, FR1 is not expressed in highly metronidazole-resistant strains. Transfection of a metronidazole-resistant clinical isolate (B7268), which does not express any detectable amounts of FR, with a plasmid bearing a functional FR1 gene nearly completely restored metronidazole sensitivity. Our results indicate that FR1 has a significant role in the emergence of metronidazole resistance in T. vaginalis. PMID:24256032

  13. The Impact of 5α-Reductase Inhibitor Use for Male Pattern Hair Loss on Men's Health.

    PubMed

    Said, Mohammed A; Mehta, Akanksha

    2018-06-16

    Male pattern hair loss, mediated by dihydrotestosterone, is a common hair loss disorder, affecting over 50% of men over the age of 50. The 5-α reductase inhibitors, finasteride and dutasteride, are Food and Drug Administration-approved drugs for the treatment of this disorder. Several recent studies have reported adverse sexual and spermatogenic events among young men using 5-α reductase inhibitors, such as erectile dysfunction, decreased ejaculate volume, decreased libido, and infertility. In this review, we summarize and analyze the literature regarding the efficacy and safety of these medications, with an overall focus on men's health. Finasteride for the treatment of male pattern hair loss was considered safe according to many previous clinical trials. However, these trials have been recently criticized for inadequate safety reporting. Comprehensive review of the current literature reveals that there is a disproportionately high number of men with 5-α reductase inhibitor-associated sexual dysfunction and infertility. Although uncommon, the use of 5-α reductase inhibitors is associated with serious and persistent sexual and reproductive side effects, such as erectile dysfunction, decreased ejaculate volume, decreased libido, and infertility.

  14. Evaluation of in vitro aldose reductase inhibitory potential of alkaloidal fractions of Piper nigrum, Murraya koenigii, Argemone mexicana, and Nelumbo nucifera.

    PubMed

    Gupta, Sakshi; Singh, Nirmal; Jaggi, Amteshwar Singh

    2014-05-01

    Aldose reductase is primarily involved in development of long-term diabetic complications due to increased polyol pathway activity. The synthetic aldose reductase inhibitors are not very successful clinically. Therefore, the natural sources may be exploited for safer and effective aldose reductase inhibitors. In the present study, the aldose reductase inhibitory potential of hydroalcoholic and alkaloidal extracts of Piper nigrum, Murraya koenigii, Argemone mexicana, and Nelumbo nucifera was evaluated. The hydroalcoholic and alkaloidal extracts of the selected plants were prepared. The different concentrations of hydroalcoholic and alkaloidal extracts of these plants were evaluated for their goat lens aldose reductase inhibitory activity using dl-glyceraldehyde as substrate. The aldose reductase inhibitory potential of extracts was assessed in terms of their IC50 value. Amongst the hydroalcoholic extracts, the highest aldose reductase inhibitory activity was shown by P. nigrum (IC50 value 35.64±2.7 μg/mL) followed by M. koenigii (IC50 value 45.67±2.57 μg/mL), A. mexicana (IC50 value 56.66±1.30 μg/mL), and N. nucifera (IC50 value 59.78±1.32 μg/mL). Among the alkaloidal extracts, highest inhibitory activity was shown by A. mexicana (IC50 value 25.67±1.25 μg/mL), followed by N. nucifera (IC50 value 28.82±1.85 μg/mL), P. nigrum (IC50 value 30.21±1.63 μg/mL), and M. koenigii (IC50 value 35.66±1.64 μg/mL). It may be concluded that the alkaloidal extracts of these plants possess potent aldose reductase inhibitory activity and may be therapeutically exploited in diabetes-related complications associated with increased activity of aldose reductase.

  15. Synthetic and Crystallographic Studies of a New Inhibitor Series Targeting Bacillus anthracis Dihydrofolate Reductase

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Beierlein, J.; Frey, K; Bolstad, D

    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 structuremore » 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.« less

  16. Decrease of Nitrate Reductase Activity in Spinach Leaves during a Light-Dark Transition 1

    PubMed Central

    Riens, Burgi; Heldt, Hans Walter

    1992-01-01

    In leaves of spinach plants (Spinacia oleracea L.) performing CO2 and NO3− assimilation, at the time of sudden darkening, which eliminates photosystem I-dependent nitrite reduction, only a minor temporary increase of the leaf nitrite content is observed. Because nitrate reduction does not depend on redox equivalents generated by photosystem I activity, a continuation of nitrate reduction after darkening would result in a large accumulation of nitrite in the leaves within a very short time, which is not observed. Measurements of the extractable nitrate reductase activity from spinach leaves assayed under standard conditions showed that in these leaves the nitrate reductase activity decreased during darkening to 15% of the control value with a half-time of only 2 minutes. Apparently, in these leaves nitrate reductase is very rapidly inactivated at sudden darkness avoiding an accumulation of the toxic nitrite in the cells. PMID:16668679

  17. The oxidation of apomorphine and other catechol compounds by horseradish peroxidase: relevance to the measurement of dihydropteridine reductase activity.

    PubMed

    Milstien, S; Kaufman, S

    1987-03-19

    It has been reported by Shen et al. (Shen, R.-S., Smith, R.V., Davis, P.J. and Abell, C.W. (1984) J. Biol. Chem. 259, 8894-9000) that apomorphine and dopamine are potent, non-competitive inhibitors of quinonoid dihydropteridine reductase. In this paper we show that apomorphine, dopamine and other catechol-containing compounds are oxidized rapidly to quinones by the horseradish peroxidase-H2O2 system which is used to generate the quinonoid dihydropterin substrate. These quinones react non-enzymatically with reduced pyridine nucleotides, depleting the other substrate of dihydropteridine reductase. When true initial rates of dihydropteridine reductase-dependent reduction of quinonoid dihydropterins are measured, neither apomorphine nor any other catechol-containing compound that has been tested has been found to inhibit dihydropteridine reductase.

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

    PubMed

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

    2017-06-01

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

  19. Synthesis, purification and crystallographic studies of the C-terminal sterol carrier protein type 2 (SCP-2) domain of human hydroxysteroid dehydrogenase-like protein 2.

    PubMed

    Cheng, Zhong; Li, Yao; Sui, Chun; Sun, Xiaobo; Xie, Yong

    2015-07-01

    Human hydroxysteroid dehydrogenase-like protein 2 (HSDL2) is a member of the short-chain dehydrogenase/reductase (SDR) subfamily of oxidoreductases and contains an N-terminal catalytic domain and a C-termianl sterol carrier protein type 2 (SCP-2) domain. In this study, the C-terminal SCP-2 domain of human HSDL2, including residues Lys318-Arg416, was produced in Escherichia coli, purified and crystallized. X-ray diffraction data were collected to 2.10 Å resolution. The crystal belonged to the trigonal space group P3(1)21 (or P3(2)21), with unit-cell parameters a = b = 70.4, c = 60.6 Å, α = β = 90, γ = 120°. Two protein molecules are present in the asymmetric unit, resulting in a Matthews coefficient of 2.16 Å(3) Da(-1) and an approximate solvent content of 43%.

  20. Antioxidant and quinone reductase-inducing constituents of black chokeberry (Aronia melanocarpa) fruits.

    PubMed

    Li, Jie; Deng, Ye; Yuan, Chunhua; Pan, Li; Chai, Heebyung; Keller, William J; Kinghorn, A Douglas

    2012-11-21

    Using in vitro hydroxyl radical-scavenging and quinone reductase-inducing assays, bioactivity-guided fractionation of an ethyl acetate-soluble extract of the fruits of the botanical dietary supplement, black chokeberry (Aronia melanocarpa), led to the isolation of 27 compounds, including a new depside, ethyl 2-[(3,4-dihydroxybenzoyloxy)-4,6-dihydroxyphenyl] acetate (1), along with 26 known compounds (2-27). The structures of the isolated compounds were identified by analysis of their physical and spectroscopic data ([α](D), NMR, IR, UV, and MS). Altogether, 17 compounds (1-4, 9, 15-17, and 19-27) showed significant antioxidant activity in the hydroxyl radical-scavenging assay, with hyperin (24, ED(50) = 0.17 μM) being the most potent. The new compound (1, ED(50) = 0.44 μM) also exhibited potent antioxidant activity in this assay. Three constituents of black chokeberry fruits doubled quinone reductase activity at concentrations <20 μM, namely, protocatechuic acid [9, concentration required to double quinone reductase activity (CD) = 4.3 μM], neochlorogenic acid methyl ester (22, CD = 6.7 μM), and quercetin (23, CD = 3.1 μM).

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

    PubMed

    Poonnachit, U; Darnell, R

    2004-04-01

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

  2. Characterization of Developmental- and Stress-Mediated Expression of Cinnamoyl-CoA Reductase in Kenaf (Hibiscus cannabinus L.)

    PubMed Central

    Lim, Hyoun-Sub; Park, Sang-Un; Bae, Hyeun-Jong; Natarajan, Savithiry

    2014-01-01

    Cinnamoyl-CoA reductase (CCR) is an important enzyme for lignin biosynthesis as it catalyzes the first specific committed step in monolignol biosynthesis. We have cloned a full length coding sequence of CCR from kenaf (Hibiscus cannabinus L.), which contains a 1,020-bp open reading frame (ORF), encoding 339 amino acids of 37.37 kDa, with an isoelectric point (pI) of 6.27 (JX524276, HcCCR2). BLAST result found that it has high homology with other plant CCR orthologs. Multiple alignment with other plant CCR sequences showed that it contains two highly conserved motifs: NAD(P) binding domain (VTGAGGFIASWMVKLLLEKGY) at N-terminal and probable catalytic domain (NWYCYGK). According to phylogenetic analysis, it was closely related to CCR sequences of Gossypium hirsutum (ACQ59094) and Populus trichocarpa (CAC07424). HcCCR2 showed ubiquitous expression in various kenaf tissues and the highest expression was detected in mature flower. HcCCR2 was expressed differentially in response to various stresses, and the highest expression was observed by drought and NaCl treatments. PMID:24723816

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

    Rižner, Tea Lanišnik; Penning, Trevor M

    2014-01-01

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

  5. The coordinated increased expression of biliverdin reductase and heme oxygenase-2 promotes cardiomyocyte survival: a reductase-based peptide counters β-adrenergic receptor ligand-mediated cardiac dysfunction

    PubMed Central

    Ding, Bo; Gibbs, Peter E. M.; Brookes, Paul S.; Maines, Mahin D.

    2011-01-01

    HO-2 oxidizes heme to CO and biliverdin; the latter is reduced to bilirubin by biliverdin reductase (BVR). In addition, HO-2 is a redox-sensitive K/Ca2-associated protein, and BVR is an S/T/Y kinase. The two enzymes are components of cellular defense mechanisms. This is the first reporting of regulation of HO-2 by BVR and that their coordinated increase in isolated myocytes and intact heart protects against cardiotoxicity of β-adrenergic receptor activation by isoproterenol (ISO). The induction of BVR mRNA, protein, and activity and HO-2 protein was maintained for ≥96 h; increase in HO-1 was modest and transient. In isolated cardiomyocytes, experiments with cycloheximide, proteasome inhibitor MG-132, and siBVR suggested BVR-mediated stabilization of HO-2. In both models, activation of BVR offered protection against the ligand's stimulation of apoptosis. Two human BVR-based peptides known to inhibit and activate the reductase, KKRILHC281 and KYCCSRK296, respectively, were tested in the intact heart. Perfusion of the heart with the inhibitory peptide blocked ISO-mediated BVR activation and augmented apoptosis; conversely, perfusion with the activating peptide inhibited apoptosis. At the functional level, peptide-mediated inhibition of BVR was accompanied by dysfunction of the left ventricle and decrease in HO-2 protein levels. Perfusion of the organ with the activating peptide preserved the left ventricular contractile function and was accompanied by increased levels of HO-2 protein. Finding that BVR and HO-2 levels, myocyte apoptosis, and contractile function of the heart can be modulated by small human BVR-based peptides offers a promising therapeutic approach for treatment of cardiac dysfunctions.—Ding, B., Gibbs, P. E. M., Brookes, P. S., Maines, M. D. The coordinated increased expression of biliverdin reductase and heme oxygenase-2 promotes cardiomyocyte survival; a reductase-based peptide counters β-adrenergic receptor ligand-mediated cardiac dysfunction

  6. Methionine sulfoxide reductase A protects hepatocytes against acetaminophen-induced toxicity via regulation of thioredoxin reductase 1 expression.

    PubMed

    Singh, Mahendra Pratap; Kwak, Geun-Hee; Kim, Ki Young; Kim, Hwa-Young

    2017-06-03

    Thioredoxin reductase 1 (TXNRD1) is associated with susceptibility to acetaminophen (APAP)-induced liver damage. Methionine sulfoxide reductase A (MsrA) is an antioxidant and protein repair enzyme that specifically catalyzes the reduction of methionine S-sulfoxide residues. We have previously shown that MsrA deficiency exacerbates acute liver injury induced by APAP. In this study, we used primary hepatocytes to investigate the underlying mechanism of the protective effect of MsrA against APAP-induced hepatotoxicity. MsrA gene-deleted (MsrA -/- ) hepatocytes showed higher susceptibility to APAP-induced cytotoxicity than wild-type (MsrA +/+ ) cells, consistent with our previous in vivo results. MsrA deficiency increased APAP-induced glutathione depletion and reactive oxygen species production. APAP treatment increased Nrf2 activation more profoundly in MsrA -/- than in MsrA +/+ hepatocytes. Basal TXNRD1 levels were significantly higher in MsrA -/- than in MsrA +/+ hepatocytes, while TXNRD1 depletion in both MsrA -/- and MsrA +/+ cells resulted in increased resistance to APAP-induced cytotoxicity. In addition, APAP treatment significantly increased TXNRD1 expression in MsrA -/- hepatocytes, while no significant change was observed in MsrA +/+ cells. Overexpression of MsrA reduced APAP-induced cytotoxicity and TXNRD1 expression levels in APAP-treated MsrA -/- hepatocytes. Collectively, our results suggest that MsrA protects hepatocytes from APAP-induced cytotoxicity through the modulation of TXNRD1 expression. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Transcriptional and metabolic regulation of denitrification in Paracoccus denitrificans allows low but significant activity of nitrous oxide reductase under oxic conditions.

    PubMed

    Qu, Zhi; Bakken, Lars R; Molstad, Lars; Frostegård, Åsa; Bergaust, Linda L

    2016-09-01

    Oxygen is known to repress denitrification at the transcriptional and metabolic levels. It has been a common notion that nitrous oxide reductase (N2 OR) is the most sensitive enzyme among the four N-oxide reductases involved in denitrification, potentially leading to increased N2 O production under suboxic or fluctuating oxygen conditions. We present detailed gas kinetics and transcription patterns from batch culture experiments with Paracoccus denitrificans, allowing in vivo estimation of e(-) -flow to O2 and N2 O under various O2 regimes. Transcription of nosZ took place concomitantly with that of narG under suboxic conditions, whereas transcription of nirS and norB was inhibited until O2 levels approached 0 μM in the liquid. Catalytically functional N2 OR was synthesized and active in aerobically raised cells transferred to vials with 7 vol% O2 in headspace, but N2 O reduction rates were 10 times higher when anaerobic pre-cultures were subjected to the same conditions. Upon oxygen exposure, there was an incomplete and transient inactivation of N2 OR that could be ascribed to its lower ability to compete for electrons compared with terminal oxidases. The demonstrated reduction of N2 O at high O2 partial pressure and low N2 O concentrations by a bacterium not known as a typical aerobic denitrifier may provide one clue to the understanding of why some soils appear to act as sinks rather than sources for atmospheric N2 O. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

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

    PubMed

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

    2009-07-01

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

  9. Phenotypic Restoration by Molybdate of Nitrate Reductase Activity in chlD Mutants of Escherichia coli

    PubMed Central

    Glaser, J. H.; DeMoss, J. A.

    1971-01-01

    ChlD mutants of Escherichia coli are pleiotropic, lacking formate-nitrate reductase activity as well as formate-hydrogenlyase activity. Whole-chain formate-nitrate reductase activity, assayed with formate as the electron donor and measuring the amount of nitrite produced, was restored to wild-type levels in the mutants by addition of 10−4m molybdate to the growth medium. Under these conditions, the activity of each of the components of the membrane-bound nitrate reductase chain increased after molybdate supplementation. In the absence of nitrate, the activities of the formate-hydrogenlyase system were also restored by molybdate. Strains deleted for the chlD gene responded in a similar way to molybdate supplementation. The concentration of molybdenum in the chlD mutant cells did not differ significantly from that in the wild-type cells at either low or high concentrations of molybdate in the medium. However, the distribution of molybdenum between the soluble protein and membrane fractions differed significantly from wild type. We conclude that the chlD gene product cannot be a structural component of the formate-hydrogenlyase pathway or the formate-nitrate reductase pathway, but that it must have an indirect role in processing molybdate to a form necessary for both electron transport systems. PMID:4942767

  10. Immunolocalisation of two tropinone reductases in potato (Solanum tuberosum L.) root, stolon, and tuber sprouts.

    PubMed

    Kaiser, Heike; Richter, Ute; Keiner, Ronald; Brabant, Anja; Hause, Bettina; Dräger, Birgit

    2006-12-01

    Tropinone reductases (TRs) are essential enzymes in the tropane alkaloid biosynthesis, providing either tropine for hyoscyamine and scopolamine formation or providing pseudotropine for calystegines. Two cDNAs coding for TRs were isolated from potato (Solanum tuberosum L.) tuber sprouts and expressed in E. coli. One reductase formed pseudotropine, the other formed tropine and showed kinetic properties typical for tropine-forming tropinone reductases (TRI) involved in hyoscyamine formation. Hyoscyamine and tropine are not found in S. tuberosum plants. Potatoes contain calystegines as the only products of the tropane alkaloid pathway. Polyclonal antibodies raised against both enzymes were purified to exclude cross reactions and were used for Western-blot analysis and immunolocalisation. The TRI (EC 1.1.1.206) was detected in protein extracts of tuber tissues, but mostly in levels too low to be localised in individual cells. The function of this enzyme in potato that does not form hyoscyamine is not clear. The pseudotropine-forming tropinone reductase (EC 1.1.1.236) was detected in potato roots, stolons, and tuber sprouts. Cortex cells of root and stolon contained the protein; additional strong immuno-labelling was located in phloem parenchyma. In tuber spouts, however, the protein was detected in companion cells.

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

    PubMed

    Scaglione, Antonella; Montemiglio, Linda Celeste; Parisi, Giacomo; Asteriti, Italia Anna; Bruni, Renato; Cerutti, Gabriele; Testi, Claudia; Savino, Carmelinda; Mancia, Filippo; Lavia, Patrizia; Vallone, Beatrice

    2017-06-01

    In humans the steroid 5alpha-reductase (SRD5A) family comprises five integral membrane enzymes that carry out reduction of a double bond in lipidic substrates: Δ 4 -3-keto steroids, polyprenol and trans-enoyl CoA. The best-characterized reaction is the conversion of testosterone into the more potent dihydrotestosterone carried out by SRD5A1-2. Some controversy exists on their possible nuclear or endoplasmic reticulum localization. We report the cloning and transient expression in HeLa cells of the five members of the human steroid 5α-reductase family as both N- and C-terminus green fluorescent protein tagged protein constructs. Following the intrinsic fluorescence of the tag, we have determined that the subcellular localization of these enzymes is in the endoplasmic reticulum, upon expression in HeLa cells. The presence of the tag at either end of the polypeptide chain can affect protein expression and, in the case of trans enoyl-CoA reductase, it induces the formation of protein aggregates.

  12. A novel aldo-keto reductase from Escherichia coli can increase resistance to methylglyoxal toxicity.

    PubMed

    Grant, Anne W; Steel, Gavin; Waugh, Hugh; Ellis, Elizabeth M

    2003-01-21

    A novel aldo-keto reductase (AKR) from Escherichia coli has been cloned, expressed and purified. This protein, YghZ, is distantly related (<40%) to mammalian aflatoxin dialdehyde reductases of the aldo-keto reductase AKR7 family and to potassium channel beta-subunits in the AKR6 family. The enzyme has been placed in a new AKR family (AKR14), with the designation AKR14A1. Sequences encoding putative homologues of this enzyme exist in many other bacteria. The enzyme can reduce several aldehyde and diketone substrates, including the toxic metabolite methylglyoxal. The K(m) for the model substrate 4-nitrobenzaldehyde is 1.06 mM and for the endogenous dicarbonyl methylglyoxal it is 3.4 mM. Overexpression of the recombinant enzyme in E. coli leads to increased resistance to methylglyoxal. It is possible that this enzyme plays a role in the metabolism of methylglyoxal, and can influence its levels in vivo.

  13. 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. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Rapid electron exchange between surface-exposed bacterial cytochromes and Fe(III) minerals

    PubMed Central

    White, Gaye F.; Shi, Zhi; Shi, Liang; Wang, Zheming; Dohnalkova, Alice C.; Marshall, Matthew J.; Fredrickson, James K.; Zachara, John M.; Butt, Julea N.; Richardson, David J.; Clarke, Thomas A.

    2013-01-01

    The mineral-respiring bacterium Shewanella oneidensis uses a protein complex, MtrCAB, composed of two decaheme cytochromes, MtrC and MtrA, brought together inside a transmembrane porin, MtrB, to transport electrons across the outer membrane to a variety of mineral-based electron acceptors. A proteoliposome system containing a pool of internalized electron carriers was used to investigate how the topology of the MtrCAB complex relates to its ability to transport electrons across a lipid bilayer to externally located Fe(III) oxides. With MtrA facing the interior and MtrC exposed on the outer surface of the phospholipid bilayer, the established in vivo orientation, electron transfer from the interior electron carrier pool through MtrCAB to solid-phase Fe(III) oxides was demonstrated. The rates were 103 times higher than those reported for reduction of goethite, hematite, and lepidocrocite by S. oneidensis, and the order of the reaction rates was consistent with those observed in S. oneidensis cultures. In contrast, established rates for single turnover reactions between purified MtrC and Fe(III) oxides were 103 times lower. By providing a continuous flow of electrons, the proteoliposome experiments demonstrate that conduction through MtrCAB directly to Fe(III) oxides is sufficient to support in vivo, anaerobic, solid-phase iron respiration. PMID:23538304

  15. A mutant of barley lacking NADH-hydroxypyruvate reductase

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    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 tomore » 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.« less

  16. Intact Plastids Are Required for Nitrate- and Light-Induced Accumulation of Nitrate Reductase Activity and mRNA in Squash Cotyledons 1

    PubMed Central

    Oelmüller, Rolf; Briggs, Winslow R.

    1990-01-01

    Induction of nitrate reductase activity and mRNA by nitrate and light is prevented if chloroplasts are destroyed by photooxidation in norflurazon-treated squash (Cucurbita maxima L.) cotyledons. The enzyme activity and mRNA can be induced if norflurazon-treated squash seedlings are kept in low-intensity red light, which minimizes photodamage to the plastids. It is concluded that induction of nitrate reductase activity and nitrate reductase mRNA requires intact plastids. If squash seedlings grown in low-intensity red light are transferred to photooxidative white light, nitrate reductase activity accumulates during the first 12 hours after the shift and declines thereafter. Thus photodamage to the plastids and the disappearance of nitrate reductase activity and mRNA are events separable in time, and disappearance of the enzyme activity is a consequence of the damage to the plastids. Images Figure 1 Figure 3 Figure 4 PMID:16667294

  17. The HIP1 initiator element plays a role in determining the in vitro requirement of the dihydrofolate reductase gene promoter for the C-terminal domain of RNA polymerase II.

    PubMed

    Buermeyer, A B; Thompson, N E; Strasheim, L A; Burgess, R R; Farnham, P J

    1992-05-01

    We examined the ability of purified RNA polymerase (RNAP) II lacking the carboxy-terminal heptapeptide repeat domain (CTD), called RNAP IIB, to transcribe a variety of promoters in HeLa extracts in which endogenous RNAP II activity was inhibited with anti-CTD monoclonal antibodies. Not all promoters were efficiently transcribed by RNAP IIB, and transcription did not correlate with the in vitro strength of the promoter or with the presence of a consensus TATA box. This was best illustrated by the GC-rich, non-TATA box promoters of the bidirectional dihydrofolate reductase (DHFR)-REP-encoding locus. Whereas the REP promoter was transcribed by RNAP IIB, the DHFR promoter remained inactive after addition of RNAP IIB to the antibody-inhibited reactions. However, both promoters were efficiently transcribed when purified RNAP with an intact CTD was added. We analyzed a series of promoter deletions to identify which cis elements determine the requirement for the CTD of RNAP II. All of the promoter deletions of both DHFR and REP retained the characteristics of their respective full-length promoters, suggesting that the information necessary to specify the requirement for the CTD is contained within approximately 65 bp near the initiation site. Furthermore, a synthetic minimal promoter of DHFR, consisting of a single binding site for Sp1 and a binding site for the HIP1 initiator cloned into a bacterial vector sequence, required RNAP II with an intact CTD for activity in vitro. Since the synthetic minimal promoter of DHFR and the smallest REP promoter deletion are both activated by Sp1, the differential response in this assay does not result from upstream activators. However, the sequences around the start sites of DHFR and REP are not similar and our data suggest that they bind different proteins. Therefore, we propose that specific initiator elements are important for determination of the requirement of some promoters for the CTD.

  18. 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.

  19. ARSENICALS INHIBIT THIOREDOXIN REDUCTASE ACTIVITY IN CULTURED RAT HEPATOCYTES

    EPA Science Inventory

    ARSENICALS INHIBIT THIOREDOXIN REDUCTASE ACTIVITY IN CULTURED RAT HEPATOCYTES.

    S. Lin1, L. M. Del Razo1, M. Styblo1, C. Wang2, W. R. Cullen2, and D.J. Thomas3. 1Univ. North Carolina, Chapel Hill, NC; 2Univ. British Columbia, Vancouver, BC, Canada; 3National Health and En...

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

    PubMed Central

    Bettiga, Maurizio; Hahn-Hägerdal, Bärbel; Gorwa-Grauslund, Marie F

    2008-01-01

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

  1. Mechanism of biological denitrification inhibition: procyanidins induce an allosteric transition of the membrane-bound nitrate reductase through membrane alteration.

    PubMed

    Bardon, Clément; Poly, Franck; Piola, Florence; Pancton, Muriel; Comte, Gilles; Meiffren, Guillaume; Haichar, Feth el Zahar

    2016-05-01

    Recently, it has been shown that procyanidins from Fallopia spp. inhibit bacterial denitrification, a phenomenon called biological denitrification inhibition (BDI). However, the mechanisms involved in such a process remain unknown. Here, we investigate the mechanisms of BDI involving procyanidins, using the model strain Pseudomonas brassicacearum NFM 421. The aerobic and anaerobic (denitrification) respiration, cell permeability and cell viability of P. brassicacearum were determined as a function of procyanidin concentration. The effect of procyanidins on the bacterial membrane was observed using transmission electronic microscopy. Bacterial growth, denitrification, NO3- and NO2-reductase activity, and the expression of subunits of NO3- (encoded by the gene narG) and NO2-reductase (encoded by the gene nirS) under NO3 or NO2 were measured with and without procyanidins. Procyanidins inhibited the denitrification process without affecting aerobic respiration at low concentrations. Procyanidins also disturbed cell membranes without affecting cell viability. They specifically inhibited NO3- but not NO2-reductase.Pseudomonas brassicacearum responded to procyanidins by over-expression of the membrane-bound NO3-reductase subunit (encoded by the gene narG). Our results suggest that procyanidins can specifically inhibit membrane-bound NO3-reductase inducing enzymatic conformational changes through membrane disturbance and that P. brassicacearum responds by over-expressing membrane-bound NO3-reductase. Our results lead the way to a better understanding of BDI. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  2. 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

  3. Structural and Biochemical Characterization of Cinnamoyl-CoA Reductases1

    PubMed Central

    Walker, Alexander M.

    2017-01-01

    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. PMID:27956488

  4. Effect of Ammonium and Nitrate on Ferric Chelate Reductase and Nitrate Reductase in Vaccinium Species

    PubMed Central

    POONNACHIT, U.; DARNELL, R.

    2004-01-01

    • Background and Aims Most Vaccinium species have strict soil requirements for optimal growth, requiring low pH, high iron availability and nitrogen primarily in the ammonium form. These soils are limited and are often located near wetlands. Vaccinium arboreum is a wild species adapted to a wide range of soils, including high pH, low iron, and nitrate‐containing soils. This broader soil adaptation in V. arboreum may be related to increased efficiency of iron or nitrate uptake compared with the cultivated Vaccinium species. • Methods Nitrate, ammonium and iron uptake, and nitrate reductase (NR) and ferric chelate reductase (FCR) activities were compared in two Vaccinium species grown hydroponically in either nitrate or ammonia, with or without iron. The species studied were the wild V. arboreum and the cultivated V. corymbosum interspecific hybrid, which exhibits the strict soil requirements of most Vaccinium species. • Key Results Ammonium uptake was significantly greater than nitrate uptake in both species, while nitrate uptake was greater in the wild species, V. arboreum, compared with the cultivated species, V. corymbosum. The increased nitrate uptake in V. arboreum was correlated with increased root NR activity compared with V. corymbosum. The lower nitrate uptake in V. corymbosum was reflected in decreased plant dry weight in this species compared with V. arboreum. Root FCR activity increased significantly in V. corymbosum grown under iron‐deficient conditions, compared with the same species grown under iron‐sufficient conditions or with V. arboreum grown under either iron condition. • Conclusions. V. arboreum appears to be more efficient in acquiring nitrate compared with V. corymbosum, possibly due to increased NR activity and this may partially explain the wider soil adaptation of V. arboreum. PMID:14980973

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

    PubMed

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

    2016-04-01

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

  6. Mercury-resistance and mercuric reductase activity in Chromobacterium, Erwinia, and Bacillus species

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Trevors, J.T.

    1987-06-01

    Mercury resistant bacteria have been the most extensively studied of all the metal-tolerant bacteria. Mercury resistance is usually mediated by two distinctly different enzymes encoded by plasmids. Mercuric reductase reduces Hg/sup 2 +/ to metallic mercury (Hg/sup 0/). Organomercurial lyases have a molecular weight of 20,000 to 40,000, are composed of 1 or 2 subunits and require the presence of thiol. Plasmic-encoded Hg/sup 2 +/ resistance and mercuric reductase activity have not been detected in many species of bacteria. A Chromobacterium, Erwinia and Bacillus species isolated from environmental samples were capable of growth in the presence of 50 ..mu..M HgCl/submore » 2/. Cell-free extracts of the 3 organisms exhibited mercuric reductase activity that oxidized NADPH in the presence of HgCl/sub 2/. Negligible oxidation of NADPH was observed in the absence of HgCl/sub 2/. The Chromobacterium sp. did not contain any plasmid DNA. This would suggest that Hg/sup 2 +/ resistance was carried on the chromosome in Chromobacterium. A single 3 Mdal plasmid in the Bacillus sp. was refractory to curing. The Erwinia sp. contained 3 plasmids which were also refractory to curing. The location of the resistance genes is unknown in the Bacillus and Erwinia isolates.« less

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

    PubMed

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

    2004-10-01

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

  8. Biological activity of aldose reductase and lipophilicity of pyrrolyl-acetic acid derivatives

    NASA Astrophysics Data System (ADS)

    Kumari, A.; Kumari, R.; Kumar, R.; Gupta, M.

    2011-12-01

    Quantitative Structure-Activity Relationship modeling is a powerful approach for correlating an organic compound to its lipophilicity. In this paper QSAR models are established for estimation of correlation of the lipophilicity of a series of pyrrolyl-acetic acid derivatives, inhibitors of the aldose reductase enzyme, in the n-octanol-water system with biological activity of aldose reductase. Lipophilicity, expressed by the logarithm of n-octnol-water partition coefficient log P and biological activity of aldose reductase inhibitory activity by log it. Result obtained by QSAR modeling of compound series reveal a definite trend in biological activity and a further improvement in quantitative relationships are established if, beside log P, Hammett electronic constant σ and connectivity index chi-3 (3 χ) term included in the regression equation. The tri-parametric model with log P, 3 χ and σ as correlating parameters have been found to be the best which gives a variance of 87% ( R 2 = 0.8743). A compound has been found to be serious outlier and when the same has been excluded the model explains about 94% variance of the data set ( R 2 = 0.9447). The topological index (3 χ) has been found to be a good parameter for modeling the biological activity.

  9. Novel aldose reductase inhibitors: a patent survey (2006--present).

    PubMed

    Chatzopoulou, Maria; Alexiou, Polyxeni; Kotsampasakou, Eleni; Demopoulos, Vassilis J

    2012-11-01

    Initially studied for its central role in the pathogenesis of chronic diabetic complications, aldose reductase (ALR2) gains more attention over the years as its implication in inflammatory diseases is being established, along with the therapeutic potential of its inhibitors. Reviewing the patents that were published since 2006, it is getting clear that the search for new chemical entities has subsided, giving rise to natural products and plant extracts with ALR2 inhibitory activity. Other aspects that were prominent were the search for proper forms of known inhibitors, in a way to improve their impaired physicochemical profile, as well as potential combination therapies with other compounds of pharmaceutical interest. On the spotlight were patents enhancing the therapeutic usage of aldose reductase inhibitors (ARIs) to various pathological conditions including cancer and inflammation-mediated diseases such as sepsis, asthma, and cancer. Although new chemical entities are scarcely registered and patented after many years of inconclusive clinical trials, the involvement of ALR2 to inflammatory pathologies might renew the interest in the field of ARIs.

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

    USDA-ARS?s Scientific Manuscript database

    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 ...

  11. Optimization of arsenic removal water treatment system through characterization of terminal electron accepting processes.

    PubMed

    Upadhyaya, Giridhar; Clancy, Tara M; Brown, Jess; Hayes, Kim F; Raskin, Lutgarde

    2012-11-06

    Terminal electron accepting process (TEAP) zones developed when a simulated groundwater containing dissolved oxygen (DO), nitrate, arsenate, and sulfate was treated in a fixed-bed bioreactor system consisting of two reactors (reactors A and B) in series. When the reactors were operated with an empty bed contact time (EBCT) of 20 min each, DO-, nitrate-, sulfate-, and arsenate-reducing TEAP zones were located within reactor A. As a consequence, sulfate reduction and subsequent arsenic removal through arsenic sulfide precipitation and/or arsenic adsorption on or coprecipitation with iron sulfides occurred in reactor A. This resulted in the removal of arsenic-laden solids during backwashing of reactor A. To minimize this by shifting the sulfate-reducing zone to reactor B, the EBCT of reactor A was sequentially lowered from 20 min to 15, 10, and 7 min. While 50 mg/L (0.81 mM) nitrate was completely removed at all EBCTs, more than 90% of 300 μg/L (4 μM) arsenic was removed with the total EBCT as low as 27 min. Sulfate- and arsenate-reducing bacteria were identified throughout the system through clone libraries and quantitative PCR targeting the 16S rRNA, dissimilatory (bi)sulfite reductase (dsrAB), and dissimilatory arsenate reductase (arrA) genes. Results of reverse transcriptase (RT) qPCR of partial dsrAB (i.e., dsrA) and arrA transcripts corresponded with system performance. The RT qPCR results indicated colocation of sulfate- and arsenate-reducing activities, in the presence of iron(II), suggesting their importance in arsenic removal.

  12. Transcription initiation from the dihydrofolate reductase promoter is positioned by HIP1 binding at the initiation site.

    PubMed

    Means, A L; Farnham, P J

    1990-02-01

    We have identified a sequence element that specifies the position of transcription initiation for the dihydrofolate reductase gene. Unlike the functionally analogous TATA box that directs RNA polymerase II to initiate transcription 30 nucleotides downstream, the positioning element of the dihydrofolate reductase promoter is located directly at the site of transcription initiation. By using DNase I footprint analysis, we have shown that a protein binds to this initiator element. Transcription initiated at the dihydrofolate reductase initiator element when 28 nucleotides were inserted between it and all other upstream sequences, or when it was placed on either side of the DNA helix, suggesting that there is no strict spatial requirement between the initiator and an upstream element. Although neither a single Sp1-binding site nor a single initiator element was sufficient for transcriptional activity, the combination of one Sp1-binding site and the dihydrofolate reductase initiator element cloned into a plasmid vector resulted in transcription starting at the initiator element. We have also shown that the simian virus 40 late major initiation site has striking sequence homology to the dihydrofolate reductase initiation site and that the same, or a similar, protein binds to both sites. Examination of the sequences at other RNA polymerase II initiation sites suggests that we have identified an element that is important in the transcription of other housekeeping genes. We have thus named the protein that binds to the initiator element HIP1 (Housekeeping Initiator Protein 1).

  13. Nitrate reductase-formate dehydrogenase couple involved in the fungal denitrification by Fusarium oxysporum.

    PubMed

    Uchimura, Hiromasa; Enjoji, Hitoshi; Seki, Takafumi; Taguchi, Ayako; Takaya, Naoki; Shoun, Hirofumi

    2002-04-01

    Dissimilatory nitrate reductase (Nar) was solubilized and partially purified from the large particle (mitochondrial) fraction of the denitrifying fungus Fusarium oxysporum and characterized. Many lines of evidence showed that the membrane-bound Nar is distinct from the soluble, assimilatory nitrate reductase. Further, the spectral and other properties of the fungal Nar were similar to those of dissimilatory Nars of Escherichia coli and denitrifying bacteria, which are comprised of a molybdoprotein, a cytochrome b, and an iron-sulfur protein. Formate-nitrate oxidoreductase activity was also detected in the mitochondrial fraction, which was shown to arise from the coupling of formate dehydrogenase (Fdh), Nar, and a ubiquinone/ubiquinol pool. This is the first report of the occurrence in a eukaryote of Fdh that is associated with the respiratory chain. The coupling with Fdh showed that the fungal Nar system is more similar to that involved in the nitrate respiration by Escherichia coli than that in the bacterial denitrifying system. Analyses of the mutant species of F. oxysporum that were defective in Nar and/or assimilatory nitrate reductase conclusively showed that Nar is essential for the fungal denitrification.

  14. Sequences required for transcription termination at the intrinsic lambdatI terminator.

    PubMed

    Martínez-Trujillo, Miguel; Sánchez-Trujillo, Alejandra; Ceja, Víctor; Avila-Moreno, Federico; Bermúdez-Cruz, Rosa María; Court, Donald; Montañez, Cecilia

    2010-02-01

    The lambdatI terminator is located approximately 280 bp beyond the lambdaint gene, and it has a typical structure of an intrinsic terminator. To identify sequences required for lambdatI transcription termination a set of deletion mutants were generated, either from the 5' or the 3' end onto the lambdatI region. The termination efficiency was determined by measuring galactokinase (galK) levels by Northern blot assays and by in vitro transcription termination. The importance of the uridines and the stability of the stem structure in the termination were demonstrated. The nontranscribed DNA beyond the 3' end also affects termination. Additionally, sequences upstream have a small effect on transcription termination. The in vivo RNA termination sites at lambdatI were determined by S1 mapping and were located at 8 different positions. Processing of transcripts from the 3' end confirmed the importance of the hairpin stem in protection against exonuclease.

  15. Structural Analysis of the Mn(IV)/Fe(III) Cofactor of Chlamydia Trachomatis Ribonucleotide Reductase By Extended X-Ray Absorption Fine Structure Spectroscopy And Density Functional Theory Calculations

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Younker, J.M.; Krest, C.M.; Jiang, W.

    2009-05-28

    The class Ic ribonucleotide reductase from Chlamydia trachomatis (C{bar A}) uses a stable Mn(lV)/ Fe(lll) cofactor to initiate nucleotide reduction by a free-radical mechanism. Extended X-ray absorption fine structure (EXAFS) spectroscopy and density functional theory (DFT) calculations are used to postulate a structure for this cofactor. Fe and Mn K-edge EXAFS data yield an intermetallic distance of -2.92 {angstrom}. The Mn data also suggest the presence of a short 1.74 {angstrom} Mn-O bond. These metrics are compared to the results of DFT calculations on 12 cofactor models derived from the crystal structure of the inactive Fe2(lll/ III) form of themore » protein. Models are differentiated by the protonation states of their bridging and terminal OH{sub x} ligands as well as the location of the Mn(lV) ion (site 1 or 2). The models that agree best with experimental observation feature a{mu}-1, 3-carboxylate bridge (E120), terminal solvent (H{sub 2}O/OH) to site 1, one {mu}-O bridge, and one {mu}-OH bridge. The site-placement of the metal ions cannot be discerned from the available data.« less

  16. Competitive folding of anti-terminator/terminator hairpins monitored by single molecule FRET.

    PubMed

    Clerte, Caroline; Declerck, Nathalie; Margeat, Emmanuel

    2013-02-01

    The control of transcription termination by RNA-binding proteins that modulate RNA-structures is an important regulatory mechanism in bacteria. LicT and SacY from Bacillus subtilis prevent the premature arrest of transcription by binding to an anti-terminator RNA hairpin that overlaps an intrinsic terminator located in the 5'-mRNA leader region of the gene to be regulated. In order to investigate the molecular determinants of this anti-termination/termination balance, we have developed a fluorescence-based nucleic acids system that mimics the competition between the LicT or SacY anti-terminator targets and the overlapping terminators. Using Förster Resonance Energy Transfer on single diffusing RNA hairpins, we could monitor directly their opening or closing state, and thus investigate the effects on this equilibrium of the binding of anti-termination proteins or terminator-mimicking oligonucleotides. We show that the anti-terminator hairpins adopt spontaneously a closed structure and that their structural dynamics is mainly governed by the length of their basal stem. The induced stability of the anti-terminator hairpins determines both the affinity and specificity of the anti-termination protein binding. Finally, we show that stabilization of the anti-terminator hairpin, by an extended basal stem or anti-termination protein binding can efficiently counteract the competing effect of the terminator-mimic.

  17. Competitive folding of anti-terminator/terminator hairpins monitored by single molecule FRET

    PubMed Central

    Clerte, Caroline; Declerck, Nathalie; Margeat, Emmanuel

    2013-01-01

    The control of transcription termination by RNA-binding proteins that modulate RNA-structures is an important regulatory mechanism in bacteria. LicT and SacY from Bacillus subtilis prevent the premature arrest of transcription by binding to an anti-terminator RNA hairpin that overlaps an intrinsic terminator located in the 5′-mRNA leader region of the gene to be regulated. In order to investigate the molecular determinants of this anti-termination/termination balance, we have developed a fluorescence-based nucleic acids system that mimics the competition between the LicT or SacY anti-terminator targets and the overlapping terminators. Using Förster Resonance Energy Transfer on single diffusing RNA hairpins, we could monitor directly their opening or closing state, and thus investigate the effects on this equilibrium of the binding of anti-termination proteins or terminator-mimicking oligonucleotides. We show that the anti-terminator hairpins adopt spontaneously a closed structure and that their structural dynamics is mainly governed by the length of their basal stem. The induced stability of the anti-terminator hairpins determines both the affinity and specificity of the anti-termination protein binding. Finally, we show that stabilization of the anti-terminator hairpin, by an extended basal stem or anti-termination protein binding can efficiently counteract the competing effect of the terminator-mimic. PMID:23303779

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

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

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

  19. Differential molecular response of monodehydroascorbate reductase and glutathione reductase by nitration and S-nitrosylation

    PubMed Central

    Begara-Morales, Juan C.; Sánchez-Calvo, Beatriz; Chaki, Mounira; Mata-Pérez, Capilla; Valderrama, Raquel; Padilla, María N.; Luque, Francisco; Corpas, Francisco J.; Barroso, Juan B.

    2015-01-01

    The ascorbate–glutathione cycle is a metabolic pathway that detoxifies hydrogen peroxide and involves enzymatic and non-enzymatic antioxidants. Proteomic studies have shown that some enzymes in this cycle such as ascorbate peroxidase (APX), monodehydroascorbate reductase (MDAR), and glutathione reductase (GR) are potential targets for post-translational modifications (PMTs) mediated by nitric oxide-derived molecules. Using purified recombinant pea peroxisomal MDAR and cytosolic and chloroplastic GR enzymes produced in Escherichia coli, the effects of peroxynitrite (ONOO–) and S-nitrosoglutathione (GSNO) which are known to mediate protein nitration and S-nitrosylation processes, respectively, were analysed. Although ONOO– and GSNO inhibit peroxisomal MDAR activity, chloroplastic and cytosolic GR were not affected by these molecules. Mass spectrometric analysis of the nitrated MDAR revealed that Tyr213, Try292, and Tyr345 were exclusively nitrated to 3-nitrotyrosine by ONOO–. The location of these residues in the structure of pea peroxisomal MDAR reveals that Tyr345 is found at 3.3 Å of His313 which is involved in the NADP-binding site. Site-directed mutagenesis confirmed Tyr345 as the primary site of nitration responsible for the inhibition of MDAR activity by ONOO–. These results provide new insights into the molecular regulation of MDAR which is deactivated by nitration and S-nitrosylation. However, GR was not affected by ONOO– or GSNO, suggesting the existence of a mechanism to conserve redox status by maintaining the level of reduced GSH. Under a nitro-oxidative stress induced by salinity (150mM NaCl), MDAR expression (mRNA, protein, and enzyme activity levels) was increased, probably to compensate the inhibitory effects of S-nitrosylation and nitration on the enzyme. The present data show the modulation of the antioxidative response of key enzymes in the ascorbate–glutathione cycle by nitric oxide (NO)-PTMs, thus indicating the close involvement

  20. Fluorescence labelling of NADPH-cytochrome P-450 reductase with the monobromomethyl derivative of syn-9,10-dioxabimane.

    PubMed Central

    Vogel, F; Lumper, L

    1983-01-01

    The kinetics of thiol-group alkylation in NADPH-cytochrome P-450 reductase during its inactivation by monobromobimane has been studied using the fluorimetric determination of S-bimane-L-cysteine by high-performance liquid chromatography. Loss of activity during the reaction of NADPH-cytochrome P-450 reductase with monobromobimane is caused by the alkylation of one single critical cysteine residue, which can be protected against thiol-specific reagents by NADP(H). The chemical stability of the bimane group allows the digestion of bimane-labelled NADPH-cytochrome P-450 reductase by CNBr. The critical cysteine residue could be located in a CNBr-cleaved peptide purified to homogeneity with Mr 10 500 +/- 1 000 and valine as N-terminus. Images Fig. 2. PMID:6414464

  1. Expression of Cyanobacterial Acyl-ACP Reductase Elevates the Triacylglycerol Level in the Red Alga Cyanidioschyzon merolae.

    PubMed

    Sumiya, Nobuko; Kawase, Yasuko; Hayakawa, Jumpei; Matsuda, Mami; Nakamura, Mami; Era, Atsuko; Tanaka, Kan; Kondo, Akihiko; Hasunuma, Tomohisa; Imamura, Sousuke; Miyagishima, Shin-ya

    2015-10-01

    Nitrogen starvation is known to induce the accumulation of triacylglycerol (TAG) in many microalgae, and potential use of microalgae as a source of biofuel has been explored. However, nitrogen starvation also stops cellular growth. The expression of cyanobacterial acyl-acyl carrier protein (ACP) reductase in the unicellular red alga Cyanidioschyzon merolae chloroplasts resulted in an accumulation of TAG, which led to an increase in the number and size of lipid droplets while maintaining cellular growth. Transcriptome and metabolome analyses showed that the expression of acyl-ACP reductase altered the activities of several metabolic pathways. The activities of enzymes involved in fatty acid synthesis in chloroplasts, such as acetyl-CoA carboxylase and pyruvate dehydrogenase, were up-regulated, while pyruvate decarboxylation in mitochondria and the subsequent consumption of acetyl-CoA by the tricarboxylic acid (TCA) cycle were down-regulated. Aldehyde dehydrogenase, which oxidizes fatty aldehydes to fatty acids, was also up-regulated in the acyl-ACP reductase expresser. This activation was required for the lipid droplet accumulation and metabolic changes observed in the acyl-ACP reductase expresser. Nitrogen starvation also resulted in lipid droplet accumulation in C. merolae, while cell growth ceased as in the case of other algal species. The metabolic changes that occur upon the expression of acyl-ACP reductase are quite different from those caused by nitrogen starvation. Therefore, there should be a method for further increasing the storage lipid level while still maintaining cell growth that is different from the metabolic response to nitrogen starvation. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  2. Androgenic correlates of genetic variation in the gene encoding 5alpha-reductase type 1.

    PubMed

    Ellis, Justine A; Panagiotopoulos, Sianna; Akdeniz, Aysel; Jerums, George; Harrap, Stephen B

    2005-01-01

    Androgens determine male secondary sexual characteristics and influence a variety of metabolic pathways. Circulating levels of androgens are highly heritable; however, the genes involved are largely unknown. The 5alpha-reductase enzymes types 1 and 2 responsible for converting testosterone to the more potent androgen dihydrotestosterone are encoded by the SRD5A1 and SRD5A2 genes, respectively. We performed indirect genetic association studies of SRD5A1 and SRD5A2 and the dihydrotestosterone/testosterone ratio that reflects the activity of 5alpha-reductase in 57 males with type 2 diabetes. We found evidence of significant association between a single nucleotide polymorphism in SRD5A1 and the dihydrotestosterone/testosterone ratio (median 0.10, interquartile range 0.08 vs. median 0.06, interquartile range 0.04, P = 0.009). The polymorphism was not associated with any diabetic phenotypes. These results suggest that functional genetic variants might exist in or around SRD5A1 that affect the activity of the 5alpha-reductase enzyme type 1 and influence androgen levels.

  3. Natural Inhibitors of HMG-CoA Reductase-An Insilico Approach Through Molecular Docking and Simulation Studies.

    PubMed

    Suganya, Subramanian; Nandagopal, Balaji; Anbarasu, Anand

    2017-01-01

    Plant products have always been considered for many important metabolic disorders due to its abundant medicinal properties. Alarming adverse effects of overuse of statins has been reported for patients with dyslipidemia. This study was aimed to identify compounds with potent anti-dyslipidemic property from selected plants and analyze them for their efficiency in binding with HMG-CoA reductase, a key enzyme in lipid metabolism. The docking studies indicate rutin as the best compound that can inhibit HMG-CoA reductase as it had strong binding affinity to the enzyme. The molecular dynamics simulation studies confirmed the stability of the HMG-CoA reductase-rutin complex. RMSD, RMSF, Rg, H-bond results indicated that the HMG-CoA reductase-rutin complex is highly stable. Presently, statins are not preferred for individuals with pre-existing liver disease. Our study identified rutin as a promising lead compound which could be further developed into an anti-dyslipidemic molecule. Our results will be a good starting point for future experimental and clinical studies and if the results from such studies match international standards plant derived rutin might emerge as a good alternative to statins. J. Cell. Biochem. 118: 52-57, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kim, Taeho; Flick, Robert; Brunzelle, Joseph

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

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

    PubMed

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

    1990-01-01

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

  6. Guinea-pig liver testosterone 17 beta-dehydrogenase (NADP+) and aldehyde reductase exhibit benzene dihydrodiol dehydrogenase activity.

    PubMed Central

    Hara, A; Hayashibara, M; Nakayama, T; Hasebe, K; Usui, S; Sawada, H

    1985-01-01

    We have kinetically and immunologically demonstrated that testosterone 17 beta-dehydrogenase (NADP+) isoenzymes (EC 1.1.1.64) and aldehyde reductase (EC 1.1.1.2) from guinea-pig liver catalyse the oxidation of benzene dihydrodiol (trans-1,2-dihydroxycyclohexa-3,5-diene) to catechol. One isoenzyme of testosterone 17 beta-dehydrogenase, which has specificity for 5 beta-androstanes, oxidized benzene dihydrodiol at a 3-fold higher rate than 5 beta-dihydrotestosterone, and showed a more than 4-fold higher affinity for benzene dihydrodiol and Vmax. value than did another isoenzyme, which exhibits specificity for 5 alpha-androstanes, and aldehyde reductase. Immunoprecipitation of guinea-pig liver cytosol with antisera against the testosterone 17 beta-dehydrogenase isoenzymes and aldehyde reductase indicated that most of the benzene dihydrodiol dehydrogenase activity in the tissue is due to testosterone 17 beta-dehydrogenase. PMID:2983661

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

    PubMed Central

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

    2009-01-01

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

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

    PubMed

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

    2011-09-01

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

  9. Multiple Mutations Modulate the Function of Dihydrofolate Reductase in Trimethoprim-Resistant Streptococcus pneumoniae

    PubMed Central

    Maskell, Jeffrey P.; Sefton, Armine M.; Hall, Lucinda M. C.

    2001-01-01

    Trimethoprim resistance in Streptococcus pneumoniae can be conferred by a single amino acid substitution (I100-L) in dihydrofolate reductase (DHFR), but resistant clinical isolates usually carry multiple DHFR mutations. DHFR genes from five trimethoprim-resistant isolates from the United Kingdom were compared to susceptible isolates and used to transform a susceptible control strain (CP1015). All trimethoprim-resistant isolates and transformants contained the I100-L mutation. The properties of DHFRs from transformants with different combinations of mutations were compared. In a transformant with only the I100-L mutation (R12/T2) and a D92-A mutation also found in the DHFRs of susceptible isolates, the enzyme was much more resistant to trimethoprim inhibition (50% inhibitory concentration [IC50], 4.2 μM) than was the DHFR from strain CP1015 (IC50, 0.09 μM). However, Km values indicated a lower affinity for the enzyme's natural substrates (Km for dihydrofolate [DHF], 3.1 μM for CP1015 and 27.5 μM for R12/T2) and a twofold decrease in the specificity constant. In transformants with additional mutations in the C-terminal portion of the enzyme, Km values for DHF were reduced (9.2 to 15.2 μM), indicating compensation for the lower affinity generated by I100-L. Additional mutations in the N-terminal portion of the enzyme were associated with up to threefold-increased resistance to trimethoprim (IC50 of up to 13.7 μM). It is postulated that carriage of the mutation M53-I—which, like I100-L, corresponds to a trimethoprim binding site in the Escherichia coli DHFR—is responsible for this increase. This study demonstrates that although the I100-L mutation alone may give rise to trimethoprim resistance, additional mutations serve to enhance resistance and modulate the effects of existing mutations on the affinity of DHFR for its natural substrates. PMID:11257022

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

    PubMed

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

    2016-07-01

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

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

    PubMed

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

    2015-12-04

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

  12. An Electron-bifurcating Caffeyl-CoA Reductase*

    PubMed Central

    Bertsch, Johannes; Parthasarathy, Anutthaman; Buckel, Wolfgang; Müller, Volker

    2013-01-01

    A low potential electron carrier ferredoxin (E0′ ≈ −500 mV) is used to fuel the only bioenergetic coupling site, a sodium-motive ferredoxin:NAD+ oxidoreductase (Rnf) in the acetogenic bacterium Acetobacterium woodii. Because ferredoxin reduction with physiological electron donors is highly endergonic, it must be coupled to an exergonic reaction. One candidate is NADH-dependent caffeyl-CoA reduction. We have purified a complex from A. woodii that contains a caffeyl-CoA reductase and an electron transfer flavoprotein. The enzyme contains three subunits encoded by the carCDE genes and is predicted to have, in addition to FAD, two [4Fe-4S] clusters as cofactor, which is consistent with the experimental determination of 4 mol of FAD, 9 mol of iron, and 9 mol of acid-labile sulfur. The enzyme complex catalyzed caffeyl-CoA-dependent oxidation of reduced methyl viologen. With NADH as donor, it catalyzed caffeyl-CoA reduction, but this reaction was highly stimulated by the addition of ferredoxin. Spectroscopic analyses revealed that ferredoxin and caffeyl-CoA were reduced simultaneously, and a stoichiometry of 1.3:1 was determined. Apparently, the caffeyl-CoA reductase-Etf complex of A. woodii uses the novel mechanism of flavin-dependent electron bifurcation to drive the endergonic ferredoxin reduction with NADH as reductant by coupling it to the exergonic NADH-dependent reduction of caffeyl-CoA. PMID:23479729

  13. Prostaglandin reductase-3 negatively modulates adipogenesis through regulation of PPARγ activity[S

    PubMed Central

    Yu, Yu-Hsiang; Chang, Yi-Cheng; Su, Tseng-Hsiung; Nong, Jiun-Yi; Li, Chao-Chin; Chuang, Lee-Ming

    2013-01-01

    Adipocyte differentiation is a multistep program under regulation by several factors. Peroxisome proliferator-activated receptor γ (PPARγ) serves as a master regulator of adipogenesis. However, the endogenous ligand for PPARγ remained elusive until 15-keto-PGE2 was identified recently as an endogenous PPARγ ligand. In this study, we demonstrate that zinc-containing alcohol dehydrogenase 2 (ZADH2; here termed prostaglandin reductase-3, PTGR-3) is a new member of prostaglandin reductase family that converts 15-keto-PGE2 to 13,14-dihydro-15-keto-PGE2. Adipogenesis is accelerated when endogenous PTGR-3 is silenced in 3T3-L1 preadipocytes, whereas forced expression of PTGR-3 significantly decreases adipogenesis. PTGR-3 expression decreased during adipocyte differentiation, accompanied by an increased level of 15-keto-PGE2. 15-keto-PGE2 exerts a potent proadipogenic effect by enhancing PPARγ activity, whereas overexpression of PTGR-3 in 3T3-L1 preadipocytes markedly suppressed the proadipogenic effect of 15-keto-PGE2 by repressing PPARγ activity. Taken together, these findings demonstrate for the first time that PTGR-3 is a novel 15-oxoprostaglandin-Δ13-reductase and plays a critical role in modulation of normal adipocyte differentiation via regulation of PPARγ activity. Thus, modulation of PTGR-3 might provide a novel avenue for treating obesity and related metabolic disorders. PMID:23821743

  14. Metabolism of the Synthetic Progestogen Norethynodrel by Human Ketosteroid Reductases of the Aldo-Keto Reductase Superfamily

    PubMed Central

    Jin, Yi; Duan, Ling; Chen, Mo; Penning, Trevor M; Kloosterboer, Helenius J.

    2012-01-01

    Human ketosteroid reductases of the aldo-keto reductase (AKR) superfamily, i.e. AKR1C1-4, are implicated in the biotransformation of synthetic steroid hormones. Norethynodrel (NOR, 17α-ethynyl-17β-hydroxy-estra-5(10)-en-3-one), the progestin component of the first marketed oral contraceptive, is known to undergo rapid and extensive metabolism to 3α- and 3β-hydroxy metabolites. The ability of the four human AKR1C enzymes to catalyze the metabolism of NOR has now been characterized. AKR1C1 and AKR1C2 almost exclusively converted NOR to 3β-hydroxy NOR, while AKR1C3 gave 3β-hydroxy NOR as the main product and AKR1C4 predominantly formed 3α-hydroxy NOR. Individual AKR1C enzymes also displayed distinct kinetic properties in the reaction of NOR. In contrast, norethindrone (NET), the Δ4-isomer of NOR and the most commonly used synthetic progestin, was not a substrate for the AKR1C enzymes. NOR is also structurally identical to the hormone replacement therapeutic tibolone (TIB), except TIB has a methyl group at the 7α-position. Product profiles and kinetic parameters for the reduction of NOR catalyzed by each individual AKR1C isoform were identical to those for the reduction of TIB catalyzed by the respective isoform. These data suggest that the presence of the 7α-methyl group has a minimal effect on the stereochemical outcome of the reaction and kinetic behavior of each enzyme. Results indicate a role of AKR1C in the hepatic and peripheral metabolism of NOR to 3α- and 3β-hydroxy NOR and provide insights into the differential pharmacological properties of NOR, NET and TIB. PMID:22210085

  15. Identification of Ser-543 as the major regulatory phosphorylation site in spinach leaf nitrate reductase

    NASA Technical Reports Server (NTRS)

    Bachmann, M.; Shiraishi, N.; Campbell, W. H.; Yoo, B. C.; Harmon, A. C.; Huber, S. C.; Davies, E. (Principal Investigator)

    1996-01-01

    Spinach leaf NADH:nitrate reductase (NR) responds to light/dark signals and photosynthetic activity in part as a result of rapid regulation by reversible protein phosphorylation. We have identified the major regulatory phosphorylation site as Ser-543, which is located in the hinge 1 region connecting the cytochrome b domain with the molybdenum-pterin cofactor binding domain of NR, using recombinant NR fragments containing or lacking the phosphorylation site sequence. Studies with NR partial reactions indicated that the block in electron flow caused by phosphorylation also could be localized to the hinge 1 region. A synthetic peptide (NR6) based on the phosphorylation site sequence was phosphorylated readily by NR kinase (NRk) in vitro. NR6 kinase activity tracked the ATP-dependent inactivation of NR during several chromatographic steps and completely inhibited inactivation/phosphorylation of native NR in vitro. Two forms of NRk were resolved by using anion exchange chromatography. Studies with synthetic peptide analogs indicated that both forms of NRk had similar specificity determinants, requiring a basic residue at P-3 (i.e., three amino acids N-terminal to the phosphorylated serine) and a hydrophobic residue at P-5. Both forms are strictly calcium dependent but belong to distinct families of protein kinases because they are distinct immunochemically.

  16. Polymorphisms in the methylene tetrahydrofolate reductase and methionine synthase reductase genes and their correlation with unexplained recurrent spontaneous abortion susceptibility.

    PubMed

    Zhu, L

    2015-07-28

    We aimed to explore the correlation between unexplained recurrent spontaneous abortion and polymorphisms in the methylene tetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR) genes. A case control study was conducted in 118 patients with unexplained recurrent spontaneous abortion (abortion group) and 174 healthy women (control group). The genetic material was extracted from the oral mucosal epithelial cells obtained from all subjects. The samples were subjected to fluorescence quantitative PCR to detect the single nucleotide polymorphisms (SNPs) in the MTHFR (C677T and A1298C) and MTRR (A66G) gene loci. The distribution frequency (18/118, 15.3%) of the MTHFR 677TT genotype was significantly higher in the abortion group (χ2 = 11.006, P = 0.004) than in the control group (2/174, 1.1%); on the other hand, the distribution frequency of the MTHFR A1298C genotype did not significantly differ between the abortion and control groups (χ(2) = 0.441, P = 0.507). The distribution frequency of the MTRR A66G genotype was also significantly higher in the abortion group (14/118, 11.9%; χ(2) = 10.503, P = 0.005) than in the control group (8/174, 4.6%). The MTHFR C677T and MTRR A66G polymorphisms are significantly correlated with the occurrence of spontaneous abortion.

  17. Crystallization of mitochondrial rhodoquinol-fumarate reductase from the parasitic nematode Ascaris suum with the specific inhibitor flutolanil

    PubMed Central

    Osanai, Arihiro; Harada, Shigeharu; Sakamoto, Kimitoshi; Shimizu, Hironari; Inaoka, Daniel Ken; Kita, Kiyoshi

    2009-01-01

    In adult Ascaris suum (roundworm) mitochondrial membrane-bound complex II acts as a rhodoquinol-fumarate reductase, which is the reverse reaction to that of mammalian complex II (succinate-ubiquinone reductase). The adult A. suum rhodoquinol-fumarate reductase was crystallized in the presence of octaethyleneglycol monododecyl ether and n-dodecyl-β-d-maltopyranoside in a 3:2 weight ratio. The crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 123.75, b = 129.08, c = 221.12 Å, and diffracted to 2.8 Å resolution using synchrotron radiation. The presence of two molecules in the asymmetric unit (120 kDa × 2) gives a crystal volume per protein mass (V M) of 3.6 Å3 Da−1. PMID:19724139

  18. Down-regulation of flavin reductase and alcohol dehydrogenase-1 (ADH1) in metronidazole-resistant isolates of Trichomonas vaginalis

    PubMed Central

    Leitsch, David; Drinić, Mirjana; Kolarich, Daniel; Duchêne, Michael

    2012-01-01

    The microaerophilic parasite Trichomonas vaginalis is a causative agent of painful vaginitis or urethritis, termed trichomoniasis, and can also cause preterm delivery or stillbirth. Treatment of trichomoniasis is almost exclusively based on the nitroimidazole drugs metronidazole and tinidazole. Metronidazole resistance in T. vaginalis does occur and is often associated with treatment failure. In most cases, metronidazole-resistant isolates remain susceptible to tinidazole, but cross resistance between the two closely related drugs can be a problem. In this study we measured activities of thioredoxin reductase and flavin reductase in four metronidazole-susceptible and five metronidazole-resistant isolates. These enzyme activities had been previously found to be downregulated in T. vaginalis with high-level metronidazole resistance induced in the laboratory. Further, we aimed at identifying factors causing metronidazole resistance and compared the protein expression profiles of all nine isolates by application of two-dimensional gel electrophoresis (2DE). Thioredoxin reductase activity was nearly equal in all strains assayed but flavin reductase activity was clearly down-regulated, or even absent, in metronidazole-resistant strains. Since flavin reductase has been shown to reduce oxygen to hydrogen peroxide, its down-regulation could significantly contribute to the impairment of oxygen scavenging as reported by others for metronidazole-resistant strains. Analysis by 2DE revealed down-regulation of alcohol dehydrogenase 1 (ADH1) in strains with reduced sensitivity to metronidazole, an enzyme that could be involved in detoxification of intracellular acetaldehyde. PMID:22449940

  19. Two Tropinone Reductases with Distinct Stereospecificities from Cultured Roots of Hyoscyamus niger1

    PubMed Central

    Hashimoto, Takashi; Nakajima, Keiji; Ongena, Godelieve; Yamada, Yasuyuki

    1992-01-01

    Tropinone is an alkamine intermediate at the branch point of biosynthetic pathways leading to various tropane alkaloids. Two stereospecifically distinct NADPH-dependent oxidoreductases, TR-I and TR-II, which, respectively, reduce tropinone to 3α-hydroxytropane (tropine) and 3β-hydroxytropane (ψ-tropine), were detected mainly in the root of tropane alkaloid-producing plants but not in nonproducing cultured root. Both reductases were purified to near homogeneity from cultured root of Hyoscyamus niger and characterized. The TR-I reaction was reversible, whereas the TR-II reaction was essentially irreversible, reduction of the ketone being highly favored over oxidation of the alcohol ψ-tropine. Marked differences were found between the two reductase in their affinities for tropinone substrate and in the effects of amino acid modification reagents. Some differences in substrate specificity were apparent. For example, N-propyl-4-piperidone was reduced by TR-II but not by TR-I. Conversely, 3-quinuclidinone and 8-thiabicyclo[3,2,1]octane-3-one were accepted as substrates by TR-I but hardly at all by TR-II. Both enzymes were shown to be class B oxidoreductases, which transfer the pro-S hydrogen of NAD(P)H to their substrates. Possible roles of these tropinone reductases in alkaloid biosynthesis are discussed. Images Figure 6 PMID:16653065

  20. Cloning, Expression, and Purification of Histidine-Tagged Escherichia coli Dihydrodipicolinate Reductase.

    PubMed

    Trigoso, Yvonne D; Evans, Russell C; Karsten, William E; Chooback, Lilian

    2016-01-01

    The enzyme dihydrodipicolinate reductase (DHDPR) is a component of the lysine biosynthetic pathway in bacteria and higher plants. DHDPR catalyzes the NAD(P)H dependent reduction of 2,3-dihydrodipicolinate to the cyclic imine L-2,3,4,5,-tetrahydropicolinic acid. The dapB gene that encodes dihydrodipicolinate reductase has previously been cloned, but the expression of the enzyme is low and the purification is time consuming. Therefore the E. coli dapB gene was cloned into the pET16b vector to improve the protein expression and simplify the purification. The dapB gene sequence was utilized to design forward and reverse oligonucleotide primers that were used to PCR the gene from Escherichia coli genomic DNA. The primers were designed with NdeI or BamHI restriction sites on the 5'and 3' terminus respectively. The PCR product was sequenced to confirm the identity of dapB. The gene was cloned into the expression vector pET16b through NdeI and BamHI restriction endonuclease sites. The resulting plasmid containing dapB was transformed into the bacterial strain BL21 (DE3). The transformed cells were utilized to grow and express the histidine-tagged reductase and the protein was purified using Ni-NTA affinity chromatography. SDS/PAGE gel analysis has shown that the protein was 95% pure and has approximate subunit molecular weight of 28 kDa. The protein purification is completed in one day and 3 liters of culture produced approximately 40-50 mgs of protein, an improvement on the previous protein expression and multistep purification.

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

    PubMed

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

    2003-03-01

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

  2. Staying green postharvest: how three mutations in the Arabidopsis chlorophyll b reductase gene NYC1 delay degreening by distinct mechanisms.

    PubMed

    Jibran, Rubina; Sullivan, Kerry L; Crowhurst, Ross; Erridge, Zoe A; Chagné, David; McLachlan, Andrew R G; Brummell, David A; Dijkwel, Paul P; Hunter, Donald A

    2015-11-01

    Stresses such as energy deprivation, wounding and water-supply disruption often contribute to rapid deterioration of harvested tissues. To uncover the genetic regulation behind such stresses, a simple assessment system was used to detect senescence mutants in conjunction with two rapid mapping techniques to identify the causal mutations. To demonstrate the power of this approach, immature inflorescences of Arabidopsis plants that contained ethyl methanesulfonate-induced lesions were detached and screened for altered timing of dark-induced senescence. Numerous mutant lines displaying accelerated or delayed timing of senescence relative to wild type were discovered. The underlying mutations in three of these were identified using High Resolution Melting analysis to map to a chromosomal arm followed by a whole-genome sequencing-based mapping method, termed 'Needle in the K-Stack', to identify the causal lesions. All three mutations were single base pair changes and occurred in the same gene, NON-YELLOW COLORING1 (NYC1), a chlorophyll b reductase of the short-chain dehydrogenase/reductase (SDR) superfamily. This was consistent with the mutants preferentially retaining chlorophyll b, although substantial amounts of chlorophyll b were still lost. The single base pair mutations disrupted NYC1 function by three distinct mechanisms, one by producing a termination codon, the second by interfering with correct intron splicing and the third by replacing a highly conserved proline with a non-equivalent serine residue. This non-synonymous amino acid change, which occurred in the NADPH binding domain of NYC1, is the first example of such a mutation in an SDR protein inhibiting a physiological response in plants. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  3. Osteogenic cell differentiation on H-terminated and O-terminated nanocrystalline diamond films

    PubMed Central

    Liskova, Jana; Babchenko, Oleg; Varga, Marian; Kromka, Alexander; Hadraba, Daniel; Svindrych, Zdenek; Burdikova, Zuzana; Bacakova, Lucie

    2015-01-01

    Nanocrystalline diamond (NCD) films are promising materials for bone implant coatings because of their biocompatibility, chemical resistance, and mechanical hardness. Moreover, NCD wettability can be tailored by grafting specific atoms. The NCD films used in this study were grown on silicon substrates by microwave plasma-enhanced chemical vapor deposition and grafted by hydrogen atoms (H-termination) or oxygen atoms (O-termination). Human osteoblast-like Saos-2 cells were used for biological studies on H-terminated and O-terminated NCD films. The adhesion, growth, and subsequent differentiation of the osteoblasts on NCD films were examined, and the extracellular matrix production and composition were quantified. The osteoblasts that had been cultivated on the O-terminated NCD films exhibited a higher growth rate than those grown on the H-terminated NCD films. The mature collagen fibers were detected in Saos-2 cells on both the H-terminated and O-terminated NCD films; however, the quantity of total collagen in the extracellular matrix was higher on the O-terminated NCD films, as were the amounts of calcium deposition and alkaline phosphatase activity. Nevertheless, the expression of genes for osteogenic markers – type I collagen, alkaline phosphatase, and osteocalcin – was either comparable on the H-terminated and O-terminated films or even lower on the O-terminated films. In conclusion, the higher wettability of the O-terminated NCD films is promising for adhesion and growth of osteoblasts. In addition, the O-terminated surface also seems to support the deposition of extracellular matrix proteins and extracellular matrix mineralization, and this is promising for better osteoconductivity of potential bone implant coatings. PMID:25670900

  4. Carboxylic acid reductase enzymes (CARs).

    PubMed

    Winkler, Margit

    2018-04-01

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

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Forlani, Giuseppe; Nocek, Boguslaw; Chakravarthy, Srinivas

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

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Forlani, Giuseppe; Nocek, Boguslaw; Chakravarthy, Srinivas

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

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

    PubMed Central

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

    2015-01-01

    Isoflavone reductase (IFR) is an enzyme involved in the biosynthetic pathway of isoflavonoid phytoalexin in plants. IFRs are unique to the plant kingdom and are considered to have crucial roles in plant response to various biotic and abiotic environmental stresses. Here, we report the characterization of a novel member of the soybean isoflavone reductase gene family GmIFR. Overexpression of GmIFR transgenic soybean exhibited enhanced resistance to Phytophthora sojae. Following stress treatments, GmIFR was significantly induced by P. sojae, ethephon (ET), abscisic acid (placeCityABA), salicylic acid (SA). It is located in the cytoplasm when transiently expressed in soybean protoplasts. The daidzein levels reduced greatly for the seeds of transgenic plants, while the relative content of glyceollins in transgenic plants was significantly higher than that of non-transgenic plants. Furthermore, we found that the relative expression levels of reactive oxygen species (ROS) of transgenic soybean plants were significantly lower than those of non-transgenic plants after incubation with P. sojae, suggesting an important role of GmIFR might function as an antioxidant to reduce ROS in soybean. The enzyme activity assay suggested that GmIFR has isoflavone reductase activity. PMID:26635848

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

    PubMed

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

    2015-01-01

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

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

    PubMed

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

    2015-12-01

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

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

    PubMed

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

    2004-05-04

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

  11. Regulation of nitrate reductase by nitric oxide in Chinese cabbage pakchoi (Brassica chinensis L.).

    PubMed

    Du, Shaoting; Zhang, Yongsong; Lin, Xianyong; Wang, Yue; Tang, Caixian

    2008-02-01

    Nitrate reductase (NR), a committed enzyme in nitrate assimilation, involves generation of nitric oxide (NO) in plants. Here we show that the NR activity was significantly enhanced by the addition of NO donors sodium nitroprusside (SNP) and NONOate (diethylamine NONOate sodium) to the culturing solution, whereas it was decreased by NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (cPTIO). Interestingly, both NO gas and SNP directly enhanced but cPTIO inhibited the NR activities of crude enzyme extracts and purified NR enzyme. The cPTIO terminated the interaction between NR-generated NO and the NR itself. Furthermore, the NR protein content was not affected by the SNP treatment. The investigation of the partial reactions catalysed by purified NR using various electron donors and acceptors indicated that the haem and molybdenum centres in NR were the two sites activated by NO. The results suggest that the activation of NR activity by NO is regulated at the post-translational level, probably via a direct interaction mechanism. Accordingly, the concentration of nitrate both in leaves and roots was decreased after 2 weeks of cultivation with SNP. The present study identifies a new mechanism of NR regulation and nitrate assimilation, which provides important new insights into the complex regulation of N-metabolism in plants.

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

    PubMed Central

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

    2015-01-01

    Pickles are popular in China and exhibits health-promoting effects. However, nitrite produced during fermentation adversely affects health due to formation of methemoglobin and conversion to carcinogenic nitrosamine. Fruiting bodies of the mushroom Boletus edulis were capable of inhibiting nitrite production during pickle fermentation. A 90-kDa nitrite reductase (NiR), demonstrating peptide sequence homology to fungal nitrite reductase, was isolated from B. edulis fruiting bodies. The optimum temperature and pH of the enzyme was 45 °C and 6.8, respectively. B. edulis NiR was capable of prolonging the lifespan of nitrite-intoxicated mice, indicating that it had the action of an antidote. The enzyme could also eliminate nitrite from blood after intragastric administration of sodium nitrite, and after packaging into capsule, this nitrite-eliminating activity could persist for at least 120 minutes thus avoiding immediate gastric degradation. B. edulis NiR represents the first nitrite reductase purified from mushrooms and may facilitate subsequent applications. PMID:26446494

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

    PubMed

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

    2015-10-08

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

  14. Streptococcus sanguinis Class Ib Ribonucleotide Reductase

    PubMed Central

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

    2014-01-01

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

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

    PubMed

    Baskaran, Gunasekaran; Salvamani, Shamala; Ahmad, Siti Aqlima; Shaharuddin, Noor Azmi; Pattiram, Parveen Devi; Shukor, Mohd Yunus

    2015-01-01

    The enzyme 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase is the key enzyme of the mevalonate pathway that produces cholesterol. Inhibition of HMG-CoA reductase reduces cholesterol biosynthesis in the liver. Synthetic drugs, statins, are commonly used for the treatment of hypercholesterolemia. Due to the side effects of statins, natural HMG-CoA reductase inhibitors of plant origin are needed. In this study, 25 medicinal plant methanol extracts were screened for anti-HMG-CoA reductase activity. Basella alba leaf extract showed the highest inhibitory effect at about 74%. Thus, B. alba was examined in order to investigate its phytochemical components. Gas chromatography with tandem mass spectrometry and reversed phase high-performance liquid chromatography analysis revealed the presence of phenol 2,6-bis(1,1-dimethylethyl), 1-heptatriacotanol, oleic acid, eicosyl ester, naringin, apigenin, luteolin, ascorbic acid, and α-tocopherol, which have been reported to possess antihypercholesterolemic effects. Further investigation of in vivo models should be performed in order to confirm its potential as an alternative treatment for hypercholesterolemia and related cardiovascular diseases.

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

    PubMed Central

    Baskaran, Gunasekaran; Salvamani, Shamala; Ahmad, Siti Aqlima; Shaharuddin, Noor Azmi; Pattiram, Parveen Devi; Shukor, Mohd Yunus

    2015-01-01

    The enzyme 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase is the key enzyme of the mevalonate pathway that produces cholesterol. Inhibition of HMG-CoA reductase reduces cholesterol biosynthesis in the liver. Synthetic drugs, statins, are commonly used for the treatment of hypercholesterolemia. Due to the side effects of statins, natural HMG-CoA reductase inhibitors of plant origin are needed. In this study, 25 medicinal plant methanol extracts were screened for anti-HMG-CoA reductase activity. Basella alba leaf extract showed the highest inhibitory effect at about 74%. Thus, B. alba was examined in order to investigate its phytochemical components. Gas chromatography with tandem mass spectrometry and reversed phase high-performance liquid chromatography analysis revealed the presence of phenol 2,6-bis(1,1-dimethylethyl), 1-heptatriacotanol, oleic acid, eicosyl ester, naringin, apigenin, luteolin, ascorbic acid, and α-tocopherol, which have been reported to possess antihypercholesterolemic effects. Further investigation of in vivo models should be performed in order to confirm its potential as an alternative treatment for hypercholesterolemia and related cardiovascular diseases. PMID:25609924

  17. Differential Effect of Irradiance and Nutrient Nitrate on the Relationship of in Vivo and in Vitro Nitrate Reductase Assay in Chlorophyllous Tissues 1

    PubMed Central

    Jones, Richard Wyn; Sheard, Robert W.

    1977-01-01

    Growth at increasing continuous irradiance (at high nutrient nitrate) and nutrient nitrate concentrations (at high continuous irradiance) furnished increases in the in vivo and in vitro nitrate reductase activities of corn (Zea mays L.), field peas (Pisum arvense L.), wheat (Triticum aestivum L.), barley (Hordeum vulgare L.), and globe amaranth (Gomphrena globosa L.) leaves and of marrow (Cucurbita pepo L.) cotyledons. Ratios of in vivo to in vitro activity declined exponentially in all species with increasing nitrate reductase levels promoted by nutrient nitrate. The ratios were more nearly independent of nitrate reductase levels generated by adjusting the irradiance; major exceptions were marrow and wheat at low (1.5 klux and less) irradiances and peas throughout the irradiance range, where decreases in the ratio were accompanied by increases in in situ nitrate concentration. The ratio also increased at the highest irradiance (39.2 klux) in wheat and barley, associated with a decline of in vitro nitrate reductase. These differences in response to irradiance and nutrient nitrate indicate that the in vivo assay does not provide a simple measure of nitrate reductase but rather yields a more composite measure of nitrate reduction, possibly related both to nitrate reductase level and to the supply of reductant for in vivo activity. PMID:16659888

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

    PubMed

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

    2010-01-15

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

  19. The ferredoxin-thioredoxin reductase variable subunit gene from Anacystis nidulans.

    PubMed

    Szekeres, M; Droux, M; Buchanan, B B

    1991-03-01

    The ferredoxin-thioredoxin reductase variable subunit gene of Anacystis nidulans was cloned, and its nucleotide sequence was determined. A single-copy 219-bp open reading frame encoded a protein of 73 amino acid residues, with a calculated Mr of 8,400. The monocistronic transcripts were represented in a 400-base and a less abundant 300-base mRNA form.

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

    PubMed Central

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

    2011-01-01

    Babesiosis is a tick-borne disease caused by eukaryotic Babesia parasites which are morphologically similar to Plasmodium falciparum, the causative agent of malaria in humans. Like Plasmodium, different species of Babesia are tuned to infect different mammalian hosts, including rats, dogs, horses and cattle. Most species of Plasmodium and Babesia possess an essential bifunctional enzyme for nucleotide synthesis and folate metabolism: dihydrofolate reductase-thymidylate synthase. Although thymidylate synthase is highly conserved across organisms, the bifunctional form of this enzyme is relatively uncommon in nature. The structural characterization of dihydrofolate reductase-thymidylate synthase in Babesia bovis, the causative agent of babesiosis in livestock cattle, is reported here. The apo state is compared with structures that contain dUMP, NADP and two different antifolate inhibitors: pemetrexed and raltitrexed. The complexes reveal modes of binding similar to that seen in drug-resistant malaria strains and point to the utility of applying structural studies with proven cancer chemotherapies towards infectious disease research. PMID:21904052

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

    PubMed

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

    2009-09-01

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

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

    PubMed

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

    1999-04-01

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

  3. Homologous expression of cytosolic dehydroascorbate reductase increases grain yield and biomass under paddy field conditions in transgenic rice (Oryza sativa L. japonica).

    PubMed

    Kim, Young-Saeng; Kim, Il-Sup; Bae, Mi-Jung; Choe, Yong-Hoe; Kim, Yul-Ho; Park, Hyang-Mi; Kang, Hong-Gyu; Yoon, Ho-Sung

    2013-06-01

    Dehydroascorbate reductase (DHAR, EC 1.8.5.1) maintains redox pools of ascorbate (AsA) by recycling oxidized AsA to reduced AsA. To investigate whether DHAR affects rice yield under normal environmental conditions, cDNA-encoding DHAR (OsDHAR1) was isolated from rice and used to develop OsDHAR1-overexpressing transgenic rice plants, under the regulation of a maize ubiquitin promoter. Incorporation and expression of the transgene in transgenic rice plants was confirmed by genomic polymerase chain reaction (PCR), semi-quantitative reverse transcription PCR (RT-PCR), western blot, and enzyme activity. The expression levels were at least twofold higher in transgenic (TG) rice plants than in control wild-type (WT) rice plants. In addition, OsDHAR1-overexpression in seven-independent homologous transgenic plants, as compared to WT plants, increased photosynthetic capacity and antioxidant enzyme activities under paddy field conditions, which led to an improved AsA pool and redox homeostasis. Furthermore, OsDHAR1 overexpression significantly improved grain yield and biomass due to the increase of culm and root weights and to enhance panicle and spikelet numbers in the same seven independent TG rice plants during the farming season (2010 and 2011) in South Korea. The OsDHAR protein contained the redox-active site (Cys20), as well as the conserved GSH-binding region, GSH-binding motif, glutathione-S-transferase (GST) N-terminal domain, C-terminal domain interface, and GST C-terminal domain. Therefore, our results indicate that OsDHAR1 overexpression, capable of functioning in AsA recycling, and protein folding increases environmental adaptation to paddy field conditions by the improving AsA pool and redox homeostasis, which enhances rice grain yield and biomass.

  4. Superconducting Cable Termination

    DOEpatents

    Sinha, Uday K.; Tolbert, Jerry

    2005-08-30

    Disclosed is a termination that connects high temperature superconducting (HTS) cable immersed in pressurized liquid nitrogen to high voltage and neutral (shield) external bushings at ambient temperature and pressure. The termination consists of a splice between the HTS power (inner) and shield (outer) conductors and concentric copper pipes which are the conductors in the termination. There is also a transition from the dielectric tape insulator used in the HTS cable to the insulators used between and around the copper pipe conductors in the termination. At the warm end of the termination the copper pipes are connected via copper braided straps to the conventional warm external bushings which have low thermal stresses. This termination allows for a natural temperature gradient in the copper pipe conductors inside the termination which enables the controlled flashing of the pressurized liquid coolant (nitrogen) to the gaseous state. Thus the entire termination is near the coolant supply pressure and the high voltage and shield cold bushings, a highly stressed component used in most HTS cables, are eliminated. A sliding seal allows for cable contraction as it is cooled from room temperature to ˜72-82 K. Seals, static vacuum, and multi-layer superinsulation minimize radial heat leak to the environment.

  5. 1,8-Dihydroxynaphthalene (DHN)-Melanin Biosynthesis Inhibitors Increase Erythritol Production in Torula corallina, and DHN-Melanin Inhibits Erythrose Reductase

    PubMed Central

    Lee, Jung-Kul; Jung, Hyung-Moo; Kim, Sang-Yong

    2003-01-01

    The yeast Torula corallina is a strong erythritol producer that is used in the industrial production of erythritol. However, melanin accumulation during culture represents a serious problem for the purification of erythritol from the fermentation broth. Melanin biosynthesis inhibitors such as 3,4-dihydroxyphenylalanine and 1,8-dihydroxynaphthalene (DHN)-melanin inhibitors were added to the T. corallina cultures. Only the DHN-melanin inhibitors showed an effect on melanin production, which suggests that the melanin formed during the culturing of T. corallina is derived from DHN. This finding was confirmed by the detection of a shunt product of the pentaketide pathway, flaviolin, and elemental analysis. Among the DHN-melanin inhibitors, tricyclazole was the most effective. Supplementation with tricyclazole enhanced the production of erythritol while significantly inhibiting the production of DHN-melanin and DHN-melanin biosynthetic enzymes, such as trihydroxynaphthalene reductase. The erythrose reductase from T. corallina was purified to homogeneity by ion-exchange and affinity chromatography. Purified erythrose reductase was significantly inhibited in vitro in a noncompetitive manner by elevated levels of DHN-melanin. In contrast, the level of erythrose reductase activity was unaffected by increasing concentrations of tricyclazole. These results suggest that supplemental tricyclazole reduces the production of DHN-melanin, which may lead to a reduction in the inhibition of erythrose reductase and a higher yield of erythritol. This is the first report to demonstrate that melanin biosynthesis inhibitors increase the production of a sugar alcohol in T. corallina. PMID:12788746

  6. The NosX and NirX Proteins of Paracoccus denitrificans Are Functional Homologues: Their Role in Maturation of Nitrous Oxide Reductase

    PubMed Central

    Saunders, Neil F. W.; Hornberg, Jorrit J.; Reijnders, Willem N. M.; Westerhoff, Hans V.; de Vries, Simon; van Spanning, Rob J. M.

    2000-01-01

    The nos (nitrous oxide reductase) operon of Paracoccus denitrificans contains a nosX gene homologous to those found in the nos operons of other denitrifiers. NosX is also homologous to NirX, which is so far unique to P. denitrificans. Single mutations of these genes did not result in any apparent phenotype, but a double nosX nirX mutant was unable to reduce nitrous oxide. Promoter-lacZ assays and immunoblotting against nitrous oxide reductase showed that the defect was not due to failure of expression of nosZ, the structural gene for nitrous oxide reductase. Electron paramagnetic resonance spectroscopy showed that nitrous oxide reductase in cells of the double mutant lacked the CuA center. A twin-arginine motif in both NosX and NirX suggests that the NosX proteins are exported to the periplasm via the TAT translocon. PMID:10960107

  7. Uptake and metabolism of fructose by rat neocortical cells in vivo and by isolated nerve terminals in vitro.

    PubMed

    Hassel, Bjørnar; Elsais, Ahmed; Frøland, Anne-Sofie; Taubøll, Erik; Gjerstad, Leif; Quan, Yi; Dingledine, Raymond; Rise, Frode

    2015-05-01

    Fructose reacts spontaneously with proteins in the brain to form advanced glycation end products (AGE) that may elicit neuroinflammation and cause brain pathology, including Alzheimer's disease. We investigated whether fructose is eliminated by oxidative metabolism in neocortex. Injection of [(14) C]fructose or its AGE-prone metabolite [(14) C]glyceraldehyde into rat neocortex in vivo led to formation of (14) C-labeled alanine, glutamate, aspartate, GABA, and glutamine. In isolated neocortical nerve terminals, [(14) C]fructose-labeled glutamate, GABA, and aspartate, indicating uptake of fructose into nerve terminals and oxidative fructose metabolism in these structures. This was supported by high expression of hexokinase 1, which channels fructose into glycolysis, and whose activity was similar with fructose or glucose as substrates. By contrast, the fructose-specific ketohexokinase was weakly expressed. The fructose transporter Glut5 was expressed at only 4% of the level of neuronal glucose transporter Glut3, suggesting transport across plasma membranes of brain cells as the limiting factor in removal of extracellular fructose. The genes encoding aldose reductase and sorbitol dehydrogenase, enzymes of the polyol pathway that forms glucose from fructose, were expressed in rat neocortex. These results point to fructose being transported into neocortical cells, including nerve terminals, and that it is metabolized and thereby detoxified primarily through hexokinase activity. We asked how the brain handles fructose, which may react spontaneously with proteins to form 'advanced glycation end products' and trigger inflammation. Neocortical cells took up and metabolized extracellular fructose oxidatively in vivo, and isolated nerve terminals did so in vitro. The low expression of fructose transporter Glut5 limited uptake of extracellular fructose. Hexokinase was a main pathway for fructose metabolism, but ketohexokinase (which leads to glyceraldehyde formation) was

  8. Characterization of a flavin reductase from a thermophilic dibenzothiophene-desulfurizing bacterium, Bacillus subtilis WU-S2B.

    PubMed

    Takahashi, Shusuke; Furuya, Toshiki; Ishii, Yoshitaka; Kino, Kuniki; Kirimura, Kohtaro

    2009-01-01

    Bacillus subtilis WU-S2B is a thermophilic dibenzothiophene (DBT)-desulfurizing bacterium and produces a flavin reductase (Frb) that couples with DBT and DBT sulfone monooxygenases. The recombinant Frb was purified from Escherichia coli cells expressing the frb gene and was characterized. The purified Frb exhibited high stability over wide temperature and pH ranges of 20-55 degrees C and 2-12, respectively. Frb contained FMN and exhibited both flavin reductase and nitroreductase activities.

  9. Cloning, Expression, and Purification of Histidine-Tagged Escherichia coli Dihydrodipicolinate Reductase

    PubMed Central

    Trigoso, Yvonne D.; Evans, Russell C.; Karsten, William E.; Chooback, Lilian

    2016-01-01

    The enzyme dihydrodipicolinate reductase (DHDPR) is a component of the lysine biosynthetic pathway in bacteria and higher plants. DHDPR catalyzes the NAD(P)H dependent reduction of 2,3-dihydrodipicolinate to the cyclic imine L-2,3,4,5,-tetrahydropicolinic acid. The dapB gene that encodes dihydrodipicolinate reductase has previously been cloned, but the expression of the enzyme is low and the purification is time consuming. Therefore the E. coli dapB gene was cloned into the pET16b vector to improve the protein expression and simplify the purification. The dapB gene sequence was utilized to design forward and reverse oligonucleotide primers that were used to PCR the gene from Escherichia coli genomic DNA. The primers were designed with NdeI or BamHI restriction sites on the 5’and 3’ terminus respectively. The PCR product was sequenced to confirm the identity of dapB. The gene was cloned into the expression vector pET16b through NdeI and BamHI restriction endonuclease sites. The resulting plasmid containing dapB was transformed into the bacterial strain BL21 (DE3). The transformed cells were utilized to grow and express the histidine-tagged reductase and the protein was purified using Ni-NTA affinity chromatography. SDS/PAGE gel analysis has shown that the protein was 95% pure and has approximate subunit molecular weight of 28 kDa. The protein purification is completed in one day and 3 liters of culture produced approximately 40–50 mgs of protein, an improvement on the previous protein expression and multistep purification. PMID:26815040

  10. Diagnostic value of succinate ubiquinone reductase activity in the identification of patients with mitochondrial DNA depletion.

    PubMed

    Hargreaves, P; Rahman, S; Guthrie, P; Taanman, J W; Leonard, J V; Land, J M; Heales, S J R

    2002-02-01

    Mitochondrial DNA (mtDNA) depletion syndrome (McKusick 251880) is characterized by a progressive quantitative loss of mtDNA resulting in severe mitochondrial dysfunction. A diagnosis of mtDNA depletion can only be confirmed after Southern blot analysis of affected tissue. Only a limited number of centres have the facilities to offer this service, and this is frequently on an irregular basis. There is therefore a need for a test that can refine sample selection as well as complementing the molecular analysis. In this study we compared the activities of the nuclear-encoded succinate ubiquinone reductase (complex II) to the activities of the combined mitochondrial and nuclear-encoded mitochondrial electron transport chain (ETC) complexes; NADH:ubiquinone reductase (complex I), ubiquinol-cytochrome-c reductase (complex III), and cytochrome-c oxidase (complex IV), in skeletal muscle biopsies from 7 patients with confirmed mtDNA depletion. In one patient there was no evidence of an ETC defect. However, the remaining 6 patients exhibited reduced complex I and IV activities. Five of these patients also displayed reduced complex II-III (succinate:cytochrome-c reductase) activity. Individual measurement of complex II and complex III activities demonstrated normal levels of complex II activity compared to complex III, which was reduced in the 5 biopsies assayed. These findings suggest a possible diagnostic value for the detection of normal levels of complex II activity in conjunction with reduced complex I, III and IV activity in the identification of likely candidates for mtDNA depletion syndrome

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

    PubMed

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

    2008-04-01

    Three Staphylococcus strains, S. carnosus, S. simulans and S. saprophyticus, selected due to their varying nitrite and/or nitrate-reductase activities, were used to initiate colour formation during sausage fermentation. During fermentation of sausages with either nitrite or nitrate added, colour was followed by L(∗)a(∗)b measurements and the content of nitrosylmyoglobin (MbFe(II)NO) quantified by electron spin resonance (ESR). MbFe(II)NO was rapidly formed in sausages with added nitrite independent of the presence of nitrite reducing bacteria, whereas the rate of MbFe(II)NO formation in sausages with added nitrate depended on the specific Staphylococcus strain. Strains with high nitrate-reductase activity showed a significantly faster rate of pigment formation, but other factors were of influence as well. Product stability for the sliced, packaged sausage was evaluated as surface colour and oxidation by autofluorescence and hexanal content, respectively. No significant direct effect of the Staphylococcus addition was observed, however, there was a clear correspondence between high initial amount of MbFe(II)NO in the different sausages and the colour stability during storage. Autofluorescence data correlated well with hexanal content, and may be used as predictive tools. Overall, nitrite- and nitrate-reductase activities of Staphylococcus strains in nitrite-cured sausages were of limited importance regarding colour development, while in nitrate-cured sausages strains with higher nitrate reductase activity were crucial for ensuring optimal colour formation during initial fermentation stages.

  12. Insights into Enzyme Catalysis and Thyroid Hormone Regulation of Cerebral Ketimine Reductase/μ-Crystallin Under Physiological Conditions.

    PubMed

    Hallen, André; Cooper, Arthur J L; Jamie, Joanne F; Karuso, Peter

    2015-06-01

    Mammalian ketimine reductase is identical to μ-crystallin (CRYM)-a protein that is also an important thyroid hormone binding protein. This dual functionality implies a role for thyroid hormones in ketimine reductase regulation and also a reciprocal role for enzyme catalysis in thyroid hormone bioavailability. In this research we demonstrate potent sub-nanomolar inhibition of enzyme catalysis at neutral pH by the thyroid hormones L-thyroxine and 3,5,3'-triiodothyronine, whereas other thyroid hormone analogues were shown to be far weaker inhibitors. We also investigated (a) enzyme inhibition by the substrate analogues pyrrole-2-carboxylate, 4,5-dibromopyrrole-2-carboxylate and picolinate, and (b) enzyme catalysis at neutral pH of the cyclic ketimines S-(2-aminoethyl)-L-cysteine ketimine (owing to the complex nomenclature trivial names are used for the sulfur-containing cyclic ketimines as per the original authors' descriptions) (AECK), Δ(1)-piperideine-2-carboxylate (P2C), Δ(1)-pyrroline-2-carboxylate (Pyr2C) and Δ(2)-thiazoline-2-carboxylate. Kinetic data obtained at neutral pH suggests that ketimine reductase/CRYM plays a major role as a P2C/Pyr2C reductase and that AECK is not a major substrate at this pH. Thus, ketimine reductase is a key enzyme in the pipecolate pathway, which is the main lysine degradation pathway in the brain. In silico docking of various ligands into the active site of the X-ray structure of the enzyme suggests an unusual catalytic mechanism involving an arginine residue as a proton donor. Given the critical importance of thyroid hormones in brain function this research further expands on our knowledge of the connection between amino acid metabolism and regulation of thyroid hormone levels.

  13. 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

  14. Bank Terminals

    NASA Technical Reports Server (NTRS)

    1978-01-01

    In the photo, employees of the UAB Bank, Knoxville, Tennessee, are using Teller Transaction Terminals manufactured by SCI Systems, Inc., Huntsville, Alabama, an electronics firm which has worked on a number of space projects under contract with NASA. The terminals are part of an advanced, computerized financial transaction system that offers high efficiency in bank operations. The key to the system's efficiency is a "multiplexing" technique developed for NASA's Space Shuttle. Multiplexing is simultaneous transmission of large amounts of data over a single transmission link at very high rates of speed. In the banking application, a small multiplex "data bus" interconnects all the terminals and a central computer which stores information on clients' accounts. The data bus replaces the maze-of wiring that would be needed to connect each terminal separately and it affords greater speed in recording transactions. The SCI system offers banks real-time data management through constant updating of the central computer. For example, a check is immediately cancelled at the teller's terminal and the computer is simultaneously advised of the transaction; under other methods, the check would be cancelled and the transaction recorded at the close of business. Teller checkout at the end of the day, conventionally a time-consuming matter of processing paper, can be accomplished in minutes by calling up a summary of the day's transactions. SCI manufactures other types of terminals for use in the system, such as an administrative terminal that provides an immediate printout of a client's account, and another for printing and recording savings account deposits and withdrawals. SCI systems have been installed in several banks in Tennessee, Arizona, and Oregon and additional installations are scheduled this year.

  15. Molecular rationale delineating the role of lycopene as a potent HMG-CoA reductase inhibitor: in vitro and in silico study.

    PubMed

    Alvi, Sahir Sultan; Iqbal, Danish; Ahmad, Saheem; Khan, M Salman

    2016-09-01

    This study initially aimed to depict the molecular rationale evolving the role of lycopene in inhibiting the enzymatic activity of β-hydroxy-β-methylglutaryl-CoA (HMG-CoA) reductase via in vitro and in silico analysis. Our results illustrated that lycopene exhibited strong HMG-CoA reductase inhibitory activity (IC50 value of 36 ng/ml) quite better than pravastatin (IC50 = 42 ng/ml) and strong DPPH free radical scavenging activity (IC50 value = 4.57 ± 0.23 μg/ml) as compared to ascorbic acid (IC50 value = 9.82 ± 0.42 μg/ml). Moreover, the Ki value of lycopene (36 ng/ml) depicted via Dixon plot was well concurred with an IC50 value of 36 ± 1.8 ng/ml. Moreover, molecular informatics study showed that lycopene exhibited binding energy of -5.62 kcal/mol indicating high affinity for HMG-CoA reductase than HMG-CoA (ΔG: -5.34 kcal/mol). Thus, in silico data clearly demonstrate and support the in vitro results that lycopene competitively inhibit HMG-CoA reductase activity by binding at the hydrophobic portion of HMG-CoA reductase.

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

    PubMed

    Samhan-Arias, Alejandro K; Fortalezas, Sofia; Cordas, Cristina M; Moura, Isabel; Moura, José J G; Gutierrez-Merino, Carlos

    2018-05-01

    In this work, we measured the effect of cytochrome c on the NADH-dependent superoxide anion production by synaptic plasma membrane vesicles from rat brain. In these membranes, the cytochrome c stimulated NADH-dependent superoxide anion production was inhibited by antibodies against cytochrome b 5 reductase linking the production to this enzyme. Measurement of the superoxide anion radical generated by purified recombinant soluble and membrane cytochrome b 5 reductase corroborates the production of the radical by different enzyme isoforms. In the presence of cytochrome c, a burst of superoxide anion as well as the reduction of cytochrome c by cytochrome b 5 reductase was measured. Complex formation between both proteins suggests that cytochrome b 5 reductase is one of the major partners of cytochrome c upon its release from mitochondria to the cytosol during apoptosis. Superoxide anion production and cytochrome c reduction are the consequences of the stimulated NADH consumption by cytochrome b 5 reductase upon complex formation with cytochrome c and suggest a major role of this enzyme as an anti-apoptotic protein during cell death. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  17. Sulfur-Mediated Electron Shuttling Sustains Microbial Long-Distance Extracellular Electron Transfer with the Aid of Metallic Iron Sulfides.

    PubMed

    Kondo, Katsuhito; Okamoto, Akihiro; Hashimoto, Kazuhito; Nakamura, Ryuhei

    2015-07-07

    In addition to serving as an energy source for microbial growth, iron sulfides are proposed to act as naturally occurring electrical wires that mediate long-distance extracellular electron transfer (EET) and bridge spatially discrete redox environments. These hypothetical EET reactions stand on the abilities of microbes to use the interfacial electrochemistry of metallic/semiconductive iron sulfides to maintain metabolisms; however, the mechanisms of these phenomena remain unexplored. To obtain insight into EET to iron sulfides, we monitored EET at the interface between Shewanella oneidensis MR-1 cells and biomineralized iron sulfides in an electrochemical cell. Respiratory current steeply increased with the concomitant formation of poorly crystalline mackinawite (FeS) minerals, indicating that S. oneidensis has the ability to exploit extracellularly formed metallic FeS for long-distance EET. Deletion of major proteins of the metal-reduction (Mtr) pathway (OmcA, MtrC, CymA, and PilD) caused only subtle effects on the EET efficiency, a finding that sharply contrasts the majority of studies that report that the Mtr pathway is indispensable for the reduction of metal oxides and electrodes. The gene expression analyses of polysulfide and thiosulfate reductase suggest the existence of a sulfur-mediated electron-shuttling mechanism by which HS(-) ions and water-soluble polysulfides (HS(n)(-), where n ≥ 2) generated in the periplasmic space deliver electrons from cellular metabolic processes to cell surface-associated FeS. The finding of this Mtr-independent pathway indicates that polysulfide reductases complement the function of outer-membrane cytochromes in EET reactions and, thus, significantly expand the number of microbial species potentially capable of long-distance EET in sulfur-rich anoxic environments.

  18. Channel and terminal description of the ACTS mobile terminal

    NASA Technical Reports Server (NTRS)

    Abbe, B. S.; Agan, M. J.; Girardey, C. C.; Jedrey, T. C.

    1994-01-01

    The Advanced Communications Technology Satellite (ACTS) Mobile Terminal (AMT) is a proof-of-concept K/Ka-band mobile satellite communications terminal under development by NASA at JPL. Currently the AMT is undergoing systems integration and testing in preparation for a July 1993 ACTS launch and the subsequent commencement of mobile experiments in the fall of 1993. The AMT objectives are presented, followed by a discussion of the AMT communications channel and the mobile terminal's design and performance.

  19. Channel and terminal description of the ACTS mobile terminal

    NASA Technical Reports Server (NTRS)

    Abbe, B. S.; Agan, M. J.; Girardey, C. C.; Jedrey, T. C.

    1993-01-01

    The Advanced Communications Technology Satellite (ACTS) Mobile Terminal (AMT) is a proof-of-concept K/Ka-band mobile satellite communications terminal under development by NASA at JPL. Currently the AMT is undergoing system integration and test in preparation for a July 1993 ACTS launch and the subsequent commencement of mobile experiments in the fall of 1993. The AMT objectives are presented followed by a discussion of the AMT communications channel and mobile terminal design and performance.

  20. The ferredoxin-thioredoxin reductase variable subunit gene from Anacystis nidulans.

    PubMed Central

    Szekeres, M; Droux, M; Buchanan, B B

    1991-01-01

    The ferredoxin-thioredoxin reductase variable subunit gene of Anacystis nidulans was cloned, and its nucleotide sequence was determined. A single-copy 219-bp open reading frame encoded a protein of 73 amino acid residues, with a calculated Mr of 8,400. The monocistronic transcripts were represented in a 400-base and a less abundant 300-base mRNA form. Images PMID:1705544

  1. Identification of an anti-MRSA dihydrofolate reductase inhibitor from a diversity-oriented synthesis.

    PubMed

    Wyatt, Emma E; Galloway, Warren R J D; Thomas, Gemma L; Welch, Martin; Loiseleur, Olivier; Plowright, Alleyn T; Spring, David R

    2008-10-28

    The screening of a diversity-oriented synthesis library followed by structure-activity relationship investigations have led to the discovery of an anti-MRSA agent which operates as an inhibitor of Staphylococcus aureus dihydrofolate reductase.

  2. Overexpression of NADH-dependent fumarate reductase improves D-xylose fermentation in recombinant Saccharomyces cerevisiae.

    PubMed

    Salusjärvi, Laura; Kaunisto, Sanna; Holmström, Sami; Vehkomäki, Maija-Leena; Koivuranta, Kari; Pitkänen, Juha-Pekka; Ruohonen, Laura

    2013-12-01

    Deviation from optimal levels and ratios of redox cofactors NAD(H) and NADP(H) is common when microbes are metabolically engineered. The resulting redox imbalance often reduces the rate of substrate utilization as well as biomass and product formation. An example is the metabolism of D-xylose by recombinant Saccharomyces cerevisiae strains expressing xylose reductase and xylitol dehydrogenase encoding genes from Scheffersomyces stipitis. This pathway requires both NADPH and NAD(+). The effect of overexpressing the glycosomal NADH-dependent fumarate reductase (FRD) of Trypanosoma brucei in D-xylose-utilizing S. cerevisiae alone and together with an endogenous, cytosol directed NADH-kinase (POS5Δ17) was studied as one possible solution to overcome this imbalance. Expression of FRD and FRD + POS5Δ17 resulted in 60 and 23 % increase in ethanol yield, respectively, on D-xylose under anaerobic conditions. At the same time, xylitol yield decreased in the FRD strain suggesting an improvement in redox balance. We show that fumarate reductase of T. brucei can provide an important source of NAD(+) in yeast under anaerobic conditions, and can be useful for metabolic engineering strategies where the redox cofactors need to be balanced. The effects of FRD and NADH-kinase on aerobic and anaerobic D-xylose and D-glucose metabolism are discussed.

  3. Kinetic mechanism and quaternary structure of Aminobacter aminovorans NADH:flavin oxidoreductase: an unusual flavin reductase with bound flavin.

    PubMed

    Russell, Thomas R; Demeler, Borries; Tu, Shiao-Chun

    2004-02-17

    The homodimeric NADH:flavin oxidoreductase from Aminobacter aminovorans is an NADH-specific flavin reductase herein designated FRD(Aa). FRD(Aa) was characterized with respect to purification yields, thermal stability, isoelectric point, molar absorption coefficient, and effects of phosphate buffer strength and pH on activity. Evidence from this work favors the classification of FRD(Aa) as a flavin cofactor-utilizing class I flavin reductase. The isolated native FRD(Aa) contained about 0.5 bound riboflavin-5'-phosphate (FMN) per enzyme monomer, but one bound flavin cofactor per monomer was obtainable in the presence of excess FMN or riboflavin. In addition, FRD(Aa) holoenzyme also utilized FMN, riboflavin, or FAD as a substrate. Steady-state kinetic results of substrate titrations, dead-end inhibition by AMP and lumichrome, and product inhibition by NAD(+) indicated an ordered sequential mechanism with NADH as the first binding substrate and reduced FMN as the first leaving product. This is contrary to the ping-pong mechanism shown by other class I flavin reductases. The FMN bound to the native FRD(Aa) can be fully reduced by NADH and subsequently reoxidized by oxygen. No NADH binding was detected using 90 microM FRD(Aa) apoenzyme and 300 microM NADH. All results favor the interpretation that the bound FMN was a cofactor rather than a substrate. It is highly unusual that a flavin reductase using a sequential mechanism would require a flavin cofactor to facilitate redox exchange between NADH and a flavin substrate. FRD(Aa) exhibited a monomer-dimer equilibrium with a K(d) of 2.7 microM. Similarities and differences between FRD(Aa) and certain flavin reductases are discussed.

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

    PubMed Central

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

    2009-01-01

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

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Hou, Feng; Miyakawa, Takuya; Kataoka, Michihiko

    2014-04-18

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

  6. 29 CFR 4043.24 - Termination or partial termination.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... REPORTABLE EVENTS AND CERTAIN OTHER NOTIFICATION REQUIREMENTS Post-Event Notice of Reportable Events § 4043.24 Termination or partial termination. (a) Reportable event. A reportable event occurs when the... within the meaning of section 411(d)(3) of the Code. (b) Waivers. Notice is waived for this event. ...

  7. 29 CFR 4043.24 - Termination or partial termination.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... REPORTABLE EVENTS AND CERTAIN OTHER NOTIFICATION REQUIREMENTS Post-Event Notice of Reportable Events § 4043.24 Termination or partial termination. (a) Reportable event. A reportable event occurs when the... within the meaning of section 411(d)(3) of the Code. (b) Waivers. Notice is waived for this event. ...

  8. 29 CFR 4043.24 - Termination or partial termination.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... REPORTABLE EVENTS AND CERTAIN OTHER NOTIFICATION REQUIREMENTS Post-Event Notice of Reportable Events § 4043.24 Termination or partial termination. (a) Reportable event. A reportable event occurs when the... within the meaning of section 411(d)(3) of the Code. (b) Waivers. Notice is waived for this event. ...

  9. 29 CFR 4043.24 - Termination or partial termination.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... REPORTABLE EVENTS AND CERTAIN OTHER NOTIFICATION REQUIREMENTS Post-Event Notice of Reportable Events § 4043.24 Termination or partial termination. (a) Reportable event. A reportable event occurs when the... within the meaning of section 411(d)(3) of the Code. (b) Waivers. Notice is waived for this event. ...

  10. 29 CFR 4043.24 - Termination or partial termination.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... REPORTABLE EVENTS AND CERTAIN OTHER NOTIFICATION REQUIREMENTS Post-Event Notice of Reportable Events § 4043.24 Termination or partial termination. (a) Reportable event. A reportable event occurs when the... within the meaning of section 411(d)(3) of the Code. (b) Waivers. Notice is waived for this event. ...

  11. Retrospective approach to methylenetetrahydrofolate reductase mutations in children.

    PubMed

    Özer, Işıl; Özçetin, Mustafa; Karaer, Hatice; Kurt, Semiha G; Şahin, Şemsettin

    2011-07-01

    Methylenetetrahydrofolate reductase reduces methyltetrahydrofolate, a cosubstrate in the remethylation of homocysteine, from methylenetetrahydrofolate. Congenital defects, hematologic tumors, and intrauterine growth retardation can occur during childhood. This study evaluated clinical and laboratory treatment approaches in children diagnosed with methylenetetrahydrofolate reductase mutations. Our group included 23 boys and 14 girls, aged 103.4 ± 70.8 months S.D. Clinical findings of patients and homocysteine, vitamin B12, folate, hemogram, electroencephalography, cranial magnetic resonance imaging, and echocardiography data were evaluated in terms of treatment approach. Our patients' findings included vitamin B12 at 400.4 ± 224.6 pg/mL S.D. (normal range, 300-700 pg/mL), folate at 10.1 ± 4.5 ng/mL S.D. (normal range, 1.8-9 ng/mL), and homocysteine at 8.4 ± 4.7 μmol/L S.D. (normal range, 5.5-17 μmol/L). Eighty-eight percent of patients demonstrated clinical findings. In comparisons involving categorical variables between groups, χ(2) tests were used. No relationship was evident between mutation type, laboratory data, and clinical severity. All mothers who had MTHFR mutations and had babies with sacral dimples had taken folate supplements during pregnancy. To avoid the risk of neural tube defects, pregnant women with a MTHFR mutation may require higher than normally recommended doses of folic acid supplementation for optimum health. Copyright © 2011 Elsevier Inc. All rights reserved.

  12. 5β-Reduced Steroids and Human Δ4-3-Ketosteroid 5β-Reductase (AKR1D1)

    PubMed Central

    Chen, Mo; Penning, Trevor M.

    2014-01-01

    5β-Reduced steroids are non-planar steroids that have 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 Δ4-3-ketosteroid-5β-reductase (AKR1D1). This enzyme is a member of the aldoketo 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. PMID:24513054

  13. Crystallization and preliminary X-ray diffraction analyses of the redox-controlled complex of terminal oxygenase and ferredoxin components in the Rieske nonhaem iron oxygenase carbazole 1,9a-dioxygenase

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Matsuzawa, Jun; Aikawa, Hiroki; Umeda, Takashi

    2014-09-25

    A crystal was obtained of the complex between reduced terminal oxygenase and oxidized ferredoxin components of carbazole 1,9a-dioxygenase. The crystal belonged to space group P2{sub 1} and diffracted to 2.25 Å resolution. The initial reaction in bacterial carbazole degradation is catalyzed by carbazole 1,9a-dioxygenase, which consists of terminal oxygenase (Oxy), ferredoxin (Fd) and ferredoxin reductase components. The electron-transfer complex between reduced Oxy and oxidized Fd was crystallized at 293 K using the hanging-drop vapour-diffusion method with PEG 3350 as the precipitant under anaerobic conditions. The crystal diffracted to a maximum resolution of 2.25 Å and belonged to space group P2{submore » 1}, with unit-cell parameters a = 97.3, b = 81.6, c = 116.2 Å, α = γ = 90, β = 100.1°. The V{sub M} value is 2.85 Å{sup 3} Da{sup −1}, indicating a solvent content of 56.8%.« less

  14. A 1,536-Well-Based Kinetic HTS Assay for Inhibitors of Schistosoma mansoni Thioredoxin Glutathione Reductase

    PubMed Central

    Lea, Wendy A.; Jadhav, Ajit; Rai, Ganesha; Sayed, Ahmed A.; Cass, Cynthia L.; Inglese, James; Williams, David L.; Austin, Christopher P.

    2008-01-01

    Abstract Schistosomiasis is a major neglected tropical disease that currently affects over 200 million people and leads to over 200,000 annual deaths. Schistosoma mansoni parasites survive in humans in part because of a set of antioxidant enzymes that continuously degrade reactive oxygen species produced by the host. A principal component of this defense system has been recently identified as thioredoxin glutathione reductase (TGR), a parasite-specific enzyme that combines the functions of two human counterparts, glutathione reductase and thioredoxin reductase, and as such this enzyme presents an attractive new target for anti-schistosomiasis drug development. Herein, we present the development of a highly miniaturized and robust screening assay for TGR. The 5-μl final volume assay is based on the Ellman reagent [5,5′-dithiobis(2-nitrobenzoic acid) (DTNB)] and utilizes a high-speed absorbance kinetic read to minimize the effect of dust, absorbance interference, and meniscus variation. This assay is further applicable to the testing of other redox enzymes that utilize DTNB as a model substrate. PMID:18665782

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

    PubMed

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

    2015-09-01

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

  16. Carbonyl reductase of dog liver: purification, properties, and kinetic mechanism.

    PubMed

    Hara, A; Nakayama, T; Deyashiki, Y; Kariya, K; Sawada, H

    1986-01-01

    A carbonyl reductase has been extracted into 0.5 M KCl from dog liver and purified to apparent homogeneity by a three-step procedure consisting of chromatography on CM-Sephadex, Matrex green A, and Sephadex G-100 in high-ionic-strength buffers. The enzyme is a dimer composed of two identical subunits of molecular weight 27,000. The pH optimum is 5.5 and the isoelectric point of the enzyme is 9.3. The enzyme reduces aromatic ketones and aldehydes; the aromatic ketones with adjacent medium alkyl chains are the best substrates. Quinones, ketosteroids, prostaglandins, and aliphatic carbonyl compounds are poor or inactive substrates for the enzyme. As a cofactor the enzyme utilizes NADPH, the pro-S hydrogen atom of which is transferred to the substrate. Two moles of NADPH bind to one mole of the enzyme molecule, causing a blue shift and enhancement of the cofactor fluorescence. The reductase reaction is reversible and the equilibrium constant determined at pH 7.0 is 12.8. Steady-state kinetic measurements in both directions suggest that the reaction proceeds through a di-iso ordered bi-bi mechanism.

  17. 14 CFR 1260.161 - Termination.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ..., Hospitals, and Other Non-Profit Organizations Termination and Enforcement § 1260.161 Termination. (a) Awards... termination conditions, including the effective date and, in the case of partial termination, the portion to... reasons for such termination, the effective date, and, in the case of partial termination, the portion to...

  18. Aldose Reductase-Deficient Mice Develop Nephrogenic Diabetes Insipidus

    PubMed Central

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

    2000-01-01

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

  19. Regulation of HMG-CoA reductase in MCF-7 cells by genistein, EPA, and DHA, alone and in combination with mevastatin.

    PubMed

    Duncan, Robin E; El-Sohemy, Ahmed; Archer, Michael C

    2005-06-28

    We investigated the regulation of HMG-CoA reductase in MCF-7 human breast cancer cells by genistein, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). All three compounds down-regulated reductase activity, primarily through post-transcriptional effects. In mevastatin-treated cells, only genistein and DHA abrogated the induction of reductase activity caused by this competitive inhibitor. Diets rich in soy isoflavones and fish oils, therefore, may exert anti-cancer effects through the inhibition of mevalonate synthesis in the breast. Genistein and DHA, in particular, may augment the efficacy of statins, increasing the potential for use of these drugs in adjuvant therapy for breast cancer.

  20. Residues in the Distal Heme Pocket of Arabidopsis Non-Symbiotic Hemoglobins: Implication for Nitrite Reductase Activity

    PubMed Central

    Kumar, Nitin; Astegno, Alessandra; Chen, Jian; Giorgetti, Alejandro; Dominici, Paola

    2016-01-01

    It is well-established that plant hemoglobins (Hbs) are involved in nitric oxide (NO) metabolism via NO dioxygenase and/or nitrite reductase activity. The ferrous-deoxy Arabidopsis Hb1 and Hb2 (AHb1 and AHb2) have been shown to reduce nitrite to NO under hypoxia. Here, to test the hypothesis that a six- to five-coordinate heme iron transition might mediate the control of the nitrite reduction rate, we examined distal pocket mutants of AHb1 and AHb2 for nitrite reductase activity, NO production and spectroscopic features. Absorption spectra of AHbs distal histidine mutants showed that AHb1 mutant (H69L) is a stable pentacoordinate high-spin species in both ferrous and ferric states, whereas heme iron in AHb2 mutant (H66L) is hexacoordinated low-spin with Lys69 as the sixth ligand. The bimolecular rate constants for nitrite reduction to NO were 13.3 ± 0.40, 7.3 ± 0.5, 10.6 ± 0.8 and 171.90 ± 9.00 M−1·s−1 for AHb1, AHb2, AHb1 H69L and AHb2 H66L, respectively, at pH 7.4 and 25 °C. Consistent with the reductase activity, the amount of NO detected by chemiluminescence was significantly higher in the AHb2 H66L mutant. Our data indicate that nitrite reductase activity is determined not only by heme coordination, but also by a unique distal heme pocket in each AHb. PMID:27136534

  1. The general mitochondrial processing peptidase from potato is an integral part of cytochrome c reductase of the respiratory chain.

    PubMed Central

    Braun, H P; Emmermann, M; Kruft, V; Schmitz, U K

    1992-01-01

    The major mitochondrial processing activity removing presequences from nuclear encoded precursor proteins is present in the soluble fraction of fungal and mammalian mitochondria. We found that in potato, this activity resides in the inner mitochondrial membrane. Surprisingly, the proteolytic activity co-purifies with cytochrome c reductase, a protein complex of the respiratory chain. The purified complex is bifunctional, as it has the ability to transfer electrons from ubiquinol to cytochrome c and to cleave off the presequences of mitochondrial precursor proteins. In contrast to the nine subunit fungal complex, cytochrome c reductase from potato comprises 10 polypeptides. Protein sequencing of peptides from individual subunits and analysis of corresponding cDNA clones reveals that subunit III of cytochrome c reductase (51 kDa) represents the general mitochondrial processing peptidase. Images PMID:1324169

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Pegan, Scott D.; Sturdy, Megan; Ferry, Gilles

    Quinone reductase 2 (QR2) is one of two members comprising the mammalian quinone reductase family of enzymes responsible for performing FAD mediated reductions of quinone substrates. In contrast to quinone reductase 1 (QR1) which uses NAD(P)H as its co-substrate, QR2 utilizes a rare group of hydride donors, N-methyl or N-ribosyl nicotinamide. Several studies have linked QR2 to the generation of quinone free radicals, several neuronal degenerative diseases, and cancer. QR2 has been also identified as the third melatonin receptor (MT3) through in cellulo and in vitro inhibition of QR2 by traditional MT3 ligands, and through recent X-ray structures of humanmore » QR2 (hQR2) in complex with melatonin and 2-iodomelatonin. Several MT3 specific ligands have been developed that exhibit both potent in cellulo inhibition of hQR2 nanomolar, affinity for MT3. The potency of these ligands suggest their use as molecular probes for hQR2. However, no definitive correlation between traditionally obtained MT3 ligand affinity and hQR2 inhibition exists limiting our understanding of how these ligands are accommodated in the hQR2 active site. To obtain a clearer relationship between the structures of developed MT3 ligands and their inhibitory properties, in cellulo and in vitro IC{sub 50} values were determined for a representative set of MT3 ligands (MCA-NAT, 2-I-MCANAT, prazosin, S26695, S32797, and S29434). Furthermore, X-ray structures for each of these ligands in complex with hQR2 were determined allowing for a structural evaluation of the binding modes of these ligands in relation to the potency of MT3 ligands.« less

  3. Mitochondrial targeting of the human peptide methionine sulfoxide reductase (MSRA), an enzyme involved in the repair of oxidized proteins.

    PubMed

    Hansel, Alfred; Kuschel, Lioba; Hehl, Solveig; Lemke, Cornelius; Agricola, Hans-Jürgen; Hoshi, Toshinori; Heinemann, Stefan H

    2002-06-01

    Peptide methionine sulfoxide reductase (MSRA) catalyzes the reduction of methionine sulfoxide to methionine. This widely expressed enzyme constitutes an important repair mechanism for oxidatively damaged proteins, which accumulate during the manifestation of certain degenerative diseases and aging processes. In addition, it is discussed to be involved in regulatory processes. Here we address the question of how the enzyme's diverse functions are reflected in its subcellular localization. Using fusions of the human version of MSRA with the enhanced green fluorescence protein expressed in various mammalian cell lines, we show a distinct localization at mitochondria. The N-terminal 23 amino acid residues contain the signal for this mitochondrial targeting. Activity tests showed that they are not required for enzyme function. Mitochondrial localization of native MSRA in mouse and rat liver slices was verified with an MSRA-specific antibody by using immunohistochemical methods. The protein was located in the mitochondrial matrix, as demonstrated by using pre-embedding immunostaining and electron microscopy. Mitochondria are the major source of reactive oxygen species (ROS). Therefore, MSRA has to be considered an important means for the general reduction of ROS release from mitochondria.

  4. Ebselen: A thioredoxin reductase-dependent catalyst for {alpha}-tocopherol quinone reduction

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Fang Jianguo; Zhong Liangwei; Zhao Rong

    2005-09-01

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

  5. Anti-HMG-CoA Reductase, Antioxidant, and Anti-Inflammatory Activities of Amaranthus viridis Leaf Extract as a Potential Treatment for Hypercholesterolemia

    PubMed Central

    Salvamani, Shamala; Gunasekaran, Baskaran; Shukor, Mohd Yunus; Shaharuddin, Noor Azmi; Sabullah, Mohd Khalizan

    2016-01-01

    Inflammation and oxidative stress are believed to contribute to the pathology of several chronic diseases including hypercholesterolemia (elevated levels of cholesterol in blood) and atherosclerosis. HMG-CoA reductase inhibitors of plant origin are needed as synthetic drugs, such as statins, which are known to cause adverse effects on the liver and muscles. Amaranthus viridis (A. viridis) has been used from ancient times for its supposedly medically beneficial properties. In the current study, different parts of A. viridis (leaf, stem, and seed) were evaluated for potential anti-HMG-CoA reductase, antioxidant, and anti-inflammatory activities. The putative HMG-CoA reductase inhibitory activity of A. viridis extracts at different concentrations was determined spectrophotometrically by NADPH oxidation, using HMG-CoA as substrate. A. viridis leaf extract revealed the highest HMG-CoA reductase inhibitory effect at about 71%, with noncompetitive inhibition in Lineweaver-Burk plot analysis. The leaf extract showed good inhibition of hydroperoxides, 2,2-diphenyl-1-picrylhydrazyl (DPPH), nitric oxide (NO), and ferric ion radicals in various concentrations. A. viridis leaf extract was proven to be an effective inhibitor of hyaluronidase, lipoxygenase, and xanthine oxidase enzymes. The experimental data suggest that A. viridis leaf extract is a source of potent antioxidant and anti-inflammatory agent and may modulate cholesterol metabolism by inhibition of HMG-CoA reductase. PMID:27051453

  6. NAD(P)H-dependent aldose reductase from the xylose-assimilating yeast Candida tenuis. Isolation, characterization and biochemical properties of the enzyme.

    PubMed Central

    Neuhauser, W; Haltrich, D; Kulbe, K D; Nidetzky, B

    1997-01-01

    During growth on d-xylose the yeast Candida tenuis produces one aldose reductase that is active with both NADPH and NADH as coenzyme. This enzyme has been isolated by dye ligand and anion-exchange chromatography in yields of 76%. Aldose reductase consists ofa single 43 kDa polypeptide with an isoelectric point of 4.70. Initial velocity, product inhibition and binding studies are consistent with a compulsory-ordered, ternary-complex mechanism with coenzyme binding first and leaving last. The catalytic efficiency (kcat/Km) in d-xylose reduction at pH 7 is more than 60-fold higher than that in xylitol oxidation and reflects significant differences in the corresponding catalytic centre activities as well as apparent substrate-binding constants. The enzyme prefers NADP(H) approx. 2-fold to NAD(H), which is largely due to better apparent binding of the phosphorylated form of the coenzyme. NADP+ is a potent competitive inhibitor of the NADH-linked aldehyde reduction (Ki 1.5 microM), whereas NAD+ is not. Unlike mammalian aldose reductase, the enzyme from C. tenuis is not subject to oxidation-induced activation. Evidence of an essential lysine residue located in or near the coenzyme binding site has been obtained from chemical modification of aldose reductase with pyridoxal 5'-phosphate. The results are discussed in the context of a comparison of the enzymic properties of yeast and mammalian aldose reductase. PMID:9307017

  7. A Plasma Display Terminal.

    ERIC Educational Resources Information Center

    Stifle, Jack

    A graphics terminal designed for use as a remote computer input/output terminal is described. Although the terminal is intended for use in teaching applications, it has several features which make it useful in many other computer terminal applications. These features include: a 10-inch square plasma display panel, permanent storage of information…

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

    PubMed

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

    2018-04-01

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

  9. Communications terminal breadboard

    NASA Technical Reports Server (NTRS)

    1972-01-01

    A baseline design is presented of a digital communications link between an advanced manned spacecraft (AMS) and an earth terminal via an Intelsat 4 type communications satellite used as a geosynchronous orbiting relay station. The fabrication, integration, and testing of terminal elements at each end of the link are discussed. In the baseline link design, the information carrying capacity of the link was estimated for both the forward direction (earth terminal to AMS) and the return direction, based upon orbital geometry, relay satellite characteristics, terminal characteristics, and the improvement that can be achieved by the use of convolutional coding/Viterbi decoding techniques.

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

    PubMed

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

    1976-03-01

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

  11. The 30 kDa protein co-purified with chick liver glutathione S-transferases is a carbonyl reductase.

    PubMed

    Tsai, S P; Wang, L Y; Yeh, H I; Tam, M F

    1996-02-08

    An unidentified 30 kDa protein was co-purified with chick liver glutathione S-transferases from S-hexylglutathione affinity column. The protein was isolated to apparent homogeneity with chromatofocusing. The molecular mass of the protein was determined to be 30 277 +/- 3 dalton by mass spectrometry. The protein was digested with Achromobacter proteinase I. Amino-acid sequence analyses of the resulting peptides show a high degree of identity with those of human carbonyl reductase. The protein is active with menadione as substrate. Thus, it is identified as chick liver carbonyl reductase.

  12. Unexpected ethical dilemmas in sex assignment in 46,XY DSD due to 5-alpha reductase type 2 deficiency.

    PubMed

    Byers, Heather M; Mohnach, Lauren H; Fechner, Patricia Y; Chen, Ming; Thomas, Inas H; Ramsdell, Linda A; Shnorhavorian, Margarett; McCauley, Elizabeth A; Amies Oelschlager, Anne-Marie E; Park, John M; Sandberg, David E; Adam, Margaret P; Keegan, Catherine E

    2017-06-01

    Sex assignment at birth remains one of the most clinically challenging and controversial topics in 46,XY disorders of sexual development (DSD). This is particularly challenging in deficiency of 5-alpha reductase type 2 given that external genitalia are typically undervirilized at birth but typically virilize at puberty to a variable degree. Historically, most individuals with 5-alpha reductase deficiency were raised females. However, reports that over half of patients who underwent a virilizing puberty adopted an adult male gender identity have challenged this practice. Consensus guidelines on assignment of sex of rearing at birth are equivocal or favor male assignment in the most virilized cases. While a male sex of rearing assignment may avoid lifelong hormonal therapy and/or allow the potential for fertility, female sex assignment may be more consistent with external anatomy in the most severely undervirilized cases. Herein, we describe five patients with 46,XY DSD due 5-alpha-reductase type 2 deficiency, all with a severe phenotype. An inter-disciplinary DSD medical team at one of two academic centers evaluated each patient. This case series illustrates the complicated decision-making process of assignment of sex of rearing at birth in 5-alpha reductase type 2 deficiency and the challenges that arise when the interests of the child, parental wishes, recommendations of the medical team, and state law collide. © 2017 Wiley Periodicals, Inc.

  13. Evolution of the Ferric Reductase Domain (FRD) Superfamily: Modularity, Functional Diversification, and Signature Motifs

    PubMed Central

    Zhang, Xuezhi; Krause, Karl-Heinz; Xenarios, Ioannis; Soldati, Thierry; Boeckmann, Brigitte

    2013-01-01

    A heme-containing transmembrane ferric reductase domain (FRD) is found in bacterial and eukaryotic protein families, including ferric reductases (FRE), and NADPH oxidases (NOX). The aim of this study was to understand the phylogeny of the FRD superfamily. Bacteria contain FRD proteins consisting only of the ferric reductase domain, such as YedZ and short bFRE proteins. Full length FRE and NOX enzymes are mostly found in eukaryotic cells and all possess a dehydrogenase domain, allowing them to catalyze electron transfer from cytosolic NADPH to extracellular metal ions (FRE) or oxygen (NOX). Metazoa possess YedZ-related STEAP proteins, possibly derived from bacteria through horizontal gene transfer. Phylogenetic analyses suggests that FRE enzymes appeared early in evolution, followed by a transition towards EF-hand containing NOX enzymes (NOX5- and DUOX-like). An ancestral gene of the NOX(1-4) family probably lost the EF-hands and new regulatory mechanisms of increasing complexity evolved in this clade. Two signature motifs were identified: NOX enzymes are distinguished from FRE enzymes through a four amino acid motif spanning from transmembrane domain 3 (TM3) to TM4, and YedZ/STEAP proteins are identified by the replacement of the first canonical heme-spanning histidine by a highly conserved arginine. The FRD superfamily most likely originated in bacteria. PMID:23505460

  14. Evolution of the ferric reductase domain (FRD) superfamily: modularity, functional diversification, and signature motifs.

    PubMed

    Zhang, Xuezhi; Krause, Karl-Heinz; Xenarios, Ioannis; Soldati, Thierry; Boeckmann, Brigitte

    2013-01-01

    A heme-containing transmembrane ferric reductase domain (FRD) is found in bacterial and eukaryotic protein families, including ferric reductases (FRE), and NADPH oxidases (NOX). The aim of this study was to understand the phylogeny of the FRD superfamily. Bacteria contain FRD proteins consisting only of the ferric reductase domain, such as YedZ and short bFRE proteins. Full length FRE and NOX enzymes are mostly found in eukaryotic cells and all possess a dehydrogenase domain, allowing them to catalyze electron transfer from cytosolic NADPH to extracellular metal ions (FRE) or oxygen (NOX). Metazoa possess YedZ-related STEAP proteins, possibly derived from bacteria through horizontal gene transfer. Phylogenetic analyses suggests that FRE enzymes appeared early in evolution, followed by a transition towards EF-hand containing NOX enzymes (NOX5- and DUOX-like). An ancestral gene of the NOX(1-4) family probably lost the EF-hands and new regulatory mechanisms of increasing complexity evolved in this clade. Two signature motifs were identified: NOX enzymes are distinguished from FRE enzymes through a four amino acid motif spanning from transmembrane domain 3 (TM3) to TM4, and YedZ/STEAP proteins are identified by the replacement of the first canonical heme-spanning histidine by a highly conserved arginine. The FRD superfamily most likely originated in bacteria.

  15. Adaptation of cytochrome-b5 reductase activity and methaemoglobinaemia in areas with a high nitrate concentration in drinking-water.

    PubMed Central

    Gupta, S. K.; Gupta, R. C.; Seth, A. K.; Gupta, A. B.; Bassin, J. K.; Gupta, A.

    1999-01-01

    An epidemiological investigation was undertaken in India to assess the prevalence of methaemoglobinaemia in areas with high nitrate concentration in drinking-water and the possible association with an adaptation of cytochrome-b5 reductase. Five areas were selected, with average nitrate ion concentrations in drinking-water of 26, 45, 95, 222 and 459 mg/l. These areas were visited and house schedules were prepared in accordance with a statistically designed protocol. A sample of 10% of the total population was selected in each of the areas, matched for age and weight, giving a total of 178 persons in five age groups. For each subject, a detailed history was documented, a medical examination was conducted and blood samples were taken to determine methaemoglobin level and cytochrome-b5 reductase activity. Collected data were subjected to statistical analysis to test for a possible relationship between nitrate concentration, cytochrome-b5 reductase activity and methaemoglobinaemia. High nitrate concentrations caused methaemoglobinaemia in infants and adults. The reserve of cytochrome-b5 reductase activity (i.e. the enzyme activity not currently being used, but which is available when needed; for example, under conditions of increased nitrate ingestion) and its adaptation with increasing water nitrate concentration to reduce methaemoglobin were more pronounced in children and adolescents. PMID:10534899

  16. 76 FR 22120 - Credit Watch Termination Initiative; Termination of Origination Approval Agreements

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-04-20

    ... DEPARTMENT OF HOUSING AND URBAN DEVELOPMENT [Docket No. FR- 5511-N-01] Credit Watch Termination Initiative; Termination of Origination Approval Agreements AGENCY: Office of the Assistant Secretary for... (FHA) against HUD-approved mortgagees through the FHA Credit Watch Termination Initiative. This notice...

  17. 75 FR 67387 - Credit Watch Termination Initiative Termination of Origination Approval Agreements

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-11-02

    ... DEPARTMENT OF HOUSING AND URBAN DEVELOPMENT [Docket No. FR-4211-N-05] Credit Watch Termination Initiative Termination of Origination Approval Agreements AGENCY: Office of the Assistant Secretary for... (FHA) against HUD-approved mortgagees through the FHA Credit Watch Termination Initiative. This notice...

  18. 77 FR 38818 - Credit Watch Termination Initiative; Termination of Origination Approval Agreements

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-06-29

    ... DEPARTMENT OF HOUSING AND URBAN DEVELOPMENT [Docket No. FR-5644-N-01] Credit Watch Termination Initiative; Termination of Origination Approval Agreements AGENCY: Office of the Assistant Secretary for... (FHA) against HUD-approved mortgagees through the FHA Credit Watch Termination Initiative. This notice...

  19. 76 FR 38406 - Credit Watch Termination Initiative; Termination of Origination Approval Agreements

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-06-30

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  20. 76 FR 4126 - Credit Watch Termination Initiative Termination of Origination Approval Agreements

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-01-24

    ... DEPARTMENT OF HOUSING AND URBAN DEVELOPMENT [Docket No. FR- 5411-N-07] Credit Watch Termination Initiative Termination of Origination Approval Agreements AGENCY: Office of the Assistant Secretary for... (FHA) against HUD-approved mortgagees through the FHA Credit Watch Termination Initiative. This notice...

  1. 77 FR 5263 - Credit Watch Termination Initiative Termination of Origination Approval Agreements

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-02-02

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  2. 75 FR 61164 - Credit Watch Termination Initiative Termination of Origination Approval Agreements

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-10-04

    ... DEPARTMENT OF HOUSING AND URBAN DEVELOPMENT [Docket No. FR-5411-N-03] Credit Watch Termination Initiative Termination of Origination Approval Agreements AGENCY: Office of the Assistant Secretary for... (FHA) against HUD-approved mortgagees through the FHA Credit Watch Termination Initiative. This notice...

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

    PubMed

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

    2010-10-05

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

  4. 10 CFR 600.161 - Termination.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... Nonprofit Organizations Post-Award Requirements § 600.161 Termination. (a) Awards may be terminated in whole... effective date and, in the case of partial termination, the portion to be terminated. (3) By the recipient upon sending to DOE written notification setting forth the reasons for such termination, the effective...

  5. 10 CFR 600.161 - Termination.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... Nonprofit Organizations Post-Award Requirements § 600.161 Termination. (a) Awards may be terminated in whole... effective date and, in the case of partial termination, the portion to be terminated. (3) By the recipient upon sending to DOE written notification setting forth the reasons for such termination, the effective...

  6. Thioredoxin Reductase and its Inhibitors

    PubMed Central

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

    2014-01-01

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

  7. Fatty acyl-CoA reductases of birds

    PubMed Central

    2011-01-01

    Background Birds clean and lubricate their feathers with waxes that are produced in the uropygial gland, a holocrine gland located on their back above the tail. The type and the composition of the secreted wax esters are dependent on the bird species, for instance the wax ester secretion of goose contains branched-chain fatty acids and unbranched fatty alcohols, whereas that of barn owl contains fatty acids and alcohols both of which are branched. Alcohol-forming fatty acyl-CoA reductases (FAR) catalyze the reduction of activated acyl groups to fatty alcohols that can be esterified with acyl-CoA thioesters forming wax esters. Results cDNA sequences encoding fatty acyl-CoA reductases were cloned from the uropygial glands of barn owl (Tyto alba), domestic chicken (Gallus gallus domesticus) and domestic goose (Anser anser domesticus). Heterologous expression in Saccharomyces cerevisiae showed that they encode membrane associated enzymes which catalyze a NADPH dependent reduction of acyl-CoA thioesters to fatty alcohols. By feeding studies of transgenic yeast cultures and in vitro enzyme assays with membrane fractions of transgenic yeast cells two groups of isozymes with different properties were identified, termed FAR1 and FAR2. The FAR1 group mainly synthesized 1-hexadecanol and accepted substrates in the range between 14 and 18 carbon atoms, whereas the FAR2 group preferred stearoyl-CoA and accepted substrates between 16 and 20 carbon atoms. Expression studies with tissues of domestic chicken indicated that FAR transcripts were not restricted to the uropygial gland. Conclusion The data of our study suggest that the identified and characterized avian FAR isozymes, FAR1 and FAR2, can be involved in wax ester biosynthesis and in other pathways like ether lipid synthesis. PMID:22151413

  8. Glutathione reductase gsr-1 is an essential gene required for Caenorhabditis elegans early embryonic development.

    PubMed

    Mora-Lorca, José Antonio; Sáenz-Narciso, Beatriz; Gaffney, Christopher J; Naranjo-Galindo, Francisco José; Pedrajas, José Rafael; Guerrero-Gómez, David; Dobrzynska, Agnieszka; Askjaer, Peter; Szewczyk, Nathaniel J; Cabello, Juan; Miranda-Vizuete, Antonio

    2016-07-01

    Glutathione is the most abundant thiol in the vast majority of organisms and is maintained in its reduced form by the flavoenzyme glutathione reductase. In this work, we describe the genetic and functional analysis of the Caenorhabditis elegans gsr-1 gene that encodes the only glutathione reductase protein in this model organism. By using green fluorescent protein reporters we demonstrate that gsr-1 produces two GSR-1 isoforms, one located in the cytoplasm and one in the mitochondria. gsr-1 loss of function mutants display a fully penetrant embryonic lethal phenotype characterized by a progressive and robust cell division delay accompanied by an aberrant distribution of interphasic chromatin in the periphery of the cell nucleus. Maternally expressed GSR-1 is sufficient to support embryonic development but these animals are short-lived, sensitized to chemical stress, have increased mitochondrial fragmentation and lower mitochondrial DNA content. Furthermore, the embryonic lethality of gsr-1 worms is prevented by restoring GSR-1 activity in the cytoplasm but not in mitochondria. Given the fact that the thioredoxin redox systems are dispensable in C. elegans, our data support a prominent role of the glutathione reductase/glutathione pathway in maintaining redox homeostasis in the nematode. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. Substrate specificity and catalytic efficiency of aldo-keto reductases with phospholipid aldehydes

    PubMed Central

    Spite, Matthew; Baba, Shahid P.; Ahmed, Yonis; Barski, Oleg A.; Nijhawan, Kanchan; Petrash, J. Mark; Bhatnagar, Aruni; Srivastava, Sanjay

    2007-01-01

    Phospholipid oxidation generates several bioactive aldehydes that remain esterified to the glycerol backbone (‘core’ aldehydes). These aldehydes induce endothelial cells to produce monocyte chemotactic factors and enhance monocyte–endothelium adhesion. They also serve as ligands of scavenger receptors for the uptake of oxidized lipoproteins or apoptotic cells. The biochemical pathways involved in phospholipid aldehyde metabolism, however, remain largely unknown. In the present study, we have examined the efficacy of the three mammalian AKR (aldo-keto reductase) families in catalysing the reduction of phospholipid aldehydes. The model phospholipid aldehyde POVPC [1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine] was efficiently reduced by members of the AKR1, but not by the AKR6 or the ARK7 family. In the AKR1 family, POVPC reductase activity was limited to AKR1A and B. No significant activity was observed with AKR1C enzymes. Among the active proteins, human AR (aldose reductase) (AKR1B1) showed the highest catalytic activity. The catalytic efficiency of human small intestinal AR (AKR1B10) was comparable with the murine AKR1B proteins 1B3 and 1B8. Among the murine proteins AKR1A4 and AKR1B7 showed appreciably lower catalytic activity as compared with 1B3 and 1B8. The human AKRs, 1B1 and 1B10, and the murine proteins, 1B3 and 1B8, also reduced C-7 and C-9 sn-2 aldehydes as well as POVPE [1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphoethanolamine]. AKR1A4, B1, B7 and B8 catalysed the reduction of aldehydes generated in oxidized C16:0-20:4 phosphatidylcholine with acyl, plasmenyl or alkyl linkage at the sn-1 position or C16:0-20:4 phosphatidylglycerol or phosphatidic acid. AKR1B1 displayed the highest activity with phosphatidic acids; AKR1A4 was more efficient with long-chain aldehydes such as 5-hydroxy-8-oxo-6-octenoyl derivatives, whereas AKR1B8 preferred phosphatidylglycerol. These results suggest that proteins of the AKR1A and B families are

  10. Regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity in murine epidermis. Modulation of enzyme content and activation state by barrier requirements.

    PubMed Central

    Proksch, E; Elias, P M; Feingold, K R

    1990-01-01

    Epidermal cholesterol biosynthesis is regulated by barrier function. We quantitated the amount and activation state (phosphorylation-dephosphorylation) of the rate-limiting enzyme, 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, in epidermis before and after barrier disruption. In murine epidermis we found high enzyme activity (1.75 +/- 0.02 nmol/min per mg protein). After acute barrier disruption, enzyme activity began to increase after 1.5 h, reaching a maximum increase by 2.5 h, and returned to normal by 15 h. Chronic barrier disruption increased total enzyme activity by 83%. In normal epidermis, measurement of HMG CoA reductase activity in microsomes isolated in NaF- vs. NaCl-containing buffers demonstrated that 46 +/- 2% of the enzyme was in the active form. After acute or chronic barrier disruption, a marked increase in the percentage of HMG CoA reductase in the active form was observed. Acute disruption increased enzyme activation state as early as 15 min, reaching a maximum after 2.5 h, with an increase still present at 15 h, indicating that changes in activation state had a close temporal relationship with barrier function. Increases in total HMG CoA reductase activity occurred only after profound barrier disruption, whereas changes in activation state occur with lesser degrees of barrier disruption. Artificial correction of barrier function prevented the increase in total HMG CoA reductase activity, and partially prevented the increase in enzyme activation. These results show that barrier requirements regulate epidermal cholesterol synthesis by modulating both the HMG CoA reductase amount and activation state. Images PMID:2312730

  11. Methionine Sulfoxide Reductase A (MsrA) and Its Function in Ubiquitin-Like Protein Modification in Archaea

    DOE PAGES

    Fu, Xian; Adams, Zachary; Liu, Rui; ...

    2017-09-05

    Methionine sulfoxide reductase A (MsrA) is an antioxidant enzyme found in all domains of life that catalyzes the reduction of methionine-S-sulfoxide (MSO) to methionine in proteins and free amino acids. We demonstrate that archaeal MsrA has a ubiquitin-like (Ubl) protein modification activity that is distinct from its stereospecific reduction of MSO residues. MsrA catalyzes this Ubl modification activity, with the Ubl-activating E1 UbaA, in the presence of the mild oxidant dimethyl sulfoxide (DMSO) and in the absence of reductant. In contrast, the MSO reductase activity of MsrA is inhibited by DMSO and requires reductant. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysismore » reveals that MsrA-dependent Ubl conjugates are associated with DNA replication, protein remodeling, and oxidative stress and include the Ubl-modified MsrA, Orc3 (Orc1/Cdc6), and Cdc48d (Cdc48/p97 AAA+ ATPase). Overall, we found archaeal MsrA to have opposing MSO reductase and Ubl modifying activities that are associated with oxidative stress responses and controlled by exposure to mild oxidant.« less

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Egerer-Sieber, Claudia; Herl, Vanessa; Müller-Uri, Frieder

    2006-03-01

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

  13. Methionine Sulfoxide Reductase A (MsrA) and Its Function in Ubiquitin-Like Protein Modification in Archaea

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Fu, Xian; Adams, Zachary; Liu, Rui

    Methionine sulfoxide reductase A (MsrA) is an antioxidant enzyme found in all domains of life that catalyzes the reduction of methionine-S-sulfoxide (MSO) to methionine in proteins and free amino acids. We demonstrate that archaeal MsrA has a ubiquitin-like (Ubl) protein modification activity that is distinct from its stereospecific reduction of MSO residues. MsrA catalyzes this Ubl modification activity, with the Ubl-activating E1 UbaA, in the presence of the mild oxidant dimethyl sulfoxide (DMSO) and in the absence of reductant. In contrast, the MSO reductase activity of MsrA is inhibited by DMSO and requires reductant. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysismore » reveals that MsrA-dependent Ubl conjugates are associated with DNA replication, protein remodeling, and oxidative stress and include the Ubl-modified MsrA, Orc3 (Orc1/Cdc6), and Cdc48d (Cdc48/p97 AAA+ ATPase). Overall, we found archaeal MsrA to have opposing MSO reductase and Ubl modifying activities that are associated with oxidative stress responses and controlled by exposure to mild oxidant.« less

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

    PubMed Central

    Azzouni, Faris; Godoy, Alejandro; Li, Yun; Mohler, James

    2012-01-01

    Despite the discovery of 5 alpha-reduction as an enzymatic step in steroid metabolism in 1951, and the discovery that dihydrotestosterone is more potent than testosterone in 1968, the significance of 5 alpha-reduced steroids in human diseases was not appreciated until the discovery of 5 alpha-reductase type 2 deficiency in 1974. Affected males are born with ambiguous external genitalia, despite normal internal genitalia. The prostate is hypoplastic, nonpalpable on rectal examination and approximately 1/10th the size of age-matched normal glands. Benign prostate hyperplasia or prostate cancer does not develop in these patients. At puberty, the external genitalia virilize partially, however, secondary sexual hair remains sparse and male pattern baldness and acne develop rarely. Several compounds have been developed to inhibit the 5 alpha-reductase isozymes and they play an important role in the prevention and treatment of many common diseases. This review describes the basic biochemical properties, functions, tissue distribution, chromosomal location, and clinical significance of the 5 alpha-reductase isozyme family. PMID:22235201

  15. 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. Copyright © 2014 Elsevier Inc. All rights reserved.

  16. Lung glutathione reductase induction in aging catalase-depleted frogs correlates with early survival throughout the life span.

    PubMed

    Perez-Campo, R; Lopez-Torres, M; Rojas, C; Cadenas, S; Barja de Quiroga, G

    1993-02-01

    A comprehensive experimental study on free radical-related parameters was performed in the lung throughout the life span of 220 initially young or old frogs. No age related differences were found transversely or longitudinally for lung superoxide dismutase, catalase, Se-dependent and -independent glutathione peroxidases, glutathione reductase, GSH, GSSG, or GSSG/GSH ratio. Continuous catalase depletion with aminotriazole led to glutathione reductase induction in the lung after 14.5 months of experimentation. This was accompanied by a great increase in survival rate of treated animals in relation to controls (especially in the old group). After 26.5 months of experimentation, glutathione reductase induction was lost and GSSG/GSH values tended to increase. This was followed by a 3-month long period of acute decrease in survival rate of treated animals. It is suggested that a high antioxidant/prooxidant balance is of protective value against causes of early death and can possibly be used in the future (when appropriately controlled) to increase the number of healthy years of the normal life span.

  17. Differential antioxidant and quinone reductase inducing activity of American, Asian, and Siberian ginseng

    USDA-ARS?s Scientific Manuscript database

    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...

  18. Applications of Carboxylic Acid Reductases in Oleaginous Microbes

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Resch, Michael G.; Linger, Jeffrey; McGeehan, John

    2016-05-26

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

  19. Ribonucleotide reductase in melanoma tissue. EPR detection in human amelanotic melanoma and quenching of the tyrosine radical by 4-hydroxyanisole.

    PubMed

    Lassmanm, G; Liermann, B; Arnold, W; Schwabe, K

    1991-01-01

    The characteristic EPR doublet of tyrosine radicals of the growth-regulating enzyme ribonucleotide reductase was detected in human melanoma tissue grown in nude mice. This was possible through the use of an amelanotic melanoma that does not exhibit disturbing EPR signals from melanin. The content of tyrosine radicals is higher in young tumor tissues than in older ones. The clinically applied antimelanotic drug, 4-hydroxyanisole, inhibits ribonucleotide reductase in Ehrlich ascites tumor cells as demonstrated by a pronounced quenching of tyrosine radicals (IC50 = 5 microM). In amelanotic melanoma tissue tyrosine radicals of the enzyme are also quenched by 4-hydroxyanisole in concentrations down to 50 microM. Thus, the inactivation of ribonucleotide reductase, which provides deoxyribonucleotides for DNA synthesis, may be a hitherto unexpected mechanism for the antitumor action of 4-hydroxyanisole.

  20. Docking into Mycobacterium tuberculosis Thioredoxin Reductase Protein Yields Pyrazolone Lead Molecules for Methicillin-Resistant Staphylococcus aureus

    PubMed Central

    Sweeney, Noreena L.; Lipker, Lauren; Hanson, Alicia M.; Bohl, Chris J.; Engel, Katie E.; Kalous, Kelsey S.; Stemper, Mary E.; Sem, Daniel S.; Schwan, William R.

    2017-01-01

    The thioredoxin/thioredoxin reductase system (Trx/TrxR) is an attractive drug target because of its involvement in a number of important physiological processes, from DNA synthesis to regulating signal transduction. This study describes the finding of pyrazolone compounds that are active against Staphylococcus aureus. Initially, the project was focused on discovering small molecules that may have antibacterial properties targeting the Mycobacterium tuberculosis thioredoxin reductase. This led to the discovery of a pyrazolone scaffold-containing compound series that showed bactericidal capability against S. aureus strains, including drug-resistant clinical isolates. The findings support continued development of the pyrazolone compounds as potential anti-S. aureus antibiotics. PMID:28134858

  1. In vitro and in silico studies of 3-hydroxy-3-methyl-glutaryl coenzyme A reductase inhibitory activity of the cowpea Gln-Asp-Phe peptide.

    PubMed

    Silva, Mariana Barros de Cerqueira E; Souza, Caio Alexandre da Cruz; Philadelpho, Biane Oliveira; Cunha, Mariana Mota Novais da; Batista, Fabiana Pacheco Reis; Silva, Jaff Ribeiro da; Druzian, Janice Izabel; Castilho, Marcelo Santos; Cilli, Eduardo Maffud; Ferreira, Ederlan S

    2018-09-01

    Previous studies have shown that cowpea protein positively interferes with cholesterol metabolism. In this study, we evaluated the ability of the fraction containing peptides of <3 kDa, as well as that of the Gln-Asp-Phe (QDF) peptide, derived from cowpea β-vignin protein, to inhibit HMG-CoA reductase activity. We established isolation and chromatography procedures to effectively obtain the protein with a purity above 95%. In silico predictions were performed to identify peptide sequences capable of interacting with HMG-CoA reductase. In vitro experiments showed that the fraction containing peptides of <3 kDa displayed inhibition of HMG-CoA reductase activity. The tripeptide QDF inhibits HMG-CoA reductase (IC 50  = 12.8 μM) in a dose-dependent manner. Furthermore, in silico studies revealed the binding profile of the QDF peptide and hinted at the molecular interactions that are responsible for its activity. Therefore, this study shows, for the first time, a peptide from cowpea β-vignin protein that inhibits HMG-CoA reductase and the chemical modifications that should be investigated to evaluate its binding profile. Copyright © 2018 Elsevier Ltd. All rights reserved.

  2. 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

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

    PubMed

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

    2005-09-01

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

  4. Towards a mechanistic and physiological understanding of a ferredoxin disulfide reductase from the domains Archaea and Bacteria.

    PubMed

    Prakash, Divya; Walters, Karim A; Martinie, Ryan J; McCarver, Addison C; Kumar, Adepu K; Lessner, Daniel J; Krebs, Carsten; Golbeck, John H; Ferry, James G

    2018-05-02

    Disulfide reductases reduce other proteins and are critically important for cellular redox signaling and homeostasis. Methanosarcina acetivorans is a methane-producing microbe from the domain Archaea that produces a ferredoxin:disulfide reductase (FDR) for which the crystal structure has been reported, yet its biochemical mechanism and physiological substrates are unknown. FDR and the extensively characterized plant-type ferredoxin:thioredoxin reductase (FTR) belong to a distinct class of disulfide reductases that contain a unique active-site [4Fe-4S] cluster. The results reported here support a mechanism for FDR similar to that reported for FTR with notable exceptions. Unlike FTR, FDR contains a rubredoxin [1Fe-0S] center postulated to mediate electron transfer from ferredoxin to the active-site [4Fe-4S] cluster.  UV-Vis, EPR and Mӧssbauer spectroscopic data indicated that two-electron reduction of the active-site disulfide in FDR involves a one-electron-reduced [4Fe-4S]1+ intermediate previously hypothesized for FTR. Our results support a role for an active-site tyrosine in FDR that occupies the equivalent position of an essential histidine in the active-site of FTR. Of note, one of seven Trxs encoded in the genome (Trx5) and methanoredoxin, a glutaredoxin-like enzyme from M. acetivorans, were reduced by FDR advancing the physiological understanding of FDRs role in the redox metabolism of methanoarchaea. Finally, bioinformatics analyses show FDR homologs are widespread in diverse microbes from the domain Bacteria. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

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

    PubMed

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

    1987-01-01

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

  6. Chloroquine Binding Reveals Flavin Redox Switch Function of Quinone Reductase 2*

    PubMed Central

    Leung, Kevin K. K.; Shilton, Brian H.

    2013-01-01

    Quinone reductase 2 (NQO2) is an FAD-linked enzyme and the only known human target of two antimalarial drugs, primaquine (PQ) and chloroquine (CQ). The structural differences between oxidized and reduced NQO2 and the structural basis for inhibition by PQ and CQ were investigated by x-ray crystallography. Structures of oxidized NQO2 in complex with PQ and CQ were solved at 1.4 Å resolution. CQ binds preferentially to reduced NQO2, and upon reduction of NQO2-CQ crystals, the space group changed from P212121 to P21, with 1-Å decreases in all three unit cell dimensions. The change in crystal packing originated in the negative charge and 4–5º bend in the reduced isoalloxazine ring of FAD, which resulted in a new mode of CQ binding and closure of a flexible loop (Phe126–Leu136) over the active site. This first structure of a reduced quinone reductase shows that reduction of the FAD cofactor and binding of a specific inhibitor lead to global changes in NQO2 structure and is consistent with a functional role for NQO2 as a flavin redox switch. PMID:23471972

  7. The endogenous adrenodoxin reductase-like flavoprotein arh1 supports heterologous cytochrome P450-dependent substrate conversions in Schizosaccharomyces pombe.

    PubMed

    Ewen, Kerstin M; Schiffler, Burkhard; Uhlmann-Schiffler, Heike; Bernhardt, Rita; Hannemann, Frank

    2008-05-01

    Mitochondrial cytochromes P450 are essential for biosynthesis of steroid hormones, vitamin D and bile acids. In mammals, the electrons needed for these reactions are provided via adrenodoxin and adrenodoxin reductase (AdR). Recently, Schizosaccharomyces pombe was introduced as a new host for the functional expression of human mitochondrial steroid hydroxylases without the coexpression of their natural redox partners. This fact qualifies S. pombe for the biotechnological production of steroids and for application as inhibitor test organism of heterologously expressed cytochromes P450. In this paper, we present evidence that the S. pombe ferredoxin reductase, arh1, and ferredoxin, etp1fd provide mammalian class I cytochromes P450 with reduction equivalents. The recombinant reductase showed an unusual weak binding of flavin adenine dinucleotide (FAD), which was mastered by modifying the FAD-binding region by site-directed mutagenesis yielding a stable holoprotein. The modified reductase arh1_A18G displayed spectroscopic characteristics similar to AdR and was shown to be capable of accepting electrons with no evident preference for NADH or NADPH, respectively. Arh1_A18G can substitute for AdR by interacting not only with its natural redox partner etp1fd but also with the mammalian homolog adrenodoxin. Cytochrome P450-dependent substrate conversion with all combinations of the mammalian and yeast redox proteins was evaluated in a reconstituted system.

  8. A site-saturated mutagenesis study of pentaerythritol tetranitrate reductase reveals that residues 181 and 184 influence ligand binding, stereochemistry and reactivity.

    PubMed

    Toogood, Helen S; Fryszkowska, Anna; Hulley, Martyn; Sakuma, Michiyo; Mansell, David; Stephens, Gill M; Gardiner, John M; Scrutton, Nigel S

    2011-03-21

    We have conducted a site-specific saturation mutagenesis study of H181 and H184 of flavoprotein pentaerythritol tetranitrate reductase (PETN reductase) to probe the role of these residues in substrate binding and catalysis with a variety of α,β-unsaturated alkenes. Single mutations at these residues were sufficient to dramatically increase the enantiopurity of products formed by reduction of 2-phenyl-1-nitropropene. In addition, many mutants exhibited a switch in reactivity to predominantly catalyse nitro reduction, as opposed to CC reduction. These mutants showed an enhancement in a minor side reaction and formed 2-phenylpropanal oxime from 2-phenyl-1-nitropropene. The multiple binding conformations of hydroxy substituted nitro-olefins in PETN reductase were examined by using both structural and catalytic techniques. These compounds were found to bind in both active and inhibitory complexes; this highlights the plasticity of the active site and the ability of the H181/H184 couple to coordinate with multiple functional groups. These properties demonstrate the potential to use PETN reductase as a scaffold in the development of industrially useful biocatalysts. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Thioredoxin Glutathione Reductase as a Novel Drug Target: Evidence from Schistosoma japonicum

    PubMed Central

    Xie, ShuYing; Qian, ChunYan; Wang, Jie; Zhang, Wei; Yin, XuRen; Hua, ZiChun; Yu, ChuanXin

    2012-01-01

    Background Schistosomiasis remains a major public health concern affecting billions of people around the world. Currently, praziquantel is the only drug of choice for treatment of human schistosomiasis. The emergence of drug resistance to praziquantel in schistosomes makes the development of novel drugs an urgent task. Thioredoxin glutathione reductase (TGR) enzymes in Schistosoma mansoni and some other platyhelminths have been identified as alternative targets. The present study was designed to confirm the existense and the potential value of TGR as a target for development of novel antischistosomal agents in Schistosoma japonicum, a platyhelminth endemic in Asia. Methods and Findings After cloning the S. japonicum TGR (SjTGR) gene, the recombinant SjTGR selenoprotein was purified and characterized in enzymatic assays as a multifunctional enzyme with thioredoxin reductase (TrxR), glutathione reductase (GR) and glutaredoxin (Grx) activities. Immunological and bioinformatic analyses confirmed that instead of having separate TrxR and GR proteins in mammalian, S. japonicum only encodes TGR, which performs the functions of both enzymes and plays a critical role in maintaining the redox balance in this parasite. These results were in good agreement with previous findings in Schistosoma mansoni and some other platyhelminths. Auranofin, a known inhibitor against TGR, caused fatal toxicity in S. japonicum adult worms in vitro and reduced worm and egg burdens in S. japonicum infected mice. Conclusions Collectively, our study confirms that a multifunctional enzyme SjTGR selenoprotein, instead of separate TrxR and GR enzymes, exists in S. japonicum. Furthermore, TGR may be a potential target for development of novel agents against schistosomes. This assumption is strengthened by our demonstration that the SjTGR is an essential enzyme for maintaining the thiol-disulfide redox homeostasis of S. japonicum. PMID:22384025

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

    PubMed

    Willies, Simon; Isupov, Misha; Littlechild, Jennifer

    2010-09-01

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

  11. Evaluation of potent inhibitors of dihydrofolate reductase in a culture model for growth of Pneumocystis carinii.

    PubMed

    Bartlett, M S; Shaw, M; Navaran, P; Smith, J W; Queener, S F

    1995-11-01

    Many antifolates are known to inhibit dihydrofolate reductase from murine Pneumocystis carinii, with 50% inhibitory concentrations (IC50s) ranging from 10(-4) to 10(-11) M. The relationship of the potency against isolated enzyme to the potency against intact murine P. carinii cells was explored with 17 compounds that had proven selectivity for or potency against P. carinii dihydrofolate reductase. Pyrimethamine and one analog were inhibitory to P. carinii in culture at concentrations two to seven times the IC50s for the enzyme, suggesting that the compounds may enter P. carinii cells in culture. Methotrexate was a potent inhibitor of P. carinii dihydrofolate reductase, but the concentrations effective in culture were more than 1,000-fold higher than IC50s for the enzyme, since P. carinii lacks an uptake system for methotrexate. Analogs of methotrexate in which chlorine, bromine, or iodine was added to the phenyl ring had improved potency against the isolated enzyme but were markedly less effective in culture; polyglutamation also lowered the activity in culture but improved activity against the enzyme. Substitution of a naphthyl group for the phenyl group of methotrexate produced a compound with improved activity against the enzyme (IC50, 0.00019 microM) and excellent activity in culture (IC50, 0.1 microM). One trimetrexate analog in which an aspartate or a chlorine replaced two of the methoxy groups of trimetrexate was much more potent and was much more selective toward P. carinii dihydrofolate reductase than trimetrexate; this analog was also as active as trimetrexate in culture. These studies suggest that modifications of antifolate structures can be made that facilitate activity against intact organisms while maintaining the high degrees of potency and the selectivities of the agents can be made.

  12. Differential Operation of Dual Protochlorophyllide Reductases for Chlorophyll Biosynthesis in Response to Environmental Oxygen Levels in the Cyanobacterium Leptolyngbya boryana1

    PubMed Central

    Yamazaki, Shoji; Nomata, Jiro; Fujita, Yuichi

    2006-01-01

    Most oxygenic phototrophs, including cyanobacteria, have two structurally unrelated protochlorophyllide (Pchlide) reductases in the penultimate step of chlorophyll biosynthesis. One is light-dependent Pchlide reductase (LPOR) and the other is dark-operative Pchlide reductase (DPOR), a nitrogenase-like enzyme assumed to be sensitive to oxygen. Very few studies have been conducted on how oxygen-sensitive DPOR operates in oxygenic phototrophic cells. Here, we report that anaerobic conditions are required for DPOR to compensate for the loss of LPOR in cyanobacterial cells. An LPOR-lacking mutant of the cyanobacterium Leptolyngbya boryana (formerly Plectonema boryanum) failed to grow in high light conditions and this phenotype was overcome by cultivating it under anaerobic conditions (2% CO2/N2). The critical oxygen level enabling the mutant to grow in high light was determined to be 3% (v/v). Oxygen-sensitive Pchlide reduction activity was successfully detected as DPOR activity in cell-free extracts of anaerobically grown mutants, whereas activity was undetectable in the wild type. The content of two DPOR subunits, ChlL and ChlN, was significantly increased in mutant cells compared with wild type. This suggests that the increase in subunits stimulates the DPOR activity that is protected efficiently from oxygen by anaerobic environments, resulting in complementation of the loss of LPOR. These results provide important concepts for understanding how dual Pchlide reductases operate differentially in oxygenic photosynthetic cells grown under natural environments where oxygen levels undergo dynamic changes. The evolutionary implications of the coexistence of two Pchlide reductases are discussed. PMID:17028153

  13. 76 FR 22119 - Credit Watch Termination Initiative; Termination of Direct Endorsement (DE) Approval

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-04-20

    ... DEPARTMENT OF HOUSING AND URBAN DEVELOPMENT [Docket No. FR-5511-N-02] Credit Watch Termination Initiative; Termination of Direct Endorsement (DE) Approval AGENCY: Office of the Assistant Secretary for... (FHA) against HUD-approved mortgagees through the FHA Credit Watch Termination Initiative. This notice...

  14. 76 FR 53148 - Credit Watch Termination Initiative; Termination of Direct Endorsement (DE) Approval

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-08-25

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  15. 77 FR 5262 - Credit Watch Termination Initiative Termination of Direct Endorsement (DE) Approval

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-02-02

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  16. 77 FR 38817 - Credit Watch Termination Initiative; Termination of Direct Endorsement (DE) Approval

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-06-29

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  17. 76 FR 38407 - Credit Watch Termination Initiative; Termination of Direct Endorsement (DE) Approval

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-06-30

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  18. 75 FR 61165 - Credit Watch Termination Initiative Termination of Direct Endorsement (DE) Approval

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-10-04

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  19. 76 FR 4364 - Credit Watch Termination Initiative; Termination of Direct Endorsement (DE) Approval

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-01-25

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  20. 75 FR 67388 - Credit Watch Termination Initiative; Termination of Direct Endorsement (DE) Approval

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-11-02

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  1. Functional expression and characterization of recombinant NADPH-P450 reductase from Malassezia globosa.

    PubMed

    Lee, Hwayoun; Park, Hyoung-Goo; Lim, Young-Ran; Lee, Im-Soon; Kim, Beom Joon; Seong, Cheul-Hun; Chun, Young-Jin; Kim, Donghak

    2012-01-01

    Malassezia globosa is a common pathogenic fungus that causes skin diseases including dandruff and seborrheic dermatitis in humans. Analysis of its genome identified a gene (MGL_1677) coding for a putative NADPH-P450 reductase (NPR) to support the fungal cytochrome P450 enzymes. The heterologously expressed recombinant M. globosa NPR protein was purified, and its functional features were characterized. The purified protein generated a single band on SDS-PAGE at 80.74 kDa and had an absorption maximum at 452 nm, indicating its possible function as an oxidized flavin cofactor. It evidenced NADPH-dependent reducing activity for cytochrome c or nitroblue tetrazolium. Human P450 1A2 and 2A6 were able to successfully catalyze the O-deethylation of 7- ethoxyresorufin and the 7-hydroxylation of coumarin, respectively, with the support of the purified NPR. These results demonstrate that purified NPR is an orthologous reductase protein that supports cytochrome P450 enzymes in M. globosa.

  2. Thermal Dependence of the Apparent Km of Glutathione Reductases from Three Plant Species

    PubMed Central

    Mahan, James R.; Burke, John J.; Orzech, Karen A.

    1990-01-01

    The thermal dependencies of the apparent Km of the glutathione reductases from spinach (Spinacia oleracea L.) corn (Zea mays L.), and cucumber (Cucumis sativus L.) were determined. The apparent Km of the enzymes were found to vary up to 9-fold between 12.5 and 45°C. Values of the apparent Km in excess of 200% of the observed minimum are suggested to be detrimental to the normal function of the enzyme. We propose the term “thermal kinetic window” to describe to the range of temperatures over which the apparent Km of the glutathione reductase is within 200% of its minimum and suggest that it may be a useful indicator of the limits of thermal stress for a given species. The thermal kinetic windows determined in this study are: <16°C for spinach, 23 to 32°C for corn, and 35 to 41°C for cucumber. PMID:16667543

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

    PubMed

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

    1988-10-15

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

  4. 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. Copyright © 2013 Elsevier Ltd. All rights reserved.

  5. Saccharomyces cerevisiae RNA Polymerase I Terminates Transcription at the Reb1 Terminator In Vivo

    PubMed Central

    Reeder, Ronald H.; Guevara, Palmira; Roan, Judith G.

    1999-01-01

    We have mapped transcription termination sites for RNA polymerase I in the yeast Saccharomyces cerevisiae. S1 nuclease mapping shows that the primary terminator is the Reb1p terminator located at +93 downstream of the 3′ end of 25S rRNA. Reverse transcription coupled with quantitative PCR shows that approximately 90% of all transcripts terminate at this site. Transcripts which read through the +93 site quantitatively terminate at a fail-safe terminator located further downstream at +250. Inactivation of Rnt1p (an RNase III involved in processing the 3′ end of 25S rRNA) greatly stabilizes transcripts extending to both sites and increases readthrough at the +93 site. In vivo assay of mutants of the Reb1p terminator shows that this site operates in vivo by the same mechanism as has previously been delineated through in vitro studies. PMID:10523625

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

    PubMed

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

    2015-06-01

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

  7. Likely gains in life expectancy of patients with coronary artery disease treated with HMG-CoA reductase inhibitors, as predicted by a decision analysis model.

    PubMed

    Kellett, J

    1997-07-01

    To estimate the likely gains in life expectancy of patients with coronary artery disease treated with HMG-CoA reductase inhibitors based on published reports and the results of the 4S and the West of Scotland Study. Decision analysis. Four likely scenarios of the effect of treatment with HMG-CoA reductase inhibitors on the life expectancy of medically and surgically managed coronary artery disease were modelled. Regardless of the scenario, treatment with HMG-CoA reductase inhibitors was estimated to provide a gain in life expectancy for medically managed patients of all ages with coronary artery disease, ranging from 4.6 to 10.1 quality adjusted life years (QALYs) for a 40 year old with three vessel disease (depending on the scenario assumed), to 0.2 QALYs for a 80 year old with two vessel disease. These gains were always greater than those predicted after bypass alone. If the use of HMG-CoA reductase inhibitors produces the same reduction in cardiac mortality after bypass as it does in medically managed patients it will increase the benefits of operation except for patients with two vessel disease over 70 years of age. Conversely, if HMG-CoA reductase inhibitors do not influence the course of coronary artery disease after bypass, the benefits of operation over medical treatment with HMG-CoA reductase inhibitors are either reduced or lost completely, ranging from a loss of -5.6 QALYs for a 40 year old with two vessel disease to a gain of 1.5 QALYs for 55 to 60 year old patients with left main stem disease. Although their effect on the progression of coronary artery disease after bypass must be defined, it is probable that HMG-CoA reductase inhibitors will produce considerable gains in life expectancy for patients with coronary artery disease.

  8. Graphics Software For VT Terminals

    NASA Technical Reports Server (NTRS)

    Wang, Caroline

    1991-01-01

    VTGRAPH graphics software tool for DEC/VT computer terminal or terminals compatible with it, widely used by government and industry. Callable in FORTRAN or C language, library program enabling user to cope with many computer environments in which VT terminals used for window management and graphic systems. Provides PLOT10-like package plus color or shade capability for VT240, VT241, and VT300 terminals. User can easily design more-friendly user-interface programs and design PLOT10 programs on VT terminals with different computer systems. Requires ReGis graphics set terminal and FORTRAN compiler.

  9. 7 CFR 947.71 - Termination.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Marketing Agreements... Effective Time and Termination § 947.71 Termination. (a) The Secretary may at any time terminate the... of such potatoes produced for market; but such termination shall be effective only if announced on or...

  10. Presence of the 5,10-methylenetetrahydrofolate reductase C677T mutation in Puerto Rican patients with neural tube defects.

    PubMed

    García-Fragoso, Lourdes; García-García, Inés; de la Vega, Alberto; Renta, Jessicca; Cadilla, Carmen L

    2002-01-01

    Folic acid supplementation can reduce the incidence of neural tube defects. The first reported genetic risk factor for neural tube defects is a C677T mutation in the 5,10-methylenetetrahydrofolate reductase gene, resulting in decreased activity of the enzyme. We examined the enzyme mutation role of methylenetetrahydrofolate reductase in the etiology of neural tube defects in our population. The study group consisted of 204 Puerto Rican individuals including 37 pregnant females with a prenatal diagnosis of neural tube defects in their fetuses, 31 newborns, 36 fathers, and 100 healthy adults. The prevalence of the C677T mutation was examined. Homozygosity for the alanine to valine substitution (TT) was observed in 9% of the controls and 19% of the mothers with children with neural tube defects. Our results indicate that the presence of the T allele at the methylenetetrahydrofolate reductase 677 position may increase the risk of giving birth to an infant with a neural tube defect.

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

    PubMed Central

    de Calais, Flávia Leme; Soardi, Fernanda Caroline; Petroli, Reginaldo José; Lusa, Ana Letícia Gori; de Paiva e Silva, Roberto Benedito; Maciel-Guerra, Andréa Trevas; Guerra-Júnior, Gil; de Mello, Maricilda Palandi

    2011-01-01

    The steroid 5α-reductase type II enzyme catalyzes the conversion of testosterone (T) to dihydrotestosterone (DHT), and its deficiency leads to undervirilization in 46,XY individuals, due to an impairment of this conversion in genital tissues. Molecular analysis in the steroid 5α-reductase type II gene (SRD5A2) was performed in two 46,XY female siblings. SRD5A2 gene sequencing revealed that the patients were homozygous for p.Gln126Arg missense mutation, which results from the CGA > CAA nucleotide substitution. The molecular result confirmed clinical diagnosis of 46,XY disorder of sex development (DSD) for the older sister and directed the investigation to other family members. Studies on SRD5A2 protein structure showed severe changes at NADPH binding region indicating that structural modeling analysis can be useful to evaluate the deleterious role of a mutation as causing 5α-reductase type II enzyme deficiency. PMID:22272144

  12. Terminating Devices in Spoken French.

    ERIC Educational Resources Information Center

    Andrews, Barry J.

    1989-01-01

    A study examines the way in which one group of discourse connectors, terminators, function in contemporary spoken French. Three types of terminators, elements used at the end of an utterance or section to indicate its completion, are investigated, including utterance terminators, interrogative tags, and terminal tags. (Author/MSE)

  13. 20 CFR 435.61 - Termination.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... INSTITUTIONS OF HIGHER EDUCATION, HOSPITALS, OTHER NON-PROFIT ORGANIZATIONS, AND COMMERCIAL ORGANIZATIONS Post... the effective date and, in the case of partial termination, the portion to be terminated. (3) By the... effective date, and, in the case of partial termination, the portion to be terminated. However, if SSA...

  14. Passive regeneration of glutathione: glutathione reductase regulation in the freeze-tolerant North American wood frog, Rana sylvatica.

    PubMed

    Dawson, Neal J; Storey, Kenneth B

    2017-09-01

    Wood frogs inhabit a broad range across North America, extending from the southern tip of the Appalachian Mountains to the northern boreal forest. Remarkably, they can survive the winter in a frozen state, where as much as 70% of their body water is converted into ice. Whilst in the frozen state, their hearts cease to pump blood, causing their cells to experience ischemia, which can dramatically increase the production of reactive oxygen species within the cell. To overcome this, wood frogs have elevated levels of glutathione, a primary antioxidant. We examined the regulation of glutathione reductase, the enzyme involved in recycling glutathione, in both the frozen and unfrozen (control) state. Glutathione reductase activity from both the control and frozen state showed a dramatic reduction in substrate specificity ( K m ) for oxidized glutathione (50%) when measured in the presence of glucose (300 mmol l -1 ) and a increase (157%) when measured in the presence of levels of urea (75 mmol l -1 ) encountered in the frozen state. However, when we tested the synergistic effect of urea and glucose simultaneously, we observed a substantial reduction in the K m for oxidized glutathione (43%) to a value similar to that with glucose alone. In fact, we found no observable differences in the kinetic and structural properties of glutathione reductase between the two states. Therefore, a significant increase in the affinity for oxidized glutathione in the presence of endogenous levels of glucose suggests that increased glutathione recycling may occur as a result of passive regulation of glutathione reductase by rising levels of glucose during freezing. © 2017. Published by The Company of Biologists Ltd.

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

    PubMed

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

    2010-01-25

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

  16. 43 CFR 12.961 - Termination.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... Institutions of Higher Education, Hospitals, and Other Non-Profit Organizations Post-Award Requirements § 12..., including the effective date and, in the case of partial termination, the portion to be terminated. (3) By... reasons for such termination, the effective date, and, in the case of partial termination, the portion to...

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

    PubMed

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

    2015-01-01

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

  18. Functional properties and structural characterization of rice δ 1-pyrroline-5-carboxylate reductase

    DOE PAGES

    Forlani, Giuseppe; Bertazzini, Michele; Zarattini, Marco; ...

    2015-07-28

    The majority of plant species accumulate high intracellular levels of proline to cope with hyperosmotic stress conditions. Proline synthesis from glutamate is tightly regulated at both the transcriptional and the translational levels, yet little is known about the mechanisms for post-translational regulation of the enzymatic activities involved. The gene coding in rice ( Oryza sativa L.) for δ 1-pyrroline-5-carboxylate (P5C) reductase, the enzyme that catalyzes the second and final step in this pathway, was isolated and expressed in Escherichia coli. The structural and functional properties of the affinity-purified protein were characterized. As for most species, rice P5C reductase was ablemore » to use in vitro either NADH or NADPH as the electron donor. However, strikingly different effects of cations and anions were found depending on the pyridine nucleotide used, namely inhibition of NADH-dependent activity and stimulation of NADPH-dependent activity. Moreover, physiological concentrations of proline and NADP + were strongly inhibitory for the NADH-dependent reaction, whereas the NADPH-dependent activity was mildly affected. Our results suggest that only NADPH may be used in vivo and that stress-dependent variations in ion homeostasis and NADPH/NADP + ratio could modulate enzyme activity, being functional in promoting proline accumulation and potentially also adjusting NADPH consumption during the defense against hyperosmotic stress. The apparent molecular weight of the native protein observed in size exclusion chromatography indicated a high oligomerization state. We also report the first crystal structure of a plant P5C reductase at 3.40-Å resolution, showing a decameric quaternary assembly. It was possible to identify dynamic structural differences among rice, human, and bacterial enzymes.« less

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

    PubMed

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

    2013-11-01

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

  20. Stimulatory effect of insulin on 5alpha-reductase type 1 (SRD5A1) expression through an Akt-dependent pathway in ovarian granulosa cells.

    PubMed

    Kayampilly, Pradeep P; Wanamaker, Brett L; Stewart, James A; Wagner, Carrie L; Menon, K M J

    2010-10-01

    Elevated levels of 5α-reduced androgens have been shown to be associated with hyperandrogenism and hyperinsulinemia, the leading causes of ovulatory dysfunction in women. 5α-Dihydrotestosterone reduces ovarian granulosa cell proliferation by inhibiting FSH-mediated mitogenic signaling pathways. The present study examined the effect of insulin on 5α-reductase, the enzyme that catalyses the conversion of androgens to their 5α-derivatives. Granulosa cells isolated from immature rat ovaries were cultured in serum-free, phenol red-free DMEM-F12 media and treated with different doses of insulin (0, 0.1, 1.0, and 10.0 μg/ml) for different time intervals up to 12 h. The expression of 5α-reductase type 1 mRNA, the predominant isoform found in granulosa cells, showed a significant (P<0.05) increase in response to the insulin treatment up to 12 h compared with control. The catalytic activity of 5α-reductase enzyme was also stimulated in a dose-depended manner (P<0.05). Inhibiting the Akt-dependent signaling pathway abolished the insulin-mediated increase in 5α-reductase mRNA expression, whereas inhibition of the ERK-dependent pathway had no effect. The dose-dependent increase in 5α-reductase mRNA expression as well as catalytic activity seen in response to insulin treatment was also demonstrated in the human granulosa cell line (KGN). In addition to increased mRNA expression, a dose-dependent increase in 5α-reductase protein expression in response to insulin was also seen in KGN cells, which corroborated well with that of mRNA expression. These results suggest that elevated levels of 5α-reduced androgens seen in hyperinsulinemic conditions might be explained on the basis of a stimulatory effect of insulin on 5α-reductase in granulosa cells. The elevated levels of these metabolites, in turn, might adversely affect growth and proliferation of granulosa cells, thereby impairing follicle growth and ovulation.

  1. Stimulatory Effect of Insulin on 5α-Reductase Type 1 (SRD5A1) Expression through an Akt-Dependent Pathway in Ovarian Granulosa Cells

    PubMed Central

    Kayampilly, Pradeep P.; Wanamaker, Brett L.; Stewart, James A.; Wagner, Carrie L.; Menon, K. M. J.

    2010-01-01

    Elevated levels of 5α-reduced androgens have been shown to be associated with hyperandrogenism and hyperinsulinemia, the leading causes of ovulatory dysfunction in women. 5α-Dihydrotestosterone reduces ovarian granulosa cell proliferation by inhibiting FSH-mediated mitogenic signaling pathways. The present study examined the effect of insulin on 5α-reductase, the enzyme that catalyses the conversion of androgens to their 5α-derivatives. Granulosa cells isolated from immature rat ovaries were cultured in serum-free, phenol red-free DMEM-F12 media and treated with different doses of insulin (0, 0.1, 1.0, and 10.0 μg/ml) for different time intervals up to 12 h. The expression of 5α-reductase type 1 mRNA, the predominant isoform found in granulosa cells, showed a significant (P < 0.05) increase in response to the insulin treatment up to 12 h compared with control. The catalytic activity of 5α-reductase enzyme was also stimulated in a dose-depended manner (P < 0.05). Inhibiting the Akt-dependent signaling pathway abolished the insulin-mediated increase in 5α-reductase mRNA expression, whereas inhibition of the ERK-dependent pathway had no effect. The dose-dependent increase in 5α-reductase mRNA expression as well as catalytic activity seen in response to insulin treatment was also demonstrated in the human granulosa cell line (KGN). In addition to increased mRNA expression, a dose-dependent increase in 5α-reductase protein expression in response to insulin was also seen in KGN cells, which corroborated well with that of mRNA expression. These results suggest that elevated levels of 5α-reduced androgens seen in hyperinsulinemic conditions might be explained on the basis of a stimulatory effect of insulin on 5α-reductase in granulosa cells. The elevated levels of these metabolites, in turn, might adversely affect growth and proliferation of granulosa cells, thereby impairing follicle growth and ovulation. PMID:20810561

  2. Novel thioredoxin-related transmembrane protein TMX4 has reductase activity.

    PubMed

    Sugiura, Yoshimi; Araki, Kazutaka; Iemura, Shun-ichiro; Natsume, Tohru; Hoseki, Jun; Nagata, Kazuhiro

    2010-03-05

    In the endoplasmic reticulum (ER), a number of thioredoxin (Trx) superfamily proteins are present to enable correct disulfide bond formation of secretory and membrane proteins via Trx-like domains. Here, we identified a novel transmembrane Trx-like protein 4 (TMX4), in the ER of mammalian cells. TMX4, a type I transmembrane protein, was localized to the ER and possessed a Trx-like domain that faced the ER lumen. A maleimide alkylation assay showed that a catalytic CXXC motif in the TMX4 Trx-like domain underwent changes in its redox state depending on cellular redox conditions, and, in the normal state, most of the endogenous TMX4 existed in the oxidized form. Using a purified recombinant protein containing the Trx-like domain of TMX4 (TMX4-Trx), we confirmed that this domain had reductase activity in vitro. The redox potential of this domain (-171.5 mV; 30 degrees C at pH 7.0) indicated that TMX4 could work as a reductase in the environment of the ER. TMX4 had no effect on the acceleration of ER-associated degradation. Because TMX4 interacted with calnexin and ERp57 by co-immunoprecipitation assay, the role of TMX4 may be to enable protein folding in cooperation with these proteins consisting of folding complex in the ER.

  3. 14 CFR 1214.108 - Termination.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... NASA. (1) The termination fee for dedicated flights will be computed as a percentage of the Shuttle... Space Shuttle Flights of Payloads for Non-U.S. Government, Reimbursable Customers § 1214.108 Termination... termination occurs Termination fee, percent of Shuttle standard flight price 18 or more 10 17 or more but less...

  4. 14 CFR 1214.108 - Termination.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... NASA. (1) The termination fee for dedicated flights will be computed as a percentage of the Shuttle... Space Shuttle Flights of Payloads for Non-U.S. Government, Reimbursable Customers § 1214.108 Termination... termination occurs Termination fee, percent of Shuttle standard flight price 18 or more 10 17 or more but less...

  5. 14 CFR 1214.108 - Termination.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... NASA. (1) The termination fee for dedicated flights will be computed as a percentage of the Shuttle... Space Shuttle Flights of Payloads for Non-U.S. Government, Reimbursable Customers § 1214.108 Termination... termination occurs Termination fee, percent of Shuttle standard flight price 18 or more 10 17 or more but less...

  6. 14 CFR 1214.108 - Termination.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... NASA. (1) The termination fee for dedicated flights will be computed as a percentage of the Shuttle... Space Shuttle Flights of Payloads for Non-U.S. Government, Reimbursable Customers § 1214.108 Termination... termination occurs Termination fee, percent of Shuttle standard flight price 18 or more 10 17 or more but less...

  7. Modeling O₂-dependent effects of nitrite reductase activity in blood and tissue on coupled NO and O₂ transport around arterioles.

    PubMed

    Buerk, Donald G; Barbee, Kenneth A; Jaron, Dov

    2011-01-01

    Recent evidence in the literature suggests that tissues play a greater role than blood in reducing nitrite to NO under ischemic or hypoxic conditions. Our previous mathematical model for coupled NO and O(2) transport around an arteriole, modified to include superoxide generation from dysfunctional endothelium, was developed further to include nitrite reductase activity in blood and tissue. Steady-state radial and axial NO and pO(2) profiles in the arteriole and surrounding tissue were simulated for different blood flow rates and arterial blood pO(2) values. The resulting computer simulations demonstrate that nitrite reductase activity in blood is not a very effective mechanism for conserving NO due to the strong scavenging of NO by hemoglobin. In contrast, nitrite reductase activity in tissue is much more effective in increasing NO bioavailability in the vascular wall and contributes progressively more NO as tissue hypoxia becomes more severe.

  8. 46 CFR 525.2 - Terminal schedules.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... scrap, new assembled motor vehicles, waste paper and paper waste in terminal schedules. (2) Marine... MARITIME COMMISSION REGULATIONS AFFECTING OCEAN SHIPPING IN FOREIGN COMMERCE MARINE TERMINAL OPERATOR SCHEDULES § 525.2 Terminal schedules. (a) Marine terminal operator schedules. A marine terminal operator, at...

  9. 46 CFR 525.2 - Terminal schedules.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... scrap, new assembled motor vehicles, waste paper and paper waste in terminal schedules. (2) Marine... MARITIME COMMISSION REGULATIONS AFFECTING OCEAN SHIPPING IN FOREIGN COMMERCE MARINE TERMINAL OPERATOR SCHEDULES § 525.2 Terminal schedules. (a) Marine terminal operator schedules. A marine terminal operator, at...

  10. Holo Structure and Steady State Kinetics of the Thiazolinyl Imine Reductases for Siderophore Biosynthesis

    PubMed Central

    Meneely, Kathleen M.; Ronnebaum, Trey A.; Riley, Andrew P.; Prisinzano, Thomas E.; Lamb, Audrey L.

    2016-01-01

    Thiazolinyl imine reductases catalyze the NADPH-dependent reduction of a thiazoline to a thiazolidine, a required step in the formation of the siderophores yersiniabactin (Yersinia spp.) and pyochelin (Pseudomonas aeruginosa). These stand-alone nonribosomal peptide tailoring domains are structural homologues of sugar oxidoreductases. Two closed structures of the thiazolinyl imine reductase from Yersinia enterocolitica (Irp3) are presented here: an NADP+-bound structure to 1.45 Å resolution and a holo structure to 1.28 Å resolution with NADP+ and a substrate analogue bound. Michaelis—Menten kinetics were measured using the same substrate analogue and the homologue from P. aeruginosa, PchG. The data presented here support the hypothesis that tyrosine 128 is the likely general acid residue for catalysis and also highlight the phosphopantetheine tunnel for tethering of the substrate to the nonribosomal peptide synthetase module during assembly line biosynthesis of the siderophore. PMID:27601130

  11. A substrate radical intermediate in the reaction between ribonucleotide reductase from Escherichia coli and 2'-azido-2'-deoxynucleoside diphosphates.

    PubMed

    Sjöberg, B M; Gräslund, A; Eckstein, F

    1983-07-10

    The B2 subunit of ribonucleotide reductase from Escherichia coli contains a tyrosine radical which is essential for enzyme activity. In the reaction between ribonucleotide reductase and the substrate analogue 2'-azido-2'-deoxycytidine 5'-diphosphate a new transient radical is formed. The EPR characteristics of this new radical species are consistent with a localization of the unpaired electron at the sugar moiety of the nucleotide. The radical shows hyperfine couplings to a hydrogen and a nitrogen nucleus, the latter probably being part of the azide substituent. The formation of the nucleotide radical in this suicidal reaction is concomitant with the decay of the tyrosine radical of the B2 subunit. Kinetic data argue for a first (pseudosecond) order decay of the B2 radical via generation of the nucleotide radical followed by a slower first order decay of the nucleotide radical. End products in the reaction are cytosine and radical-free protein B2. In the reaction between bacteriophage T4 ribonucleotide reductase and 2'-azido-2'-deoxycytidine 5'-diphosphate an identical nucleotide radical is formed. The present results are consistent with the hypothesis that the appearance and structure of the transient radical mimic stages in the normal reaction pathway of ribonucleotide reductase, postulated to proceed via 3'-hydrogen abstraction and cation radical formation of the substrate nucleotide (Stubbe, J., and Ackles, D. (1980) J. Biol. Chem. 255, 8027-8030). The nucleotide radical described here might be equivalent to such a cation radical intermediate.

  12. Ferric reductase activity of low molecular weight human milk fraction is associated with enhanced iron solubility and uptake in Caco-2 cells.

    PubMed

    Pullakhandam, Raghu; Nair, Madhavan Krishnapillai; Kasula, Sunanda; Kilari, Sreenivasulu; Thippande, Tippeswamy Gowda

    2008-09-19

    It is known that the fractional absorption of extrinsic iron from human milk is higher in infants and adults. A low molecular weight milk fraction has been proposed to increase the bioavailability of iron from human milk. Nevertheless, the mechanisms remained elusive. Here in we demonstrate ferric reductase activity (Km7.73x10(-6)M) in low molecular weight human milk fraction (10kF, filtrate derived from ultra filtration of milk whey through 10kDa cutoff membrane), which increased ferric iron solubility and iron uptake in Caco-2 cells. The 10kF fraction was as effective as ascorbic acid (1:20 iron to ascorbic acid) in increasing the ferric iron solubility and uptake in Caco-2 cells. Further, gel filtration chromatography on peptide column led to co-elution of ferric reductase and iron solubilization activities at an apparent molecular mass of <1500Da. Interestingly, only these fractions containing ferric reductase activity also stimulated the uptake of iron in Caco-2 cells. Thus, it is concluded that human milk possesses ferric reductase activity and is associated with ferric iron solubilization and enhanced absorption.

  13. Mechanism of ribonucleotide reductase from Herpes simplex virus type 1. Evidence for 3' carbon-hydrogen bond cleavage and inactivation by nucleotide analogs

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ator, M.A.; Stubbe, J.; Spector, T.

    1986-03-15

    Isotope effects of 2.5, 2.1, and 1.0 were measured on the conversion of (3'-3H)ADP, (3'-H)UDP, and (5-3H) UDP to the corresponding 2'-deoxynucleotides by herpes simplex virus type 1 ribonucleotide reductase. These results indicate that the reduction of either purine or pyrimidine nucleotides requires cleavage of the 3' carbon-hydrogen bond of the substrate. The substrate analogs 2'-chloro-2'-deoxyuridine 5'-diphosphate (ClUDP), 2'-deoxy-2'-fluorouridine 5'-diphosphate, and 2'-azido-2'-deoxyuridine 5'-diphosphate were time-dependent inactivators of the herpes simplex virus type 1 ribonucleotide reductase. Incubation of (3'-3H)ClUDP with the enzyme was accompanied by time-dependent release of 3H to the solvent. Reaction of (beta-32P)ClUDP with the reductase resulted in themore » production of inorganic pyrophosphate. These results are consistent with the enzyme-mediated cleavage of the 3' carbon-hydrogen bond of ClUDP and the subsequent conversion of the nucleotide to 2-methylene-3(2H)furanone, as previously reported with the Escherichia coli ribonucleotide reductase.« less

  14. Spectroscopic Definition of the CuZ° Intermediate in Turnover of Nitrous Oxide Reductase and Molecular Insight into the Catalytic Mechanism.

    PubMed

    Johnston, Esther M; Carreira, Cíntia; Dell'Acqua, Simone; Dey, Somdatta Ghosh; Pauleta, Sofia R; Moura, Isabel; Solomon, Edward I

    2017-03-29

    Spectroscopic methods and density functional theory (DFT) calculations are used to determine the geometric and electronic structure of Cu Z °, an intermediate form of the Cu 4 S active site of nitrous oxide reductase (N 2 OR) that is observed in single turnover of fully reduced N 2 OR with N 2 O. Electron paramagnetic resonance (EPR), absorption, and magnetic circular dichroism (MCD) spectroscopies show that Cu Z ° is a 1-hole (i.e., 3Cu I Cu II ) state with spin density delocalized evenly over Cu I and Cu IV . Resonance Raman spectroscopy shows two Cu-S vibrations at 425 and 413 cm -1 , the latter with a -3 cm -1 O 18 solvent isotope shift. DFT calculations correlated to these spectral features show that Cu Z ° has a terminal hydroxide ligand coordinated to Cu IV , stabilized by a hydrogen bond to a nearby lysine residue. Cu Z ° can be reduced via electron transfer from Cu A using a physiologically relevant reductant. We obtain a lower limit on the rate of this intramolecular electron transfer (IET) that is >10 4 faster than the unobserved IET in the resting state, showing that Cu Z ° is the catalytically relevant oxidized form of N 2 OR. Terminal hydroxide coordination to Cu IV in the Cu Z ° intermediate yields insight into the nature of N 2 O binding and reduction, specifying a molecular mechanism in which N 2 O coordinates in a μ-1,3 fashion to the fully reduced state, with hydrogen bonding from Lys397, and two electrons are transferred from the fully reduced μ 4 S 2- bridged tetranuclear copper cluster to N 2 O via a single Cu atom to accomplish N-O bond cleavage.

  15. Subcellular Localization of Arabidopsis 3-Hydroxy-3-Methylglutaryl-Coenzyme A Reductase1

    PubMed Central

    Leivar, Pablo; González, Víctor M.; Castel, Susanna; Trelease, Richard N.; López-Iglesias, Carmen; Arró, Montserrat; Boronat, Albert; Campos, Narciso; Ferrer, Albert; Fernàndez-Busquets, Xavier

    2005-01-01

    Plants produce diverse isoprenoids, which are synthesized in plastids, mitochondria, endoplasmic reticulum (ER), and the nonorganellar cytoplasm. 3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) catalyzes the synthesis of mevalonate, a rate-limiting step in the cytoplasmic pathway. Several branches of the pathway lead to the synthesis of structurally and functionally varied, yet essential, isoprenoids. Several HMGR isoforms have been identified in all plants examined. Studies based on gene expression and on fractionation of enzyme activity suggested that subcellular compartmentalization of HMGR is an important intracellular channeling mechanism for the production of the specific classes of isoprenoids. Plant HMGR has been shown previously to insert in vitro into the membrane of microsomal vesicles, but the final in vivo subcellular localization(s) remains controversial. To address the latter in Arabidopsis (Arabidopsis thaliana) cells, we conducted a multipronged microscopy and cell fractionation approach that included imaging of chimeric HMGR green fluorescent protein localizations in transiently transformed cell leaves, immunofluorescence confocal microscopy in wild-type and stably transformed seedlings, immunogold electron microscopy examinations of endogenous HMGR in seedling cotyledons, and sucrose density gradient analyses of HMGR-containing organelles. Taken together, the results reveal that endogenous Arabidopsis HMGR is localized at steady state within ER as expected, but surprisingly also predominantly within spherical, vesicular structures that range from 0.2- to 0.6-μm diameter, located in the cytoplasm and within the central vacuole in differentiated cotyledon cells. The N-terminal region, including the transmembrane domain of HMGR, was found to be necessary and sufficient for directing HMGR to ER and the spherical structures. It is believed, although not directly demonstrated, that these vesicle-like structures are derived from segments of HMGR

  16. Enhancement of Ganoderic Acid Accumulation by Overexpression of an N-Terminally Truncated 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Gene in the Basidiomycete Ganoderma lucidum

    PubMed Central

    Xu, Jun-Wei; Xu, Yi-Ning

    2012-01-01

    Ganoderic acids produced by Ganoderma lucidum, a well-known traditional Chinese medicinal mushroom, exhibit antitumor and antimetastasis activities. Genetic modification of G. lucidum is difficult but critical for the enhancement of cellular accumulation of ganoderic acids. In this study, a homologous genetic transformation system for G. lucidum was developed for the first time using mutated sdhB, encoding the iron-sulfur protein subunit of succinate dehydrogenase, as a selection marker. The truncated G. lucidum gene encoding the catalytic domain of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) was overexpressed by using the Agrobacterium tumefaciens-mediated transformation system. The results showed that the mutated sdhB successfully conferred carboxin resistance upon transformation. Most of the integrated transfer DNA (T-DNA) appeared as a single copy in the genome. Moreover, deregulated constitutive overexpression of the HMGR gene led to a 2-fold increase in ganoderic acid content. It also increased the accumulation of intermediates (squalene and lanosterol) and the upregulation of downstream genes such as those of farnesyl pyrophosphate synthase, squalene synthase, and lanosterol synthase. This study demonstrates that transgenic basidiomycete G. lucidum is a promising system to achieve metabolic engineering of the ganoderic acid pathway. PMID:22941092

  17. Identification of a Noroxomaritidine Reductase with Amaryllidaceae Alkaloid Biosynthesis Related Activities*

    PubMed Central

    Kilgore, Matthew B.; Holland, Cynthia K.; Jez, Joseph M.; Kutchan, Toni M.

    2016-01-01

    Amaryllidaceae alkaloids are a large group of plant natural products with over 300 documented structures and diverse biological activities. Several groups of Amaryllidaceae alkaloids including the hemanthamine- and crinine-type alkaloids show promise as anticancer agents. Two reduction reactions are required for the production of these compounds: the reduction of norcraugsodine to norbelladine and the reduction of noroxomaritidine to normaritidine, with the enantiomer of noroxomaritidine dictating whether the derivatives will be the crinine-type or hemanthamine-type. It is also possible for the carbon-carbon double bond of noroxomaritidine to be reduced, forming the precursor for maritinamine or elwesine depending on the enantiomer reduced to an oxomaritinamine product. In this study, a short chain alcohol dehydrogenase/reductase that co-expresses with the previously discovered norbelladine 4′-O-methyltransferase from Narcissus sp. and Galanthus spp. was cloned and expressed in Escherichia coli. Biochemical analyses and x-ray crystallography indicates that this protein functions as a noroxomaritidine reductase that forms oxomaritinamine from noroxomaritidine through a carbon-carbon double bond reduction. The enzyme also reduces norcraugsodine to norbelladine with a 400-fold lower specific activity. These studies identify a missing step in the biosynthesis of this pharmacologically important class of plant natural products. PMID:27252378

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

    PubMed

    Alshogran, Osama Y

    2017-10-01

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

  19. Nonleaking battery terminals.

    NASA Technical Reports Server (NTRS)

    Snider, W. E.; Nagle, W. J.

    1972-01-01

    Three different terminals were designed for usage in a 40 ampere/hour silver zinc battery which has a 45% KOH by weight electrolyte in a plastic battery case. Life tests, including thermal cycling, electrical charge and discharge for up to three years duration, were conducted on these three different terminal designs. Tests for creep rate and tensile strength were conducted on the polyphenylene oxide plastic battery cases. Some cases were unused and others containing KOH electrolyte were placed on life tests. The design and testing of nonleaking battery terminals for use with a KOH electrolyte in a plastic case are considered.

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

    PubMed Central

    Vasconcelos, Marta W.; Clemente, Thomas E.; Grusak, Michael A.

    2014-01-01

    Iron is an important micronutrient in human and plant nutrition. Adequate iron nutrition during crop production is central for assuring appropriate iron concentrations in the harvestable organs, for human food or animal feed. The whole-plant movement of iron involves several processes, including the reduction of ferric to ferrous iron at several locations throughout the plant, prior to transmembrane trafficking of ferrous iron. In this study, soybean plants that constitutively expressed the AtFRO2 iron reductase gene were analyzed for leaf iron reductase activity, as well as the effect of this transgene’s expression on root, leaf, pod wall, and seed mineral concentrations. High Fe supply, in combination with the constitutive expression of AtFRO2, resulted in significantly higher concentrations of different minerals in roots (K, P, Zn, Ca, Ni, Mg, and Mo), pod walls (Fe, K, P, Cu, and Ni), leaves (Fe, P, Cu, Ca, Ni, and Mg) and seeds (Fe, Zn, Cu, and Ni). Leaf and pod wall iron concentrations increased as much as 500% in transgenic plants, while seed iron concentrations only increased by 10%, suggesting that factors other than leaf and pod wall reductase activity were limiting the translocation of iron to seeds. Protoplasts isolated from transgenic leaves had three-fold higher reductase activity than controls. Expression levels of the iron storage protein, ferritin, were higher in the transgenic leaves than in wild-type, suggesting that the excess iron may be stored as ferritin in the leaves and therefore unavailable for phloem loading and delivery to the seeds. Also, citrate and malate levels in the roots and leaves of transgenic plants were significantly higher than in wild-type, suggesting that organic acid production could be related to the increased accumulation of minerals in roots, leaves, and pod walls, but not in the seeds. All together, these results suggest a more ubiquitous role for the iron reductase in whole-plant mineral accumulation and

  1. Cloning, functional characterization, and expression profiles of NADPH-cytochrome P450 reductase gene from the Asiatic rice striped stem borer, Chilo suppressalis (Lepidoptera: Pyralidae).

    PubMed

    Liu, Su; Liang, Qing-Mei; Huang, Yuan-Jie; Yuan, Xin; Zhou, Wen-Wu; Qiao, Fei; Cheng, Jiaan; Gurr, Geoff M; Zhu, Zeng-Rong

    2013-01-01

    NADPH-cytochrome P450 reductase (CPR) is one of the most important components of the cytochrome P450 enzyme system. It catalyzes electron transfer from NADPH to all known P450s, thus plays central roles not only in the metabolism of exogenous xenobiotics but also in the regulation of endogenous hormones in insects. In this study, a full-length cDNA encoding of a CPR (named CsCPR) was isolated from the Asiatic rice striped stem borer, Chilo suppressalis, by using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods. The cDNA contains a 2061 bp open reading frame, which encodes an enzyme of 686 amino acid residues, with a calculated molecular mass of 77.6 kDa. The deduced peptide has hallmarks of typical CPR, including an N-terminal membrane anchor and the FMN, FAD and NADPH binding domains. The N-terminal-truncated protein fused with a 6 × His·tag was heterologously expressed in Escherichia coli Rosetta (DE3) cells and purified, specific activity and the Km values of the recombinant enzyme were determined. Tissue- and developmental stage-dependent expression of CsCPR mRNA was investigated by real-time quantitative PCR. The CsCPR mRNA was noticeably expressed in the digestive, metabolic, and olfactory organs of the larvae and adults of C. suppressalis. Our initial results would provide valuable information for further study on the interactions between CPR and cytochrome P450 enzyme systems. © 2013.

  2. Effects of cooperation between translating ribosome and RNA polymerase on termination efficiency of the Rho-independent terminator

    PubMed Central

    Li, Rui; Zhang, Qing; Li, Junbai; Shi, Hualin

    2016-01-01

    An experimental system was designed to measure in vivo termination efficiency (TE) of the Rho-independent terminator and position–function relations were quantified for the terminator tR2 in Escherichia coli. The terminator function was almost completely repressed when tR2 was located several base pairs downstream from the gene, and TE gradually increased to maximum values with the increasing distance between the gene and terminator. This TE–distance relation reflected a stochastic coupling of the ribosome and RNA polymerase (RNAP). Terminators located in the first 100 bp of the coding region can function efficiently. However, functional repression was observed when the terminator was located in the latter part of the coding region, and the degree of repression was determined by transcriptional and translational dynamics. These results may help to elucidate mechanisms of Rho-independent termination and reveal genomic locations of terminators and functions of the sequence that precedes terminators. These observations may have important applications in synthetic biology. PMID:26602687

  3. Outer membrane cytochromes/flavin interactions in Shewanella spp.—A molecular perspective

    DOE PAGES

    Babanova, Sofia; Matanovic, Ivana; Cornejo, Jose; ...

    2017-05-31

    Extracellular electron transfer (EET) is intrinsically associated with the core phenomena of energy harvesting/energy conversion in natural ecosystems and biotechnology applications. But, the mechanisms associated with EET are complex and involve molecular interactions that take place at the “bionano interface” where biotic/abiotic interactions are usually explored. Our work provides molecular perspective on the electron transfer mechanism(s) employed by Shewanella oneidensis MR-1. Molecular docking simulations were used to explain the interfacial relationships between two outer-membrane cytochromes (OMC) OmcA and MtrC and riboflavin (RF) and flavin mononucleotide (FMN), respectively. OMC-flavin interactions were analyzed by studying the electrostatic potential, the hydrophilic/hydrophobic surface properties,more » and the van der Waals surface of the OMC proteins. As a result, it was proposed that the interactions between flavins and OMCs are based on geometrical recognition event. The possible docking positions of RF and FMN to OmcA and MtrC were also shown.« less

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

    PubMed

    Stapelfeld, Claudia; Maser, Edmund

    2017-10-01

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

  5. Trigger loop dynamics can explain stimulation of intrinsic termination by bacterial RNA polymerase without terminator hairpin contact.

    PubMed

    Ray-Soni, Ananya; Mooney, Rachel A; Landick, Robert

    2017-10-31

    In bacteria, intrinsic termination signals cause disassembly of the highly stable elongating transcription complex (EC) over windows of two to three nucleotides after kilobases of RNA synthesis. Intrinsic termination is caused by the formation of a nascent RNA hairpin adjacent to a weak RNA-DNA hybrid within RNA polymerase (RNAP). Although the contributions of RNA and DNA sequences to termination are largely understood, the roles of conformational changes in RNAP are less well described. The polymorphous trigger loop (TL), which folds into the trigger helices to promote nucleotide addition, also is proposed to drive termination by folding into the trigger helices and contacting the terminator hairpin after invasion of the hairpin in the RNAP main cleft [Epshtein V, Cardinale CJ, Ruckenstein AE, Borukhov S, Nudler E (2007) Mol Cell 28:991-1001]. To investigate the contribution of the TL to intrinsic termination, we developed a kinetic assay that distinguishes effects of TL alterations on the rate at which ECs terminate from effects of the TL on the nucleotide addition rate that indirectly affect termination efficiency by altering the time window in which termination can occur. We confirmed that the TL stimulates termination rate, but found that stabilizing either the folded or unfolded TL conformation decreased termination rate. We propose that conformational fluctuations of the TL (TL dynamics), not TL-hairpin contact, aid termination by increasing EC conformational diversity and thus access to favorable termination pathways. We also report that the TL and the TL sequence insertion (SI3) increase overall termination efficiency by stimulating pausing, which increases the flux of ECs into the termination pathway. Published under the PNAS license.

  6. Trigger loop dynamics can explain stimulation of intrinsic termination by bacterial RNA polymerase without terminator hairpin contact

    PubMed Central

    Ray-Soni, Ananya; Mooney, Rachel A.; Landick, Robert

    2017-01-01

    In bacteria, intrinsic termination signals cause disassembly of the highly stable elongating transcription complex (EC) over windows of two to three nucleotides after kilobases of RNA synthesis. Intrinsic termination is caused by the formation of a nascent RNA hairpin adjacent to a weak RNA−DNA hybrid within RNA polymerase (RNAP). Although the contributions of RNA and DNA sequences to termination are largely understood, the roles of conformational changes in RNAP are less well described. The polymorphous trigger loop (TL), which folds into the trigger helices to promote nucleotide addition, also is proposed to drive termination by folding into the trigger helices and contacting the terminator hairpin after invasion of the hairpin in the RNAP main cleft [Epshtein V, Cardinale CJ, Ruckenstein AE, Borukhov S, Nudler E (2007) Mol Cell 28:991–1001]. To investigate the contribution of the TL to intrinsic termination, we developed a kinetic assay that distinguishes effects of TL alterations on the rate at which ECs terminate from effects of the TL on the nucleotide addition rate that indirectly affect termination efficiency by altering the time window in which termination can occur. We confirmed that the TL stimulates termination rate, but found that stabilizing either the folded or unfolded TL conformation decreased termination rate. We propose that conformational fluctuations of the TL (TL dynamics), not TL-hairpin contact, aid termination by increasing EC conformational diversity and thus access to favorable termination pathways. We also report that the TL and the TL sequence insertion (SI3) increase overall termination efficiency by stimulating pausing, which increases the flux of ECs into the termination pathway. PMID:29078293

  7. Acrolein-induced activation of mitogen-activated protein kinase signaling is mediated by alkylation of thioredoxin reductase and thioredoxin 1.

    PubMed

    Randall, Matthew J; Spiess, Page C; Hristova, Milena; Hondal, Robert J; van der Vliet, Albert

    2013-01-01

    Cigarette smoking remains a major health concern worldwide, and many of the adverse effects of cigarette smoke (CS) can be attributed to its abundant electrophilic aldehydes, such as acrolein (2-propenal). Previous studies indicate that acrolein readily reacts with thioredoxin reductase 1 (TrxR1), a critical enzyme involved in regulation of thioredoxin (Trx)-mediated redox signaling, by alkylation at its selenocysteine (Sec) residue. Because alkylation of Sec within TrxR1 has significant implications for its enzymatic function, we explored the potential importance of TrxR1 alkylation in acrolein-induced activation or injury of bronchial epithelial cells. Exposure of human bronchial epithelial HBE1 cells to acrolein (1-30 μM) resulted in dose-dependent loss of TrxR thioredoxin reductase activity, which coincided with its alkylation, as determined by biotin hydrazide labeling, and was independent of initial GSH status. To test the involvement of TrxR1 in acrolein responses in HBE1 cells, we suppressed TrxR1 using siRNA silencing or augmented TrxR1 by cell supplementation with sodium selenite. Acrolein exposure of HBE1 cells induced dose-dependent activation of the MAP kinases, extracellular regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38, and activation of JNK was markedly enhanced after selenite-mediated induction of TrxR1, and was associated with increased alkylation of TrxR1. Conversely, siRNA silencing of TrxR1 significantly suppressed the ability of acrolein to activate JNK, and also appeared to attenuate acrolein-dependent activation of ERK and p38. Alteration of initial TrxR1 levels by siRNA or selenite supplementation also affected initial Trx1 redox status and acrolein-mediated alkylation of Trx1, but did not significantly affect acrolein-mediated activation of HO-1 or cytotoxicity. Collectively, our findings indicate that alkylation of TrxR1 and/or Trx1 may contribute directly to acrolein-mediated activation of MAP kinases such as JNK, and

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

    PubMed

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

    2016-10-04

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

  10. Identification of the missing trans-acting enoyl reductase required for phthiocerol dimycocerosate and phenolglycolipid biosynthesis in Mycobacterium tuberculosis.

    PubMed

    Siméone, Roxane; Constant, Patricia; Guilhot, Christophe; Daffé, Mamadou; Chalut, Christian

    2007-07-01

    Phthiocerol dimycocerosates (DIM) and phenolglycolipids (PGL) are functionally important surface-exposed lipids of Mycobacterium tuberculosis. Their biosynthesis involves the products of several genes clustered in a 70-kb region of the M. tuberculosis chromosome. Among these products is PpsD, one of the modular type I polyketide synthases responsible for the synthesis of the lipid core common to DIM and PGL. Bioinformatic analyses have suggested that this protein lacks a functional enoyl reductase activity domain required for the synthesis of these lipids. We have identified a gene, Rv2953, that putatively encodes an enoyl reductase. Mutation in Rv2953 prevents conventional DIM formation and leads to the accumulation of a novel DIM-like product. This product is unsaturated between C-4 and C-5 of phthiocerol. Consistently, complementation of the mutant with a functional pks15/1 gene from Mycobacterium bovis BCG resulted in the accumulation of an unsaturated PGL-like substance. When an intact Rv2953 gene was reintroduced into the mutant strain, the phenotype reverted to the wild type. These findings indicate that Rv2953 encodes a trans-acting enoyl reductase that acts with PpsD in phthiocerol and phenolphthiocerol biosynthesis.

  11. Effects of cooperation between translating ribosome and RNA polymerase on termination efficiency of the Rho-independent terminator.

    PubMed

    Li, Rui; Zhang, Qing; Li, Junbai; Shi, Hualin

    2016-04-07

    An experimental system was designed to measure in vivo termination efficiency (TE) of the Rho-independent terminator and position-function relations were quantified for the terminator tR2 in Escherichia coli The terminator function was almost completely repressed when tR2 was located several base pairs downstream from the gene, and TE gradually increased to maximum values with the increasing distance between the gene and terminator. This TE-distance relation reflected a stochastic coupling of the ribosome and RNA polymerase (RNAP). Terminators located in the first 100 bp of the coding region can function efficiently. However, functional repression was observed when the terminator was located in the latter part of the coding region, and the degree of repression was determined by transcriptional and translational dynamics. These results may help to elucidate mechanisms of Rho-independent termination and reveal genomic locations of terminators and functions of the sequence that precedes terminators. These observations may have important applications in synthetic biology. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  12. Nonleaking battery terminals

    NASA Technical Reports Server (NTRS)

    Snider, W. E.; Nagle, W. J.

    1972-01-01

    Three different terminals were designed for usage in a 40 ampere/hour silver zinc battery which has a 45 percent KOH by weight electrolyte in a plastic battery case. Life tests, including thermal cycling, electrical charge and discharge for up to three years duration, were conducted on these three different terminal designs. Tests for creep rate and tensile strength were conducted on the polyphenylene oxide (PPO) plastic battery cases. Some cases were unused and others containing KOH electrolyte were placed on life tests. The design and testing of nonleaking battery terminals for use with a potassium hydroxide (KOH) electrolyte in a plastic case are discussed.

  13. Defective Pollen Wall is Required for Anther and Microspore Development in Rice and Encodes a Fatty Acyl Carrier Protein Reductase

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Shi, J.; Shanklin, J.; Tan, H.

    Aliphatic alcohols naturally exist in many organisms as important cellular components; however, their roles in extracellular polymer biosynthesis are poorly defined. We report here the isolation and characterization of a rice (Oryza sativa) male-sterile mutant, defective pollen wall (dpw), which displays defective anther development and degenerated pollen grains with an irregular exine. Chemical analysis revealed that dpw anthers had a dramatic reduction in cutin monomers and an altered composition of cuticular wax, as well as soluble fatty acids and alcohols. Using map-based cloning, we identified the DPW gene, which is expressed in both tapetal cells and microspores during anther development.more » Biochemical analysis of the recombinant DPW enzyme shows that it is a novel fatty acid reductase that produces 1-hexadecanol and exhibits >270-fold higher specificity for palmiltoyl-acyl carrier protein than for C16:0 CoA substrates. DPW was predominantly targeted to plastids mediated by its N-terminal transit peptide. Moreover, we demonstrate that the monocot DPW from rice complements the dicot Arabidopsis thaliana male sterile2 (ms2) mutant and is the probable ortholog of MS2. These data suggest that DPWs participate in a conserved step in primary fatty alcohol synthesis for anther cuticle and pollen sporopollenin biosynthesis in monocots and dicots.« less

  14. Expression, purification and enzymatic characterization of Brugia malayi dihydrofolate reductase.

    PubMed

    Perez-Abraham, Romy; Sanchez, Karla Garabiles; Alfonso, Melany; Gubler, Ueli; Siekierka, John J; Goodey, Nina M

    2016-12-01

    Brugia malayi (B. malayi) is one of the three causative agents of lymphatic filariasis, a neglected parasitic disease. Current literature suggests that dihydrofolate reductase is a potential drug target for the elimination of B. malayi. Here we report the recombinant expression and purification of a ∼20 kDa B. malayi dihydrofolate reductase (BmDHFR). A His6-tagged construct was expressed in E. coli and purified by affinity chromatography to yield active and homogeneous enzyme for steady-state kinetic characterization and inhibition studies. The catalytic activity kcat was found to be 1.4 ± 0.1 s(-1), the Michaelis Menten constant KM for dihydrofolate 14.7 ± 3.6 μM, and the equilibrium dissociation constant KD for NADPH 25 ± 24 nM. For BmDHFR, IC50 values for a six DHFR inhibitors were determined to be 3.1 ± 0.2 nM for methotrexate, 32 ± 22 μM for trimethoprim, 109 ± 34 μM for pyrimethamine, 154 ± 46 μM for 2,4-diaminoquinazoline, 771 ± 44 μM for cycloguanil, and >20,000 μM for 2,4-diaminopyrimidine. Our findings suggest that antifolate compounds can serve as inhibitors of BmDHFR. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. 29 CFR 95.61 - Termination.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ...-PROFIT ORGANIZATIONS, AND WITH COMMERCIAL ORGANIZATIONS, FOREIGN GOVERNMENTS, ORGANIZATIONS UNDER THE JURISDICTION OF FOREIGN GOVERNMENTS, AND INTERNATIONAL ORGANIZATIONS Post-Award Requirements Termination and... effective date and, in the case of partial termination, the portion to be terminated. (3) By the recipient...

  16. 29 CFR 95.61 - Termination.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ...-PROFIT ORGANIZATIONS, AND WITH COMMERCIAL ORGANIZATIONS, FOREIGN GOVERNMENTS, ORGANIZATIONS UNDER THE JURISDICTION OF FOREIGN GOVERNMENTS, AND INTERNATIONAL ORGANIZATIONS Post-Award Requirements Termination and... effective date and, in the case of partial termination, the portion to be terminated. (3) By the recipient...

  17. Activity-guided isolation of the chemical constituents of Muntingia calabura using a quinone reductase induction assay.

    PubMed

    Su, Bao-Ning; Jung Park, Eun; Vigo, Jose Schunke; Graham, James G; Cabieses, Fernando; Fong, Harry H S; Pezzuto, John M; Kinghorn, A Douglas

    2003-06-01

    Activity-guided fractionation of an EtOAc-soluble extract of the leaves of Muntingia calabura collected in Peru, using an in vitro quinone reductase induction assay with cultured Hepa 1c1c7 (mouse hepatoma) cells, resulted in the isolation of a flavanone with an unsubstituted B-ring, (2R,3R)-7-methoxy-3,5,8-trihydroxyflavanone (5), as well as 24 known compounds, which were mainly flavanones and flavones. The structure including absolute stereochemistry of compound 5 was determined by spectroscopic (HRMS, 1D and 2D NMR, and CD spectra) methods. Of the isolates obtained, in addition to 5, (2S)-5-hydroxy-7-methoxyflavanone, 2',4'-dihydroxychalcone, 4,2',4'-trihydroxychalcone, 7-hydroxyisoflavone and 7,3',4'-trimethoxyisoflavone were found to induce quinone reductase activity.

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

    PubMed

    Frías, José E; Flores, Enrique

    2015-07-01

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

  19. Measurement and modeling of intrinsic transcription terminators

    PubMed Central

    Cambray, Guillaume; Guimaraes, Joao C.; Mutalik, Vivek K.; Lam, Colin; Mai, Quynh-Anh; Thimmaiah, Tim; Carothers, James M.; Arkin, Adam P.; Endy, Drew

    2013-01-01

    The reliable forward engineering of genetic systems remains limited by the ad hoc reuse of many types of basic genetic elements. Although a few intrinsic prokaryotic transcription terminators are used routinely, termination efficiencies have not been studied systematically. Here, we developed and validated a genetic architecture that enables reliable measurement of termination efficiencies. We then assembled a collection of 61 natural and synthetic terminators that collectively encode termination efficiencies across an ∼800-fold dynamic range within Escherichia coli. We simulated co-transcriptional RNA folding dynamics to identify competing secondary structures that might interfere with terminator folding kinetics or impact termination activity. We found that structures extending beyond the core terminator stem are likely to increase terminator activity. By excluding terminators encoding such context-confounding elements, we were able to develop a linear sequence-function model that can be used to estimate termination efficiencies (r = 0.9, n = 31) better than models trained on all terminators (r = 0.67, n = 54). The resulting systematically measured collection of terminators should improve the engineering of synthetic genetic systems and also advance quantitative modeling of transcription termination. PMID:23511967

  20. Termination unit

    DOEpatents

    Traeholt, Chresten; Willen, Dag; Roden, Mark; Tolbert, Jerry C.; Lindsay, David; Fisher, Paul W.; Nielsen, Carsten Thidemann

    2016-05-03

    Cable end section comprises end-parts of N electrical phases/neutral, and a thermally-insulation envelope comprising cooling fluid. The end-parts each comprises a conductor and are arranged with phase 1 innermost, N outermost surrounded by the neutral, electrical insulation being between phases and N and neutral. The end-parts comprise contacting surfaces located sequentially along the longitudinal extension of the end-section. A termination unit has an insulating envelope connected to a cryostat, special parts at both ends comprising an adapter piece at the cable interface and a closing end-piece terminating the envelope in the end-section. The special parts houses an inlet and/or outlet for cooling fluid. The space between an inner wall of the envelope and a central opening of the cable is filled with cooling fluid. The special part at the end connecting to the cryostat houses an inlet or outlet, splitting cooling flow into cable annular flow and termination annular flow.

  1. Molecular coevolution of mammalian ribosomal gene terminator sequences and the transcription termination factor TTF-I.

    PubMed Central

    Evers, R; Grummt, I

    1995-01-01

    Both the DNA elements and the nuclear factors that direct termination of ribosomal gene transcription exhibit species-specific differences. Even between mammals--e.g., human and mouse--the termination signals are not identical and the respective transcription termination factors (TTFs) which bind to the terminator sequence are not fully interchangeable. To elucidate the molecular basis for this species-specificity, we have cloned TTF-I from human and mouse cells and compared their structural and functional properties. Recombinant TTF-I exhibits species-specific DNA binding and terminates transcription both in cell-free transcription assays and in transfection experiments. Chimeric constructs of mouse TTF-I and human TTF-I reveal that the major determinant for species-specific DNA binding resides within the C terminus of TTF-I. Replacing 31 C-terminal amino acids of mouse TTF-I with the homologous human sequences relaxes the DNA-binding specificity and, as a consequence, allows the chimeric factor to bind the human terminator sequence and to specifically stop rDNA transcription. Images Fig. 2 Fig. 3 Fig. 4 PMID:7597036

  2. 45 CFR 74.61 - Termination.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... AND SUBAWARDS TO INSTITUTIONS OF HIGHER EDUCATION, HOSPITALS, OTHER NONPROFIT ORGANIZATIONS, AND COMMERCIAL ORGANIZATIONS Post-Award Requirements Termination and Enforcement § 74.61 Termination. (a) Awards... two parties shall agree upon the termination conditions, including the effective date and, in the case...

  3. 7 CFR 1206.22 - Terminate.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 10 2010-01-01 2010-01-01 false Terminate. 1206.22 Section 1206.22 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (MARKETING AGREEMENTS... INFORMATION Mango Promotion, Research, and Information Order Definitions § 1206.22 Terminate. Terminate means...

  4. Isolation and Expression Analysis of CYP9A11 and Cytochrome P450 Reductase Gene in the Beet Armyworm (Lepidoptera: Noctuidae)

    PubMed Central

    Zhao, Chunqing; Feng, Xiaoyun; Tang, Tao; Qiu, Lihong

    2015-01-01

    Cytochrome P450 monooxygenases (CYPs), as an enzyme superfamily, is widely distributed in organisms and plays a vital function in the metabolism of exogenous and endogenous compounds by interacting with its obligatory redox partner, CYP reductase (CPR). A novel CYP gene (CYP9A11) and CPR gene from the agricultural pest insect Spodoptera exigua were cloned and characterized. The complete cDNA sequences of SeCYP9A11 and SeCPR are 1,931 and 3,919 bp in length, respectively, and contain open reading frames of 1,593 and 2,070 nucleotides, respectively. Analysis of the putative protein sequences indicated that SeCYP9A11 contains a heme-binding domain and the unique characteristic sequence (SRFALCE) of the CYP9 family, in addition to a signal peptide and transmembrane segment at the N-terminal. Alignment analysis revealed that SeCYP9A11 shares the highest sequence similarity with CYP9A13 from Mamestra brassicae, which is 66.54%. The putative protein sequence of SeCPR has all of the classical CPR features, such as an N-terminal membrane anchor; three conserved domain flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), and nicotinamide adenine dinucleotide phosphate (NADPH) domain; and characteristic binding motifs. Phylogenetic analysis revealed that SeCPR shares the highest identity with HaCPR, which is 95.21%. The SeCYP9A11 and SeCPR genes were detected in the midgut, fat body, and cuticle tissues, and throughout all of the developmental stages of S. exigua. The mRNA levels of SeCYP9A11 and SeCPR decreased remarkably after exposure to plant secondary metabolites quercetin and tannin. The results regarding SeCYP9A11 and SeCPR genes in the current study provide foundation for the further study of S. exigua P450 system. PMID:26320261

  5. Structure of a short-chain dehydrogenase/reductase from Bacillus anthracis

    PubMed Central

    Hou, Jing; Wojciechowska, Kamila; Zheng, Heping; Chruszcz, Maksymilian; Cooper, David R.; Cymborowski, Marcin; Skarina, Tatiana; Gordon, Elena; Luo, Haibin; Savchenko, Alexei; Minor, Wladek

    2012-01-01

    The crystal structure of a short-chain dehydrogenase/reductase from Bacillus anthracis strain ‘Ames Ancestor’ complexed with NADP has been determined and refined to 1.87 Å resolution. The structure of the enzyme consists of a Rossmann fold composed of seven parallel β-strands sandwiched by three α-­helices on each side. An NADP molecule from an endogenous source is bound in the conserved binding pocket in the syn conformation. The loop region responsible for binding another substrate forms two perpendicular short helices connected by a sharp turn. PMID:22684058

  6. Purification and properties of dihydrofolate reductase from cultured mammalian cells

    PubMed Central

    Gauldie, Jack; Marshall, Lyse; Hillcoat, Brian L.

    1973-01-01

    Dihydrofolate reductase was purified quickly and simply from small quantities of cultured mammalian cells by affinity chromatography. On gel electrophoresis of the purified enzyme, multiple bands of activity resulted from enzyme–buffer interaction at low but not high buffer concentration. A Ferguson plot (Ferguson, 1964) showed that this heterogeneity was due to a charge difference with no alteration in the size of the enzyme. Stimulation of enzyme activity by KCl, urea and p-hydroxymercuribenzoate, and inhibition by methotrexate and trimethoprim, showed only minor differences between the various enzymes. PMID:4723779

  7. 48 CFR 49.105 - Duties of termination contracting officer after issuance of notice of termination.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... Regulations System FEDERAL ACQUISITION REGULATION CONTRACT MANAGEMENT TERMINATION OF CONTRACTS General Principles 49.105 Duties of termination contracting officer after issuance of notice of termination. (a... principles relating to the settlement of any settlement proposal, including obligations of the contractor...

  8. 7 CFR 953.66 - Termination.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 8 2010-01-01 2010-01-01 false Termination. 953.66 Section 953.66 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Marketing Agreements... STATES Order Regulating Handling Effective Time and Termination § 953.66 Termination. (a) The Secretary...

  9. Monodehydroascorbate reductase mediates TNT toxicity in plants.

    PubMed

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

    2015-09-04

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

  10. The regulation of 3-hydroxy-3-methylglutaryl-CoA reductase activity, cholesterol esterification and the expression of low-density lipoprotein receptors in cultured monocyte-derived macrophages.

    PubMed Central

    Knight, B L; Patel, D D; Soutar, A K

    1983-01-01

    Human blood monocytes cultured in medium containing 20% whole serum showed the greatest activity of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase and [14C]acetate incorporation into non-saponifiable lipids around the 7th day after seeding, the period of greatest growth. Although there was enough low-density lipoprotein (LDL) in the medium to saturate the LDL receptors that were expressed by normal cells at that time, HMG-CoA reductase activity and acetate incorporation were as high in normal cells as in cells from familial-hypercholesterolaemic (FH) patients. Both the addition of extra LDL, which interacted with the cells by non-saturable processes, and receptor-mediated uptake of acetylated LDL significantly reduced reductase activity and increased incorporation of [14C]oleate into cholesteryl esters in normal cells and cells from FH patients ('FH cells'), and reduced the expression of LDL receptors in normal cells. Pre-incubation for 20h in lipoprotein-deficient medium apparently increased the number of LDL receptors expressed by normal cells but reduced the activity of HMG-CoA reductase in both normal and FH cells. During subsequent incubations the same rate of degradation of acetylated LDL and of non-saturable degradation of LDL by FH cells was associated with the same reduction in HMG-CoA reductase activity, although LDL produced a much smaller stimulation of oleate incorporation into cholesteryl esters. In normal cells pre-incubated without lipoproteins, receptor-mediated uptake of LDL could abolish reductase activity and the expression of LDL receptors. The results suggested that in these cells, receptor-mediated uptake of LDL might have a greater effect on reductase activity and LDL receptors than the equivalent uptake of acetylated LDL. It is proposed that endogenous synthesis is an important source of cholesterol for growth of normal cells, and that the site at which cholesterol is deposited in the cells may determine the nature and extent of the

  11. 22 CFR 518.61 - Termination.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... INSTITUTIONS OF HIGHER EDUCATION, HOSPITALS, AND OTHER NON-PROFIT ORGANIZATIONS Post-Award Requirements... conditions, including the effective date and, in the case of partial termination, the portion to be... forth the reasons for such termination, the effective date, and, in the case of partial termination, the...

  12. 22 CFR 145.61 - Termination.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ..., HOSPITALS, AND OTHER NON-PROFIT ORGANIZATIONS Post-Award Requirements Termination and Enforcement § 145.61... parties shall agree upon the termination conditions, including the effective date and, in the case of... written notification setting forth the reasons for such termination, the effective date, and, in the case...

  13. 36 CFR 1210.61 - Termination.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ..., HOSPITALS, AND OTHER NON-PROFIT ORGANIZATIONS Post-Award Requirements Termination and Enforcement § 1210.61... agree upon the termination conditions, including the effective date and, in the case of partial... setting forth the reasons for such termination, the effective date, and, in the case of partial...

  14. 28 CFR 70.61 - Termination.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... (INCLUDING SUBAWARDS) WITH INSTITUTIONS OF HIGHER EDUCATION, HOSPITALS AND OTHER NON-PROFIT ORGANIZATIONS... conditions, including the effective date and, in the case of partial termination, the portion to be... reasons for such termination, the effective date, and, in the case of partial termination, the portion to...

  15. 22 CFR 518.61 - Termination.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... INSTITUTIONS OF HIGHER EDUCATION, HOSPITALS, AND OTHER NON-PROFIT ORGANIZATIONS Post-Award Requirements... conditions, including the effective date and, in the case of partial termination, the portion to be... forth the reasons for such termination, the effective date, and, in the case of partial termination, the...

  16. 22 CFR 145.61 - Termination.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ..., HOSPITALS, AND OTHER NON-PROFIT ORGANIZATIONS Post-Award Requirements Termination and Enforcement § 145.61... parties shall agree upon the termination conditions, including the effective date and, in the case of... written notification setting forth the reasons for such termination, the effective date, and, in the case...

  17. 33 CFR 159.79 - Terminals.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 33 Navigation and Navigable Waters 2 2011-07-01 2011-07-01 false Terminals. 159.79 Section 159.79 Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) POLLUTION MARINE SANITATION DEVICES Design, Construction, and Testing § 159.79 Terminals. Terminals must be solderless lugs...

  18. 42 CFR 66.109 - Termination.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 42 Public Health 1 2013-10-01 2013-10-01 false Termination. 66.109 Section 66.109 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES FELLOWSHIPS, INTERNSHIPS, TRAINING NATIONAL RESEARCH SERVICE AWARDS Direct Awards § 66.109 Termination. (a) The Secretary may terminate an...

  19. 42 CFR 66.109 - Termination.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 42 Public Health 1 2014-10-01 2014-10-01 false Termination. 66.109 Section 66.109 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES FELLOWSHIPS, INTERNSHIPS, TRAINING NATIONAL RESEARCH SERVICE AWARDS Direct Awards § 66.109 Termination. (a) The Secretary may terminate an...

  20. 42 CFR 66.109 - Termination.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 42 Public Health 1 2010-10-01 2010-10-01 false Termination. 66.109 Section 66.109 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES FELLOWSHIPS, INTERNSHIPS, TRAINING NATIONAL RESEARCH SERVICE AWARDS Direct Awards § 66.109 Termination. (a) The Secretary may terminate an...

  1. 42 CFR 66.109 - Termination.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 42 Public Health 1 2011-10-01 2011-10-01 false Termination. 66.109 Section 66.109 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES FELLOWSHIPS, INTERNSHIPS, TRAINING NATIONAL RESEARCH SERVICE AWARDS Direct Awards § 66.109 Termination. (a) The Secretary may terminate an...

  2. 42 CFR 66.109 - Termination.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 42 Public Health 1 2012-10-01 2012-10-01 false Termination. 66.109 Section 66.109 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES FELLOWSHIPS, INTERNSHIPS, TRAINING NATIONAL RESEARCH SERVICE AWARDS Direct Awards § 66.109 Termination. (a) The Secretary may terminate an...

  3. Molecular modelling and synthesis of spiroimidazolidine-2,4-diones with dual activities as hypoglycemic agents and selective inhibitors of aldose reductase.

    PubMed

    Salem, Manar G; Abdel Aziz, Yasmine M; Elewa, Marwa; Elshihawy, Hosam A; Said, Mohamed M

    2018-05-02

    Novel derivatives of spiroimidazolidinedione were synthesized and evaluated as hypoglycemic agents through binding to sulfonylurea receptor 1 (SUR1) in pancreatic beta-cells. Their selectivity index was calculated against both aldehyde reductase (ALR1) and aldose reductase (ALR2). Aldehyde reductase is a key enzyme in the polyol pathway that is involved in the etiology of the secondary diabetic complications. All structures were confirmed by microanalysis and by IR, 1 H NMR, 13 C NMR and EI-MS spectroscopy. The investigated compounds were subjected to molecular docking and an in silico prediction study to determine their free energy of binding (ΔG) values and predict their physicochemical properties and drug-likeness scores. Compound 1'-(5-chlorothiophene-2-ylsulfonyl)spiro[cyclohexane-1,5'-imidazolidine]-2',4'-dione showed IC 50 0.47 µM and 79% reduction in blood glucose level with a selectivity index 127 for ALR2. Copyright © 2018 Elsevier Inc. All rights reserved.

  4. Molecular docking studies of selected tricyclic and quinone derivatives on trypanothione reductase of Leishmania infantum.

    PubMed

    Venkatesan, Santhosh Kannan; Shukla, Anil Kumar; Dubey, Vikash Kumar

    2010-10-01

    Visceral leishmaniasis, most lethal form of Leishmaniasis, is caused by Leishmania infantum in the Old world. Current therapeutics for the disease is associated with a risk of high toxicity and development of drug resistant strains. Thiol-redox metabolism involving trypanothione and trypanothione reductase, key for survival of Leishmania, is a validated target for rational drug design. Recently published structure of trypanothione reductase (TryR) from L. infantum, in oxidized and reduced form along with Sb(III), provides vital clues on active site of the enzyme. In continuation with our attempts to identify potent inhibitors of TryR, we have modeled binding modes of selected tricyclic compounds and quinone derivatives, using AutoDock4. Here, we report a unique binding mode for quinone derivatives and 9-aminoacridine derivatives, at the FAD binding domain. A conserved hydrogen bonding pattern was observed in all these compounds with residues Thr335, Lys60, His461. With the fact that these residues aid in the orientation of FAD towards the active site forming the core of the FAD binding domain, designing selective and potent compounds that could replace FAD in vivo during the synthesis of Trypanothione reductase can be deployed as an effective strategy in designing new drugs towards Leishmaniasis. We also report the binding of Phenothiazine and 9-aminoacridine derivatives at the Z site of the protein. The biological significance and possible mode of inhibition by quinone derivatives, which binds to FAD binding domain, along with other compounds are discussed. (c) 2010 Wiley Periodicals, Inc.

  5. The Role of Large-Scale Motions in Catalysis by Dihydrofolate Reductase

    PubMed Central

    2011-01-01

    Dihydrofolate reductase has long been used as a model system to study the coupling of protein motions to enzymatic hydride transfer. By studying environmental effects on hydride transfer in dihydrofolate reductase (DHFR) from the cold-adapted bacterium Moritella profunda (MpDHFR) and comparing the flexibility of this enzyme to that of DHFR from Escherichia coli (EcDHFR), we demonstrate that factors that affect large-scale (i.e., long-range, but not necessarily large amplitude) protein motions have no effect on the kinetic isotope effect on hydride transfer or its temperature dependence, although the rates of the catalyzed reaction are affected. Hydrogen/deuterium exchange studies by NMR-spectroscopy show that MpDHFR is a more flexible enzyme than EcDHFR. NMR experiments with EcDHFR in the presence of cosolvents suggest differences in the conformational ensemble of the enzyme. The fact that enzymes from different environmental niches and with different flexibilities display the same behavior of the kinetic isotope effect on hydride transfer strongly suggests that, while protein motions are important to generate the reaction ready conformation, an optimal conformation with the correct electrostatics and geometry for the reaction to occur, they do not influence the nature of the chemical step itself; large-scale motions do not couple directly to hydride transfer proper in DHFR. PMID:22060818

  6. Rapid X-ray Photoreduction of Dimetal-Oxygen Cofactors in Ribonucleotide Reductase

    PubMed Central

    Sigfridsson, Kajsa G. V.; Chernev, Petko; Leidel, Nils; Popović-Bijelić, Ana; Gräslund, Astrid; Haumann, Michael

    2013-01-01

    Prototypic dinuclear metal cofactors with varying metallation constitute a class of O2-activating catalysts in numerous enzymes such as ribonucleotide reductase. Reliable structures are required to unravel the reaction mechanisms. However, protein crystallography data may be compromised by x-ray photoreduction (XRP). We studied XPR of Fe(III)Fe(III) and Mn(III)Fe(III) sites in the R2 subunit of Chlamydia trachomatis ribonucleotide reductase using x-ray absorption spectroscopy. Rapid and biphasic x-ray photoreduction kinetics at 20 and 80 K for both cofactor types suggested sequential formation of (III,II) and (II,II) species and similar redox potentials of iron and manganese sites. Comparing with typical x-ray doses in crystallography implies that (II,II) states are reached in <1 s in such studies. First-sphere metal coordination and metal-metal distances differed after chemical reduction at room temperature and after XPR at cryogenic temperatures, as corroborated by model structures from density functional theory calculations. The inter-metal distances in the XPR-induced (II,II) states, however, are similar to R2 crystal structures. Therefore, crystal data of initially oxidized R2-type proteins mostly contain photoreduced (II,II) cofactors, which deviate from the native structures functional in O2 activation, explaining observed variable metal ligation motifs. This situation may be remedied by novel femtosecond free electron-laser protein crystallography techniques. PMID:23400774

  7. Rapid X-ray photoreduction of dimetal-oxygen cofactors in ribonucleotide reductase.

    PubMed

    Sigfridsson, Kajsa G V; Chernev, Petko; Leidel, Nils; Popovic-Bijelic, Ana; Gräslund, Astrid; Haumann, Michael

    2013-04-05

    Prototypic dinuclear metal cofactors with varying metallation constitute a class of O2-activating catalysts in numerous enzymes such as ribonucleotide reductase. Reliable structures are required to unravel the reaction mechanisms. However, protein crystallography data may be compromised by x-ray photoreduction (XRP). We studied XPR of Fe(III)Fe(III) and Mn(III)Fe(III) sites in the R2 subunit of Chlamydia trachomatis ribonucleotide reductase using x-ray absorption spectroscopy. Rapid and biphasic x-ray photoreduction kinetics at 20 and 80 K for both cofactor types suggested sequential formation of (III,II) and (II,II) species and similar redox potentials of iron and manganese sites. Comparing with typical x-ray doses in crystallography implies that (II,II) states are reached in <1 s in such studies. First-sphere metal coordination and metal-metal distances differed after chemical reduction at room temperature and after XPR at cryogenic temperatures, as corroborated by model structures from density functional theory calculations. The inter-metal distances in the XPR-induced (II,II) states, however, are similar to R2 crystal structures. Therefore, crystal data of initially oxidized R2-type proteins mostly contain photoreduced (II,II) cofactors, which deviate from the native structures functional in O2 activation, explaining observed variable metal ligation motifs. This situation may be remedied by novel femtosecond free electron-laser protein crystallography techniques.

  8. Fumarate Reductase Activity Maintains an Energized Membrane in Anaerobic Mycobacterium tuberculosis

    PubMed Central

    Watanabe, Shinya; Zimmermann, Michael; Goodwin, Michael B.; Sauer, Uwe; Barry, Clifton E.; Boshoff, Helena I.

    2011-01-01

    Oxygen depletion of Mycobacterium tuberculosis engages the DosR regulon that coordinates an overall down-regulation of metabolism while up-regulating specific genes involved in respiration and central metabolism. We have developed a chemostat model of M. tuberculosis where growth rate was a function of dissolved oxygen concentration to analyze metabolic adaptation to hypoxia. A drop in dissolved oxygen concentration from 50 mmHg to 0.42 mmHg led to a 2.3 fold decrease in intracellular ATP levels with an almost 70-fold increase in the ratio of NADH/NAD+. This suggests that re-oxidation of this co-factor becomes limiting in the absence of a terminal electron acceptor. Upon oxygen limitation genes involved in the reverse TCA cycle were upregulated and this upregulation was associated with a significant accumulation of succinate in the extracellular milieu. We confirmed that this succinate was produced by a reversal of the TCA cycle towards the non-oxidative direction with net CO2 incorporation by analysis of the isotopomers of secreted succinate after feeding stable isotope (13C) labeled precursors. This showed that the resulting succinate retained both carbons lost during oxidative operation of the TCA cycle. Metabolomic analyses of all glycolytic and TCA cycle intermediates from 13C-glucose fed cells under aerobic and anaerobic conditions showed a clear reversal of isotope labeling patterns accompanying the switch from normoxic to anoxic conditions. M. tuberculosis encodes three potential succinate-producing enzymes including a canonical fumarate reductase which was highly upregulated under hypoxia. Knockout of frd, however, failed to reduce succinate accumulation and gene expression studies revealed a compensatory upregulation of two homologous enzymes. These major realignments of central metabolism are consistent with a model of oxygen-induced stasis in which an energized membrane is maintained by coupling the reductive branch of the TCA cycle to succinate

  9. Molecular cloning and catalytic characterization of a recombinant tropine biosynthetic tropinone reductase from Withania coagulans leaf.

    PubMed

    Kushwaha, Amit K; Sangwan, Neelam S; Tripathi, Sandhya; Sangwan, Rajender S

    2013-03-10

    Tropinone reductases (TRs) are small proteins belonging to the SDR (short chain dehydrogenase/reductase) family of enzymes. TR-I and TR-II catalyze the conversion of tropinone into tropane alcohols (tropine and pseudotropine, respectively). The steps are intermediary enroute to biosynthesis of tropane esters of medicinal importance, hyoscyamine/scopolamine, and calystegins, respectively. Biosynthesis of tropane alkaloids has been proposed to occur in roots. However, in the present report, a tropine forming tropinone reductase (TR-I) cDNA was isolated from the aerial tissue (leaf) of a medicinal plant, Withania coagulans. The ORF was deduced to encode a polypeptide of 29.34 kDa. The complete cDNA (WcTRI) was expressed in E. coli and the recombinant His-tagged protein was purified for functional characterization. The enzyme had a narrow pH range of substantial activity with maxima at 6.6. Relatively superior thermostability of the enzyme (30% retention of activity at 60 °C) was catalytic novelty in consonance with the desert area restricted habitat of the plant. The in vitro reaction kinetics predominantly favoured the forward reaction. The enzyme had wide substrate specificity but did not cover the substrates of other well-known plant SDR related to menthol metabolism. To our knowledge, this pertains to be the first report on any gene and enzyme of secondary metabolism from the commercially and medicinally important vegetable rennet species. Copyright © 2012 Elsevier B.V. All rights reserved.

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

    PubMed

    Byshneva, L N; Senchuk, V V

    2002-01-01

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

  11. Trypanosoma brucei DHFR-TS Revisited: Characterisation of a Bifunctional and Highly Unstable Recombinant Dihydrofolate Reductase-Thymidylate Synthase.

    PubMed

    Gibson, Marc W; Dewar, Simon; Ong, Han B; Sienkiewicz, Natasha; Fairlamb, Alan H

    2016-05-01

    Bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) is a chemically and genetically validated target in African trypanosomes, causative agents of sleeping sickness in humans and nagana in cattle. Here we report the kinetic properties and sensitivity of recombinant enzyme to a range of lipophilic and classical antifolate drugs. The purified recombinant enzyme, expressed as a fusion protein with elongation factor Ts (Tsf) in ThyA- Escherichia coli, retains DHFR activity, but lacks any TS activity. TS activity was found to be extremely unstable (half-life of 28 s) following desalting of clarified bacterial lysates to remove small molecules. Stability could be improved 700-fold by inclusion of dUMP, but not by other pyrimidine or purine (deoxy)-nucleosides or nucleotides. Inclusion of dUMP during purification proved insufficient to prevent inactivation during the purification procedure. Methotrexate and trimetrexate were the most potent inhibitors of DHFR (Ki 0.1 and 0.6 nM, respectively) and FdUMP and nolatrexed of TS (Ki 14 and 39 nM, respectively). All inhibitors showed a marked drop-off in potency of 100- to 1,000-fold against trypanosomes grown in low folate medium lacking thymidine. The most potent inhibitors possessed a terminal glutamate moiety suggesting that transport or subsequent retention by polyglutamylation was important for biological activity. Supplementation of culture medium with folate markedly antagonised the potency of these folate-like inhibitors, as did thymidine in the case of the TS inhibitors raltitrexed and pemetrexed.

  12. Trichomonas vaginalis: metronidazole and other nitroimidazole drugs are reduced by the flavin enzyme thioredoxin reductase and disrupt the cellular redox system. Implications for nitroimidazole toxicity and resistance.

    PubMed

    Leitsch, David; Kolarich, Daniel; Binder, Marina; Stadlmann, Johannes; Altmann, Friedrich; Duchêne, Michael

    2009-04-01

    Infections with the microaerophilic parasite Trichomonas vaginalis are treated with the 5-nitroimidazole drug metronidazole, which is also in use against Entamoeba histolytica, Giardia intestinalis and microaerophilic/anaerobic bacteria. Here we report that in T. vaginalis the flavin enzyme thioredoxin reductase displays nitroreductase activity with nitroimidazoles, including metronidazole, and with the nitrofuran drug furazolidone. Reactive metabolites of metronidazole and other nitroimidazoles form covalent adducts with several proteins that are known or assumed to be associated with thioredoxin-mediated redox regulation, including thioredoxin reductase itself, ribonucleotide reductase, thioredoxin peroxidase and cytosolic malate dehydrogenase. Disulphide reducing activity of thioredoxin reductase was greatly diminished in extracts of metronidazole-treated cells and intracellular non-protein thiol levels were sharply decreased. We generated a highly metronidazole-resistant cell line that displayed only minimal thioredoxin reductase activity, not due to diminished expression of the enzyme but due to the lack of its FAD cofactor. Reduction of free flavins, readily observed in metronidazole-susceptible cells, was also absent in the resistant cells. On the other hand, iron-depleted T. vaginalis cells, expressing only minimal amounts of PFOR and hydrogenosomal malate dehydrogenase, remained fully susceptible to metronidazole. Thus, taken together, our data suggest a flavin-based mechanism of metronidazole activation and thereby challenge the current model of hydrogenosomal activation of nitroimidazole drugs.

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

    PubMed

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

    2014-11-01

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

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

    PubMed

    Moon, Jaewoong; Liu, Z Lewis

    2015-04-01

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

  15. High dose androgen therapy in male pseudohermaphroditism due to 5 alpha-reductase deficiency and disorders of the androgen receptor.

    PubMed

    Price, P; Wass, J A; Griffin, J E; Leshin, M; Savage, M O; Large, D M; Bu'Lock, D E; Anderson, D C; Wilson, J D; Besser, G M

    1984-10-01

    We describe the clinical and biochemical features of six men with male pseudohermaphroditism due to androgen resistance. Each of the subjects had male-gender behavior but incomplete virilization. The underlying defects in androgen metabolism were defined by studies of the 5 alpha-reductase enzyme and the androgen receptor in fibroblasts cultured from biopsies of genital skin. Four of the six have 5 alpha-reductase deficiency, and two have defects of the androgen receptor (the Reifenstein syndrome). The responses of these men to androgen treatment were assessed by monitoring nitrogen balance, plasma luteinizing hormone (LH) values, and clinical parameters of virilization including penile growth, potency and ejaculatory volume, muscle bulk, and growth of body and facial hair. In all of the subjects with 5 alpha-reductase deficiency and one man with the Reifenstein syndrome significant response occurred, as evidence by nitrogen retention, lowered plasma LH levels, and improved virilization, with doses of parenteral testosterone esters that raised plasma testosterone levels above the normal male range and brought plasma dihydrotestosterone levels into the normal male range. The subject who did not respond with clinical virilization nevertheless showed nitrogen retention in response to acute testosterone administration. This patient had a profound deficiency of the androgen receptor, whereas the man with a receptor defect who did respond clinically to therapy had normal amounts of a qualitatively abnormal receptor. We conclude that high dose androgen therapy may be of benefit in improving virilization, self-image, and sexual performance in subjects with 5 alpha-reductase deficiency who have male-gender behavior and in some subjects with defects of the androgen receptor.

  16. 25 CFR 11.1114 - Termination.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... functions; (iii) The parent(s) has subjected the minor to willful and repeated acts of sexual abuse; (iv... Minor-in-Need-of-Care Procedure § 11.1114 Termination. (a) Parental rights to a child may be terminated by the children's court according to the procedures in this section. (b) Proceedings to terminate...

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

    PubMed

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

    2014-01-01

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

  18. Anaerobic respiration of Escherichia coli in the mouse intestine.

    PubMed

    Jones, Shari A; Gibson, Terri; Maltby, Rosalie C; Chowdhury, Fatema Z; Stewart, Valley; Cohen, Paul S; Conway, Tyrrell

    2011-10-01

    The intestine is inhabited by a large microbial community consisting primarily of anaerobes and, to a lesser extent, facultative anaerobes, such as Escherichia coli, which we have shown requires aerobic respiration to compete successfully in the mouse intestine (S. A. Jones et al., Infect. Immun. 75:4891-4899, 2007). If facultative anaerobes efficiently lower oxygen availability in the intestine, then their sustained growth must also depend on anaerobic metabolism. In support of this idea, mutants lacking nitrate reductase or fumarate reductase have extreme colonization defects. Here, we further explore the role of anaerobic respiration in colonization using the streptomycin-treated mouse model. We found that respiratory electron flow is primarily via the naphthoquinones, which pass electrons to cytochrome bd oxidase and the anaerobic terminal reductases. We found that E. coli uses nitrate and fumarate in the intestine, but not nitrite, dimethyl sulfoxide, or trimethylamine N-oxide. Competitive colonizations revealed that cytochrome bd oxidase is more advantageous than nitrate reductase or fumarate reductase. Strains lacking nitrate reductase outcompeted fumarate reductase mutants once the nitrate concentration in cecal mucus reached submillimolar levels, indicating that fumarate is the more important anaerobic electron acceptor in the intestine because nitrate is limiting. Since nitrate is highest in the absence of E. coli, we conclude that E. coli is the only bacterium in the streptomycin-treated mouse large intestine that respires nitrate. Lastly, we demonstrated that a mutant lacking the NarXL regulator (activator of the NarG system), but not a mutant lacking the NarP-NarQ regulator, has a colonization defect, consistent with the advantage provided by NarG. The emerging picture is one in which gene regulation is tuned to balance expression of the terminal reductases that E. coli uses to maximize its competitiveness and achieve the highest possible population in

  19. Chloroplast SRP43 acts as a chaperone for glutamyl-tRNA reductase, the rate-limiting enzyme in tetrapyrrole biosynthesis.

    PubMed

    Wang, Peng; Liang, Fu-Cheng; Wittmann, Daniel; Siegel, Alex; Shan, Shu-Ou; Grimm, Bernhard

    2018-04-10

    Assembly of light-harvesting complexes requires synchronization of chlorophyll (Chl) biosynthesis with biogenesis of light-harvesting Chl a/b-binding proteins (LHCPs). The chloroplast signal recognition particle (cpSRP) pathway is responsible for transport of nucleus-encoded LHCPs in the stroma of the plastid and their integration into the thylakoid membranes. Correct folding and assembly of LHCPs require the incorporation of Chls, whose biosynthesis must therefore be precisely coordinated with membrane insertion of LHCPs. How the spatiotemporal coordination between the cpSRP machinery and Chl biosynthesis is achieved is poorly understood. In this work, we demonstrate a direct interaction between cpSRP43, the chaperone that mediates LHCP targeting and insertion, and glutamyl-tRNA reductase (GluTR), a rate-limiting enzyme in tetrapyrrole biosynthesis. Concurrent deficiency for cpSRP43 and the GluTR-binding protein (GBP) additively reduces GluTR levels, indicating that cpSRP43 and GBP act nonredundantly to stabilize GluTR. The substrate-binding domain of cpSRP43 binds to the N-terminal region of GluTR, which harbors aggregation-prone motifs, and the chaperone activity of cpSRP43 efficiently prevents aggregation of these regions. Our work thus reveals a function of cpSRP43 in Chl biosynthesis and suggests a striking mechanism for posttranslational coordination of LHCP insertion with Chl biosynthesis.

  20. Structure-Based Insight into the Asymmetric Bioreduction of the C=C Double Bond of α,β-Unsaturated Nitroalkenes by Pentaerythritol Tetranitrate Reductase

    PubMed Central

    Toogood, Helen S.; Fryszkowska, Anna; Hare, Victoria; Fisher, Karl; Roujeinikova, Anna; Leys, David; Gardiner, John M.; Stephens, Gill M.; Scrutton, Nigel S.

    2009-01-01

    Biocatalytic reduction of α- or β-alkyl-β-arylnitroalkenes provides a convenient and efficient method to prepare chiral substituted nitroalkanes. Pentaerythritol tetranitrate reductase (PETN reductase) from Enterobacter cloacae st. PB2 catalyses the reduction of nitroolefins such as 1-nitrocyclohexene (1) with steady state and rapid reaction kinetics comparable to other old yellow enzyme homologues. Furthermore, it reduces 2-aryl-1-nitropropenes (4a-d) to their equivalent (S)-nitropropanes 9a-d. The enzyme shows a preference for the (Z)-isomer of substrates 4a-d, providing almost pure enantiomeric products 9a-d (ees up to > 99%) in quantitative yield, whereas the respective (E)-isomers are reduced with lower enantioselectivity (63-89% ee) and lower product yields. 1-Aryl-2-nitropropenes (5a, b) are also reduced efficiently, but the products (R)-10 have lower optical purities. The structure of the enzyme complex with 1-nitrocyclohexene (1) was determined by X-ray crystallography, revealing two substrate-binding modes, with only one compatible with hydride transfer. Models of nitropropenes 4 and 5 in the active site of PETN reductase predicted that the enantioselectivity of the reaction was dependent on the orientation of binding of the (E)- and (Z)-substrates. This work provides a structural basis for understanding the mechanism of asymmetric bioreduction of nitroalkenes by PETN reductase. PMID:20396603