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Sample records for aldose reductase rlar

  1. Aldose reductase inhibitory activity of compounds from Zea mays L.

    PubMed

    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 (IC(50), 4.78 μ M). 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

  2. Structure of aldose reductase from Giardia lamblia

    PubMed Central

    Ferrell, M.; Abendroth, J.; Zhang, Y.; Sankaran, B.; Edwards, T. E.; Staker, B. L.; Van Voorhis, W. C.; Stewart, L. J.; Myler, P. J.

    2011-01-01

    Giardia lamblia is an anaerobic aerotolerant eukaryotic parasite of the intestines. It is believed to have diverged early from eukarya during evolution and is thus lacking in many of the typical eukaryotic organelles and biochemical pathways. Most conspicuously, mitochondria and the associated machinery of oxidative phosphorylation are absent; instead, energy is derived from substrate-level phosphorylation. Here, the 1.75 Å resolution crystal structure of G. lamblia aldose reductase heterologously expressed in Escherichia coli is reported. As in other oxidoreductases, G. lamblia aldose reductase adopts a TIM-barrel conformation with the NADP+-binding site located within the eight β-strands of the interior. PMID:21904059

  3. 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

  4. Esculetin, a Coumarin Derivative, Inhibits Aldose Reductase Activity in vitro and Cataractogenesis in Galactose-Fed Rats

    PubMed Central

    Kim, Chan-Sik; Kim, Junghyun; Lee, Yun Mi; Sohn, Eunjin; Kim, Jin Sook

    2016-01-01

    Naturally occurring coumarin compounds have received substantial attention due to their pharmaceutical effects. Esculetin is a coumarin derivative and a polyphenol compound that is used in a variety of therapeutic and pharmacological strategies. However, its effect on aldose reductase activity remains poorly understood. In this study, the potential beneficial effects of esculetin on lenticular aldose reductase were investigated in galactose-fed (GAL) rats, an animal model of sugar cataracts. Cataracts were induced in Sprague-Dawley (SD) rats via a 50% galactose diet for 2 weeks, and groups of GAL rats were orally treated with esculetin (10 or 50 mg/kg body weight). In vehicle-treated GAL rats, lens opacification was observed, and swelling and membrane rupture of the lens fiber cells were increased. Additionally, aldose reductase was highly expressed in the lens epithelium and superficial cortical fibers during cataract development in the GAL rats. Esculetin reduced rat lens aldose reductase (RLAR) activity in vitro, and esculetin treatment significantly inhibited lens opacity, as well as morphological alterations, such as swelling, vacuolation and liquefaction of lens fibers, via the inhibition of aldose reductase in the GAL rats. These results indicate that esculetin is a useful treatment for galactose-induced cataracts. PMID:26902086

  5. Esculetin, a Coumarin Derivative, Inhibits Aldose Reductase Activity in vitro and Cataractogenesis in Galactose-Fed Rats.

    PubMed

    Kim, Chan-Sik; Kim, Junghyun; Lee, Yun Mi; Sohn, Eunjin; Kim, Jin Sook

    2016-03-01

    Naturally occurring coumarin compounds have received substantial attention due to their pharmaceutical effects. Esculetin is a coumarin derivative and a polyphenol compound that is used in a variety of therapeutic and pharmacological strategies. However, its effect on aldose reductase activity remains poorly understood. In this study, the potential beneficialeffects of esculetin on lenticular aldose reductase were investigated in galactose-fed (GAL) rats, an animal model of sugar cataracts. Cataracts were induced in Sprague-Dawley (SD) rats via a 50% galactose diet for 2 weeks, and groups of GAL rats were orally treated with esculetin (10 or 50 mg/kg body weight). In vehicle-treated GAL rats, lens opacificationwas observed, and swelling and membrane rupture of the lens fibercells were increased. Additionally, aldose reductase was highly expressed in the lens epithelium and superficialcortical fibersduring cataract development in the GAL rats. Esculetin reduced rat lens aldose reductase (RLAR) activity in vitro, and esculetin treatment significanty inhibited lens opacity, as well as morphological alterations, such as swelling, vacuolation and liquefaction of lens fibers,via the inhibition of aldose reductase in the GAL rats. These results indicate that esculetin is a useful treatment for galactose-induced cataracts. PMID:26902086

  6. Low apparent aldose reductase activity produced by monosaccharide autoxidation.

    PubMed Central

    Wolff, S P; Crabbe, M J

    1985-01-01

    Low apparent aldose reductase activity, as measured by NADPH oxidation, can be produced by the spontaneous autoxidation of monosaccharides. NADPH is oxidized to metabolically active NADP+ in a solution of autoxidizing DL-glyceraldehyde at rates of up to 15 X 10(-4) A340/min. The close parallelism between the effects of buffer salt type and concentration, monosaccharide structure and temperature activation on autoxidation and NADPH oxidation imply that autoxidation is a prerequisite for the NADPH oxidation, probably via the hydroperoxy radical. Nucleotide-binding proteins enhanced NADPH oxidation induced by DL-glyceraldehyde, up to 10.6-fold with glucose-6-phosphate dehydrogenase. Glutathione reductase-catalysed NADPH oxidation in the presence of autoxidizing monosaccharide showed many characteristics of the aldose reductase reaction. Aldose reductase inhibitors acted as antioxidants in inhibiting this NADPH oxidation. These results indicate that low apparent aldose reductase activities may be due to artifacts of monosaccharide autoxidation, and could provide an explanation for the non-linear steady-state kinetics observed with DL-glyceraldehyde and aldose reductase. PMID:2985042

  7. Aldose reductase mediates retinal microglia activation.

    PubMed

    Chang, Kun-Che; Shieh, Biehuoy; Petrash, J Mark

    2016-04-29

    Retinal microglia (RMG) are one of the major immune cells in charge of surveillance of inflammatory responses in the eye. In the absence of an inflammatory stimulus, RMG reside predominately in the ganglion layer and inner or outer plexiform layers. However, under stress RMG become activated and migrate into the inner nuclear layer (INL) or outer nuclear layer (ONL). Activated RMG in cell culture secrete pro-inflammatory cytokines in a manner sensitive to downregulation by aldose reductase inhibitors. In this study, we utilized CX3CR1(GFP) mice carrying AR mutant alleles to evaluate the role of AR on RMG activation and migration in vivo. When tested on an AR(WT) background, IP injection of LPS induced RMG activation and migration into the INL and ONL. However, this phenomenon was largely prevented by AR inhibitors or in AR null mice, or was exacerbated in transgenic mice that over-express AR. LPS-induced increases in ocular levels of TNF-α and CX3CL-1 in WT mice were substantially lower in AR null mice or were reduced by AR inhibitor treatment. These studies demonstrate that AR expression in RMG may contribute to the proinflammatory phenotypes common to various eye diseases such as uveitis and diabetic retinopathy. PMID:27033597

  8. Aldose reductase, oxidative stress, and diabetic mellitus.

    PubMed

    Tang, Wai Ho; Martin, Kathleen A; Hwa, John

    2012-01-01

    Diabetes mellitus (DM) is a complex metabolic disorder arising from lack of insulin production or insulin resistance (Diagnosis and classification of diabetes mellitus, 2007). DM is a leading cause of morbidity and mortality in the developed world, particularly from vascular complications such as atherothrombosis in the coronary vessels. Aldose reductase (AR; ALR2; EC 1.1.1.21), a key enzyme in the polyol pathway, catalyzes nicotinamide adenosine dinucleotide phosphate-dependent reduction of glucose to sorbitol, leading to excessive accumulation of intracellular reactive oxygen species (ROS) in various tissues of DM including the heart, vasculature, neurons, eyes, and kidneys. As an example, hyperglycemia through such polyol pathway induced oxidative stress, may have dual heart actions, on coronary blood vessel (atherothrombosis) and myocardium (heart failure) leading to severe morbidity and mortality (reviewed in Heather and Clarke, 2011). In cells cultured under high glucose conditions, many studies have demonstrated similar AR-dependent increases in ROS production, confirming AR as an important factor for the pathogenesis of many diabetic complications. Moreover, recent studies have shown that AR inhibitors may be able to prevent or delay the onset of cardiovascular complications such as ischemia/reperfusion injury, atherosclerosis, and atherothrombosis. In this review, we will focus on describing pivotal roles of AR in the pathogenesis of cardiovascular diseases as well as other diabetic complications, and the potential use of AR inhibitors as an emerging therapeutic strategy in preventing DM complications. PMID:22582044

  9. Aldose reductase inhibitory compounds from Xanthium strumarium.

    PubMed

    Yoon, Ha Na; Lee, Min Young; Kim, Jin-Kyu; Suh, Hong-Won; Lim, Soon Sung

    2013-09-01

    As part of our ongoing search for natural sources of therapeutic and preventive agents for diabetic complications, we evaluated the inhibitory effects of components of the fruit of Xanthium strumarium (X. strumarium) on aldose reductase (AR) and galactitol formation in rat lenses with high levels of glucose. To identify the bioactive components of X. strumarium, 7 caffeoylquinic acids and 3 phenolic compounds were isolated and their chemical structures were elucidated on the basis of spectroscopic evidence and comparison with published data. The abilities of 10 X. strumarium-derived components to counteract diabetic complications were investigated by means of inhibitory assays with rat lens AR (rAR) and recombinant human AR (rhAR). From the 10 isolated compounds, methyl-3,5-di-O-caffeoylquinate showed the most potent inhibition, with IC₅₀ values of 0.30 and 0.67 μM for rAR and rhAR, respectively. In the kinetic analyses using Lineweaver-Burk plots of 1/velocity and 1/substrate, methyl-3,5-di-O-caffeoylquinate showed competitive inhibition of rhAR. Furthermore, methyl-3,5-di-O-caffeoylquinate inhibited galactitol formation in the rat lens and in erythrocytes incubated with a high concentration of glucose, indicating that this compound may be effective in preventing diabetic complications. PMID:23604720

  10. The inhibitory activity of aldose reductase in vitro by constituents of Garcinia mangostana Linn.

    PubMed

    Fatmawati, Sri; Ersam, Taslim; Shimizu, Kuniyoshi

    2015-01-15

    We investigated aldose reductase inhibition of Garcinia mangostana Linn. from Indonesia. Dichloromethane extract of the root bark of this tree was found to demonstrate an IC50 value of 11.98 µg/ml for human aldose reductase in vitro. From the dichloromethane fraction, prenylated xanthones were isolated as potent human aldose reductase inhibitors. We discovered 3-isomangostin to be most potent against aldose reductase, with an IC50 of 3.48 µM. PMID:25636870

  11. Activated and unactivated forms of human erythrocyte aldose reductase.

    PubMed Central

    Srivastava, S K; Hair, G A; Das, B

    1985-01-01

    Aldose reductase (alditol:NADP+ 1-oxidoreductase, EC 1.1.1.21) has been partially purified from human erythrocytes by DEAE-cellulose (DE-52) column chromatography. This enzyme is activated severalfold upon incubation with 10 microM each glucose 6-phosphate, NADPH, and glucose. The activation of the enzyme was confirmed by following the oxidation of NADPH as well as the formation of sorbitol with glucose as substrate. The activated form of aldose reductase exhibited monophasic kinetics with both glyceraldehyde and glucose (Km of glucose = 0.68 mM and Km of glyceraldehyde = 0.096 mM), whereas the native (unactivated) enzyme exhibited biphasic kinetics (Km of glucose = 9.0 and 0.9 mM and Km of glyceraldehyde = 1.1 and 0.14 mM). The unactivated enzyme was strongly inhibited by aldose reductase inhibitors such as sorbinil, alrestatin, and quercetrin, and by phosphorylated intermediates such as ADP, glycerate 3-phosphate, glycerate 1,3-bisphosphate, and glycerate 2,3-trisphosphate. The activated form of the enzyme was less susceptible to inhibition by aldose reductase inhibitors and phosphorylated intermediates. PMID:3933003

  12. Human brain aldehyde reductases: relationship to succinic semialdehyde reductase and aldose reductase.

    PubMed

    Hoffman, P L; Wermuth, B; von Wartburg, J P

    1980-08-01

    Human brain contains multiple forms of aldehyde-reducing enzymes. One major form (AR3), as previously shown, has properties that indicate its identity with NADPH-dependent aldehyde reductase isolated from brain and other organs of various species; i.e., low molecular weight, use of NADPH as the preferred cofactor, and sensitivity to inhibition by barbiturates. A second form of aldehyde reductase ("SSA reductase") specifically reduces succinic semialdehyde (SSA) to produce gamma-hydroxybutyrate. This enzyme form has a higher molecular weight than AR3, and uses NADH as well as NADPH as cofactor. SSA reductase was not inhibited by pyrazole, oxalate, or barbiturates, and the only effective inhibitor found was the flavonoid quercetine. Although AR3 can also reduce SSA, the relative specificity of SSA reductase may enhance its in vivo role. A third form of human brain aldehyde reductase, AR2, appears to be comparable to aldose reductases characterized in several species, on the basis of its activity pattern with various sugar aldehydes and its response to characteristic inhibitors and activators, as well as kinetic parameters. This enzyme is also the most active in reducing the aldehyde derivatives of biogenic amines. These studies suggest that the various forms of human brain aldehyde reductases may have specific physiological functions. PMID:6778961

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

    PubMed

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

    2003-09-01

    Isoaffinetin (5,7,3',4',5'-pentahydroxyflavone-6-C-glucoside) was isolated from Manilkara indica as a potent inhibitor of lens aldose reductase by bioassay-directed fractionation. This C-glucosyl flavone showed specific inhibition against aldose reductases (rat lens, porcine lens and recombinant human) with no inhibition against aldehyde reductase and NADH oxidase. Kinetic analysis showed that isoaffinetin exhibited uncompetitive inhibition against both dl-glyceraldehyde and NADPH. A structure-activity relationship study revealed that the increasing number of hydroxy groups in the B-ring contributes to the increase in aldose reductase inhibition by C-glucosyl flavones. PMID:14598214

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

    SciTech Connect

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

    1999-09-03

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

  15. Molecular cloning and characterization of Schistosoma japonicum aldose reductase.

    PubMed

    Liu, Jian; Wang, Jipeng; Wang, Shuqi; Xu, Bin; Liu, Xiufeng; Wang, Xiaoning; Hu, Wei

    2013-02-01

    Antioxidant defense is an essential mechanism for schistosomes to cope with damage from host immune-generated reactive oxygen species. The evaluation of the effects of aldose reductase, an important enzyme that may be involved in this system, has long been neglected. In the present study, aldose reductase of Schistosoma japonicum (SjAR) was cloned and characterized. The activity of SjAR was assessed in vitro and was suppressed by the reported inhibitor, epalrestat. RT-PCR analysis revealed that SjAR was expressed at each of the development stages analyzed with increased levels in cercariae. The results also showed that SjAR was expressed at higher levels in adult male worms than in adult female worms. Indirect enzyme-linked immunosorbent assay and western blot analysis indicated that the purified recombinant SjAR (rSjAR) protein displayed a significant level of antigenicity. Immunolocalization analysis revealed that SjAR was mainly distributed in the gynecophoral canal of adult male worms. BALB/c mice immunized with rSjAR induced a 32.91 % worm reduction compared to the adjuvant group (P < 0.01). Moreover, a 28.27 % reduction in egg development in the liver (P > 0.05) and a 42.75 % reduction in egg development in the fecal samples (P < 0.05) were also observed. These results suggested that SjAR may be a potential new drug target or vaccine candidate for schistosomes. PMID:23160889

  16. Virtual screening of plant derived compounds for aldose reductase inhibition using molecular docking.

    PubMed

    Muppalaneni, Naresh Babu; Rao, Allam Appa

    2012-01-01

    The role of the aldose reductase in type 2 diabetes is widely described. Therefore, it is of interest to identify plant derived compounds to inhibit its activity. We studied the protein-ligand interaction of 267 compounds from different parts of seven plants (Allium sativum, Coriandrum sativum, Dacus carota, Murrayyakoneigii, Eucalyptus, Calendula officinalis and Lycopersicon esculentum) with aldose reductase as the target protein. Molecular docking and re-scoring of top ten compounds (using GOLD, AutoDock Vina, eHiTS, PatchDock and MEDock) followed by rank-sum technique identified compound allium38 with high binding affinity for aldose reductase. PMID:23275691

  17. Studies on WF-3681, a novel aldose reductase inhibitor. I. Taxonomy, fermentation, isolation and characterization.

    PubMed

    Nishikawa, M; Tsurumi, Y; Namiki, T; Yoshida, K; Okuhara, M

    1987-10-01

    WF-3681 was isolated from a cultured filtrate of Chaetomella raphigera as a novel inhibitor of aldose reductase. It was extracted with ethyl acetate and then purified with silica gel chromatography. Its molecular formula was determined to be C13H12O5 by elemental analysis and high resolution electron impact mass spectrometry. IC50 of WF-3681 was 2.5 X 10(-7) M for partially purified aldose reductase of rabbit lens. PMID:3119547

  18. In vitro expression of rat lens aldose reductase in Escherichia coli.

    PubMed Central

    Old, S E; Sato, S; Kador, P F; Carper, D A

    1990-01-01

    Aldose reductase (alditol:NADP+ oxidoreductase, EC 1.1.1.21), an enzyme that converts glucose to sorbitol, the first step of the polyol pathway, has been implicated in secondary complications of diabetes, such as cataracts, retinopathy, neuropathy, and nephropathy. Aldose reductase inhibitors have been observed to prevent or delay the onset of these complications; however, more potent and specific inhibitors are needed. Development of new inhibitors necessitates a better understanding of the molecular structure of this protein. To elucidate the structure-function relationships of aldose reductase and to develop methods of regulating this enzyme, large and homogeneous quantities of rat lens aldose reductase have been expressed in bacterial cells. A construction of the complete coding sequence and 3' untranslated region for rat lens aldose reductase was assembled in the expression vector pKK233-2 (Pharmacia). This construction expresses an active enzyme that has been purified and demonstrates kinetic, immunological, and inhibitory properties similar to rat lens aldose reductase. Images PMID:2114645

  19. Isoquinoline alkaloids from Tinospora cordifolia inhibit rat lens aldose reductase.

    PubMed

    Patel, Mayurkumar B; Mishra, Shrihari

    2012-09-01

    The inhibitory activity of Tinospora cordifolia stem-derived alkaloids was evaluated against lens aldose reductase (AR) isolated from male Wistar rats. Anticataract potential of the alkaloids of T. cordifolia was evaluated in vitro in rat lenses, considering the activity of normal rat lenses as 100%. The biologically active constituents of T. cordifolia extract were characterized as the isoquinoline alkaloids, jatrorrhizine, palmatine and magnoflorine, by spectral analysis. The inhibitory effects varied with all chemicals and concentrations used. The inhibitory concentration (IC₅₀) values of jatrorrhizine, palmatine and magnoflorine are 3.23, 3.45 and 1.25 µg/mL respectively. The concentration of maximum activity was selected for its effect on galactose-induced polyol accumulation in vitro. The percentage inhibition of galactose-induced polyol accumulation was 62.6, 58.8 and 27.7% in the presence of jatrorrhizine, palmatine and magnoflorine, respectively. Magnoflorine may be useful as lead compounds and new agents for AR inhibition. PMID:22294283

  20. Autoimmunity in Membranous Nephropathy Targets Aldose Reductase and SOD2

    PubMed Central

    Prunotto, Marco; Carnevali, Maria Luisa; Candiano, Giovanni; Murtas, Corrado; Bruschi, Maurizio; Corradini, Emilia; Trivelli, Antonella; Magnasco, Alberto; Petretto, Andrea; Santucci, Laura; Mattei, Silvia; Gatti, Rita; Scolari, Francesco; Kador, Peter; Allegri, Landino

    2010-01-01

    Glomerular targets of autoimmunity in human membranous nephropathy are poorly understood. Here, we used a combined proteomic approach to identify specific antibodies against podocyte proteins in both serum and glomeruli of patients with membranous nephropathy (MN). We detected specific anti–aldose reductase (AR) and anti–manganese superoxide dismutase (SOD2) IgG4 in sera of patients with MN. We also eluted high titers of anti-AR and anti-SOD2 IgG4 from microdissected glomeruli of three biopsies of MN kidneys but not from biopsies of other glomerulonephritides characterized by IgG deposition (five lupus nephritis and two membranoproliferative glomerulonephritis). We identified both antigens in MN biopsies but not in other renal pathologies or normal kidney. Confocal and immunoelectron microscopy (IEM) showed co-localization of anti-AR and anti-SOD2 with IgG4 and C5b-9 in electron-dense podocyte immune deposits. Preliminary in vitro experiments showed an increase of SOD2 expression on podocyte plasma membrane after treatment with hydrogen peroxide. In conclusion, our data support AR and SOD2 as renal antigens of human MN and suggest that oxidative stress may drive glomerular SOD2 expression. PMID:20150532

  1. Analysis of enzyme-catalyzed nucleotide modification by aldose reductase

    SciTech Connect

    Grimshaw, C.E.

    1987-05-01

    Homogeneous bovine kidney aldose reductase catalyzes two reactions in addition to the normal aldehyde-dependent oxidation of NADPH. First, adduct formation between the oxidized nucleotide and the oxidized substrate is observed during turnover due to initial formation of a reversible E:NADP/sup +/:R-CHO ternary complex, which subsequently reacts to give the covalent complex (E:NADP/sup +/-R-CHO). The reaction is enzyme-catalyzed with substantial enhancement of both the pseudo-first order rate constant and the overall K/sub eq/ relative to the reaction with free NADP/sup +/ in aqueous buffer. Analysis of the concentration dependence and time-course for reversible dead-end and covalent complex formation are described for several aldehyde and nucleotide substrates. Non-linear time courses for aldehyde reduction and substrate inhibition by the aldehyde substrate in initial velocity studies are completely accounted for by this mechanism, thereby eliminating a simple Dalziel-type explanation for the substrate activation by aldehyde which is also observed. Second, enzyme-catalyzed oxidation of NADPH occurs in the absence of aldehyde substrate with a rate equal to .03% of V/sub max/ for the normal reduction of glyceraldehyde. By 500 MHz /sup 1/H-NMR, the enzyme-catalyzed oxidation of (4-/sup 2/H)NADPH appears to be greater than 95% stereospecific. Spectroscopic evidence for a similar oxidation reaction is observed for the covalent E:NADP/sup +/-R-CHO adduct with glyceraldehyde, but not with glycolaldehyde.

  2. Aldose reductase inhibition improves nerve conduction velocity in diabetic patients.

    PubMed

    Judzewitsch, R G; Jaspan, J B; Polonsky, K S; Weinberg, C R; Halter, J B; Halar, E; Pfeifer, M A; Vukadinovic, C; Bernstein, L; Schneider, M; Liang, K Y; Gabbay, K H; Rubenstein, A H; Porte, D

    1983-01-20

    To assess the potential role of polyol-pathway activity in diabetic neuropathy, we measured the effects of sorbinil--a potent inhibitor of the key polyol-pathway enzyme aldose reductase--on nerve conduction velocity in 39 stable diabetics in a randomized, double-blind, cross-over trial. During nine weeks of treatment with sorbinil (250 mg per day), nerve conduction velocity was greater than during a nine-week placebo period for all three nerves tested: the peroneal motor nerve (mean increase [+/- S.E.M.], 0.70 +/- 0.24 m per second, P less than 0.008), the median motor nerve (mean increase, 0.66 +/- 0.27, P less than 0.005), and the median sensory nerve (mean increase, 1.16 +/- 0.50, P less than 0.035). Conduction velocity for all three nerves declined significantly within three weeks after cessation of the drug. These effects of sorbinil were not related to glycemic control, which was constant during the study. Although the effect of sorbinil in improving nerve conduction velocity in diabetics was small, the findings suggest that polyol-pathway activity contributes to slowed nerve conduction in diabetics. The clinical applicability of these observations remains to be determined, but they encourage further exploration of this approach to the treatment or prevention of diabetic neuropathy. PMID:6401351

  3. Overexpression of Aldose Reductase Render Mouse Hepatocytes More Sensitive to Acetaminophen Induced Oxidative Stress and Cell Death.

    PubMed

    Ahmed, Munzir M E; Al-Obosi, J A S; Osman, H M; Shayoub, M E

    2016-04-01

    Acetaminophen (APAP) a commonly used drug for decrease the fever and pain but is capable to induced hepatotoxicity at over dose. This study was carried out to investigate the effect of APAP on the expression of anti-apoptotic and antioxidative defense genes, and whether aldose reductase over-expressing plasmid capable to protect against APAP-induced oxidative stress and cell death. APAP treatment induced oxidative stress and hepatotoxicity, and significantly increased aldose reductase mRNA and protein expression in mouse hepatocyte (AML-12). Unexpectedly, AML-12 cells over-expressing aldose reductase augmented APAP-induced reduction in cell viability, reactive oxygen species (ROS) production, glutathione (GSH) depletion and glutathione S-transferase A2 expression. Moreover, over-expression of aldose reductase potentiated APAP induced reduction on proliferating cell nuclear antigen, B cell lymphoma-extra large (bcl-xL), catalase, glutathione peroxidase-1 (GPx-1) and abolished APAP-induced B-cell lymphoma 2 (bcl-2) inductions. Further, over-expression of aldose reductase significantly abolished AMP activated protein kinase (AMPK) activity in APAP-treated cells and induced p53 expression. This results demonstrate that APAP induced toxicity in AML-12, increased aldose reductase expression, and over-expression of aldose reductase render this cell more susceptible to APAP induced oxidative stress and cell death, this probably due to inhibition AMPK or bcl-2 activity, or may due to competition between aldose reductase and glutathione reductase for NADPH. PMID:27069324

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

    PubMed

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

    2016-07-01

    To find aldose reductase inhibitors, two previously unreported compounds, grandifolias H and I, and five known compounds, including rosmarinic acid and rosmarinic acid derivatives, were isolated from the roots of Salvia grandifolia. A series of rosmarinic acid derivatives was obtained from rosmarinic acid using simple synthetic methods. The aldose reductase inhibitory activity of the isolated and synthesized compounds was assessed. Seven of the tested compounds showed moderate aldose reductase inhibition (IC50=0.06-0.30μM). The structure-activity relationship of aldose reductase inhibitory activity of rosmarinic acid derivatives was discussed for the first time. This study provided useful information that will facilitate the development of aldose reductase inhibitors. PMID:27233987

  5. Affinity purifications of aldose reductase and xylitol dehydrogenase from the xylose-fermenting yeast Pachysolen tannophilus

    SciTech Connect

    Bolen, P.L.; Roth, K.A.; Freer, S.N.

    1986-10-01

    Although xylose is a major product of hydrolysis of lignocellulosic materials, few yeasts are able to convert it to ethanol. In Pachysolen tannophilus, one of the few xylose-fermenting yeasts found, aldose reductase and xylitol dehydrogenase were found to be key enzymes in the metabolic pathway for xylose fermentation. This paper presents a method for the rapid and simultaneous purification of both aldose reductase and xylitol dehydrogenase from P. tannophilus. Preliminary studies indicate that this method may be easily adapted to purify similar enzymes from other xylose-fermenting yeasts.

  6. Aldose reductase expression as a risk factor for cataract

    PubMed Central

    Snow, Anson; Shieh, Biehuoy; Chang, Kun-Che; Pal, Arttatrana; Lenhart, Patricia; Ammar, David; Ruzycki, Philip; Palla, Suryanarayana; Reddy, G. Bhanuprakesh; Petrash, J. Mark

    2015-01-01

    Aldose reductase (AR) is thought to play a role in the pathogenesis of diabetic eye diseases, including cataract and retinopathy. However, not all diabetics develop ocular complications. Paradoxically, some diabetics with poor metabolic control appear to be protected against retinopathy, while others with a history of excellent metabolic control develop severe complications. These observations indicate that one or more risk factors may influence the likelihood that an individual with diabetes will develop cataracts and/or retinopathy. We hypothesize that an elevated level of AR gene expression could confer higher risk for development of diabetic eye disease. To investigate this hypothesis, we examined the onset and severity of diabetes-induced cataract in transgenic mice, designated AR-TG, that were either heterozygous or homozygous for the human AR (AKR1B1) transgene construct. AR-TG mice homozygous for the transgene demonstrated a conditional cataract phenotype, whereby they developed lens vacuoles and cataract-associated structural changes only after induction of experimental diabetes; no such changes were observed in AR-TG heterozygotes or nontransgenic mice with or without experimental diabetes induction. We observed that nondiabetic AR-TG mice did not show lens structural changes even though they had lenticular sorbitol levels almost as high as the diabetic AR-TG lenses that showed early signs of cataract. Over-expression of AR led to increases in the ratio of activated to total levels of extracellular signal-regulated kinase (ERK1/2) and c-Jun N-terminal (JNK1/2), which are known to be involved in cell growth and apoptosis respectively. After diabetes induction, AR-TG but not WT controls had decreased levels of phosphorylated as well as total ERK1/2 and JNK1/2 compared to their nondiabetic counterparts. These results indicate that high AR expression in the context of hyperglycemia and insulin deficiency may constitute a risk factor that could predispose the

  7. Aldose reductase expression as a risk factor for cataract.

    PubMed

    Snow, Anson; Shieh, Biehuoy; Chang, Kun-Che; Pal, Arttatrana; Lenhart, Patricia; Ammar, David; Ruzycki, Philip; Palla, Suryanarayana; Reddy, G Bhanuprakesh; Petrash, J Mark

    2015-06-01

    Aldose reductase (AR) is thought to play a role in the pathogenesis of diabetic eye diseases, including cataract and retinopathy. However, not all diabetics develop ocular complications. Paradoxically, some diabetics with poor metabolic control appear to be protected against retinopathy, while others with a history of excellent metabolic control develop severe complications. These observations indicate that one or more risk factors may influence the likelihood that an individual with diabetes will develop cataracts and/or retinopathy. We hypothesize that an elevated level of AR gene expression could confer higher risk for development of diabetic eye disease. To investigate this hypothesis, we examined the onset and severity of diabetes-induced cataract in transgenic mice, designated AR-TG, that were either heterozygous or homozygous for the human AR (AKR1B1) transgene construct. AR-TG mice homozygous for the transgene demonstrated a conditional cataract phenotype, whereby they developed lens vacuoles and cataract-associated structural changes only after induction of experimental diabetes; no such changes were observed in AR-TG heterozygotes or nontransgenic mice with or without experimental diabetes induction. We observed that nondiabetic AR-TG mice did not show lens structural changes even though they had lenticular sorbitol levels almost as high as the diabetic AR-TG lenses that showed early signs of cataract. Over-expression of AR led to increases in the ratio of activated to total levels of extracellular signal-regulated kinase (ERK1/2) and c-Jun N-terminal (JNK1/2), which are known to be involved in cell growth and apoptosis, respectively. After diabetes induction, AR-TG but not WT controls had decreased levels of phosphorylated as well as total ERK1/2 and JNK1/2 compared to their nondiabetic counterparts. These results indicate that high AR expression in the context of hyperglycemia and insulin deficiency may constitute a risk factor that could predispose the

  8. In vitro antidiabetic effects of selected fruits and vegetables against glycosidase and aldose reductase.

    PubMed

    Wu, Tong; Luo, Jiaqiang; Xu, Baojun

    2015-11-01

    In vitro antidiabetic effect of fruits and vegetables with reports as folk remedies were investigated. The antidiabetic effects were evaluated by comparing the inhibitory properties of α-glycosidase, aldose reductase, and antioxidant activity. The results indicated that lychee extract exhibited the best dose-dependent inhibitory activity against α-glycosidase with IC 50 of 10.4 mg/mL, and lemon peel extract exhibited aldose reductase inhibitory potential with IC 50 value at 3.63 mg/mL. Besides, the result also showed that the inhibitory effects of blueberry and plum against α-glycosidase were strong among the fruits samples. Bitter gourd and eggplant demonstrated significant inhibitory potential against aldose reductase, with IC 50 values at 8.55 mg/mL and 8.06 mg/mL, respectively. The result from correlation analysis part showed that the antioxidant activities of selected fruits and vegetables were found related to their health beneficial effects, as there was positive correlations between total flavonoids content (TFC) and aldose reductase inhibitory activity (r (2) = 0.556). PMID:26788291

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

    SciTech Connect

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

    2007-11-01

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

  10. Kinetic and molecular docking studies of loganin and 7-O-galloyl-D-sedoheptulose from Corni Fructus as therapeutic agents for diabetic complications through inhibition of aldose reductase.

    PubMed

    Lee, Chan Mee; Jung, Hyun Ah; Oh, Sang Ho; Park, Chan Hum; Tanaka, Takashi; Yokozawa, Takako; Choi, Jae Sue

    2015-06-01

    Aldose reductase (AR) is a key enzyme in the polyol pathway that is strongly implicated in the pathogenesis of diabetic complications. AR inhibitors have been proposed as therapeutic agents for diabetic complications through suppression of sorbitol formation and accumulation. In this study, we evaluated whether two major compounds of Corni Fructus, loganin and 7-O-galloyl-D-sedoheptulose, had an inhibitory effect on diabetic complications through AR inhibition. Because the iridoid glycoside loganin and the low-molecular-weight polyphenol 7-O-galloyl-D-sedoheptulose showed marginal inhibitory activities against rat lens AR (RLAR) and human recombinant AR (HRAR) in inhibition assays, we performed enzyme kinetic analyses and molecular simulation of the interaction of these two compounds with AR to further investigate their potential as inhibitors of diabetic complications. In kinetic analysis using Lineweaver-Burk plots and Dixon plots, loganin and 7-O-galloyl-D-sedoheptulose were both mixed inhibitors of RLAR with inhibition constants (K i) of 27.99 and 128.68 μΜ, respectively. Moreover, molecular docking simulation of both compounds demonstrated negative binding energies (Autodock 4.0 = -6.7; -7.5 kcal/mol; Fred 2.0 = -59.4; -63.2 kcal/mol) indicating a high affinity and tight binding capacity for the active site of the enzyme. Iridoid nucleus and aromatic ring systems and glycoside and sedoheptulose moieties were found to bind tightly to the specificity pocket and the anion binding pocket in RLAR through Phe123, His111, Trp21, Tyr49, His111, and Trp112 residues. Our results clearly indicate that loganin and 7-O-galloyl-D-sedoheptulose have great promise for the treatment of diabetic complications through inhibition of AR. PMID:25315636

  11. Mapping of aldose reductase gene sequences to human chromosomes 1, 3, 7, 9, 11, and 13

    SciTech Connect

    Bateman, J.B.; Kojis, T. UCLA School of Medicine, Los Angeles, CA ); Heinzmann, C.; Sparkes, R.S.; Klisak, I.; Diep, A. ); Carper, D. ); Nishimura, Chihiro ); Mohandas, T. )

    1993-09-01

    Aldose reductase (alditol:NAD(P)+ 1-oxidoreductase; EC 1.1.1.21) (AR) catalyzes the reduction of several aldehydes, including that of glucose, to the corresponding sugar alcohol. Using a complementary DNA clone encoding human AR, the authors mapped the gene sequences to human chromosomes 1, 3, 7, 9, 11, 13, 14, and 18 by somatic cell hybridization. By in situ hybridization analysis, sequences were localized to human chromosomes 1q32-q43, 3p12, 7q31-q35, 9q22, 11p14-p15, and 13q14-q21. As a putative functional AR gene has been mapped to chromosome 7 and a putative pseudogene to chromosome 3, the sequences on the other seven chromosomes may represent other active genes, non-aldose reductase homologous sequences, or pseudogenes. 24 refs., 3 figs., 2 tabs.

  12. Effects of aldose reductase inhibitor treatment in diabetic polyneuropathy - a clinical and neurophysiological study.

    PubMed Central

    Fagius, J; Jameson, S

    1981-01-01

    The efficacy of treatment with an aldose reductase inhibitor (1,3-dioxo-1 H-benz-de-isoquinoline-2(3H)-acetic acid, AY-22,284, Alrestatin) on peripheral nerve function in diabetic polyneuropathy was assessed. Thirty patients with long-standing diabetes and slight to moderate neuropathy participated in the double-blind placebo trial. Clinical examination, sensory threshold determinations for vibratory, tactile and thermal stimuli, conduction velocity measurements and studies of automatic function were performed to evaluate the treatment. Significant differences favouring Alrestatin over placebo were found for many of the measured variables, whereas no changes occurred on placebo. The apparent improvement of neuropathy occurred despite persisting hyperglycaemia. The results indicate that aldose reductase inhibitor treatment may be of value in diabetic polyneuropathy, and provide support for the sorbitol pathway hypothesis of diabetic polyneuropathy. PMID:6801211

  13. Inhibitory effects of Colocasia esculenta (L.) Schott constituents on aldose reductase.

    PubMed

    Li, Hong Mei; Hwang, Seung Hwan; Kang, Beom Goo; Hong, Jae Seung; Lim, Soon Sung

    2014-01-01

    The goal of this study was to determine the rat lens aldose reductase-inhibitory effects of 95% ethanol extracts from the leaves of C. esculenta and, its organic solvent soluble fractions, including the dichloromethane (CH2Cl2), ethyl acetate (EtOAc), n-butanol (BuOH) and water (H2O) layers, using dl-glyceraldehyde as a substrate. Ten compounds, namely tryptophan (1), orientin (2), isoorientin (3), vitexin (4), isovitexin (5), luteolin-7-O-glucoside (6), luteolin-7-O-rutinoside (7), rosmarinic acid (8), 1-O-feruloyl-d-glucoside (9) and 1-O-caffeoyl-d-glucoside (10) were isolated from the EtOAc and BuOH fractions of C. esculenta. The structures of compounds 1-10 were elucidated by spectroscopic methods and comparison with previous reports. All the isolates were subjected to an in vitro bioassay to evaluate their inhibitory activity against rat lens aldose reductase. Among tested compounds, compounds 2 and 3 significantly inhibited rat lens aldose reductase, with IC50 values of 1.65 and 1.92 μM, respectively. Notably, the inhibitory activity of orientin was 3.9 times greater than that of the positive control, quercetin (4.12 μM). However, the isolated compounds showed only moderate ABTS+ [2,29-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)] activity. These results suggest that flavonoid derivatives from Colocasia esculenta (L.) Schott represent potential compounds for the prevention and/or treatment of diabetic complications. PMID:25255750

  14. GP-1447, an inhibitor of aldose reductase, prevents the progression of diabetic cataract in rats.

    PubMed

    Kawakubo, Ken; Mori, Asami; Sakamoto, Kenji; Nakahara, Tsutomu; Ishii, Kunio

    2012-01-01

    We examined the effects of GP-1447 (3-[(4,5,7-trifluorobenzothiazol-2-yl)methyl]-5-methylphenyl acetic acid) on existing cataracts and sorbitol content in the lens in rats with streptozotocin-induced diabetes. GP-1447 is an inhibitor of aldose reductase, which is the first enzyme in the polyol pathway. Cataracts in the central region of the lens were observed in 7 of 14 eyes (50%) by the fifth week after induction of diabetes, and development of mature cataracts was observed in most lenses by the ninth week. In diabetic rats that received GP-1447 treatment beginning in the fifth week after induction of diabetes, progression of cataracts was observed for 1 week after initiation of treatment. Thereafter, the severity of cataracts did not change substantially. Sorbitol levels in the lens peaked during the first week of diabetes, and this increase was maintained during the 9-week observation period. Elevated sorbitol levels in the lenses of diabetic rats gradually declined after GP-1447 treatment was started on the fifth week after induction of diabetes. Cataracts and sorbitol elevation were not observed in the lenses of controls or diabetic rats treated with GP-1447 immediately after induction of diabetes. These results suggest that the polyol pathway plays an important role in both the appearance and progression of cataracts in diabetic rats. Inhibition of aldose reductase could significantly prevent progression of existing cataracts. PMID:22687477

  15. Clinical and neurophysiological studies of aldose reductase inhibitor ponalrestat in chronic symptomatic diabetic peripheral neuropathy.

    PubMed

    Florkowski, C M; Rowe, B R; Nightingale, S; Harvey, T C; Barnett, A H

    1991-01-01

    Increased flux through the polyol pathway mediated by the enzyme aldose reductase may be associated with the development of diabetic neuropathy. Fifty-four diabetic patients (median age 56 yr, range 25-65 yr) with chronic neuropathic symptoms were randomly allocated to placebo or aldose reductase inhibition (300 or 600 mg ponalrestat ICI 128436) groups for 24 wk. Patients with vibration perception thresholds (VPTs) greater than 35 V at the great toe or thermal difference thresholds (TTs) greater than 10 degrees C on the dorsum of the foot were excluded from the trial. No significant changes were observed in symptoms of pain, numbness, or paresthesia between ponalrestat and placebo groups, and there were no improvements in VPT or TT at several sites. Posterior tibial nerve conduction velocity changed from 35.3 +/- 4.9 m/s at baseline to 33.4 +/- 4.0 m/s at 24 wk (NS) with placebo compared with 37.6 +/- 5.6 vs. 37.2 +/- 8.7 m/s (NS) with 300 mg ponalrestat and 34.5 +/- 6.1 vs. 36.2 +/- 6.8 m/s (NS) with 600 mg ponalrestat. Further studies are indicated with intervention at an earlier stage in the evolution of neuropathy and for longer periods. PMID:1901808

  16. Phytochemical analysis with the antioxidant and aldose reductase inhibitory capacities of Tephrosia humilis aerial parts' extracts.

    PubMed

    Plioukas, Michael; Gabrieli, Chrysi; Lazari, Diamanto; Kokkalou, Eugene

    2016-06-01

    The aerial parts of Tephrosia humilis were tested about their antioxidant potential, their ability to inhibit the aldose/aldehyde reductase enzymes and their phenolic content. The plant material was exhaustively extracted with petroleum ether, dichloromethane and methanol, consecutively. The concentrated methanol extract was re-extracted, successively, with diethyl ether, ethyl acetate and n-butanol. All extracts showed significant antioxidant capacity, but the most effective was the ethyl acetate extract. As about the aldose reductase inhibition, all fractions, except the aqueous, were strong inhibitors of the enzyme, with the n-butanolic and ethyl acetate fractions to inhibit the enzyme above 75%. These findings provide support to the ethnopharmacological usage of the plant as antioxidant and validate its potential to act against the long-term diabetic complications. The phytochemical analysis showed the presence of 1,4-dihydroxy-3,4-(epoxyethano)-5-cyclohexene(1), cleroindicin E(2), lupeol(3), methyl p-coumarate(4), methyl 4-hydroxybenzoate(5), prunin(6), 5,7,2',5'-tetrahydroxyflavanone 7-rutinoside(7), protocatechuic acid(8), luteolin 7-glucoside(9), apigenin(10), naringin(11), rhoifolin(12) and luteolin 7-glucuronate(13). PMID:26209262

  17. Inhibitory effects of Zingiber officinale Roscoe derived components on aldose reductase activity in vitro and in vivo.

    PubMed

    Kato, Atsushi; Higuchi, Yasuko; Goto, Hirozo; Kizu, Haruhisa; Okamoto, Tadashi; Asano, Naoki; Hollinshead, Jackie; Nash, Robert J; Adachi, Isao

    2006-09-01

    Ginger (Zingiber officinale Roscoe) continues to be used as an important cooking spice and herbal medicine around the world. Scientific research has gradually verified the antidiabetic effects of ginger. Especially gingerols, which are the major components of ginger, are known to improve diabetes including the effect of enhancement against insulin-sensitivity. Aldose reductase inhibitors have considerable potential for the treatment of diabetes, without increased risk of hypoglycemia. The assay for aldose reductase inhibitors in ginger led to the isolation of five active compounds including 2-(4-hydroxy-3-methoxyphenyl)ethanol (2) and 2-(4-hydroxy-3-methoxyphenyl)ethanoic acid (3). Compounds 2 and 3 were good inhibitors of recombinant human aldose reductase, with IC50 values of 19.2 +/- 1.9 and 18.5 +/- 1.1 microM, respectively. Furthermore, these compounds significantly suppressed not only sorbitol accumulation in human erythrocytes but also lens galactitol accumulation in 30% of galactose-fed cataract rat model. A structure-activity relationship study revealed that the applicable side alkyl chain length and the presence of a C3 OCH3 group in the aromatic ring are essential features for enzyme recognition and binding. These results suggested that it would contribute to the protection against or improvement of diabetic complications for a dietary supplement of ginger or its extract containing aldose reductase inhibitors. PMID:16939321

  18. Model of the catalytic mechanism of human aldose reductase based on quantum chemical calculations.

    SciTech Connect

    Cachau, R. C.; Howard, E. H.; Barth, P. B.; Mitschler, A. M.; Chevrier, B. C.; Lamour, V.; Joachimiak, A.; Sanishvili, R.; Van Zandt, M.; Sibley, E.; Moras, D.; Podjarny, A.; UPR de Biologie Structurale; National Cancer Inst.; Univ. Louis Pasteur; Inst. for Diabetes Discovery, Inc.

    2000-01-01

    Aldose Reductase is an enzyme involved in diabetic complications, thoroughly studied for the purpose of inhibitor development. The structure of an enzyme-inhibitor complex solved at sub-atomic resolution has been used to develop a model for the catalytic mechanism. This model has been refined using a combination of Molecular Dynamics and Quantum calculations. It shows that the proton donation, the subject of previous controversies, is the combined effect of three residues: Lys 77, Tyr 48 and His 110. Lys 77 polarises the Tyr 48 OH group, which donates the proton to His 110, which becomes doubly protonated. His 110 then moves and donates the proton to the substrate. The key information from the sub-atomic resolution structure is the orientation of the ring and the single protonafion of the His 110 in the enzyme-inhibitor complex. This model is in full agreement with all available experimental data.

  19. Osmolarity and glucose differentially regulate aldose reductase activity in cultured mouse podocytes.

    PubMed

    Lewko, Barbara; Latawiec, Elżbieta; Maryn, Anna; Barczyńska, Anna; Pikuła, Michał; Zieliński, Maciej; Rybczyńska, Apolonia

    2011-01-01

    Podocyte injury is associated with progression of many renal diseases, including diabetic nephropathy. In this study we examined whether aldose reductase (AR), the enzyme implicated in diabetic complications in different tissues, is modulated by high glucose and osmolarity in podocyte cells. AR mRNA, protein expression, and activity were measured in mouse podocytes cultured in both normal and high glucose and osmolarity for 6 hours to 5 days. Hyperosmolarity acutely stimulated AR expression and activity, with subsequent increase of AR expression but decrease of activity. High glucose also elevated AR protein level; however, this was not accompanied by respective enzyme activation. Furthermore, high glucose appeared to counteract the osmolarity-dependent activation of AR. In conclusion, in podocytes AR is modulated by high glucose and increased osmolarity in a different manner. Posttranslational events may affect AR activity independent of enzyme protein amount. Activation of AR in podocytes may be implicated in diabetic podocytopathy. PMID:22253613

  20. Synthesis of benzothiadiazine derivatives exhibiting dual activity as aldose reductase inhibitors and antioxidant agents.

    PubMed

    Zhu, Shaojuan; Hao, Xin; Zhang, Shuzhen; Qin, Xiangyu; Chen, Xin; Zhu, Changjin

    2016-06-15

    Several multifunctional benzothiadiazine derivatives were synthesized and examined for their inhibition to the enzyme aldose reductase and in vitro antioxidant activity to identify novel drugs for diabetes and its complications. Most of them exhibited good inhibitory activity. Importantly, a number of compounds demonstrated strong antioxidant activity and one compound in particular was extremely active in the DPPH radical scavenging and MDA inhibition analysis. The DPPH radical scavenging rate with this compound was 98.0%, 92.3% and 42.1% at concentrations of 100μM, 10μM, and 1μM, respectively, and the initial reaction rate was faster than Trolox at a concentration of 10μM. PMID:27156769

  1. Aldose reductase, ocular diabetic complications and the development of topical Kinostat(®).

    PubMed

    Kador, Peter F; Wyman, Milton; Oates, Peter J

    2016-09-01

    Diabetes mellitus (DM) is a major health problem with devastating effects on ocular health in both industrialized and developing countries. The control of hyperglycemia is critical to minimizing the impact of DM on ocular tissues because inadequate glycemic control leads to ocular tissue changes that range from a temporary blurring of vision to permanent vision loss. The biochemical mechanisms that promote the development of diabetic complications have been extensively studied. As a result, a number of prominent biochemical pathways have been identified. Among these, the two-step sorbitol pathway has been the most extensively investigated; nevertheless, it remains controversial. To date, long-term pharmacological studies in animal models of diabetes have demonstrated that the onset and development of ocular complications that include keratopathy, retinopathy and cataract can be ameliorated by the control of excess metabolic flux through aldose reductase (AR). Clinically the alleles of AR have been linked to the rapidity of onset and severity of diabetic ocular complications in diabetic patient populations around the globe. In spite of these promising preclinical and human genetic rationales, several clinical trials of varying durations with structurally diverse aldose reductase inhibitors (ARIs) have shown limited success or failure in preventing or arresting diabetic retinopathy. Despite these clinical setbacks, topical ARI Kinostat(®) promises to find a home in clinical veterinary ophthalmology where its anticipated approval by the FDA will present an alternative treatment paradigm to cataract surgery in diabetic dogs. Here, we critically review the role of AR in diabetes mellitus-linked ocular disease and highlight the development of Kinostat(®) for cataract prevention in diabetic dogs. In addition to the veterinary market, we speculate that with further safety and efficacy studies in humans, Kinostat(®) or a closely related product could have a future role

  2. Development of novel pyrazolone derivatives as inhibitors of aldose reductase: an eco-friendly one-pot synthesis, experimental screening and in silico analysis.

    PubMed

    Kadam, Aparna; Dawane, Bhaskar; Pawar, Manisha; Shegokar, Harshala; Patil, Kapil; Meshram, Rohan; Gacche, Rajesh

    2014-04-01

    Aldose reductase is the key enzyme of polypol pathway leading to accumulation of sorbitol. Sorbitol does not diffuse across the cell membranes easily and therefore accumulates within the cell, causing osmotic damage which leads to retinopathy (cataractogenesis), neuropathy and other diabetic complications. Currently, aldose reductase inhibitors like epalrestat, ranirestat and fidarestat are used for the amelioration of diabetic complications. However, such drugs are effective in patients having good glycemic control and less severe diabetic complications. In present study we have designed novel pyrazolone derivative and performed eco-friendly synthesis approach and tested the synthesized compounds as potential inhibitors of aldose reductase activity. Additional in silico analysis in current study indicates presence of highly conserved chemical environment in active site of goat lens aldose reductase. The reported data is expected to be useful for developing novel pyrazolone derivatives as lead compounds in the management of diabetic complications. PMID:24607578

  3. Aldose reductase C-106T polymorphism is associated with the risk of essential hypertension.

    PubMed

    Wang, Yaqin; Yu, Min; Mo, Long; Li, Zhenyu; Wang, Junjie; Zhou, Hong-Hao; Ouyang, Dong-Sheng

    2016-10-10

    Aldose Reductase (AR), encoded by AKR1B1, is a member of NADPH-dependent aldo-keto reductase superfamily. The C-106T polymorphism of AKR1B1 is closely related to the diabetic complications. Our previous studies have indicated that the expression of AR was increased in spontaneously hypertensive rats, suggesting the effect of AR in hypertension. Here we investigated whether AKR1B1 C-106T polymorphism was associated with essential hypertension (EH). AKR1B1 C-106T polymorphism was genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and the direct sequencing methods. 383 healthy subjects and 383 essential hypertensive patients were recruited in this study. The polymorphism of AKR1B1 C-106T in EH and normal tensive (NT) groups was in agreement with Hardy-Weinberg equilibrium. -106T allele of AKR1B1 C-106T variants was more frequent in EH patients compared with normal tensive subjects, indicating that -106T allele was a risk factor of EH (OR=1.841, 95%CI=1.366-2.481). In male patients, C-106T polymorphism was associated significantly with decreased serum high density lipoprotein cholesterol and higher systolic blood pressure levels. Our results suggest that -106T allele of AKR1B1 C-106T polymorphism may be associated with increased risk for EH in Chinese Han population. PMID:27343777

  4. Aldose reductase participates in the downregulation of T cell functions due to suppressor macrophages

    PubMed Central

    Shimizu, Toshiaki; Tatano, Yutaka; Tomioka, Haruaki

    2016-01-01

    The cell-to-cell contact of T lymphocytes with immunosuppressive macrophages causes marked changes in the tyrosine phosphorylation of some cytosolic proteins of T cells. By phosphoproteome analysis, we identified a 36-kDa protein as aldose reductase (AR). The AR expression in T cells was not changed by TCR stimulation or due to cell-to-cell transmission of suppressor signals from immunosuppressive macrophages. Therefore, AR phosphorylation/dephosphorylation is essential for the transduction of TCR-mediated T-cell stimulatory signals, and moreover plays important roles for the cross-talk of immunosuppressive macrophage-derived suppressor signals with the signaling pathways for T-cell activation. Moreover, AR played important roles in the upregulation of ERK1/2-mediated signaling pathways in T lymphocytes. Notably, the enzymatic activity of AR was not required for its signaling action. Taken together, it is concluded that AR mediates intracellular transmission of the suppressor signal of immunosuppressive macrophages toward downstream ERK1/2 pathways, possibly through its direct interaction with acceptor proteins. PMID:26868163

  5. Bioactive fraction of Saraca indica prevents diabetes induced cataractogenesis: An aldose reductase inhibitory activity

    PubMed Central

    Somani, Gauresh; Sathaye, Sadhana

    2015-01-01

    Background: The present study was designed to investigate the effect of Saraca indica (SI) flowers extract and different bioactive fraction on in vitro aldose reductase (AR) inhibitory activity, high glucose-induced cataract in goat lens and in vivo streptozotocin (STZ; 45 mg/kg, i.p) induced cataract in rats. Methods: Extract of flowers of SI tested for inhibition against rat lens AR. Furthermore, bioactive fraction was investigated against high glucose-induced opacification of the lens in vitro lens culture and STZ induced diabetic cataract in rats. Identification of the bioactive component was attempted through high-performance thin-layer chromatography, high-performance liquid chromatography and liquid chromatography-mass spectrometry analysis. Results: Ethyl acetate fraction of S. indica (EASI) produced maximum inhibition that may be due to high phenolic content. Goat lenses in media containing glucose developed a distinctly opaque ring in 72 h and treatment with EASI fraction lowered lens opacity in 72 h. Prolonged treatment with EASI to STZ-induced diabetic rats inhibited the AR activity and delayed cataract progression in a dose dependent manner. Conclusion: Ethyl acetate fraction of S. indica fraction has potential to inhibit rat lens AR enzyme and prevent cataractogenesis not only in goat lens model (in vitro), but also in STZ induced diabetic rats (in vivo). This study is suggestive of the anticataract activity of EASI fraction that could be attributed to the phytoconstituents present in the same. PMID:25709218

  6. Glucose and collagen regulate human platelet activity through aldose reductase induction of thromboxane

    PubMed Central

    Tang, Wai Ho; Stitham, Jeremiah; Gleim, Scott; Di Febbo, Concetta; Porreca, Ettore; Fava, Cristiano; Tacconelli, Stefania; Capone, Marta; Evangelista, Virgilio; Levantesi, Giacomo; Wen, Li; Martin, Kathleen; Minuz, Pietro; Rade, Jeffrey; Patrignani, Paola; Hwa, John

    2011-01-01

    Diabetes mellitus is associated with platelet hyperactivity, which leads to increased morbidity and mortality from cardiovascular disease. This is coupled with enhanced levels of thromboxane (TX), an eicosanoid that facilitates platelet aggregation. Although intensely studied, the mechanism underlying the relationship among hyperglycemia, TX generation, and platelet hyperactivity remains unclear. We sought to identify key signaling components that connect high levels of glucose to TX generation and to examine their clinical relevance. In human platelets, aldose reductase synergistically modulated platelet response to both hyperglycemia and collagen exposure through a pathway involving ROS/PLCγ2/PKC/p38α MAPK. In clinical patients with platelet activation (deep vein thrombosis; saphenous vein graft occlusion after coronary bypass surgery), and particularly those with diabetes, urinary levels of a major enzymatic metabolite of TX (11-dehydro-TXB2 [TX-M]) were substantially increased. Elevated TX-M persisted in diabetic patients taking low-dose aspirin (acetylsalicylic acid, ASA), suggesting that such patients may have underlying endothelial damage, collagen exposure, and thrombovascular disease. Thus, our study has identified multiple potential signaling targets for designing combination chemotherapies that could inhibit the synergistic activation of platelets by hyperglycemia and collagen exposure. PMID:22005299

  7. Glucose and collagen regulate human platelet activity through aldose reductase induction of thromboxane.

    PubMed

    Tang, Wai Ho; Stitham, Jeremiah; Gleim, Scott; Di Febbo, Concetta; Porreca, Ettore; Fava, Cristiano; Tacconelli, Stefania; Capone, Marta; Evangelista, Virgilio; Levantesi, Giacomo; Wen, Li; Martin, Kathleen; Minuz, Pietro; Rade, Jeffrey; Patrignani, Paola; Hwa, John

    2011-11-01

    Diabetes mellitus is associated with platelet hyperactivity, which leads to increased morbidity and mortality from cardiovascular disease. This is coupled with enhanced levels of thromboxane (TX), an eicosanoid that facilitates platelet aggregation. Although intensely studied, the mechanism underlying the relationship among hyperglycemia, TX generation, and platelet hyperactivity remains unclear. We sought to identify key signaling components that connect high levels of glucose to TX generation and to examine their clinical relevance. In human platelets, aldose reductase synergistically modulated platelet response to both hyperglycemia and collagen exposure through a pathway involving ROS/PLCγ2/PKC/p38α MAPK. In clinical patients with platelet activation (deep vein thrombosis; saphenous vein graft occlusion after coronary bypass surgery), and particularly those with diabetes, urinary levels of a major enzymatic metabolite of TX (11-dehydro-TXB2 [TX-M]) were substantially increased. Elevated TX-M persisted in diabetic patients taking low-dose aspirin (acetylsalicylic acid, ASA), suggesting that such patients may have underlying endothelial damage, collagen exposure, and thrombovascular disease. Thus, our study has identified multiple potential signaling targets for designing combination chemotherapies that could inhibit the synergistic activation of platelets by hyperglycemia and collagen exposure. PMID:22005299

  8. Design and synthesis of potent and multifunctional aldose reductase inhibitors based on quinoxalinones.

    PubMed

    Qin, Xiangyu; Hao, Xin; Han, Hui; Zhu, Shaojuan; Yang, Yanchun; Wu, Bobin; Hussain, Saghir; Parveen, Shagufta; Jing, Chaojun; Ma, Bing; Zhu, Changjin

    2015-02-12

    Quinoxalin-2(1H)-one based design and synthesis produced several series of aldose reductase (ALR2) inhibitor candidates. In particular, phenolic structure was installed in the compounds for the combination of antioxidant activity and strengthening the ability to fight against diabetic complications. Most of the series 6 showed potent and selective effects on ALR2 inhibition with IC50 values in the range of 0.032-0.468 μM, and 2-(3-(2,4-dihydroxyphenyl)-7-fluoro-2-oxoquinoxalin-1(2H)-yl)acetic acid (6e) was the most active. More significantly, most of the series 8 revealed not only good activity in the ALR2 inhibition but also potent antioxidant activity, and 2-(3-(3-methoxy-4-hydroxystyryl)-2-oxoquinoxalin-1(2H)-yl)acetic acid (8d) was even as strong as the well-known antioxidant Trolox at a concentration of 100 μM, verifying the C3 p-hydroxystyryl side chain as the key structure for alleviating oxidative stress. These results therefore suggest an achievement of multifunctional ALR2 inhibitors having both potency for ALR2 inhibition and as antioxidants. PMID:25602762

  9. Aldose Reductase acts as a Selective Derepressor of PPARγ and Retinoic Acid Receptor

    PubMed Central

    Thiagarajan, Devi; Ananthakrishnan, Radha; Zhang, Jinghua; O’Shea, Karen M.; Quadri, Nosirudeen; Li, Qing; Sas, Kelli; Jing, Xiao; Rosario, Rosa; Pennathur, Subramaniam; Schmidt, Ann Marie; Ramasamy, Ravichandran

    2016-01-01

    Summary Histone deacetylase 3 (HDAC3), a chromatin modifying enzyme, requires association with the deacetylase containing domain (DAD) of the nuclear receptor co-repressors NCOR1 and SMRT for its stability and activity. Here we show that aldose reductase (AR), the rate-limiting enzyme of the polyol pathway, competes with HDAC3 to bind the NCOR1/SMRT DAD. Increased AR expression leads to HDAC3 degradation followed by increased PPARγ signaling resulting in lipid accumulation in the heart. AR also downregulates expression of nuclear corepressor complex cofactors including Gps2 and Tblr1, thus affecting activity of the nuclear corepressor complex itself. Though AR reduces HDAC3-corepressor complex formation, it specifically de-represses the retinoic acid receptor (RAR), but not other nuclear receptors such as the thyroid receptor (TR) and liver X receptor (LXR). In summary, this work defines a distinct role for AR in lipid and retinoid metabolism through HDAC3 regulation and consequent de-repression of PPARγ and RAR. PMID:27052179

  10. Stress tolerance of transgenic barley accumulating the alfalfa aldose reductase in the cytoplasm and the chloroplast.

    PubMed

    Nagy, Bettina; Majer, Petra; Mihály, Róbert; Pauk, János; Horváth, Gábor V

    2016-09-01

    Barley represents one of the major crops grown worldwide; its genetic transformation provides an important tool for the improvement of crop quality and tolerance to environmental stress factors. Biotic and abiotic stresses produce reactive oxygen species in the plant cells that can directly oxidize the cellular components including lipid membranes; resulting in lipid peroxidation and subsequently the accumulation of reactive carbonyl compounds. In order to protect barley plants from the effects of stress-produced reactive carbonyls, an Agrobacterium-mediated transformation was carried out using the Medicago sativa aldose reductase (MsALR) gene. In certain transgenic lines the produced MsALR enzyme was targeted to the chloroplasts to evaluate its protective effect in these organelles. The dual fluorescent protein-based method was used for the evaluation of tolerance of young seedlings to diverse stresses; our results demonstrated that this technique could be reliably applied for the detection of cellular stress in a variety of conditions. The chlorophyll and carotenoid content measurements also supported the results of the fluorescent protein-based method and the stress-protective effect of the MsALR enzyme. Targeting of MsALR into the chloroplast has also resulted in increased stress tolerance, similarly to the observed effect of the cytosolic MsALR accumulation. The results of the DsRed/GFP fluorescent protein-based method indicated that both the cytosol and chloroplast accumulation of MsALR can increase the abiotic stress tolerance of transgenic barley lines. PMID:27469099

  11. Diallyl sulfide protects against N-nitrosodiethylamine-induced liver tumorigenesis: Role of aldose reductase

    PubMed Central

    Ibrahim, Safinaz S; Nassar, Noha N

    2008-01-01

    AIM: To evaluate the protective effect of diallyl sulfide (DAS) against N-nitrosodiethylamine (NDEA)-induced liver carcinogenesis. METHODS: Male Wistar rats received either NDEA or NDEA together with DAS as protection. Liver energy metabolism was assessed in terms of lactate, pyruvate, lactate/pyruvate, ATP levels, lactate dehydrogenase (LDH) and glucose-6-phosphate dehydrogenase (G6PD) activities. In addition, membrane disintegration of the liver cells was evaluated by measuring lipid-peroxidation products, measured as malondialdehyde (MDA); nitric oxide (NO) levels; glucose-6-phosphatase (G6Pase), catalase (CAT) and superoxide dismutase (SOD) activities. Liver DNA level, glutathione-S-transferase (GST) and cytochrome c oxidase activities were used as DNA fragmentation indices. Aldose reductase (AR) activity was measured as an index for cancer cells resistant to chemotherapy and histopathological examination was performed on liver sections from different groups. RESULTS: NDEA significantly disturbed liver functions and most of the aforementioned indices. Treatment with DAS significantly restored liver functions and hepatocellular integrity; improved parameters of energy metabolism and suppressed free-radical generation. CONCLUSION: We provide evidence that DAS exerts a protective role on liver functions and tissue integrity in face of enhanced tumorigenesis caused by NDEA, as well as improving cancer-cell sensitivity to chemotherapy. This is mediated through combating oxidative stress of free radicals, improving the energy metabolic state of the cell, and enhancing the activity of G6Pase, GST and AR enzymes. PMID:18985804

  12. Altered aldose reductase gene regulation in cultured human retinal pigment epithelial cells.

    PubMed Central

    Henry, D N; Del Monte, M; Greene, D A; Killen, P D

    1993-01-01

    Aldose reductase (AR2), a putative "hypertonicity stress protein" whose gene is induced by hyperosmolarity, protects renal medullary cells against the interstitial hyperosmolarity of antidiuresis by catalyzing the synthesis of millimolar concentrations of intracellular sorbitol from glucose. Although AR2 gene induction has been noted in a variety of renal and nonrenal cells subjected to hypertonic stress in vitro, the functional significance of AR2 gene expression in cells not normally exposed to a hyperosmolar milieu is not fully understood. The physiological impact of basal AR2 expression in such cells may be limited to hyperglycemic states in which AR2 promotes pathological polyol accumulation, a mechanism invoked in the pathogenesis of diabetic complications. Since AR2 overexpression in the retinal pigment epithelium has been associated with diabetic retinopathy, the regulation of AR2 gene expression and associated changes in sorbitol and myo-inositol were studied in human retinal pigment epithelial cells in culture. The relative abundance of aldehyde reductase (AR1) and AR2 mRNA was quantitated by filter hybridization of RNA from several human retinal pigment epithelial cell lines exposed to hyperglycemic and hyperosmolar conditions in vitro. AR2 but not AR1 mRNA was significantly increased some 11- to 18-fold by hyperosmolarity in several retinal pigment epithelial cell lines. A single cell line with a 15-fold higher basal level of AR2 mRNA than other cell lines tested demonstrated no significant increase in AR2 mRNA in response to hypertonic stress. This cell line demonstrated accelerated and exaggerated production of sorbitol and depletion of myo-inositol upon exposure to 20 mM glucose. Therefore, abnormal AR2 expression may enhance the sensitivity of cells to the biochemical consequences of hyperglycemia potentiating the development of diabetic complications. Images PMID:8349800

  13. Melatonin Reduces Cataract Formation and Aldose Reductase Activity in Lenses of Streptozotocin-induced Diabetic Rat

    PubMed Central

    Khorsand, Marjan; Akmali, Masoumeh; Sharzad, Sahab; Beheshtitabar, Mojtaba

    2016-01-01

    Background: The relationship between the high activity of aldose reductase (AR) and diabetic cataract formation has been previously investigated. The purpose of the present study was to determine the preventing effect of melatonin on streptozotocin (STZ)-induced diabetic cataract in rats. Methods: 34 adult healthy male Sprague-Dawely rats were divided into four groups. Diabetic control and diabetic+melatonin received a single dose of STZ (50 mg/kg, intraperitoneally), whereas the normal control and normal+melatonin received vehicle. The melatonin groups were gavaged with melatonin (5 mg/kg) daily for a period of 8 weeks, whereas the rats in the normal control and diabetic control groups received only the vehicle. The rats’ eyes were examined every week and cataract formation scores (0-4) were determined by slit-lamp microscope. At the end of the eighth week, the rats were sacrificed and markers of the polyol pathway and antioxidative (Glutathione, GSH) in their lens were determined. The levels of blood glucose, HbA1c and plasma malondialdhyde (MDA), as a marker of lipid peroxidation, were also measured. Results: Melatonin prevented STZ-induced hyperglycemia by decreased blood glucose and HbA1c levels. Slit lamp examination indicated that melatonin delayed cataract progression in diabetic rats. The results revealed that melatonin feeding increased the GSH levels, decreased the activities of AR and sorbitol dehydrogenase (SDH) and sorbitol formation in catractous lenses as well as plasma MDA content. Conclusion: In summary, for the first time we demonstrated that melatonin delayed the formation and progression of cataract in diabetic rat lenses. PMID:27365552

  14. Aldose Reductase Is Involved in the Development of Murine Diet-Induced Nonalcoholic Steatohepatitis

    PubMed Central

    Qiu, Longxin; Lin, Jianhui; Ying, Miao; Chen, Weiqiang; Yang, Jinmei; Deng, Tiantian; Chen, Jinfeng; Shi, Duanyu; Yang, James Y.

    2013-01-01

    Hepatic aldose reductase (AR) expression is known to be induced in liver diseases, including hepatitis and hepatocellular carcinoma. However, the role of AR in the development of these diseases remains unclear. We performed this current study to determine whether and how AR might be involved in the development of diet-induced nonalcoholic steatohepatitis. Our results showed that the level of AR protein expression was significantly higher in db/db mice fed the methionine-choline-deficient (MCD) diet than in mice fed the control diet. In parallel with the elevation in AR, steatohepatitis was observed in MCD diet-fed mice, and this diet-induced steatohepatitis was significantly attenuated by lentiviral-mediated knock-down of the AR gene. This suppressive effect of AR knock-down was associated with repressed levels of serum alanine aminotransferase and hepatic lipoperoxides, reduced mRNA and protein expression of hepatic cytochrome P450 2E1 (CYP2E1), and decreased mRNA expression of pro-inflammatory tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Moreover, AR-induced elevations on the level of CYP2E1 expression, reactive oxygen species, mRNA expression of TNF-α and IL-6 were confirmed in AML12 hepatocytes. Further, lentiviral-mediated knock-down of AR ameliorated MCD diet-induced collagen deposition in the livers of db/db mice. With the improvement in liver fibrosis, the mRNA levels of tissue inhibitor of metalloproteinase-1 (TIMP-1) and matrix metalloproteinase-2 (MMP-2), two genes involved in hepatic fibrogenesis, were found to be significantly suppressed, while TIMP-2 and MMP-13 were unaffected. Together these data indicate that inhibition of AR alleviates the MCD diet-induced liver inflammation and fibrosis in db/db mice, probably through dampening CYP2E1 mediated-oxidative stress and ameliorating the expression of pro-inflammatory cytokines. PMID:24066058

  15. Highly selective aldose reductase inhibitors. 3. Structural diversity of 3-(arylmethyl)-2,4,5-trioxoimidazolidine-1-acetic acids.

    PubMed

    Kotani, T; Nagaki, Y; Ishii, A; Konishi, Y; Yago, H; Suehiro, S; Okukado, N; Okamoto, K

    1997-02-28

    Accumulation of intracellular sorbitol, the reduced product of glucose, catalyzed by aldose reductase (AR) (EC 1.1.1.21), is thought to be the cause of the development of diabetic complications. Our attention is focused on finding compounds which inhibit AR without significantly inhibiting aldehyde reductase (ALR) (EC 1.1.1.2). The uracil or 2,4-dioxoimidazolidine skeleton having the benzothiazolyl or 4-chloro-3-nitrophenyl group as an aryl part indicated not only extremely high AR inhibitory activity but also AR selectivity. The ratio of IC50(ALR)/IC50(AR) of 3-[(5-chlorobenzothiazol-2-yl)methyl]-1,2,3,4-tetrahydro-2,4- dioxopyrimidine-1-acetic acid (47d) was more than 17 500. The uracil skeleton with the benzothiazolyl moiety seemed to be the best combination for selective AR inhibition. PMID:9057855

  16. Structural and kinetic modifications of aldose reductase by S-nitrosothiols.

    PubMed Central

    Srivastava, S; Dixit, B L; Ramana, K V; Chandra, A; Chandra, D; Zacarias, A; Petrash, J M; Bhatnagar, A; Srivastava, S K

    2001-01-01

    Modification of aldose reductase (AR) by the nitrosothiols S-nitroso-N-acetyl penicillamine (SNAP) and N-(beta-glucopyranosyl)-N(2)-acetyl-S-nitrosopenicillamide (glyco-SNAP) resulted in a 3-7-fold increase in its k(cat) and a 25-40-fold increase in its K(m) for glyceraldehyde. In comparison with the native protein, the modified enzyme was less sensitive to inhibition by sorbinil and was not activated by SO(2-)(4) anions. The active-site residue, Cys-298, was identified as the main site of modification, because the site-directed mutant in which Cys-298 was replaced by serine was insensitive to glyco-SNAP. The extent of modification was not affected by P(i) or O(2), indicating that it was not due to spontaneous release of nitric oxide (NO) by the nitrosothiols. Electrospray ionization MS revealed that the modification reaction proceeds via the formation of an N-hydroxysulphenamide-like adduct between glyco-SNAP and AR. In time, the adduct dissociates into either nitrosated AR (AR-NO) or a mixed disulphide between AR and glyco-N-acetylpenicillamine (AR-S-S-X). Removal of the mixed-disulphide form of the protein by lectin-column chromatography enriched the preparation in the high-K(m)-high-k(cat) form of the enzyme, suggesting that the kinetic changes are due to the formation of AR-NO, and that the AR-S-S-X form of the enzyme is catalytically inactive. Modification of AR by the non-thiol NO donor diethylamine NONOate (DEANO) increased enzyme activity and resulted in the formation of AR-NO. However, no adducts between AR and DEANO were formed. These results show that nitrosothiols cause multiple structural and functional changes in AR. Our observations also suggest the general possibility that transnitrosation reactions can generate both nitrosated and thiolated products, leading to non-unique changes in protein structure and function. PMID:11485558

  17. Structural and thermodynamic study on aldose reductase: nitro-substituted inhibitors with strong enthalpic binding contribution.

    PubMed

    Steuber, Holger; Heine, Andreas; Klebe, Gerhard

    2007-05-01

    To prevent diabetic complications derived from enhanced glucose flux via the polyol pathway the development of aldose reductase inhibitors (ARIs) has been established as a promising therapeutic concept. In order to identify novel lead compounds, a virtual screening (VS) was performed successfully suggesting carboxylate-type inhibitors of sub-micromolar to micromolar affinity. Here, we combine a structural characterization of the binding modes observed by X-ray crystallography with isothermal titration calorimetry (ITC) measurements providing insights into the driving forces of inhibitor binding, particularly of the first leads from VS. Characteristic features of this novel inhibitor type include a carboxylate head group connected via an alkyl spacer to a heteroaromatic moiety, which is linked to a further nitro-substituted aromatic portion. The crystal structures of two enzyme-inhibitor complexes have been determined at resolutions of 1.43 A and 1.55 A. Surprisingly, the carboxylic group of the most potent VS lead occupies the catalytic pocket differently compared to the interaction geometry observed in almost all other crystal structures with structurally related ligands and obtained under similar conditions, as an interstitial water molecule is picked up upon ligand binding. The nitro-aromatic moiety of both leads occupies the specificity pocket of the enzyme, however, adopting a different geometry compared to the docking prediction: unexpectedly, the nitro group binds to the bottom of the specificity pocket and provokes remarkable induced-fit adaptations. A peptide group located at the active site orients in such a way that H-bond formation to one nitro group oxygen atom is enabled, whereas a neighbouring tyrosine side-chain performs a slight rotation off from the binding cavity to accommodate the nitro group. Identically constituted ligands, lacking this nitro group, exhibit an affinity drop of one order of magnitude. In addition, thermodynamic data suggest a

  18. Aldose reductase modulates cardiac glycogen synthase kinase-3β phosphorylation during ischemia-reperfusion

    PubMed Central

    Abdillahi, Mariane; Ananthakrishnan, Radha; Vedantham, Srinivasan; Shang, Linshan; Zhu, Zhengbin; Rosario, Rosa; Zirpoli, Hylde; Bohren, Kurt M.; Gabbay, Kenneth H.

    2012-01-01

    Earlier studies have demonstrated that aldose reductase (AR) plays a key role in mediating ischemia-reperfusion (I/R) injury. Our objective was to investigate if AR mediates I/R injury by influencing phosphorylation of glycogen synthase kinase-3β (p-GSK3β). To investigate this issue, we used three separate models to study the effects of stress injury on the heart. Hearts isolated from wild-type (WT), human expressing AR transgenic (ARTg), and AR knockout (ARKO) mice were perfused with/without GSK3β inhibitors (SB-216763 and LiCl) and subjected to I/R. Ad-human AR (Ad-hAR)-expressing HL-1 cardiac cells were exposed to hypoxia (0.5% O2) and reoxygenation (20.9% O2) conditions. I/R in a murine model of transient occlusion and reperfusion of the left anterior descending coronary artery (LAD) was used to study if p-GSK3β was affected through increased AR flux. Lactate dehydrogenase (LDH) release and left ventricular developed pressure (LVDP) were measured. LVDP was decreased in hearts from ARTg mice compared with WT and ARKO after I/R, whereas LDH release and apoptotic markers were increased (P < 0.05). p-GSK3β was decreased in ARTg hearts compared with WT and ARKO (P < 0.05). In ARKO, p-GSK3β and apoptotic markers were decreased compared with WT (P < 0.05). WT and ARTg hearts perfused with GSK3β inhibitors improved p-GSK3β expression and LVDP and exhibited decreased LDH release, apoptosis, and mitochondrial pore opening (P < 0.05). Ad-hAR-expressing HL-1 cardiac cells, exposed to hypoxia (0.5% O2) and reoxygenation (20.9% O2), had greater LDH release compared with control HL-1 cells (P < 0.05). p-GSK3β was decreased and correlated with increased apoptotic markers in Ad-hAR HL-1 cells (P < 0.05). Treatment with phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) inhibitor increased injury demonstrated by increased LDH release in ARTg, WT, and ARKO hearts and in Ad-hAR-expressing HL-1 cells. Cells treated with protein kinase C (PKC) α/β inhibitor

  19. Aldose reductase modulates cardiac glycogen synthase kinase-3β phosphorylation during ischemia-reperfusion.

    PubMed

    Abdillahi, Mariane; Ananthakrishnan, Radha; Vedantham, Srinivasan; Shang, Linshan; Zhu, Zhengbin; Rosario, Rosa; Zirpoli, Hylde; Bohren, Kurt M; Gabbay, Kenneth H; Ramasamy, Ravichandran

    2012-08-01

    Earlier studies have demonstrated that aldose reductase (AR) plays a key role in mediating ischemia-reperfusion (I/R) injury. Our objective was to investigate if AR mediates I/R injury by influencing phosphorylation of glycogen synthase kinase-3β (p-GSK3β). To investigate this issue, we used three separate models to study the effects of stress injury on the heart. Hearts isolated from wild-type (WT), human expressing AR transgenic (ARTg), and AR knockout (ARKO) mice were perfused with/without GSK3β inhibitors (SB-216763 and LiCl) and subjected to I/R. Ad-human AR (Ad-hAR)-expressing HL-1 cardiac cells were exposed to hypoxia (0.5% O(2)) and reoxygenation (20.9% O(2)) conditions. I/R in a murine model of transient occlusion and reperfusion of the left anterior descending coronary artery (LAD) was used to study if p-GSK3β was affected through increased AR flux. Lactate dehydrogenase (LDH) release and left ventricular developed pressure (LVDP) were measured. LVDP was decreased in hearts from ARTg mice compared with WT and ARKO after I/R, whereas LDH release and apoptotic markers were increased (P < 0.05). p-GSK3β was decreased in ARTg hearts compared with WT and ARKO (P < 0.05). In ARKO, p-GSK3β and apoptotic markers were decreased compared with WT (P < 0.05). WT and ARTg hearts perfused with GSK3β inhibitors improved p-GSK3β expression and LVDP and exhibited decreased LDH release, apoptosis, and mitochondrial pore opening (P < 0.05). Ad-hAR-expressing HL-1 cardiac cells, exposed to hypoxia (0.5% O(2)) and reoxygenation (20.9% O(2)), had greater LDH release compared with control HL-1 cells (P < 0.05). p-GSK3β was decreased and correlated with increased apoptotic markers in Ad-hAR HL-1 cells (P < 0.05). Treatment with phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) inhibitor increased injury demonstrated by increased LDH release in ARTg, WT, and ARKO hearts and in Ad-hAR-expressing HL-1 cells. Cells treated with protein kinase C (PKC)

  20. High-resolution neutron protein crystallography with radically small crystal volumes: application of perdeuteration to human aldose reductase.

    PubMed

    Hazemann, I; Dauvergne, M T; Blakeley, M P; Meilleur, F; Haertlein, M; Van Dorsselaer, A; Mitschler, A; Myles, D A A; Podjarny, A

    2005-10-01

    Neutron diffraction data have been collected to 2.2 Angstrom resolution from a small (0.15 mm(3)) crystal of perdeuterated human aldose reductase (h-AR; MW = 36 kDa) in order to help to determine the protonation state of the enzyme. h-AR belongs to the aldo-keto reductase family and is implicated in diabetic complications. Its ternary complexes (h-AR-coenzyme NADPH-selected inhibitor) provide a good model to study both the enzymatic mechanism and inhibition. Here, the successful production of fully deuterated human aldose reductase [h-AR(D)], subsequent crystallization of the ternary complex h-AR(D)-NADPH-IDD594 and neutron Laue data collection at the LADI instrument at ILL using a crystal volume of just 0.15 mm(3) are reported. Neutron data were recorded to 2 Angstrom resolution, with subsequent data analysis using data to 2.2 Angstrom. This is the first fully deuterated enzyme of this size (36 kDa) to be solved by neutron diffraction and represents a milestone in the field, as the crystal volume is at least one order of magnitude smaller than those usually required for other high-resolution neutron structures determined to date. This illustrates the significant increase in the signal-to-noise ratio of data collected from perdeuterated crystals and demonstrates that good-quality neutron data can now be collected from more typical protein crystal volumes. Indeed, the signal-to-noise ratio is then dominated by other sources of instrument background, the nature of which is under investigation. This is important for the design of future instruments, which should take maximum advantage of the reduction in the intrinsic diffraction pattern background from fully deuterated samples. PMID:16204895

  1. High-resolution neutron protein crystallography with radically small crystal volumes: Application of perdeuteration to human aldose reductase

    SciTech Connect

    Hazemann, I.; Dauvergne, M. T.; Blakeley, M. P.; Meilleur, Flora; Haertlein, M.; Van Dorsselaer, A.; Mitschler, A.; Myles, Dean A A; Podjarny, A.

    2005-08-01

    Neutron diffraction data have been collected to 2.2 {angstrom} resolution from a small (0.15 mm{sup 3}) crystal of perdeuterated human aldose reductase (h-AR; MW = 36 kDa) in order to help to determine the protonation state of the enzyme. h-AR belongs to the aldo-keto reductase family and is implicated in diabetic complications. Its ternary complexes (h-AR-coenzyme NADPH-selected inhibitor) provide a good model to study both the enzymatic mechanism and inhibition. Here, the successful production of fully deuterated human aldose reductase [h-AR(D)], subsequent crystallization of the ternary complex h-AR(D)-NADPH-IDD594 and neutron Laue data collection at the LADI instrument at ILL using a crystal volume of just 0.15 mm{sup 3} are reported. Neutron data were recorded to 2 {angstrom} resolution, with subsequent data analysis using data to 2.2 {angstrom}. This is the first fully deuterated enzyme of this size (36 kDa) to be solved by neutron diffraction and represents a milestone in the field, as the crystal volume is at least one order of magnitude smaller than those usually required for other high-resolution neutron structures determined to date. This illustrates the significant increase in the signal-to-noise ratio of data collected from perdeuterated crystals and demonstrates that good-quality neutron data can now be collected from more typical protein crystal volumes. Indeed, the signal-to-noise ratio is then dominated by other sources of instrument background, the nature of which is under investigation. This is important for the design of future instruments, which should take maximum advantage of the reduction in the intrinsic diffraction pattern background from fully deuterated samples.

  2. Endotoxin causes pulmonary hypertension by upregulating smooth muscle endothelin type-B receptors: role of aldose reductase.

    PubMed

    Dschietzig, Thomas; Alexiou, Kosta; Richter, Christoph; Bobzin, Martin; Baumann, Gert; Stangl, Karl; Brunner, Friedrich

    2008-08-01

    Endothelin-1 (ET-1), a potent vasoconstrictor and mitogen, is upregulated in pulmonary tissue during endotoxemia and contributes markedly to endotoxin-induced pulmonary hypertension. It is, however, unknown whether the ET receptors, ET(A) and ET(B), are differentially regulated in endotoxemic pulmonary vasculature and how this may impact on pulmonary vascular tone. To investigate this topic, we used isolated perfused lungs, pulmonary endothelial cells (ECs), and pulmonary vascular smooth muscle cells (SMCs) of the rat. During a 6-h endotoxin exposure, isolated perfused lungs developed significant pulmonary hypertension that was markedly attenuated by antagonizing ET(A) or ET(B) receptors using subtype-selective or a mixed ET(A/B) receptor antagonist. Peptide levels of big ET-1 and ET-1 and gene expression of prepro-ET-1 were increased after endotoxin challenge in all tissues. In endotoxemic isolated perfused lungs and ECs, the significant rise of mature ET-1 seen in controls after ET(B) receptor or mixed antagonism disappeared completely. However, this effect was preserved in endotoxemic SMCs. In ECs, endotoxin markedly downregulated maximum ET(B) receptor sites and ET(B) mRNA levels, whereas in SMCs, it generated substantial ET(B) receptor upregulation and moderate ET(A) receptor downregulation. The aldose reductase inhibitors sorbinil and zopolrestat mitigated endotoxin-induced pulmonary hypertension, ET-1 stimulation, and differential ET(B) receptor regulation. We conclude that endotoxin-induced pulmonary hypertension in the rat results from a loss of endothelial and concomitant gain of vascular smooth muscle ET(B) receptors. These changes are at least partly mediated by aldose reductase. PMID:18091567

  3. Osmotic Stress, not Aldose Reductase Activity, Directly induces Growth Factors and MAPK Signaling changes during Sugar Cataract Formation

    PubMed Central

    Zhang, Peng; Xing, Kuiyi; Randazzo, James; Blessing, Karen; Lou, Marjorie F.; Kador, Peter F.

    2012-01-01

    In sugar cataract formation in rats, aldose reductase (AR) actitvity is not only linked to lenticular sorbitol (diabetic) or galactitol (galactosemic) formation but also to signal transduction changes, cytotoxic signals and activation of apoptosis. Using both in vitro and in vivo techniques, the interrelationship between AR activity, polyol (sorbitol and galactitol) formation, osmotic stress, growth factor induction, and cell signaling changes have been investigated. For in vitro studies, lenses from Sprague Dawley rats were cultured for up to 48 hrs in TC-199-bicarbonate media containing either 30 mM fructose (control), or 30 mM glucose or galctose with/without the aldose reductase inhibitors AL1576 or tolrestat, the sorbitol dehydrogenase inhibitor (SDI) CP-470,711, or 15 mM mannitol (osmotic-compensated media). For in vivo studies, lenses were obtained from streptozotocin-induced diabetic Sprague Dawley rats fed diet with/without the ARIs AL1576 or tolrestat for 10 weeks. As expected, lenses cultured in high glucose / galactose media or from untreated diabetic rats all showed a decrease in the GSH pool that was lessened by ARI treatment. Lenses either from diabetic rats or from glucose/galactose culture conditions showed increased expression of basic-FGF, TGF-β, and increased signaling through P-Akt, P-ERK1/2 and P-SAPK/JNK which were also normalized by ARIs to the expression levels observed in non-diabetic controls. Culturing rat lenses in osomotically compensated media containing 30 mM glucose or galactose did not lead to increased growth factor expression or altered signaling. These studies indicate that it is the biophysical response of the lens to osmotic stress that results in an increased intralenticular production of basic-FGF and TGF-β and the altered cytotoxic signaling that is observed during sugar cataract formation. PMID:22710095

  4. Bioactivity Focus of α-Cyano-4-hydroxycinnamic acid (CHCA) Leads to Effective Multifunctional Aldose Reductase Inhibitors.

    PubMed

    Zhang, Laitao; Li, Yi-Fang; Yuan, Sheng; Zhang, Shijie; Zheng, Huanhuan; Liu, Jie; Sun, Pinghua; Gu, Yijun; Kurihara, Hiroshi; He, Rong-Rong; Chen, Heru

    2016-01-01

    Bioactivity focus on α-cyano-4-hydroxycinnamic acid (CHCA) scaffold results in a small library of novel multifunctional aldose reductase (ALR2) inhibitors. All the entities displayed good to excellent inhibition with IC50 72-405 nM. (R,E)-N-(3-(2-acetamido-3-(benzyloxy)propanamido)propyl)-2-cyano-3-(4-hydroxy phenyl)acrylamide (5f) was confirmed as the most active inhibitor (IC50 72.7 ± 1.6 nM), and the best antioxidant. 5f bound to ALR2 with new mode without affecting the aldehyde reductase (ALR1) activity, implicating high selectivity to ALR2. 5f was demonstrated as both an effective ALR2 inhibitor (ARI) and antioxidant in a chick embryo model of hyperglycemia. It attenuated hyperglycemia-induced incidence of neural tube defects (NTD) and death rate, and significantly improved the body weight and morphology of the embryos. 5f restored the expression of paired box type 3 transcription factor (Pax3), and reduced the hyperglycemia-induced increase of ALR2 activity, sorbitol accumulation, and the generation of ROS and MDA to normal levels. All the evidences support that 5f may be a potential agent to treat diabetic complications. PMID:27109517

  5. Bioactivity Focus of α-Cyano-4-hydroxycinnamic acid (CHCA) Leads to Effective Multifunctional Aldose Reductase Inhibitors

    PubMed Central

    Zhang, Laitao; Li, Yi-Fang; Yuan, Sheng; Zhang, Shijie; Zheng, Huanhuan; Liu, Jie; Sun, Pinghua; Gu, Yijun; Kurihara, Hiroshi; He, Rong-Rong; Chen, Heru

    2016-01-01

    Bioactivity focus on α-cyano-4-hydroxycinnamic acid (CHCA) scaffold results in a small library of novel multifunctional aldose reductase (ALR2) inhibitors. All the entities displayed good to excellent inhibition with IC50 72–405 nM. (R,E)-N-(3-(2-acetamido-3-(benzyloxy)propanamido)propyl)-2-cyano-3-(4-hydroxy phenyl)acrylamide (5f) was confirmed as the most active inhibitor (IC50 72.7 ± 1.6 nM), and the best antioxidant. 5f bound to ALR2 with new mode without affecting the aldehyde reductase (ALR1) activity, implicating high selectivity to ALR2. 5f was demonstrated as both an effective ALR2 inhibitor (ARI) and antioxidant in a chick embryo model of hyperglycemia. It attenuated hyperglycemia-induced incidence of neural tube defects (NTD) and death rate, and significantly improved the body weight and morphology of the embryos. 5f restored the expression of paired box type 3 transcription factor (Pax3), and reduced the hyperglycemia-induced increase of ALR2 activity, sorbitol accumulation, and the generation of ROS and MDA to normal levels. All the evidences support that 5f may be a potential agent to treat diabetic complications. PMID:27109517

  6. Synthesis and biological evaluation of some new pyrazoline substituted benzenesulfonylurea/thiourea derivatives as anti-hyperglycaemic agents and aldose reductase inhibitors.

    PubMed

    Ovais, Syed; Pushpalatha, H; Reddy, G Bhanuprakash; Rathore, Pooja; Bashir, Rafia; Yaseen, Shafiya; Dheyaa, Alhamza; Yaseen, Raed; Tanwar, Omprakash; Akthar, Mymoona; Samim, Mohammed; Javed, Kalim

    2014-06-10

    Seventeen new pyrazoline substituted benzenesulfonylurea/thiourea derivatives (2a-q) were synthesized and characterized by elemental analysis and various spectroscopic techniques viz; IR, (1)H NMR, (13)C NMR, and MS data. Thirteen compounds showed moderate to good anti-hyperglycaemic activity in glucose fed hyperglycaemic normal rats at the dose of 0.05 mM/kg b.w. On the basis of docking results nine compounds (2a, 2c, 2e, 2h, 2k, 2l, 2n, 2o and 2q) were evaluated for their ability to inhibit rat lens aldose reductase. Out of these six compounds (2h, 2k, 2l, 2n, 2o and 2q) were found more effective than the known ARI sorbinil. Five compounds (2h, 2k, 2l, 2n and 2o) showed significant dual action (anti-hyperglycaemic and aldose reductase inhibition). PMID:24780598

  7. Anti-neuroinflammatory efficacy of the aldose reductase inhibitor FMHM via phospholipase C/protein kinase C-dependent NF-κB and MAPK pathways

    SciTech Connect

    Zeng, Ke-Wu; Li, Jun; Dong, Xin; Wang, Ying-Hong; Ma, Zhi-Zhong; Jiang, Yong; Jin, Hong-Wei; Tu, Peng-Fei

    2013-11-15

    Aldose reductase (AR) has a key role in several inflammatory diseases: diabetes, cancer and cardiovascular diseases. Therefore, AR inhibition seems to be a useful strategy for anti-inflammation therapy. In the central nervous system (CNS), microglial over-activation is considered to be a central event in neuroinflammation. However, the effects of AR inhibition in CNS inflammation and its underlying mechanism of action remain unknown. In the present study, we found that FMHM (a naturally derived AR inhibitor from the roots of Polygala tricornis Gagnep.) showed potent anti-neuroinflammatory effects in vivo and in vitro by inhibiting microglial activation and expression of inflammatory mediators. Mechanistic studies showed that FMHM suppressed the activity of AR-dependent phospholipase C/protein kinase C signaling, which further resulted in downstream inactivation of the IκB kinase/IκB/nuclear factor-kappa B (NF-κB) inflammatory pathway. Therefore, AR inhibition-dependent NF-κB inactivation negatively regulated the transcription and expression of various inflammatory genes. AR inhibition by FMHM exerted neuroprotective effects in lipopolysaccharide-induced neuron–microglia co-cultures. These findings suggested that AR is a potential target for neuroinflammation inhibition and that FMHM could be an effective agent for treating or preventing neuroinflammatory diseases. - Highlights: • FMHM is a natural-derived aldose reductase (AR) inhibitor. • FMHM inhibits various neuroinflammatory mediator productions in vitro and in vivo. • FMHM inhibits neuroinflammation via aldose reductase/PLC/PKC-dependent NF-κB pathway. • FMHM inhibits neuroinflammation via aldose reductase/PLC/PKC-dependent MAPK pathway. • FMHM protects neurons against inflammatory injury in microglia-neuron co-cultures.

  8. Quantum mechanical calculation of electric fields and vibrational Stark shifts at active site of human aldose reductase.

    PubMed

    Wang, Xianwei; Zhang, John Z H; He, Xiao

    2015-11-14

    Recent advance in biophysics has made it possible to directly measure site-specific electric field at internal sites of proteins using molecular probes with C = O or C≡N groups in the context of vibrational Stark effect. These measurements directly probe changes of electric field at specific protein sites due to, e.g., mutation and are very useful in protein design. Computational simulation of the Stark effect based on force fields such as AMBER and OPLS, while providing good insight, shows large errors in comparison to experimental measurement due to inherent difficulties associated with point charge based representation of force fields. In this study, quantum mechanical calculation of protein's internal electrostatic properties and vibrational Stark shifts was carried out by using electrostatically embedded generalized molecular fractionation with conjugate caps method. Quantum calculated change of mutation-induced electric field and vibrational Stark shift is reported at the internal probing site of enzyme human aldose reductase. The quantum result is in much better agreement with experimental data than those predicted by force fields, underscoring the deficiency of traditional point charge models describing intra-protein electrostatic properties. PMID:26567650

  9. Quantum mechanical calculation of electric fields and vibrational Stark shifts at active site of human aldose reductase

    NASA Astrophysics Data System (ADS)

    Wang, Xianwei; Zhang, John Z. H.; He, Xiao

    2015-11-01

    Recent advance in biophysics has made it possible to directly measure site-specific electric field at internal sites of proteins using molecular probes with C = O or C≡N groups in the context of vibrational Stark effect. These measurements directly probe changes of electric field at specific protein sites due to, e.g., mutation and are very useful in protein design. Computational simulation of the Stark effect based on force fields such as AMBER and OPLS, while providing good insight, shows large errors in comparison to experimental measurement due to inherent difficulties associated with point charge based representation of force fields. In this study, quantum mechanical calculation of protein's internal electrostatic properties and vibrational Stark shifts was carried out by using electrostatically embedded generalized molecular fractionation with conjugate caps method. Quantum calculated change of mutation-induced electric field and vibrational Stark shift is reported at the internal probing site of enzyme human aldose reductase. The quantum result is in much better agreement with experimental data than those predicted by force fields, underscoring the deficiency of traditional point charge models describing intra-protein electrostatic properties.

  10. Aldose Reductase-Mediated Phosphorylation of p53 Leads to Mitochondrial Dysfunction, and Damage in Diabetic Platelets

    PubMed Central

    Tang, Wai Ho; Stitham, Jeremiah; Jin, Yu; Liu, Renjing; Lee, Seung Hee; Du, Jing; Atteya, Gourg; Gleim, Scott; Spollett, Geralyn; Martin, Kathleen; Hwa, John

    2014-01-01

    Background Platelet abnormalities are well-recognized complications of diabetes mellitus (DM). Mitochondria play a central role in platelet metabolism and activation. Mitochondrial dysfunction is evident in DM. The molecular pathway for hyperglycemia-induced mitochondrial dysfunction in DM platelets is unknown. Methods and Results Using both human and humanized mouse models, we report that hyperglycemia-induced aldose reductase (AR) activation, and subsequent reactive oxygen species (ROS) production, leads to increased p53 phosphorylation (Ser15), which promotes mitochondrial dysfunction, damage and rupture by sequestration of the anti-apoptotic protein Bcl-xL. In a glucose dose dependent manner, severe mitochondrial damage leads to loss of mitochondrial membrane potential and platelet apoptosis (cytochrome c release, caspase 3 activation and phosphatidylserine exposure). Although platelet hyperactivation, mitochondrial dysfunction, AR activation, ROS production and p53 phosphorylation are all induced by hyperglycemia, we demonstrate that platelet apoptosis and hyperactivation are two distinct states, dependent upon the severity of the hyperglycemia and mitochondrial damage. Combined, both lead to increased thrombus formation in a mouse blood stasis model. Conclusions AR contributes to diabetes-mediated mitochondrial dysfunction and damage through the activation of p53. The degree of mitochondrial dysfunction and damage determines whether hyperactivity (mild damage) or apoptosis (severe damage) will ensue. These signaling components provide novel therapeutic targets for DM thrombotic complications. PMID:24474649

  11. Quantum mechanical calculation of electric fields and vibrational Stark shifts at active site of human aldose reductase

    SciTech Connect

    Wang, Xianwei; Zhang, John Z. H.; He, Xiao

    2015-11-14

    Recent advance in biophysics has made it possible to directly measure site-specific electric field at internal sites of proteins using molecular probes with C = O or C≡N groups in the context of vibrational Stark effect. These measurements directly probe changes of electric field at specific protein sites due to, e.g., mutation and are very useful in protein design. Computational simulation of the Stark effect based on force fields such as AMBER and OPLS, while providing good insight, shows large errors in comparison to experimental measurement due to inherent difficulties associated with point charge based representation of force fields. In this study, quantum mechanical calculation of protein’s internal electrostatic properties and vibrational Stark shifts was carried out by using electrostatically embedded generalized molecular fractionation with conjugate caps method. Quantum calculated change of mutation-induced electric field and vibrational Stark shift is reported at the internal probing site of enzyme human aldose reductase. The quantum result is in much better agreement with experimental data than those predicted by force fields, underscoring the deficiency of traditional point charge models describing intra-protein electrostatic properties.

  12. Exclusion of aldose reductase as a mediator of ERG deficits in a mouse model of diabetic eye disease

    PubMed Central

    SAMUELS, IVY S.; LEE, CHIEH-ALLEN; PETRASH, J. MARK; PEACHEY, NEAL S.; KERN, TIMOTHY S.

    2013-01-01

    Streptozotocin (STZ)-induced diabetes is associated with reductions in the electrical response of the outer retina and retinal pigment epithelium (RPE) to light. Aldose reductase (AR) is the first enzyme required in the polyol-mediated metabolism of glucose, and AR inhibitors have been shown to improve diabetes-induced electroretinogram (ERG) defects. Here, we used control and AR−/− mice to determine if genetic inactivation of this enzyme likewise inhibits retinal electrophysiological defects observed in a mouse model of type 1 diabetes. STZ was used to induce hyperglycemia and type 1 diabetes. Diabetic and age-matched nondiabetic controls of each genotype were maintained for 22 weeks, after which ERGs were used to measure the light-evoked components of the RPE (dc-ERG) and the neural retina (a-wave, b-wave). In comparison to their nondiabetic controls, wildtype (WT) and AR−/− diabetic mice displayed significant decreases in the c-wave, fast oscillation, and off response components of the dc-ERG but not in the light peak response. Nondiabetic AR−/− mice displayed larger ERG component amplitudes than did nondiabetic WT mice; however, the amplitude of dc-ERG components in diabetic AR−/− animals were similar to WT diabetics. ERG a-wave amplitudes were not reduced in either diabetic group, but b-wave amplitudes were lower in WT and AR−/− diabetic mice. These findings demonstrate that the light-induced responses of the RPE and outer retina are disrupted in diabetic mice, but these defects are not due to photoreceptor dysfunction, nor are they ameliorated by deletion of AR. This latter finding suggests that benefits observed in other studies utilizing pharmacological inhibitors of AR might have been secondary to off-target effects of the drugs. PMID:23101909

  13. SIRT1 contributes to aldose reductase expression through modulating NFAT5 under osmotic stress: In vitro and in silico insights.

    PubMed

    Timucin, Ahmet Can; Bodur, Cagri; Basaga, Huveyda

    2015-11-01

    So far, a myriad of molecules were characterized to modulate NFAT5 and its downstream targets. Among these NFAT5 modifiers, SIRT1 was proposed to have a promising role in NFAT5 dependent events, yet the exact underlying mechanism still remains obscure. Hence, the link between SIRT1 and NFAT5-aldose reductase (AR) axis under osmotic stress, was aimed to be delineated in this study. A unique osmotic stress model was generated and its mechanistic components were deciphered in U937 monocytes. In this model, AR expression and nuclear NFAT5 stabilization were revealed to be positively regulated by SIRT1 through utilization of pharmacological modulators. Overexpression and co-transfection studies of NFAT5 and SIRT1 further validated the contribution of SIRT1 to AR and NFAT5. The involvement of SIRT1 activity in these events was mediated via modification of DNA binding of NFAT5 to AR ORE region. Besides, NFAT5 and SIRT1 were also shown to co-immunoprecipitate under isosmotic conditions and this interaction was disrupted by osmotic stress. Further in silico experiments were conducted to investigate if SIRT1 directly targets NFAT5. In this regard, certain lysine residues of NFAT5, when kept deacetylated, were found to contribute to its DNA binding and SIRT1 was shown to directly bind K282 of NFAT5. Based on these in vitro and in silico findings, SIRT1 was identified, for the first time, as a novel positive regulator of NFAT5 dependent AR expression under osmotic stress in U937 monocytes. PMID:26297866

  14. Amelioration of Acute Kidney Injury in Lipopolysaccharide-Induced Systemic Inflammatory Response Syndrome by an Aldose Reductase Inhibitor, Fidarestat

    PubMed Central

    Takahashi, Kazunori; Mizukami, Hiroki; Kamata, Kosuke; Inaba, Wataru; Kato, Noriaki; Hibi, Chihiro; Yagihashi, Soroku

    2012-01-01

    Background Systemic inflammatory response syndrome is a fatal disease because of multiple organ failure. Acute kidney injury is a serious complication of systemic inflammatory response syndrome and its genesis is still unclear posing a difficulty for an effective treatment. Aldose reductase (AR) inhibitor is recently found to suppress lipopolysaccharide (LPS)-induced cardiac failure and its lethality. We studied the effects of AR inhibitor on LPS-induced acute kidney injury and its mechanism. Methods Mice were injected with LPS and the effects of AR inhibitor (Fidarestat 32 mg/kg) before or after LPS injection were examined for the mortality, severity of renal failure and kidney pathology. Serum concentrations of cytokines (interleukin-1β, interleukin-6, monocyte chemotactic protein-1 and tumor necrosis factor-α) and their mRNA expressions in the lung, liver, spleen and kidney were measured. We also evaluated polyol metabolites in the kidney. Results Mortality rate within 72 hours was significantly less in LPS-injected mice treated with AR inhibitor both before (29%) and after LPS injection (40%) than untreated mice (90%). LPS-injected mice showed marked increases in blood urea nitrogen, creatinine and cytokines, and AR inhibitor treatment suppressed the changes. LPS-induced acute kidney injury was associated with vacuolar degeneration and apoptosis of renal tubular cells as well as infiltration of neutrophils and macrophages. With improvement of such pathological findings, AR inhibitor treatment suppressed the elevation of cytokine mRNA levels in multiple organs and renal sorbitol accumulation. Conclusion AR inhibitor treatment ameliorated LPS-induced acute kidney injury, resulting in the lowered mortality. PMID:22253906

  15. Effect of SG-210, a novel aldose reductase inhibitor, on impaired polyol pathway in rats received diabetic manipulations.

    PubMed

    Horie, S; Nagai, H; Yuuki, T; Narita, Y; Tsuda, Y; Nakajima, T; Nakamura, N

    1998-01-01

    To investigate the effect of SG-210, a potent inhibitor selective to aldose reductase (ARI), on the impaired polyol pathway, we examined biochemically and histologically the potencies of this compound in streptozotocin-induced diabetic or galactosemic rats. The study with diabetic rats showed that SG-210 (1-10 mg x kg(-1)) dose-dependently inhibited sorbitol accumulations in erythrocytes, sciatic nerves, lens, and retina with ED50 values of 1.4, 1.3, 3.5, and 4.6 mg x kg(-1), respectively. Zenarestat, currently under clinical trials both in Japan and the United States, was about two or over five times less potent than SG-210 in suppressing sorbitol contents of erythrocytes or other tissues, respectively. Epalrestat, commercially available, was much less potent in reducing the contents with ED50 values of more than 30 mg x kg(-1) in all of the cells and the tissues examined. An extensive study using galactosemic rats indicated that SG-210 (3-30 mg x kg(-1)) inhibited galactitol accumulations in lens and retina as well as in erythrocytes, preventing the progression of histological abnormalities in lens accompanied by the reduction in galactitol contents. Epalrestat (3-30 mg x kg(-1)) failed to show any significant effects. Pharmacokinetic studies suggested that SG-210 has a high bioavailability and possesses a long half-life in rats (ca. 10 h). Taken together with its excellent pharmacokinetic profiles, the potent suppressive effects of SG-210 observed in this study may be available as a new treatment of diabetic complications. PMID:9618072

  16. Deficiency of aldose reductase attenuates inner retinal neuronal changes in a mouse model of retinopathy of prematurity.

    PubMed

    Fu, Zhongjie; Nian, Shen; Li, Suk-Yee; Wong, David; Chung, Sookja K; Lo, Amy C Y

    2015-09-01

    Retinopathy of prematurity (ROP) is a leading cause of childhood blindness where vascular abnormality and retinal dysfunction are reported. We showed earlier that genetic deletion of aldose reductase (AR), the rate-limiting enzyme in the polyol pathway, reduced the neovascularization through attenuating oxidative stress induction in the mouse oxygen-induced retinopathy (OIR) modeling ROP. In this study, we further investigated the effects of AR deficiency on retinal neurons in the mouse OIR. Seven-day-old wild-type and AR-deficient mice were exposed to 75 % oxygen for 5 days and then returned to room air. Electroretinography was used to assess the neuronal function at postnatal day (P) 30. On P17 and P30, retinal cytoarchitecture was examined by morphometric analysis and immunohistochemistry for calbindin, protein kinase C alpha, calretinin, Tuj1, and glial fibrillary acidic protein. In OIR, attenuated amplitudes and delayed implicit time of a-wave, b-wave, and oscillatory potentials were observed in wild-type mice, but they were not significantly changed in AR-deficient mice. The morphological changes of horizontal, rod bipolar, and amacrine cells were shown in wild-type mice and these changes were partly preserved with AR deficiency. AR deficiency attenuated the Müller cell gliosis induced in OIR. Our observations demonstrated AR deficiency preserved retinal functions in OIR and AR deficiency could partly reduce the extent of retinal neuronal histopathology. These findings suggested a therapeutic potential of AR inhibition in ROP treatment with beneficial effects on the retinal neurons. PMID:25921391

  17. Design of an Amide N-glycoside Derivative of β-Glucogallin: A Stable, Potent, and Specific Inhibitor of Aldose Reductase

    PubMed Central

    Li, Linfeng; Chang, Kun-Che; Zhou, Yaming; Shieh, Biehuoy; Ponder, Jessica; Abraham, Adedoyin D.; Ali, Hadi; Snow, Anson; Petrash, J. Mark; LaBarbera, Daniel V.

    2014-01-01

    β-glucogallin (BGG), a major component of the Emblica officinalis medicinal plant, is a potent and selective inhibitor of aldose-reductase (AKR1B1). New linkages (ether/triazole/amide) were introduced via high yielding, efficient syntheses to replace the labile ester, and an original 2-step (90%) preparation of BGG was developed. Inhibition of AKR1B1was assessed in vitro and using transgenic lens organ cultures, which identified the amide linked glucoside (BGA) as a stable, potent and selective lead therapeutic toward the treatment of diabetic eye disease. PMID:24341381

  18. Quantum Model of Catalysis Based on a Mobile Proton Revealed by Subatomic X-ray and Neutron Diffraction Studies of h-aldose Reductase

    SciTech Connect

    Blakeley, M. P.; Ruiz, Fredrico; Cachau, Raul; Hazemann, I.; Meilleur, Flora; Mitschler, A.; Ginell, Stephan; Afonine, Pavel; Ventura, Oscar; Cousido-Siah, Alexandra; Haertlein, M.; Joachimiak, Andrzej; Myles, Dean A A; Podjarny, A.

    2008-01-01

    We present results of combined studies of the enzyme human aldose reductase (h-AR, 36 kDa) using single-crystal x-ray data (0.66 Angstroms, 100K; 0.80 Angstroms, 15K; 1.75 Angstroms, 293K), neutron Laue data (2.2 Angstroms, 293K), and quantum mechanical modeling. These complementary techniques unveil the internal organization and mobility of the hydrogen bond network that defines the properties of the catalytic engine, explaining how this promiscuous enzyme overcomes the simultaneous requirements of efficiency and promiscuity offering a general mechanistic view for this class of enzymes.

  19. [Role of heat shock proteins, aldose reductase, Bcl-2 protein and microRNA in the mechanism of delayed preconditioning of heart].

    PubMed

    Lishmanov, Iu B; Maslov, L N; Khaliulin, I G; Zhang, Y; Pei, J -M

    2010-05-01

    Analysis of published data allows affirming that heat shock proteins (HSP) play an important role in the mechanism of cardioprotective effect of delayed preconditioning. However, HSP in all probability are non-end effectors but mediators of preconditioning because a peak of their levels in myocardium does not concur with maximal elevation of cardiac tolerance to impact of ischemia and reperfusion. There are bases to think that aldose reductase and Bcl-2 protein are claimants to the role of end-effectors of delayed preconditioning but microRNAs serve as mediators of forming increased cardiac tolerance to ischemia-reperfusion. PMID:20583571

  20. A defect in sodium-dependent amino acid uptake in diabetic rabbit peripheral nerve. Correction by an aldose reductase inhibitor or myo-inositol administration.

    PubMed Central

    Greene, D A; Lattimer, S A; Carroll, P B; Fernstrom, J D; Finegold, D N

    1990-01-01

    A myo-inositol-related defect in nerve sodium-potassium ATPase activity in experimental diabetes has been suggested as a possible pathogenetic factor in diabetic neuropathy. Because the sodium-potassium ATPase is essential for other sodium-cotransport systems, and because myo-inositol-derived phosphoinositide metabolites regulate multiple membrane transport processes, sodium gradient-dependent amino acid uptake was examined in vitro in endoneurial preparations derived from nondiabetic and 14-d alloxan diabetic rabbits. Untreated alloxan diabetes reduced endoneurial sodium-gradient dependent uptake of the nonmetabolized amino acid 2-aminoisobutyric acid by greater than 50%. Administration of an aldose reductase inhibitor prevented reductions in both nerve myo-inositol content and endoneurial sodium-dependent 2-aminoisobutyric acid uptake. Myo-inositol supplementation that produced a transient pharmacological elevation in plasma myo-inositol concentration, but did not raise nerve myo-inositol content, reproduced the effect of the aldose reductase inhibitor on endoneurial sodium-dependent 2-aminoisobutyric acid uptake. Phorbol myristate acetate, which acutely normalizes sodium-potassium ATPase activity in diabetic nerve, did not acutely correct 2-aminoisobutyric uptake when added in vitro. These data suggest that depletion of a small myo-inositol pool may be implicated in the pathogenesis of defects in amino acid uptake in diabetic nerve and that rapid correction of sodium-potassium ATPase activity with protein kinase C agonists in vitro does not acutely normalize sodium-dependent 2-aminoisobutyric acid uptake. PMID:2185278

  1. Preventive effect of long-term aldose reductase inhibition (ponalrestat) on nerve conduction and sural nerve structure in the spontaneously diabetic Bio-Breeding rat.

    PubMed Central

    Sima, A A; Prashar, A; Zhang, W X; Chakrabarti, S; Greene, D A

    1990-01-01

    To test the hypothesis that aldose reductase inhibition may prevent or delay the development of functional and structural neuropathy in the insulin-deficient diabetic Bio-Breeding rat (BB-rat), hyperglycemic rats were begun on the aldose reductase inhibitor (ARI) ponalrestat 25 mg/kg body wt soon after the onset of diabetes and followed for 4 or 6 mo. Ponalrestat treatment completely prevented the characteristic nerve conduction slowing and structural abnormalities of the node of Ranvier for 4 mo despite only partial preservation of axonal integrity. Ponalrestat treatment for 6 mo achieved a partial but significant prevention of nerve conduction slowing, axoglial dysjunction, and axonal degenerative changes. This incomplete but significant prevention of neuropathy by ponalrestat suggests that additional mechanisms besides polyol-pathway activation may be of importance in the pathogenesis of diabetic neuropathy. Alternatively, the dosage used in the present study may not have been sufficient to achieve a complete prevention. Despite the only partial protective effect of ARI treatment on degenerative peripheral nerve changes in hyperglycemic BB-rats, 6 mo of treatment resulted in a more than threefold increase in regenerating nerve fibers. These data suggest that prophylactic ARI treatment may be efficacious in delaying the development of diabetic neuropathy. Images PMID:2110189

  2. A series of pyrido[2,3-b]pyrazin-3(4H)-one derivatives as aldose reductase inhibitors with antioxidant activity.

    PubMed

    Han, Zhongfei; Hao, Xin; Ma, Bing; Zhu, Changjin

    2016-10-01

    A series of pyrido[2,3-b]pyrazin-3(4H)-one based derivatives were designed as inhibitors of aldose reductase (ALR2), the enzyme which plays a key role in the development of diabetes complications as well as in the oxidative stress processes associated with diabetes and other pathologies. Most of the derivatives, having a substituted C2 aromatic group and a N4 acetic acid group on the core structure, were found to be potent and selective aldose reductase inhibitors with submicromolar IC50 values, and 9c was the most active with IC50 value 0.009 μM. Particularly, a number of the designed compounds bearing phenolic hydroxyl substituted C2-styryl side chain showed excellent not only in ALR2 inhibition but also in antioxidant, and among these 11i was proved to be the top one with an antioxidant ability even comparable to that of the well-known antioxidant Trolox. Structure-activity relationship and molecular docking studies highlighted the importance of phenolic hydroxyl substituents and vinyl spacer in C2 side chain of the scaffold for the construction of efficient and multifunctional ALR2 inhibitors. PMID:27267001

  3. Phenolic Compounds from the Leaves and Twigs of Osteomeles schwerinae That Inhibit Rat Lens Aldose Reductase and Vessel Dilation in Zebrafish Larvae.

    PubMed

    Lee, Ik-Soo; Jung, Seung-Hyun; Lee, Yun Mi; Choi, So-Jin; Sun, Hang; Kim, Jin Sook

    2015-09-25

    Three new phenolic biphenyl derivatives (1-3) and one new lignan glycoside (4) were isolated from the leaves and twigs of Osteomeles schwerinae. The structures of the new compounds were established by spectroscopic data interpretation. The inhibitory effects of 1-4 on rat lens aldose reductase in vitro were examined, and compounds 1-3 markedly inhibited the enzyme with IC50 values of 3.8 to 13.8 μM. In addition, the effects of these isolates on the dilation of hyaloid-retinal vessels induced by high glucose (HG) in zebrafish larvae were investigated. Compound 1 was the most effective in reducing HG-induced dilation of hyaloid-retinal vessels. PMID:26331986

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  6. Part 1: synthesis of irreversible inhibitors of aldose reductase with subsequent development of a carbon-13 NMR protein probe. Part 2: synthesis of selenium analogs of dopamine as potential dopamine receptor agonists

    SciTech Connect

    Ares, J.J.

    1986-01-01

    Aldose reductase converts glucose into sorbitol using NADPH as a cofactor. Sorbitol accumulation in various tissues is believed to play a major role in the development of debilitating complications of diabetes; thus, much effort has been directed toward the preparation of aldose reductase inhibitors. Of the compounds prepared, the most active are the isothiocyanate and azide analogs of the reversible aldose reductase inhibitor alrestatin. The potency of the alrestatin isothiocyanate prompted the authors to examine the possibility that isothiocyanates enriched with carbon-13 could be used as carbon-13 NMR protein probes. Toward this end, a synthesis of carbon-13 enriched phenylisothiocyanate has been developed. This reagent has been successfully utilized to study peptides via carbon-13 NMR spectroscopy. Research in their laboratory over the years has focused on answering two fundamental questions regarding the interaction of dopamine with its receptor. First, can the concept of bioisosterism be applied to dopamine agonists. Secondly, what is the actual molecular species of dopamine which interacts with the dopamine receptor. In an effort to answer these questions, methyl selenide and dimethyl selenonium analogs of dopamine have been synthesized.

  7. The Prostaglandin F Synthase Activity of the Human Aldose Reductase AKR1B1 Brings New Lenses to Look at Pathologic Conditions

    PubMed Central

    Bresson, Eva; Lacroix-Pépin, Nicolas; Boucher-Kovalik, Sofia; Chapdelaine, Pierre; Fortier, Michel A.

    2012-01-01

    Prostaglandins are important regulators of female reproductive functions to which aldose reductases exhibiting hydroxysteroid dehydrogenase activity also contribute. Our work on the regulation of reproductive function by prostaglandins (PGs), lead us to the discovery that AKR1B5 and later AKR1B1were highly efficient and physiologically relevant PGF synthases. PGE2 and PGF2α are the main prostanoids produced in the human endometrium and proper balance in their relative production is important for normal menstruation and optimal fertility. Recent evidence suggests that PGE2/EP2 and PGF2α/FP may constitute a functional dyad with physiological relevance comparable to the prostacyclin-thromboxane dyad in the vascular system. We have recently reported that AKR1B1 was expressed and modulated in association with PGF2α production in response to IL-1β in the human endometrium. In the present study, we show that the human AKR1B1 (gene ID: 231) also known as ALDR1 or ALR2 is a functional PGF2α synthase in different models of living cells and tissues. Using human endometrial cells, prostate, and vascular smooth muscle cells, cardiomyocytes and endothelial cells we demonstrate that IL-1β is able to up regulate COX-2 and AKR1B1 proteins as well as PGF2α production under normal glucose concentrations. We show that the promoter activity of AKR1B1 gene is increased by IL-1β particularly around the multiple stress response region containing two putative antioxidant response elements adjacent to TonE and AP1. We also show that AKR1B1 is able to regulate PGE2 production through PGF2α acting on its FP receptor and that aldose reductase inhibitors like alrestatin, Statil (ponalrestat), and EBPC exhibit distinct and characteristic inhibition of PGF2α production in different cell models. The PGF synthase activity of AKR1B1 represents a new and important target to regulate ischemic and inflammatory responses associated with several human pathologies. PMID:22654757

  8. Excretion of tectoridin metabolites in rat urine and bile orally administrated at different dosages and their inhibitory activity against aldose reductase.

    PubMed

    Qu, Jialin; Wu, Zhizhen; Gao, Jie; Wen, Hao; Wang, Tao; Yuan, Dan

    2014-12-01

    This study investigated the urinary and biliary excretion of tectoridin, a major active isoflavonoid found in the flowers of Pueraria thomsonii Benth. and the rhizomes of Belamcanda chinensis (L.) DC. Using UHPLC/Q-TOFMS, seven glucuronides and/or sulfated metabolites and four Phase I metabolites were simultaneously quantified in rat urine after oral administration of tectoridin at 100 and 200 mg/kg. Over a 72-h period, 14.2% and 14.7% of the tectoridin were excreted as eleven metabolites in urine, among which, two major metabolites tectorigenin-7-O-β-D-glucuronide (Te-7G) and tectorigenin accounted for 5.5-5.5% and 4.3-4.4%. Furthermore, the cumulative excretion of four glucuronides and sulfated metabolites in bile accounted for 7.3% and 3.9% of the dose within 60 h, among which, Te-7G and tectorigenin-7-O-glucuronide-4'-O-sulfate (Te-7G-4'S) accounted for 2.3-3.0% and 1.4-3.9%, respectively. The results indicate that the urine was the primary elimination route, and glucuronidation after deglycosylation at C-7 position was the major metabolic pathway of tectoridin in vivo. Moreover, the inhibitory activities of tectoridin and its five metabolites on rat lens aldose reductase were confirmed (IC₅₀: 1.4-15.5 μM), whereas irisolidone-7-O-glucuronide (Ir-7G) and irisolidone showed little activity. PMID:25256063

  9. SIRT6 Is a Positive Regulator of Aldose Reductase Expression in U937 and HeLa cells under Osmotic Stress: In Vitro and In Silico Insights.

    PubMed

    Timucin, Ahmet Can; Basaga, Huveyda

    2016-01-01

    SIRT6 is a protein deacetylase, involved in various intracellular processes including suppression of glycolysis and DNA repair. Aldose Reductase (AR), first enzyme of polyol pathway, was proposed to be indirectly associated to these SIRT6 linked processes. Despite these associations, presence of SIRT6 based regulation of AR still remains ambiguous. Thus, regulation of AR expression by SIRT6 was investigated under hyperosmotic stress. A unique model of osmotic stress in U937 cells was used to demonstrate the presence of a potential link between SIRT6 and AR expression. By overexpressing SIRT6 in HeLa cells under hyperosmotic stress, its role on upregulation of AR was revealed. In parallel, increased SIRT6 activity was shown to upregulate AR in U937 cells under hyperosmotic milieu by using pharmacological modulators. Since these modulators also target SIRT1, binding of the inhibitor, Ex-527, specifically to SIRT6 was analyzed in silico. Computational observations indicated that Ex-527 may also target SIRT6 active site residues under high salt concentration, thus, validating in vitro findings. Based on these evidences, a novel regulatory step by SIRT6, modifying AR expression under hyperosmotic stress was presented and its possible interactions with intracellular machinery was discussed. PMID:27536992

  10. 3D-QSAR (CoMFA and CoMSIA) and pharmacophore (GALAHAD) studies on the differential inhibition of aldose reductase by flavonoid compounds.

    PubMed

    Caballero, Julio

    2010-11-01

    Inhibitory activities of flavonoid derivatives against aldose reductase (AR) enzyme were modelled by using CoMFA, CoMSIA and GALAHAD methods. CoMFA and CoMSIA methods were used for deriving quantitative structure-activity relationship (QSAR) models. All QSAR models were trained with 55 compounds, after which they were evaluated for predictive ability with additional 14 compounds. The best CoMFA model included both steric and electrostatic fields, meanwhile, the best CoMSIA model included steric, hydrophobic and H-bond acceptor fields. These models had a good predictive quality according to both internal and external validation criteria. On the other hand, GALAHAD was used for deriving a 3D pharmacophore model. Twelve active compounds were used for deriving this model. The obtained model included hydrophobe, hydrogen bond acceptor and hydrogen bond donor features; it was able to identify the active AR inhibitors from the remaining compounds. These in silico tools might be useful in the rational design of new AR inhibitors. PMID:20863730

  11. Three-dimensional quantitative structure-activity relationships and docking studies of some structurally diverse flavonoids and design of new aldose reductase inhibitors

    PubMed Central

    Chandra De, Utpal; Debnath, Tanusree; Sen, Debanjan; Debnath, Sudhan

    2015-01-01

    Aldose reductase (AR) plays an important role in the development of several long-term diabetic complications. Inhibition of AR activities is a strategy for controlling complications arising from chronic diabetes. Several AR inhibitors have been reported in the literature. Flavonoid type compounds are shown to have significant AR inhibition. The objective of this study was to perform a computational work to get an idea about structural insight of flavonoid type compounds for developing as well as for searching new flavonoid based AR inhibitors. The data-set comprising 68 flavones along with their pIC50 values ranging from 0.44 to 4.59 have been collected from literature. Structure of all the flavonoids were drawn in Chembiodraw Ultra 11.0, converted into corresponding three-dimensional structure, saved as mole file and then imported to maestro project table. Imported ligands were prepared using LigPrep option of maestro 9.6 version. Three-dimensional quantitative structure-activity relationships and docking studies were performed with appropriate options of maestro 9.6 version installed in HP Z820 workstation with CentOS 6.3 (Linux). A model with partial least squares factor 5, standard deviation 0.2482, R2 = 0.9502 and variance ratio of regression 122 has been found as the best statistical model. PMID:25709964

  12. SIRT6 Is a Positive Regulator of Aldose Reductase Expression in U937 and HeLa cells under Osmotic Stress: In Vitro and In Silico Insights

    PubMed Central

    Timucin, Ahmet Can; Basaga, Huveyda

    2016-01-01

    SIRT6 is a protein deacetylase, involved in various intracellular processes including suppression of glycolysis and DNA repair. Aldose Reductase (AR), first enzyme of polyol pathway, was proposed to be indirectly associated to these SIRT6 linked processes. Despite these associations, presence of SIRT6 based regulation of AR still remains ambiguous. Thus, regulation of AR expression by SIRT6 was investigated under hyperosmotic stress. A unique model of osmotic stress in U937 cells was used to demonstrate the presence of a potential link between SIRT6 and AR expression. By overexpressing SIRT6 in HeLa cells under hyperosmotic stress, its role on upregulation of AR was revealed. In parallel, increased SIRT6 activity was shown to upregulate AR in U937 cells under hyperosmotic milieu by using pharmacological modulators. Since these modulators also target SIRT1, binding of the inhibitor, Ex-527, specifically to SIRT6 was analyzed in silico. Computational observations indicated that Ex-527 may also target SIRT6 active site residues under high salt concentration, thus, validating in vitro findings. Based on these evidences, a novel regulatory step by SIRT6, modifying AR expression under hyperosmotic stress was presented and its possible interactions with intracellular machinery was discussed. PMID:27536992

  13. Tonicity-responsive enhancer binding protein regulates the expression of aldose reductase and protein kinase C δ in a mouse model of diabetic retinopathy.

    PubMed

    Park, Jeongsook; Kim, Hwajin; Park, So Yun; Lim, Sun Woo; Kim, Yoon Sook; Lee, Dong Hoon; Roh, Gu Seob; Kim, Hyun Joon; Kang, Sang Soo; Cho, Gyeong Jae; Jeong, Bo-Young; Kwon, H Moo; Choi, Wan Sung

    2014-05-01

    Recent studies revealed that Tonicity-responsive enhancer binding protein (TonEBP) directly regulates the transcription of aldose reductase (AR), which catalyzes the first step of the polyol pathway of glucose metabolism. Activation of protein kinase C δ (PKCδ) is dependent on AR and it has been linked to diabetic complications. However, whether TonEBP affects expressions of AR and PKCδ in diabetic retinopathy was not clearly shown. In this study, we used TonEBP heterozygote mice to study the role of TonEBP in streptozotocin (STZ)-induced diabetic retinopathy. We performed immunofluorescence staining and found that retinal expressions of AR and PKCδ were significantly reduced in the heterozygotes compared to wild type littermates, particularly in ganglion cell layer. To examine further the effect of TonEBP reduction in retinal tissues, we performed intravitreal injection of TonEBP siRNA and confirmed the decrease in AR and PKCδ levels. In addition, we found that a proapoptotic factor, Bax level was reduced and a survival factor, Bcl2 level was increased after injection of TonEBP siRNA, indicating that TonEBP mediates apoptotic cell death. In parallel, TonEBP siRNA was applied to the in vitro human retinal pigment epithelial (ARPE-19) cells cultured in high glucose media. We have consistently found the decrease in AR and PKCδ levels and changes in apoptotic factors for survival. Together, these results clearly demonstrated that hyperglycemia-induced TonEBP plays a crucial role in increasing AR and PKCδ levels and leading to apoptotic death. Our findings suggest that TonEBP reduction is an effective therapeutic strategy for diabetic retinopathy. PMID:24631337

  14. Aldose Reductase Regulates Microglia/Macrophages Polarization Through the cAMP Response Element-Binding Protein After Spinal Cord Injury in Mice.

    PubMed

    Zhang, Qian; Bian, Ganlan; Chen, Peng; Liu, Ling; Yu, Caiyong; Liu, Fangfang; Xue, Qian; Chung, Sookja K; Song, Bing; Ju, Gong; Wang, Jian

    2016-01-01

    Inflammatory reactions are the most critical pathological processes occurring after spinal cord injury (SCI). Activated microglia/macrophages have either detrimental or beneficial effects on neural regeneration based on their functional polarized M1/M2 subsets. However, the mechanism of microglia/macrophage polarization to M1/M2 at the injured spinal cord environment remains unknown. In this study, wild-type (WT) or aldose reductase (AR)-knockout (KO) mice were subjected to SCI by a spinal crush injury model. The expression pattern of AR, behavior tests for locomotor activity, and lesion size were assessed at between 4 h and 28 days after SCI. We found that the expression of AR is upregulated in microglia/macrophages after SCI in WT mice. In AR KO mice, SCI led to smaller injury lesion areas compared to WT. AR deficiency-induced microglia/macrophages induce the M2 rather than the M1 response and promote locomotion recovery after SCI in mice. In the in vitro experiments, microglia cell lines (N9 or BV2) were treated with the AR inhibitor (ARI) fidarestat. AR inhibition caused 4-hydroxynonenal (HNE) accumulation, which induced the phosphorylation of the cAMP response element-binding protein (CREB) to promote Arg1 expression. KG501, the specific inhibitor of phosphorylated CREB, could cancel the upregulation of Arg1 by ARI or HNE stimulation. Our results suggest that AR works as a switch which can regulate microglia by polarizing cells to either the M1 or the M2 phenotype under M1 stimulation based on its states of activity. We suggest that inhibiting AR may be a promising therapeutic method for SCI in the future. PMID:25520004

  15. Rabbit corneal and conjunctival permeability of the novel aldose reductase inhibitors: N-[[4-(benzoylamino)phenyl] sulphonyl]glycines and N-benzoyl-N-phenylglycines.

    PubMed

    Kompella, U B; Sunkara, G; Thomas, E; Clark, C R; Deruiter, J

    1999-08-01

    Corneal and conjunctival permeability has been investigated for novel aldose reductase inhibitors (ARIs) of the N{[4-(benzoylamino)phenyl]sulphonyl}glycine (benzoylaminophenylsulphonylglycine) and N-benzoyl-N-phenylglycine (benzoylphenylglycine) series, compounds developed for prevention of cataract formation in diabetic subjects. Six benzoylaminophenylsulphonylglycines were synthesized with modifications either of the phenyl group or of the glycine structure and three benzoylphenylglycines were synthesized with modification in the phenyl group of the benzoyl moiety. Transport of ARIs in the mucosal to serosal direction was evaluated across rabbit cornea and conjunctiva bathed in glutathione-bicarbonate Ringer's solution maintained at pH 7.4 and 37 degrees C. The permeability coefficients of the novel ARIs across cornea and conjunctiva ranged from 1.87 to 8.95 x 10(-6) cm s(-1) and from 4.6 to 19.15 x 10(-6) cm s(-1), respectively. The ratio of corneal to conjunctival permeability ranged from 0.12 to 0.79. The calculated log partition coefficient (log P) values for the ARIs were in the range 0.84 to 2.78. The log distribution coefficients (log D) were in the range -2.87 to -0.89. There was no apparent relationship between log P or log D and the permeability coefficients of the ARIs for either tissue. Cornea was more resistant to ARI transport than was conjunctiva. Substitution of a phenyl group for hydrogen in the glycine methylene group reduced the permeability coefficient. Permeability coefficients were different for different stereoisomers. Compared with the permeability coefficient of benzoylaminophenylsulphonylglycine, that of 4-fluorobenzoylaminophenylsulphonylglycine was lower in the cornea but similar in the conjunctiva. In both tissues, the permeability coefficient of 2-nitrobenzoylaminophenylsulphonylglycine was less than that of 4-nitrobenzoylaminophenylsulphonylglycine. There was no significant difference between the permeability coefficients of 3-nitro

  16. Gedunin abrogates aldose reductase, PI3K/Akt/mToR, and NF-κB signaling pathways to inhibit angiogenesis in a hamster model of oral carcinogenesis.

    PubMed

    Kishore T, Kranthi Kiran; Ganugula, Raghu; Gade, Deepak Reddy; Reddy, Geereddy Bhanuprakash; Nagini, Siddavaram

    2016-02-01

    Aberrant activation of oncogenic signaling pathways plays a central role in tumor development and progression. The aim of this present study was to investigate the chemopreventive effects of the neem limonoid gedunin in the hamster model of oral cancer based on its ability to modulate aldose reductase (AR), phosphatidyl inositol-3-kinase (PI3K)/Akt, and nuclear factor kappa B (NF-κB) pathways to block angiogenesis. Administration of gedunin suppressed the development of HBP carcinomas by inhibiting PI3K/Akt and NF-κB pathways through the inactivation of Akt and inhibitory kappa B kinase (IKK), respectively. Immunoblot and molecular docking interactions revealed that inhibition of these signaling pathways may be mediated via inactivation of AR by gedunin. Gedunin blocked angiogenesis by downregulating the expression of miR-21 and the pro-angiogenic factors vascular endothelial growth factor and hypoxia inducible factor-1 alpha (HIF-1α). In conclusion, the results of the present study provide compelling evidence that gedunin prevents progression of hamster buccal pouch (HBP) carcinomas via inhibition of the kinases Akt, IKK, and AR, and the oncogenic transcription factors NF-κB and HIF-1α to block angiogenesis. PMID:26342697

  17. Inhibition of aldose reductase prevents growth factor-induced G1-S phase transition through the AKT/phosphoinositide 3-kinase/E2F-1 pathway in human colon cancer cells.

    PubMed

    Ramana, Kota V; Tammali, Ravinder; Srivastava, Satish K

    2010-04-01

    Colon cancer is the leading cause of cancer death in both men and women worldwide. The deregulated cell cycle control or decreased apoptosis of normal epithelial cells leading to uncontrolled proliferation is one of the major features of tumor progression. We have previously shown that aldose reductase (AR), a NADPH-dependent aldo-keto reductase, has been shown to be involved in growth factor-induced proliferation of colon cancer cells. Herein, we report that inhibition of AR prevents epidermal growth factor (EGF)- and basic fibroblast growth factor (bFGF)-induced HT29 cell proliferation by accumulating cells at G(1) phase of cell cycle. Similar results were observed in SW480 and HCT-116 colon cancer cells. Treatment of HT29 cells with AR inhibitor, sorbinil or zopolrestat, prevented the EGF- and bFGF-induced DNA binding activity of E2F-1 and phosphorylation of retinoblastoma protein. Inhibition of AR also prevented EGF- and bFGF-induced phosphorylation of cyclin-dependent kinase (cdk)-2 and expression of G(1)-S transition regulatory proteins such as cyclin D1, cdk4, proliferating cell nuclear antigen, cyclin E, and c-myc. More importantly, inhibition of AR prevented the EGF- and bFGF-induced activation of phosphoinositide 3-kinase/AKT and reactive oxygen species generation in colon cancer cells. Further, inhibition of AR also prevented the tumor growth of human colon cancer cells in nude mouse xenografts. Collectively, these results show that AR mediates EGF- and bFGF-induced colon cancer cell proliferation by activating or expressing G(1)-S phase proteins such as E2F-1, cdks, and cyclins through the reactive oxygen species/phosphoinositide 3-kinase/AKT pathway, indicating the use of AR inhibitors in the prevention of colon carcinogenesis. Mol Cancer Ther; 9(4); 813-24. (c)2010 AACR. PMID:20354121

  18. Structure and function of Caulobacter crescentus aldose-aldose oxidoreductase.

    PubMed

    Taberman, Helena; Andberg, Martina; Koivula, Anu; Hakulinen, Nina; Penttilä, Merja; Rouvinen, Juha; Parkkinen, Tarja

    2015-12-15

    Aldose-aldose oxidoreductase (Cc AAOR) is a recently characterized enzyme from the bacterial strain Caulobacter crescentus CB15 belonging to the glucose-fructose oxidoreductase/inositol dehydrogenase/rhizopine catabolism protein (Gfo/Idh/MocA) family. Cc AAOR catalyses the oxidation and reduction of a panel of aldose monosaccharides using a tightly bound NADP(H) cofactor that is regenerated in the catalytic cycle. Furthermore, Cc AAOR can also oxidize 1,4-linked oligosaccharides. In the present study, we present novel crystal structures of the dimeric Cc AAOR in complex with the cofactor and glycerol, D-xylose, D-glucose, maltotriose and D-sorbitol determined to resolutions of 2.0, 1.8, 1.7, 1.9 and 1.8 Å (1 Å=0.1 nm), respectively. These complex structures allowed for a detailed analysis of the ligand-binding interactions. The structures showed that the C1 carbon of a substrate, which is either reduced or oxidized, is close to the reactive C4 carbon of the nicotinamide ring of NADP(H). In addition, the O1 hydroxy group of the substrate, which is either protonated or deprotonated, is unexpectedly close to both Lys(104) and Tyr(189), which may both act as a proton donor or acceptor. This led us to hypothesize that this intriguing feature could be beneficial for Cc AAOR to catalyse the reduction of a linear form of a monosaccharide substrate and the oxidation of a pyranose form of the same substrate in a reaction cycle, during which the bound cofactor is regenerated. PMID:26438878

  19. Purification and partial characterization of an aldo-keto reductase from Saccharomyces cerevisiae.

    PubMed Central

    Kuhn, A; van Zyl, C; van Tonder, A; Prior, B A

    1995-01-01

    A cytosolic aldo-keto reductase was purified from Saccharomyces cerevisiae ATCC 26602 to homogeneity by affinity chromatography, chromatofocusing, and hydroxylapatite chromatography. The relative molecular weights of the aldo-keto reductase as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size exclusion chromatography were 36,800 and 35,000, respectively, indicating that the enzyme is monomeric. Amino acid composition and N-terminal sequence analysis revealed that the enzyme is closely related to the aldose reductases of xylose-fermenting yeasts and mammalian tissues. The enzyme was apparently immunologically unrelated to the aldose reductases of other xylose-fermenting yeasts. The aldo-keto reductase is NADPH specific and catalyzes the reduction of a variety of aldehydes. The best substrate for the enzyme is the aromatic aldehyde p-nitrobenzaldehyde (Km = 46 microM; kcat/Km = 52,100 s-1 M-1), whereas among the aldoses, DL-glyceraldehyde was the preferred substrate (Km = 1.44 mM; kcat/Km = 1,790 s-1 M-1). The enzyme failed to catalyze the reduction of menadione and p-benzoquinone, substrates for carbonyl reductase. The enzyme was inhibited only slightly by 2 mM sodium valproate and was activated by pyridoxal 5'-phosphate. The optimum pH of the enzyme is 5. These data indicate that the S. cerevisiae aldo-keto reductase is a monomeric NADPH-specific reductase with strong similarities to the aldose reductases. PMID:7747971

  20. Functional studies of aldo-keto reductases in Saccharomyces cerevisiae*

    PubMed Central

    Chang, Qing; Griest, Terry A.; Harter, Theresa M.; Petrash, J. Mark

    2007-01-01

    SUMMARY We utilized the budding yeast Saccharomyces cerevisiae as a model to systematically explore physiological roles for yeast and mammalian aldo-keto reductases. Six open reading frames encoding putative aldo-keto reductases were identified when the yeast genome was queried against the sequence for human aldose reductase, the prototypical mammalian aldo-keto reductase. Recombinant proteins produced from five of these yeast open reading frames demonstrated NADPH-dependent reductase activity with a variety of aldehyde and ketone substrates. A triple aldo-keto reductase null mutant strain demonstrated a glucose-dependent heat shock phenotype which could be rescued by ectopic expression of human aldose reductase. Catalytically-inactive mutants of human or yeast aldo-keto reductases failed to effect a rescue of the heat shock phenotype, suggesting that the phenotype results from either an accumulation of one or more unmetabolized aldo-keto reductase substrates or a synthetic deficiency of aldo-keto reductase products generated in response to heat shock stress. These results suggest that multiple aldo-keto reductases fulfill functionally redundant roles in the stress response in yeast. PMID:17140678

  1. Extreme ultraviolet photoionization of aldoses and ketoses

    NASA Astrophysics Data System (ADS)

    Shin, Joong-Won; Dong, Feng; Grisham, Michael E.; Rocca, Jorge J.; Bernstein, Elliot R.

    2011-04-01

    Gas phase monosaccharides (2-deoxyribose, ribose, arabinose, xylose, lyxose, glucose galactose, fructose, and tagatose), generated by laser desorption of solid sample pellets, are ionized with extreme ultraviolet photons (EUV, 46.9 nm, 26.44 eV). The resulting fragment ions are analyzed using a time of flight mass spectrometer. All aldoses yield identical fragment ions regardless of size, and ketoses, while also generating same ions as aldoses, yields additional features. Extensive fragmentation of the monosaccharides is the result the EUV photons ionizing various inner valence orbitals. The observed fragmentation patterns are not dependent upon hydrogen bonding structure or OH group orientation.

  2. Catalytic anomeric aminoalkynylation of unprotected aldoses.

    PubMed

    Kimura, Yasuaki; Ito, Soichi; Shimizu, Yohei; Kanai, Motomu

    2013-08-16

    A copper(I)-catalyzed anomeric aminoalkynylation reaction of unprotected aldoses was realized. Use of an electron-deficient phosphine ligand, boric acid to stabilize the iminium intermediate, and a protic additive (IPA) to presumably enhance reversible carbohydrate-boron complexation were all essential for efficient conversion. The reaction proceeded well even with a natural disaccharide substrate, suggesting that the developed catalytic reaction could be useful for the synthesis of glycoconjugates with minimum use of protecting groups. PMID:23901780

  3. Medicinal flowers. XXXX . Structures of dihydroisocoumarin glycosides and inhibitory effects on aldose reducatase from the flowers of Hydrangea macrophylla var.thunbergii.

    PubMed

    Liu, Jiang; Nakamura, Seikou; Zhuang, Yan; Yoshikawa, Masayuki; Hussein, Ghazi Mohamed Eisa; Matsuo, Kyohei; Matsuda, Hisashi

    2013-01-01

    Six dihydroisocoumarin glycosides, florahydrosides I and II, thunberginol G 8-O-β-d-glucopyranoside, thunberginol C 8-O-β-d-glucopyranoside, 4-hydroxythunberginol G 3'-O-β-d-glucopyranoside, and thunberginol D 3'-O-β-d-glucopyranoside, have been isolated from the flowers of Hydrangea macrophylla Seringe var. thunbergii Makino (Saxifragaceae) together with 20 known compounds. The chemical structures of the new compounds were elucidated on the basis of chemical and physicochemical evidence. Among the constituents, acylated quinic acid analog, neochlorogenic acid, was shown to substantially inhibit aldose reductase [IC50=5.6 µm]. In addition, the inhibitory effects on aldose reductase of several caffeoylquinic acid analogs were examined for structure-activity relationship study. As the results, 4,5-O-trans-p-dicaffeoyl-d-quinic acid was found to exhibit a potent inhibitory effect [IC50=0.29 µm]. PMID:23727779

  4. Characterization of a unique Caulobacter crescentus aldose-aldose oxidoreductase having dual activities.

    PubMed

    Andberg, Martina; Maaheimo, Hannu; Kumpula, Esa-Pekka; Boer, Harry; Toivari, Mervi; Penttilä, Merja; Koivula, Anu

    2016-01-01

    We describe here the characterization of a novel enzyme called aldose-aldose oxidoreductase (Cc AAOR; EC 1.1.99) from Caulobacter crescentus. The Cc AAOR exists in solution as a dimer, belongs to the Gfo/Idh/MocA family and shows homology with the glucose-fructose oxidoreductase from Zymomonas mobilis. However, unlike other known members of this protein family, Cc AAOR is specific for aldose sugars and can be in the same catalytic cycle both oxidise and reduce a panel of monosaccharides at the C1 position, producing in each case the corresponding aldonolactone and alditol, respectively. Cc AAOR contains a tightly-bound nicotinamide cofactor, which is regenerated in this oxidation-reduction cycle. The highest oxidation activity was detected on D-glucose but significant activity was also observed on D-xylose, L-arabinose and D-galactose, revealing that both hexose and pentose sugars are accepted as substrates by Cc AAOR. The configuration at the C2 and C3 positions of the saccharides was shown to be especially important for the substrate binding. Interestingly, besides monosaccharides, Cc AAOR can also oxidise a range of 1,4-linked oligosaccharides having aldose unit at the reducing end, such as lactose, malto- and cello-oligosaccharides as well as xylotetraose. (1)H NMR used to monitor the oxidation and reduction reaction simultaneously, demonstrated that although D-glucose has the highest affinity and is also oxidised most efficiently by Cc AAOR, the reduction of D-glucose is clearly not as efficient. For the overall reaction catalysed by Cc AAOR, the L-arabinose, D-xylose and D-galactose were the most potent substrates. PMID:26428243

  5. Multiple aldehyde reductases of human brain.

    PubMed

    Hoffman, P L; Wermuth, B; von Wartburg, J P

    1980-01-01

    Human brain contains four forms of aldehyde reducing enzymes. One major activity, designated AR3, has properties indicating its identity with the NADPH-dependent aldehyde reductase, EC 1.1.1.2. The other major form of human brain enzyme, AR1, which is also NADPH-dependent, reduces both aldehyde and ketone-containing substrates, including vitamin K3 (menadione) and daunorubicin, a cancer chemotherapeutic agent. This enzyme is very sensitive to inhibition by the flavonoids quercitrin and quercetine, and may be analogous to a daunorubicin reductase previously described in liver of other species. One minor form of human brain aldehyde reductase, AR2, demonstrates substrate specificity and inhibitor sensitivity which suggest its similarity to aldose reductases found in lens and other tissues of many species. This enzyme, which can also use NADH as cofactor to some extent, is the most active in reducing the aldehyde derivatives of the biogenic amines. The fourth human brain enzyme ("SSA reductase") differs from the other forms in its ability to use NADH as well as or better than NADPH as cofactor, and in its molecular weight, which is nearly twice that of the other forms. It is quite specific for succinic semialdehyde (SSA) as substrate, and was found to be significantly inhibited only by quercetine and quercitrin. AR3 can also reduce SSA, and both enzymes may contribute to the production of gamma-hydroxybutyric acid in vivo. These results indicate that the human brain aldehyde reductases can play relatively specific physiologic roles. PMID:7424738

  6. Catalytic Isomerization of Biomass-Derived Aldoses: A Review.

    PubMed

    Delidovich, Irina; Palkovits, Regina

    2016-03-21

    Selected aldohexoses (d-glucose, d-mannose, and d-galactose) and aldopentoses (d-xylose, l-arabinose, and d-ribose) are readily available components of biopolymers. Isomerization reactions of these substances are very attractive as carbon-efficient processes to broaden the portfolio of abundant monosaccharides. This review focuses on the chemocatalytic isomerization of aldoses into the corresponding ketoses as well as epimerization of aldoses at C2. Recent advances in the fields of catalysis by bases and Lewis acids are considered. The emphasis is laid on newly uncovered catalytic systems and mechanisms of carbohydrate transformations. PMID:26948404

  7. Dehydration of different ketoses and aldoses to 5-hydroxymethylfurfural.

    PubMed

    van Putten, Robert-Jan; Soetedjo, Jenny N M; Pidko, Evgeny A; van der Waal, Jan C; Hensen, Emiel J M; de Jong, Ed; Heeres, Hero J

    2013-09-01

    5-Hydroxymethylfurfural (HMF) is considered an important building block for future bio-based chemicals. Here, we present an experimental study using different ketoses (fructose, sorbose, tagatose) and aldoses (glucose, mannose, galactose) under aqueous acidic conditions (65 g L(-1) substrate, 100-160 °C, 33-300 mM H2 SO4 ) to gain insights into reaction pathways for hexose dehydration to HMF. Both reaction rates and HMF selectivities were significantly higher for ketoses than for aldoses, which is in line with literature. Screening and kinetic experiments showed that the reactivity of the different ketoses is a function of the hydroxyl group orientation at the C3 and C4 positions. These results, in combination with DFT calculations, point to a dehydration mechanism involving cyclic intermediates. For aldoses, no influence of the hydroxyl group orientation was observed, indicating a different rate-determining step. The combination of the knowledge from the literature and the findings in this work indicates that aldoses require an isomerization to ketose prior to dehydration to obtain high HMF yields. PMID:24039165

  8. A novel aldose-aldose oxidoreductase for co-production of D-xylonate and xylitol from D-xylose with Saccharomyces cerevisiae.

    PubMed

    Wiebe, Marilyn G; Nygård, Yvonne; Oja, Merja; Andberg, Martina; Ruohonen, Laura; Koivula, Anu; Penttilä, Merja; Toivari, Mervi

    2015-11-01

    An open reading frame CC1225 from the Caulobacter crescentus CB15 genome sequence belongs to the Gfo/Idh/MocA protein family and has 47 % amino acid sequence identity with the glucose-fructose oxidoreductase from Zymomonas mobilis (Zm GFOR). We expressed the ORF CC1225 in the yeast Saccharomyces cerevisiae and used a yeast strain expressing the gene coding for Zm GFOR as a reference. Cell extracts of strains overexpressing CC1225 (renamed as Cc aaor) showed some Zm GFOR type of activity, producing D-gluconate and D-sorbitol when a mixture of D-glucose and D-fructose was used as substrate. However, the activity in Cc aaor expressing strain was >100-fold lower compared to strains expressing Zm gfor. Interestingly, C. crescentus AAOR was clearly more efficient than the Zm GFOR in converting in vitro a single sugar substrate D-xylose (10 mM) to xylitol without an added cofactor, whereas this type of activity was very low with Zm GFOR. Furthermore, when cultured in the presence of D-xylose, the S. cerevisiae strain expressing Cc aaor produced nearly equal concentrations of D-xylonate and xylitol (12.5 g D-xylonate l(-1) and 11.5 g D-xylitol l(-1) from 26 g D-xylose l(-1)), whereas the control strain and strain expressing Zm gfor produced only D-xylitol (5 g l(-1)). Deletion of the gene encoding the major aldose reductase, Gre3p, did not affect xylitol production in the strain expressing Cc aaor, but decreased xylitol production in the strain expressing Zm gfor. In addition, expression of Cc aaor together with the D-xylonolactone lactonase encoding the gene xylC from C. crescentus slightly increased the final concentration and initial volumetric production rate of both D-xylonate and D-xylitol. These results suggest that C. crescentus AAOR is a novel type of oxidoreductase able to convert the single aldose substrate D-xylose to both its oxidized and reduced product. PMID:26264136

  9. Aldose-ketose interconversion in pyridine in the presence of aluminium oxide.

    PubMed

    Ekeberg, Dag; Morgenlie, Svein; Stenstrøm, Yngve

    2007-10-15

    The reaction rate of the Lobry de Bruyn-Alberda van Ekenstein transformation of aldoses to ketoses in boiling pyridine was strongly increased by the addition of aluminium oxide. In addition to aldose-ketose transformation, 2-epimers of the starting aldoses and 3-epimers of the primarily produced ketoses were formed to some extent, as reported also when these reactions are carried out without aluminium oxide. The relative amounts of the primary ketose and the starting aldose in the reaction mixtures may be explained on the basis of their stability, predicted from reported free energy calculations. Isomerisation of ketoses to aldoses was much slower than the reverse reaction. The relative free energies are also in these cases important, the very stable xylo-2-hexulose gave only 7% and 6% of the aldoses gulose and idose, respectively, after boiling for 7h in pyridine in the presence of aluminium oxide. PMID:17606255

  10. 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

  11. A kinetic estimate of the free aldehyde content of aldoses

    NASA Technical Reports Server (NTRS)

    Dworkin, J. P.; Miller, S. L.; Bada, J. L. (Principal Investigator)

    2000-01-01

    The relative free aldehyde content of eight hexoses and four pentoses has been estimated within about 10% from the rate constants for their reaction with urazole (1,2,4-triazole-3,5-dione). These values of the percent free aldehyde are in agreement with those estimated from CD measurements, but are more accurate. The relative free aldehyde contents for the aldoses were then correlated to various literature NMR measurements to obtain the absolute values. This procedure was also done for three deoxyaldoses, which react much more rapidly than can be accounted for by the free aldehyde content. This difference in reactivity between aldoses and deoxyaldoses is due to the inductive effect of the H versus the OH on C-2'. This may help explain why deoxyribonucleosides hydrolyze much more rapidly than ribonucleosides.

  12. Color polymorphs of aldose reductase inhibitor epalrestat: configurational, conformational and synthon differences.

    PubMed

    Swapna, Battini; Suresh, Kuthuru; Nangia, Ashwini

    2016-03-01

    We report five crystalline polymorphs and an amorphous phase of epalrestat together with configurational isomerism and color behavior: form I (deep red), form II (deep orange), form III (bright yellow), form IV (yellow), and form V (orange) are in the E,Z configuration of the drug, and a Z,Z isomer (bright yellow). Two pathways are identified for polymorph conversion: direct transformation of the E,Z isomer and another pathway via the Z,Z isomer to the E,Z polymorphs. From a pharmaceutical perspective, the stability of polymorphs was established under grinding, solvent slurry and thermal conditions: form I (thermodynamic) > form II > form V > form III > form IV (least stable). PMID:26889760

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

    PubMed Central

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

    1997-01-01

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

  14. Thioredoxin reductase.

    PubMed

    Mustacich, D; Powis, G

    2000-02-15

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

  15. Aldo–Keto Reductase 1B10 and Its Role in Proliferation Capacity of Drug-Resistant Cancers

    PubMed Central

    Matsunaga, Toshiyuki; Wada, Yasuhiro; Endo, Satoshi; Soda, Midori; El-Kabbani, Ossama; Hara, Akira

    2011-01-01

    The human aldo–keto reductase AKR1B10, originally identified as an aldose reductase-like protein and human small intestine aldose reductase, is a cytosolic NADPH-dependent reductase that metabolizes a variety of endogenous compounds, such as aromatic and aliphatic aldehydes and dicarbonyl compounds, and some drug ketones. The enzyme is highly expressed in solid tumors of several tissues including lung and liver, and as such has received considerable interest as a relevant biomarker for the development of those tumors. In addition, AKR1B10 has been recently reported to be significantly up-regulated in some cancer cell lines (medulloblastoma D341 and colon cancer HT29) acquiring resistance toward chemotherapeutic agents (cyclophosphamide and mitomycin c), suggesting the validity of the enzyme as a chemoresistance marker. Although the detailed information on the AKR1B10-mediated mechanisms leading to the drug resistance process is not well understood so far, the enzyme has been proposed to be involved in functional regulations of cell proliferation and metabolism of drugs and endogenous lipids during the development of chemoresistance. This article reviews the current literature focusing mainly on expression profile and roles of AKR1B10 in the drug resistance of cancer cells. Recent developments of AKR1B10 inhibitors and their usefulness in restoring sensitivity to anticancer drugs are also reviewed. PMID:22319498

  16. The C-terminal loop of aldehyde reductase determines the substrate and inhibitor specificity.

    PubMed

    Barski, O A; Gabbay, K H; Bohren, K M

    1996-11-12

    Human aldehyde reductase has a preference for carboxyl group-containing negatively charged substrates. It belongs to the NADPH-dependent aldo-keto reductase superfamily whose members are in part distinguished by unique C-terminal loops. To probe the role of the C-terminal loops in determining substrate specificities in these enzymes, two arginine residues, Arg308 and Arg311, located in the C-terminal loop of aldehyde reductase, and not found in any other C-terminal loop, were replaced with alanine residues. The catalytic efficiency of the R311A mutant for aldehydes containing a carboxyl group is reduced 150-250-fold in comparison to that of the wild-type enzyme, while substrates not containing a negative charge are unaffected. The R311A mutant is also significantly less sensitive to inhibition by dicarboxylic acids, indicating that Arg311 interacts with one of the carboxyl groups. The inhibition pattern indicates that the other carboxyl group binds to the anion binding site formed by Tyr49, His112, and the nicotinamide moiety of NADP+. The correlation between inhibitor potency and the length of the dicarboxylic acid molecules suggests a distance of approximately 10 A between the amino group of Arg311 and the anion binding site in the aldehyde reductase molecule. The sensitivity of inhibition of the R311A mutant by several commercially available aldose reductase inhibitors (ARIs) was variable, with tolrestat and zopolrestat becoming more potent inhibitors (30- and 5-fold, respectively), while others remained the same or became less potent. The catalytic properties, substrate specificity, and susceptibility to inhibition of the R308A mutant remained similar to that of the wild-type enzyme. The data provide direct evidence for C-terminal loop participation in determining substrate and inhibitor specificity of aldo-keto reductases and specifically identifies Arg311 as the basis for the carboxyl-containing substrate preference of aldehyde reductase. PMID:8916913

  17. Quinone Reductase 2 Is a Catechol Quinone Reductase

    SciTech Connect

    Fu, Yue; Buryanovskyy, Leonid; Zhang, Zhongtao

    2008-09-05

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

  18. Molecular cloning of mannose-6-phosphate reductase and its developmental expression in celery.

    PubMed Central

    Everard, J D; Cantini, C; Grumet, R; Plummer, J; Loescher, W H

    1997-01-01

    Compared with other primary photosynthetic products (e.g. sucrose and starch), little is known about sugar alcohol metabolism, its regulation, and the manner in which it is integrated with other pathways. Mannose-6-phosphate reductase (M6PR) is a key enzyme that is involved in mannitol biosynthesis in celery (Apium graveolens L.). The M6PR gene was cloned from a leaf cDNA library, and clonal authenticity was established by assays of M6PR activity, western blots, and comparisons of the deduced amino acid sequence with a celery M6PR tryptic digestion product. Recombinant M6PR, purified from Escherichia coli, had specific activity, molecular mass, and kinetic characteristics indistinguishable from those of authentic celery M6PR. Sequence analyses showed M6PR to be a member of the aldo-keto reductase superfamily, which includes both animal and plant enzymes. The greatest sequence similarity was with aldose-6-phosphate reductase (EC 1.1.1.200), a key enzyme in sorbitol synthesis in Rosaceae. Developmental studies showed M6PR to be limited to green tissues and to be under tight transcriptional regulation during leaf initiation, expansion, and maturation. These data confirmed a close relationship between the development of photosynthetic capacity, mannitol synthesis, and M6PR activity. PMID:9112783

  19. Kinetics and molecular docking studies of an anti-diabetic complication inhibitor fucosterol from edible brown algae Eisenia bicyclis and Ecklonia stolonifera.

    PubMed

    Jung, Hyun Ah; Islam, Md Nurul; Lee, Chan Mi; Oh, Sang Ho; Lee, Sanghyuk; Jung, Jee H; Choi, Jae Sue

    2013-10-25

    In the present study, we investigated the anti-diabetic potential of fucosterol by evaluating the ability of this compound to inhibit rat lens aldose reductase (RLAR), human recombinant aldose reductase (HRAR), protein tyrosine phosphatase 1B (PTP1B), and α-glucosidase. Fucosterol displayed moderate inhibitory activity against RLAR, HRAR, and PTP1B. However, it showed weak or no activity against AGE formation and α-glucosidase. In addition, our kinetic study revealed that fucosterol showed a mixed type inhibition against RLAR and HRAR, while it noncompetitively inhibited PTP1B. Since fucosterol inhibited aldose reductase (AR), it holds great promise for use in the treatment of diabetic complications. Therefore, we predicted the 3D structure of AR in rat and human using the Autodock program to simulate binding between AR and fucosterol and evaluate the binding site-directed inhibition of AR by fucosterol. Results of the docking simulations of fucosterol demonstrated negative binding energies (-8.2 kcal/mol for RLAR and -8.5 kcal/mol for HRAR), which indicated a higher affinity and tighter binding capacity of fucosterol for the active site of the enzyme. In particular, the hydrophobic ring system and the aliphatic side chain of fucosterol were found to be tightly bound in a specificity pocket through apolar amino acid residues on AR, while the anion binding site on AR interacts with the 3-hydroxyl group and the double bond on the side chain of fucosterol. The results of the present study clearly demonstrated the potential of using fucosterol for the management and treatment of diabetes and diabetes-associated complications. PMID:23994501

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

    PubMed

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

    2015-12-25

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

  1. An Expeditious Synthesis of Sialic Acid Derivatives by Copper(I)-Catalyzed Stereodivergent Propargylation of Unprotected Aldoses

    PubMed Central

    2016-01-01

    We developed a copper(I)-catalyzed stereodivergent anomeric propargylation of unprotected aldoses as a facile synthetic pathway to a broad variety of sialic acid derivatives. The soft allenylcopper(I) species, catalytically generated from stable allenylboronic acid pinacolate (2), is unusually inert to protonolysis by the multiple hydroxy groups of the substrates and thereby functions as a carbon nucleophile. The key additive B(OMe)3 facilitated ring-opening of the nonelectrophilic cyclic hemiacetal forms of aldoses to the reactive aldehyde forms. The chirality of the catalyst, and not the internal stereogenic centers of substrates, predominantly controlled the stereochemistry of the propargylation step; i.e., the diastereoselectivity was switched simply by changing the catalyst chirality. This is the first nonenzyme catalyst-controlled stereodivergent C–C bond elongation at the anomeric center of unprotected aldoses, which contain multiple protic functional groups and stereogenic centers. The propargylation products can be expeditiously transformed into naturally occurring and synthetic sialic acid derivatives in a simple three-step sequence. This synthetic method, which requires no protecting groups, can be performed on a gram-scale and thus offers general and practical access to various sialic acid derivatives from unprotected aldoses. PMID:27163022

  2. An Expeditious Synthesis of Sialic Acid Derivatives by Copper(I)-Catalyzed Stereodivergent Propargylation of Unprotected Aldoses.

    PubMed

    Wei, Xiao-Feng; Shimizu, Yohei; Kanai, Motomu

    2016-01-27

    We developed a copper(I)-catalyzed stereodivergent anomeric propargylation of unprotected aldoses as a facile synthetic pathway to a broad variety of sialic acid derivatives. The soft allenylcopper(I) species, catalytically generated from stable allenylboronic acid pinacolate (2), is unusually inert to protonolysis by the multiple hydroxy groups of the substrates and thereby functions as a carbon nucleophile. The key additive B(OMe)3 facilitated ring-opening of the nonelectrophilic cyclic hemiacetal forms of aldoses to the reactive aldehyde forms. The chirality of the catalyst, and not the internal stereogenic centers of substrates, predominantly controlled the stereochemistry of the propargylation step; i.e., the diastereoselectivity was switched simply by changing the catalyst chirality. This is the first nonenzyme catalyst-controlled stereodivergent C-C bond elongation at the anomeric center of unprotected aldoses, which contain multiple protic functional groups and stereogenic centers. The propargylation products can be expeditiously transformed into naturally occurring and synthetic sialic acid derivatives in a simple three-step sequence. This synthetic method, which requires no protecting groups, can be performed on a gram-scale and thus offers general and practical access to various sialic acid derivatives from unprotected aldoses. PMID:27163022

  3. Asymmetric assembly of aldose carbohydrates from formaldehyde and glycolaldehyde by tandem biocatalytic aldol reactions.

    PubMed

    Szekrenyi, Anna; Garrabou, Xavier; Parella, Teodor; Joglar, Jesús; Bujons, Jordi; Clapés, Pere

    2015-09-01

    The preparation of multifunctional chiral molecules can be greatly simplified by adopting a route via the sequential catalytic assembly of achiral building blocks. The catalytic aldol assembly of prebiotic compounds into stereodefined pentoses and hexoses is an as yet unmet challenge. Such a process would be of remarkable synthetic utility and highly significant with regard to the origin of life. Pursuing an expedient enzymatic approach, here we use engineered D-fructose-6-phosphate aldolase from Escherichia coli to prepare a series of three- to six-carbon aldoses by sequential one-pot additions of glycolaldehyde. Notably, the pertinent selection of the aldolase variant provides control of the sugar size. The stereochemical outcome of the addition was also altered to allow the synthesis of L-glucose and related derivatives. Such engineered biocatalysts may offer new routes for the straightforward synthesis of natural molecules and their analogues that circumvent the intricate enzymatic pathways forged by evolution. PMID:26291944

  4. Asymmetric assembly of aldose carbohydrates from formaldehyde and glycolaldehyde by tandem biocatalytic aldol reactions

    NASA Astrophysics Data System (ADS)

    Szekrenyi, Anna; Garrabou, Xavier; Parella, Teodor; Joglar, Jesús; Bujons, Jordi; Clapés, Pere

    2015-09-01

    The preparation of multifunctional chiral molecules can be greatly simplified by adopting a route via the sequential catalytic assembly of achiral building blocks. The catalytic aldol assembly of prebiotic compounds into stereodefined pentoses and hexoses is an as yet unmet challenge. Such a process would be of remarkable synthetic utility and highly significant with regard to the origin of life. Pursuing an expedient enzymatic approach, here we use engineered D-fructose-6-phosphate aldolase from Escherichia coli to prepare a series of three- to six-carbon aldoses by sequential one-pot additions of glycolaldehyde. Notably, the pertinent selection of the aldolase variant provides control of the sugar size. The stereochemical outcome of the addition was also altered to allow the synthesis of L-glucose and related derivatives. Such engineered biocatalysts may offer new routes for the straightforward synthesis of natural molecules and their analogues that circumvent the intricate enzymatic pathways forged by evolution.

  5. 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

  6. Aldo-Keto Reductases 1B in Endocrinology and Metabolism

    PubMed Central

    Pastel, Emilie; Pointud, Jean-Christophe; Volat, Fanny; Martinez, Antoine; Lefrançois-Martinez, Anne-Marie

    2012-01-01

    The aldose reductase (AR; human AKR1B1/mouse Akr1b3) has been the focus of many research because of its role in diabetic complications. The starting point of these alterations is the massive entry of glucose in polyol pathway where it is converted into sorbitol by this enzyme. However, the issue of AR function in non-diabetic condition remains unresolved. AR-like enzymes (AKR1B10, Akr1b7, and Akr1b8) are highly related isoforms often co-expressed with bona fide AR, making functional analysis of one or the other isoform a challenging task. AKR1B/Akr1b members share at least 65% protein identity and the general ability to reduce many redundant substrates such as aldehydes provided from lipid peroxidation, steroids and their by-products, and xenobiotics in vitro. Based on these properties, AKR1B/Akr1b are generally considered as detoxifying enzymes. Considering that divergences should be more informative than similarities to help understanding their physiological functions, we chose to review specific hallmarks of each human/mouse isoforms by focusing on tissue distribution and specific mechanisms of gene regulation. Indeed, although the AR shows ubiquitous expression, AR-like proteins exhibit tissue-specific patterns of expression. We focused on three organs where certain isoforms are enriched, the adrenal gland, enterohepatic, and adipose tissues and tried to connect recent enzymatic and regulation data with endocrine and metabolic functions of these organs. We presented recent mouse models showing unsuspected physiological functions in the regulation of glucido-lipidic metabolism and adipose tissue homeostasis. Beyond the widely accepted idea that AKR1B/Akr1b are detoxification enzymes, these recent reports provide growing evidences that they are able to modify or generate signal molecules. This conceptually shifts this class of enzymes from unenviable status of scavenger to upper class of messengers. PMID:22876234

  7. Molecular distribution and degradation status of combined aldoses in sinking particulate organic matter

    NASA Astrophysics Data System (ADS)

    Panagiotopoulos, C.; Sempéré, R.

    2003-04-01

    Particulate samples were collected by using floating sediment traps (50--300 m) and in situ pumps (30 and 200 m) in the Southern Indian Ocean (Polar Front Zone (PFZ) and Sub-Tropical Zone (STZ)), Mediterranean Sea (Ligurian and Ionian Seas) and Atlantic Ocean (Upwelling (UPW) of Agadir-Morocco). They were studied for monosaccharide composition after acid hydrolysis (HCl 0.09 M, 20 h, 100^oC) by using High Performance Anion Exchange Chromatography followed by Pulsed Amperometric Detection (HPAEC-PAD). Our results indicated that higher PCHO yields (calculated as PCHO-C/POC ratios) were associated to higher C:N ratios (Med. Sea sample, PCHO yields = 12.7 ± 7.7%; C:N ratios = 8.3 ± 1.6; n = 12) whether the opposite trend was found for Southern Ocean samples (PCHO yields = 3.3 ± 0.75%; C:N ratios = 5.7 ± 0.59, n = 5) indicating significant variability in the sugar content of particles which might be due to the degradation degree of the particles as well as to the initial chemical composition of plankton. Alternatively, other processes such as high production of extracellular polysaccharides (type transparent exopolymer polysaccharides (TEP)) due to phosphorus limitation of some phytoplanktonic species may increase the sugar content in Mediterranean particles and the C/N ratio. In any case, glucose appeared to be the most abundant monosaccharide in Mediterranean Sea or UPW samples (range 23--59 wt% of the total aldoses) whereas ribose (17--39 wt%) and galactose (range 10--28 wt%) were the predominant aldoses in Southern Indian Ocean. These sugars (glucose + ribose) exhibited a strong negative relationship with C:N (r = -0.53, p >0.01; n = 30) in sediment traps (data from this study) and sediment (data from literature) particulate material which further indicates that these two monosaccharides are selectively extracted from the carbohydrate pool in sediment. In vitro biodegradation experiments performed with large particles (>60 μm) sampled using in situ pumps in

  8. Bioactive constituents of Clausena lansium and a method for discrimination of aldose enantiomers.

    PubMed

    Shen, De-Yang; Chao, Chih-Hua; Chan, Hsiu-Hui; Huang, Guan-Jhong; Hwang, Tsong-Long; Lai, Chin-Yu; Lee, Kuo-Hsiung; Thang, Tran Dinh; Wu, Tian-Shung

    2012-10-01

    Glycosides, clausenosides A and B, and carbazole alkaloids, clausenaline A, claulamine A, and claulamine B, together with 50 known compounds, were isolated from the stems of Clausena lansium. Their structures were determined by means of spectroscopic methods, including that of CD and 1D/2D NMR analysis. Claulamine A has a 1-oxygenated carbazole skeleton with a rare 2,3-lactone ring, and claulamine B represents an hitherto unknown acetal carbazole alkaloid. Thirty-one of the isolated known compounds were evaluated in various assays for anti-inflammatory activity. Among them, imperatorin, isoheraclenin, and osthol exhibited selective and potent inhibition of formyl-l-methionyl-l-leucyl-l-phenylalanine/cytochalasin B (fMLP/CB)-induced superoxide anion generation, and lansiumarin C also decreased nitric oxide (NO) and tumor necrosis factor-α (TNF-α) production in lipopolysaccharide (LPS)-induced macrophages. In addition, a modified HPLC method of pre-column derivatization was developed that is more practical for simultaneous analysis of aldose enantiomers as compared to the literature method. The absolute configurations of the sugar moieties in clausenosides A and B were determined with this modified method. PMID:22818357

  9. Isolated menthone reductase and nucleic acid molecules encoding same

    DOEpatents

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

    2013-04-23

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

  10. Zeatin reductase in Phaseolus embryos

    SciTech Connect

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

    1989-04-01

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

  11. Genetics Home Reference: 5-alpha reductase deficiency

    MedlinePlus

    ... gene provides instructions for making an enzyme called steroid 5-alpha reductase 2. This enzyme is involved ... external genitalia. Mutations in the SRD5A2 gene prevent steroid 5-alpha reductase 2 from effectively converting testosterone ...

  12. Identification, cloning and regulation of cDNA encoding aldo-keto reductase 1B7 in the adrenal gland of two Saharan rodents Meriones libycus (Libyan jird) and Gerbillus gerbillus (gerbil).

    PubMed

    Mataoui-Mazari, Houria; Amirat, Zaïna; Khammar, Farida; Martinez, Antoine

    2011-12-01

    Aldo-Keto Reductase 1B7 (AKR1B7) is a mouse aldose reductase-like protein with two major sites of expression, the vas deferens and the adrenal cortex. In the adrenal cortex, Akr1b7 is an adrenocorticotropin (ACTH)-responsive-gene whose product scavenges harmful byproducts of steroidogenesis and limits stress response through the biosynthesis of prostaglandin F2α. The purpose of the present study was to explore the possible expression of AKR1B7 in the adrenal glands of two saharan rodents, Libyan jird and Lesser Egyptian gerbil. Western blot analyses demonstrated that a protein related to murine/rat AKR1B7 was highly expressed in adrenals and absent from vas deferens of both saharan species. Based on conserved sequences between mouse and rat, full length cDNA were cloned and sequenced in both species while hormonal regulation and tissue localization were explored in Libyan jird. Both cDNA encoded the expected 316 amino acids protein typical of AKR1B subfamily and contained the highly conserved catalytic tetrad consisting in Asp-44, Tyr-49, Lys-78 and His-111 residues. The deduced proteins shared higher identities with aldose reductase-like, i.e. AKR1B7 (86-94%), AKR1B8 and AKR1B10 (83-86%) than with aldose reductase group, i.e. AKR1B1 and AKR1B3 (70%). Phylogenetic analysis showed that the Libyan jird and gerbil enzymes were more closely related to murine and rat AKR1B7 than to the other AKR1B members. Northern blot analyses of total RNA from Libyan jird adrenals showed a single mRNA transcript of 1.4 kb whose expression was dependent on circulating ACTH levels. In conclusion, we demonstrate here that adrenal glands of Libyan jird and gerbil express both an ortholog of the murine/rat Akr1b7 gene and that ACTH-responsiveness is at least conserved in Libyan jird. PMID:21963864

  13. Comparison of the xylose reductase-xylitol dehydrogenase and the xylose isomerase pathways for xylose fermentation by recombinant Saccharomyces cerevisiae

    PubMed Central

    Karhumaa, Kaisa; Sanchez, Rosa Garcia; Hahn-Hägerdal, Bärbel; Gorwa-Grauslund, Marie-F

    2007-01-01

    Background Two heterologous pathways have been used to construct recombinant xylose-fermenting Saccharomyces cerevisiae strains: i) the xylose reductase (XR) and xylitol dehydrogenase (XDH) pathway and ii) the xylose isomerase (XI) pathway. In the present study, the Pichia stipitis XR-XDH pathway and the Piromyces XI pathway were compared in an isogenic strain background, using a laboratory host strain with genetic modifications known to improve xylose fermentation (overexpressed xylulokinase, overexpressed non-oxidative pentose phosphate pathway and deletion of the aldose reductase gene GRE3). The two isogenic strains and the industrial xylose-fermenting strain TMB 3400 were studied regarding their xylose fermentation capacity in defined mineral medium and in undetoxified lignocellulosic hydrolysate. Results In defined mineral medium, the xylose consumption rate, the specific ethanol productivity, and the final ethanol concentration were significantly higher in the XR- and XDH-carrying strain, whereas the highest ethanol yield was achieved with the strain carrying XI. While the laboratory strains only fermented a minor fraction of glucose in the undetoxified lignocellulose hydrolysate, the industrial strain TMB 3400 fermented nearly all the sugar available. Xylitol was formed by the XR-XDH-carrying strains only in mineral medium, whereas in lignocellulose hydrolysate no xylitol formation was detected. Conclusion Despite by-product formation, the XR-XDH xylose utilization pathway resulted in faster ethanol production than using the best presently reported XI pathway in the strain background investigated. The need for robust industrial yeast strains for fermentation of undetoxified spruce hydrolysates was also confirmed. PMID:17280608

  14. 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

  15. Prostaglandin (PG) F2 Alpha Synthesis in Human Subcutaneous and Omental Adipose Tissue: Modulation by Inflammatory Cytokines and Role of the Human Aldose Reductase AKR1B1

    PubMed Central

    Michaud, Andréanne; Lacroix-Pépin, Nicolas; Pelletier, Mélissa; Veilleux, Alain; Noël, Suzanne; Bouchard, Céline; Marceau, Picard; Fortier, Michel A.; Tchernof, André

    2014-01-01

    Introduction PGF2α may be involved in the regulation of adipose tissue function. Objectives 1) To examine PGF2α release by primary preadipocytes, mature adipocytes and whole tissue explants from the subcutaneous and omental fat compartments; 2) To assess which PGF synthase is the most relevant in human adipose tissue. Methods Fat samples were obtained by surgery in women. PGF2α release by preadipocytes, adipocytes and explants under stimulation by TNF-α, IL-1β or both was measured. Messenger RNA expression levels of AKR1B1 and AKR1C3 were measured by RT-PCR in whole adipose tissue and cytokine-treated preadipocytes. The effect of AKR1B1 inhibitor ponalrestat on PGF2α synthesis was investigated. Results PGF2α release was significantly induced in response to cytokines compared to control in omental (p = 0.01) and to a lesser extent in subcutaneous preadipocytes (p = 0.02). Messenger RNA of COX-2 was significantly higher in omental compared to subcutaneous preadipocytes in response to combined TNF-α and IL-1β (p = 0.01). Inflammatory cytokines increased AKR1B1 mRNA expression and protein levels (p≤0.05), but failed to increase expression levels of AKR1C3 in cultured preadipocytes. Accordingly, ponalrestat blunted PGF2α synthesis by preadipocytes in basal and stimulated conditions (p≤0.05). Women with the highest PGF2α release by omental adipocytes had a higher BMI (p = 0.05), waist circumference (p≤0.05) and HOMAir index (p≤0.005) as well as higher mRNA expression of AKR1B1 in omental (p<0.10) and subcutaneous (p≤0.05) adipose tissue compared to women with low omental adipocytes PGF2α release. Positive correlations were observed between mRNA expression of AKR1B1 in both compartments and BMI, waist circumference as well as HOMAir index (p≤0.05 for all). Conclusion PGF2α release by omental mature adipocytes is increased in abdominally obese women. Moreover, COX-2 expression and PGF2α release is particularly responsive to inflammatory stimulation in omental preadipocytes. Yet, blockade of PGF synthase AKR1B1 inhibits most of the PGF2α release. PMID:24663124

  16. LC-MS-MS Characterization of Forced Degradation Products of Fidarestat, a Novel Aldose Reductase Inhibitor: Development and Validation of a Stability-Indicating RP-HPLC Method.

    PubMed

    Talluri, M V N Kumar; Khatoon, Lubna; Kalariya, Pradipbhai D; Chavan, Balasaheb B; Ragampeta, Srinivas

    2015-10-01

    An accurate, precise, robust and selective stability-indicating liquid chromatographic (LC) method has been developed for the monitoring of fidarestat in the presence of its forced degradants. The drug was subjected to hydrolysis (acid, alkali and neutral degradation), oxidation, photolysis and thermal stress conditions. The drug degraded significantly under hydrolytic (basic, acidic and neutral) and oxidative stress conditions, whereas it was found to be stable in photolytic and thermal conditions. The chromatographic separation was achieved on a Grace C18, (250 mm × 4.6 mm × 5 μm) column using gradient mobile phase system consisting of 10 mM of ammonium acetate buffer at pH 4 and acetonitrile at a flow rate of 1 mL/min with UV detection at 283 nm. The developed method was extended to liquid chromatography quadrupole time-of-flight tandem mass spectrometry (LC-QTOF-MS-MS) for characterization of all the degradation products. A total of five new degradation products were identified and characterized by LC-QTOF-MS-MS. The developed LC method was validated as per ICH guideline Q2 (R1). The proposed method was found to be successively applied for the quality control of fidarestat in bulk drug analysis. PMID:26014964

  17. 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. PMID:12626517

  18. Fatty acyl-CoA reductase

    SciTech Connect

    Reiser, Steven E.; Somerville, Chris R.

    1998-12-01

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

  19. Dihydropteridine reductase from Escherichia coli.

    PubMed Central

    Vasudevan, S G; Shaw, D C; Armarego, W L

    1988-01-01

    A dihydropteridine reductase from Escherichia coli was purified to apparent homogeneity. It is a dimeric enzyme with identical subunits (Mr 27000) and a free N-terminal group. It can use NADH (Vmax./Km 3.36 s-1) and NADPH (Vmax./Km 1.07 s-1) when 6-methyldihydro-(6H)-pterin is the second substrate, as well as quinonoid dihydro-(6H)-biopterin (Vmax./Km 0.69 s-1), dihydro-(6H)-neopterin (Vmax./Km 0.58 s-1), dihydro-(6H)-monapterin 0.66 s-1), 6-methyldihydro-(6H)-pterin and cis-6,7-dimethyldihydro-(6H)-pterin (Vmax./Km 0.66 s-1) when NADH is the second substrate. The pure reductase has a yellow colour and contains bound FAD. The enzyme also has pterin-independent NADH and NADPH oxidoreductase activities when potassium ferricyanide is the electron acceptor. Images Fig. 2. PMID:3060113

  20. An electrogenic nitric oxide reductase.

    PubMed

    Al-Attar, Sinan; de Vries, Simon

    2015-07-22

    Nitric oxide reductases (Nors) are members of the heme-copper oxidase superfamily that reduce nitric oxide (NO) to nitrous oxide (N₂O). In contrast to the proton-pumping cytochrome oxidases, Nors studied so far have neither been implicated in proton pumping nor have they been experimentally established as electrogenic. The copper-A-dependent Nor from Bacillus azotoformans uses cytochrome c₅₅₁ as electron donor but lacks menaquinol activity, in contrast to our earlier report (Suharti et al., 2001). Employing reduced phenazine ethosulfate (PESH) as electron donor, the main NO reduction pathway catalyzed by Cu(A)Nor reconstituted in liposomes involves transmembrane cycling of the PES radical. We show that Cu(A)Nor reconstituted in liposomes generates a proton electrochemical gradient across the membrane similar in magnitude to cytochrome aa₃, highlighting that bacilli using Cu(A)Nor can exploit NO reduction for increased cellular ATP production compared to organisms using cNor. PMID:26149211

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

    SciTech Connect

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

    1986-04-29

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

  2. Genetics Home Reference: sepiapterin reductase deficiency

    MedlinePlus

    ... reductase enzyme. This enzyme is involved in the production of a molecule called tetrahydrobiopterin (also known as ... is responsible for the last step in the production of tetrahydrobiopterin. Tetrahydrobiopterin helps process several building blocks ...

  3. A dissimilatory nitrite reductase in Paracoccus halodenitrificans

    NASA Technical Reports Server (NTRS)

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

    1984-01-01

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

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

    PubMed Central

    2010-01-01

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

  5. 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

  6. Characterization of thyroidal glutathione reductase

    SciTech Connect

    Raasch, R.J.

    1989-01-01

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

  7. Promising anti-diabetic potential of capillin and capillinol isolated from Artemisia capillaris.

    PubMed

    Islam, Md Nurul; Choi, Ran Joo; Jung, Hyun Ah; Oh, Sang Ho; Choi, Jae Sue

    2016-03-01

    Caffeoylquinic acids, flavonoids, and coumarins isolated from Artemisia capillaris have recently emerged as therapeutic candidates for diabetes and diabetic complications; however, there have been very few studies of the anti-diabetic potential of polyacetylenes. In the present study, we investigated the anti-diabetic potential of two polyacetylenes isolated from A. capillaris, namely capillin and capillinol by investigating their ability to inhibit α-glucosidase, protein tyrosine phosphatase 1B (PTP1B), and rat lens aldose reductase (RLAR). Capillin displayed potent inhibitory activity against α-glucosidase, PTP1B, and RLAR, while capillinol showed moderate inhibitory activity against α-glucosidase and PTP1B at the concentrations tested. In addition, a kinetic study revealed that capillin inhibited α-glucosidase and RLAR in a noncompetitive manner, while inhibited PTP1B in a mixed-type manner. Capillinol inhibited α-glucosidase and PTP1B in a mixed-type manner. Docking simulations of these compounds demonstrated negative binding energies and close proximity to residues in the binding pocket of PTP1B, indicating that these polyacetylenes have a high affinity and tight binding capacity for the active site of the enzyme. Furthermore, capillin dose-dependently inhibited peroxynitrite (ONOO(-))-mediated tyrosine nitration. The results clearly demonstrate the promising potential of capillin and capillinol as therapeutic interventions for the management of diabetes as well as diabetes-associated complications. PMID:26832324

  8. Alteration of organic matter during infaunal polychaete gut passage and links to sediment organic geochemistry. Part II: Fatty acids and aldoses

    NASA Astrophysics Data System (ADS)

    Woulds, Clare; Middelburg, Jack J.; Cowie, Greg L.

    2014-07-01

    The activities of sediment-dwelling fauna are known to influence the rates of and pathways through which organic matter is cycled in marine sediments, and thus to influence eventual organic carbon burial or decay. However, due to methodological constraints, the role of faunal gut passage in determining the subsequent composition and thus degradability of organic matter is relatively little studied. Previous studies of organic matter digestion by benthic fauna have been unable to detect uptake and retention of specific biochemicals in faunal tissues, and have been of durations too short to fit digestion into the context of longer-term sedimentary degradation processes. Therefore this study aimed to investigate the aldose and fatty acid compositional alterations occurring to organic matter during gut passage by the abundant and ubiquitous polychaetes Hediste diversicolor and Arenicola marina, and to link these to longer-term changes typically observed during organic matter decay. This aim was approached through microcosm experiments in which selected polychaetes were fed with 13C-labelled algal detritus, and organisms, sediments, and faecal pellets were sampled at three timepoints over ∼6 weeks. Samples were analysed for their 13C-labelled aldose and fatty acid contents using GC-MS and GC-IRMS. Compound-selective net accumulation of biochemicals in polychaete tissues was observed for both aldoses and fatty acids, and the patterns of this were taxon-specific. The dominant patterns included an overall loss of glucose and polyunsaturated fatty acids; and preferential preservation or production of arabinose, microbial compounds (rhamnose, fucose and microbial fatty acids), and animal-synthesised fatty acids. These patterns may have been driven by fatty acid essentiality, preferential metabolism of glucose, and A. marina grazing on bacteria. Fatty acid suites in sediments from faunated microcosms showed greater proportions of saturated fatty acids and bacterial markers

  9. Structural and mechanistic insights on nitrate reductases.

    PubMed

    Coelho, Catarina; Romão, Maria João

    2015-12-01

    Nitrate reductases (NR) belong to the DMSO reductase family of Mo-containing enzymes and perform key roles in the metabolism of the nitrogen cycle, reducing nitrate to nitrite. Due to variable cell location, structure and function, they have been divided into periplasmic (Nap), cytoplasmic, and membrane-bound (Nar) nitrate reductases. The first crystal structure obtained for a NR was that of the monomeric NapA from Desulfovibrio desulfuricans in 1999. Since then several new crystal structures were solved providing novel insights that led to the revision of the commonly accepted reaction mechanism for periplasmic nitrate reductases. The two crystal structures available for the NarGHI protein are from the same organism (Escherichia coli) and the combination with electrochemical and spectroscopic studies also lead to the proposal of a reaction mechanism for this group of enzymes. Here we present an overview on the current advances in structural and functional aspects of bacterial nitrate reductases, focusing on the mechanistic implications drawn from the crystallographic data. PMID:26362109

  10. 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.

  11. Respiratory arsenate reductase as a bidirectional enzyme

    SciTech Connect

    Richey, Christine; Chovanec, Peter; Department of Chemistry and Biochemistry, Duquesne University, Pittsburgh, PA 15282 ; Hoeft, Shelley E.; Oremland, Ronald S.; Basu, Partha; Stolz, John F.

    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 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.

  12. Phylogenomics of Mycobacterium Nitrate Reductase Operon.

    PubMed

    Huang, Qinqin; Abdalla, Abualgasim Elgaili; Xie, Jianping

    2015-07-01

    NarGHJI operon encodes a nitrate reductase that can reduce nitrate to nitrite. This process enhances bacterial survival by nitrate respiration under anaerobic conditions. NarGHJI operon exists in many bacteria, especially saprophytic bacteria living in soil which play a key role in the nitrogen cycle. Most actinomycetes, including Mycobacterium tuberculosis, possess NarGHJI operons. M. tuberculosis is a facultative intracellular pathogen that expands in macrophages and has the ability to persist in a non-replicative form in granuloma lifelong. Nitrogen and nitrogen compounds play crucial roles in the struggle between M. tuberculosis and host. M. tuberculosis can use nitrate as a final electron acceptor under anaerobic conditions to enhance its survival. In this article, we reviewed the mechanisms regulating nitrate reductase expression and affecting its activity. Potential genes involved in regulating the nitrate reductase expression in M. tuberculosis were identified. The conserved NarG might be an alternative mycobacterium taxonomic marker. PMID:25980349

  13. IN VITRO INHIBITION OF GLUTATHIONE REDUCTASE BY ARSENOTRI-GLUTATHIONE

    EPA Science Inventory

    Arsenotriglutathione, a product of the reduction of arsenate and the complexation of arsenite by glutathione, is a mixed type inhibitor of the reduction of glutathione disulfide by purified yeast glutathione reductase or the glutathione reductase activity in rabbit erythrocyte ly...

  14. Evaluation of nitrate reductase activity in Rhizobium japonicum

    SciTech Connect

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

    1983-08-01

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

  15. Post-translational Regulation of Nitrate Reductase

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  16. Promiscuity and diversity in 3-ketosteroid reductases.

    PubMed

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

    2015-07-01

    Many steroid hormones contain a Δ(4)-3-ketosteroid functionality that undergoes sequential reduction by 5α- or 5β- steroid reductases to produce 5α- or 5β-dihydrosteroids; and a subsequent 3-keto-reduction to produce a series of isomeric tetrahydrosteroids. Apart from steroid 5α-reductase all the remaining enzymes involved in the two step reduction process in humans belong to the aldo-keto reductase (AKR) superfamily. The enzymes involved in 3-ketosteroid reduction are AKR1C1-AKR1C4. These enzymes are promiscuous and also catalyze 20-keto- and 17-keto-steroid reduction. Interest in these reactions exist since they regulate steroid hormone metabolism in the liver, and in steroid target tissues, they may regulate steroid hormone receptor occupancy. In addition many of the dihydrosteroids are not biologically inert. The same enzymes are also involved in the metabolism of synthetic steroids e.g., hormone replacement therapeutics, contraceptive agents and inhaled glucocorticoids, and may regulate drug efficacy at their cognate receptors. This article reviews these reactions and the structural basis for substrate diversity in AKR1C1-AKR1C4, ketosteroid reductases. This article is part of a Special Issue entitled 'Steroid/Sterol signaling'. PMID:25500069

  17. Promiscuity and diversity in 3-ketosteroid reductases

    PubMed Central

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

    2014-01-01

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

  18. Ferrisiderophore reductase activity in Agrobacterium tumefaciens.

    PubMed Central

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

    1982-01-01

    Reduction of the iron in ferriagrobactin by the cytoplasmic fraction of Agrobacterium tumefaciens strictly required NaDH as the reductant. Addition of flavin mononucleotide and anaerobic conditions were necessary for the reaction; when added with flavin mononucleotide, magnesium was stimulatory. This ferrisiderophore reductase activity may be a part of the iron assimilation process in A. tumefaciens. PMID:7056702

  19. Methylenetetrahydrofolate reductase deficiency: importance of early diagnosis.

    PubMed

    Fattal-Valevski, A; Bassan, H; Korman, S H; Lerman-Sagie, T; Gutman, A; Harel, S

    2000-08-01

    Methylenetetrahydrofolate reductase deficiency is the most common inborn error of folate metabolism and should be suspected when homocystinuria is combined with hypomethioninemia. The main clinical findings are neurologic signs such as severe developmental delay, marked hypotonia, seizures, microcephaly, apnea, and coma. Most patients present in early life. The infantile form is severe, with rapid deterioration leading to death usually within 1 year. Treatment with betaine has been shown to be efficient in lowering homocysteine concentrations and returning methionine to normal, but the clinical response is variable. We report two brothers with methylenetetrahydrofolate reductase deficiency: the first was undiagnosed and died at 8 months of age from neurologic deterioration and apnea, while his brother, who was treated with betaine from the age of 4 months, is now 3 years old and has developmental delay. PMID:10961793

  20. Characterization of erythrose reductases from filamentous fungi.

    PubMed

    Jovanović, Birgit; Mach, Robert L; Mach-Aigner, Astrid R

    2013-01-01

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

  1. Role of the Dinitrogenase Reductase Arginine 101 Residue in Dinitrogenase Reductase ADP-Ribosyltransferase Binding, NAD Binding, and Cleavage

    PubMed Central

    Ma, Yan; Ludden, Paul W.

    2001-01-01

    Dinitrogenase reductase is posttranslationally regulated by dinitrogenase reductase ADP-ribosyltransferase (DRAT) via ADP-ribosylation of the arginine 101 residue in some bacteria. Rhodospirillum rubrum strains in which the arginine 101 of dinitrogenase reductase was replaced by tyrosine, phenylalanine, or leucine were constructed by site-directed mutagenesis of the nifH gene. The strain containing the R101F form of dinitrogenase reductase retains 91%, the strain containing the R101Y form retains 72%, and the strain containing the R101L form retains only 28% of in vivo nitrogenase activity of the strain containing the dinitrogenase reductase with arginine at position 101. In vivo acetylene reduction assays, immunoblotting with anti-dinitrogenase reductase antibody, and [adenylate-32P]NAD labeling experiments showed that no switch-off of nitrogenase activity occurred in any of the three mutants and no ADP-ribosylation of altered dinitrogenase reductases occurred either in vivo or in vitro. Altered dinitrogenase reductases from strains UR629 (R101Y) and UR630 (R101F) were purified to homogeneity. The R101F and R101Y forms of dinitrogenase reductase were able to form a complex with DRAT that could be chemically cross-linked by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. The R101F form of dinitrogenase reductase and DRAT together were not able to cleave NAD. This suggests that arginine 101 is not critical for the binding of DRAT to dinitrogenase reductase but that the availability of arginine 101 is important for NAD cleavage. Both DRAT and dinitrogenase reductase can be labeled by [carbonyl-14C]NAD individually upon UV irradiation, but most 14C label is incorporated into DRAT when both proteins are present. The ability of R101F dinitrogenase reductase to be labeled by [carbonyl-14C]NAD suggested that Arg 101 is not absolutely required for NAD binding. PMID:11114923

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

    SciTech Connect

    Iyanagi, Takashi . E-mail: iyanagi@spring8.or.jp

    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 can 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.

  3. Reduction of tetrathionate by mammalian thioredoxin reductase

    PubMed Central

    Narayan, Vivek; Kudva, Avinash K.; Prabhu, K. Sandeep

    2016-01-01

    Tetrathionate, a polythionate oxidation product of microbial hydrogen sulfide and reactive oxygen species from immune cells in the gut, serves as a terminal electron acceptor to confer growth advantage for Salmonella and other enterobacteria. Here we show that the rat liver selenoen-zyme thioredoxin reductase (Txnrd1; TR1) efficiently reduces tetrathionate in vitro. Furthermore, lysates of selenium-supplemented murine macrophages also displayed activity towards tetrathionate, while cells lacking TR1 were unable to reduce tetrathionate. These studies suggest that upregulation of TR1 expression, via selenium supplementation, may modulate the gut microbiome, particularly during inflammation, by regulating the levels of tetrathionate. PMID:26252619

  4. Biliverdin reductase: a target for cancer therapy?

    PubMed Central

    Gibbs, Peter E. M.; Miralem, Tihomir; Maines, Mahin D.

    2015-01-01

    Biliverdin reductase (BVR) is a multifunctional protein that is the primary source of the potent antioxidant, bilirubin. BVR regulates activities/functions in the insulin/IGF-1/IRK/PI3K/MAPK pathways. Activation of certain kinases in these pathways is/are hallmark(s) of cancerous cells. The protein is a scaffold/bridge and intracellular transporter of kinases that regulate growth and proliferation of cells, including PKCs, ERK and Akt, and their targets including NF-κB, Elk1, HO-1, and iNOS. The scaffold and transport functions enable activated BVR to relocate from the cytosol to the nucleus or to the plasma membrane, depending on the activating stimulus. This enables the reductase to function in diverse signaling pathways. And, its expression at the transcript and protein levels are increased in human tumors and the infiltrating T-cells, monocytes and circulating lymphocytes, as well as the circulating and infiltrating macrophages. These functions suggest that the cytoprotective role of BVR may be permissive for cancer/tumor growth. In this review, we summarize the recent developments that define the pro-growth activities of BVR, particularly with respect to its input into the MAPK signaling pathway and present evidence that BVR-based peptides inhibit activation of protein kinases, including MEK, PKCδ, and ERK as well as downstream targets including Elk1 and iNOS, and thus offers a credible novel approach to reduce cancer cell proliferation. PMID:26089799

  5. Flavodiiron Oxygen Reductase from Entamoeba histolytica

    PubMed Central

    Gonçalves, Vera L.; Vicente, João B.; Pinto, Liliana; Romão, Célia V.; Frazão, Carlos; Sarti, Paolo; Giuffrè, Alessandro; Teixeira, Miguel

    2014-01-01

    Flavodiiron proteins (FDPs) are a family of enzymes endowed with bona fide oxygen- and/or nitric-oxide reductase activity, although their substrate specificity determinants remain elusive. After a comprehensive comparison of available three-dimensional structures, particularly of FDPs with a clear preference toward either O2 or NO, two main differences were identified near the diiron active site, which led to the construction of site-directed mutants of Tyr271 and Lys53 in the oxygen reducing Entamoeba histolytica EhFdp1. The biochemical and biophysical properties of these mutants were studied by UV-visible and electron paramagnetic resonance (EPR) spectroscopies coupled to potentiometry. Their reactivity with O2 and NO was analyzed by stopped-flow absorption spectroscopy and amperometric methods. These mutations, whereas keeping the overall properties of the redox cofactors, resulted in increased NO reductase activity and faster inactivation of the enzyme in the reaction with O2, pointing to a role of the mutated residues in substrate selectivity. PMID:25151360

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

    SciTech Connect

    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 be used independently for detecting nitrite residues/contamination in environmental/food samples. 10 refs., 2 figs.

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

    PubMed

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

    2014-01-01

    Anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR) play an important role in the monomeric units biosynthesis of proanthocyanidins (PAs) such as catechin and epicatechin in several plants. The aim of this study was to clone ANR and LAR genes involved in PAs biosynthesis and examine the expression of these two genes in different organs under different growth conditions in two tartary buckwheat cultivars, Hokkai T8 and T10. Gene expression was carried out by quantitative real-time RT-PCR, and catechin and epicatechin content was analyzed by high performance liquid chromatography. The expression pattern of ANR and LAR did not match the accumulation pattern of PAs in different organs of two cultivars. Epicatechin content was the highest in the flowers of both cultivars and it was affected by light in only Hokkai T8 sprouts. ANR and LAR levels in tartary buckwheat might be regulated by different mechanisms for catechin and epicatechin biosynthesis under light and dark conditions. PMID:24605062

  8. 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

  9. Chaperone properties of Escherichia coli thioredoxin and thioredoxin reductase.

    PubMed Central

    Kern, Renée; Malki, Abderrahim; Holmgren, Arne; Richarme, Gilbert

    2003-01-01

    Thioredoxin, thioredoxin reductase and NADPH form the thioredoxin system and are the major cellular protein disulphide reductase. We report here that Escherichia coli thioredoxin and thioredoxin reductase interact with unfolded and denatured proteins, in a manner similar to that of molecular chaperones that are involved in protein folding and protein renaturation after stress. Thioredoxin and/or thioredoxin reductase promote the functional folding of citrate synthase and alpha-glucosidase after urea denaturation. They also promote the functional folding of the bacterial galactose receptor, a protein without any cysteines. Furthermore, redox cycling of thioredoxin/thioredoxin reductase in the presence of NADPH and cystine stimulates the renaturation of the galactose receptor, suggesting that the thioredoxin system functions like a redox-powered chaperone machine. Thioredoxin reductase prevents the aggregation of citrate synthase under heat-shock conditions. It forms complexes that are more stable than those formed by thioredoxin with several unfolded proteins such as reduced carboxymethyl alpha-lactalbumin and unfolded bovine pancreatic trypsin inhibitor. These results suggest that the thioredoxin system, in addition to its protein disulphide isomerase activity possesses chaperone-like properties, and that its thioredoxin reductase component plays a major role in this function. PMID:12549977

  10. Ribonucleotide reductase metallocofactor: assembly, maintenance and inhibition

    PubMed Central

    ZHANG, Caiguo; LIU, Guoqi; HUANG, Mingxia

    2014-01-01

    Ribonucleotide reductase (RNR) supplies cellular deoxyribonucleotide triphosphates (dNTP) pools by converting ribonucleotides to the corresponding deoxy forms using radical-based chemistry. Eukaryotic RNR comprises α and β subunits: α contains the catalytic and allosteric sites; β houses a diferric-tyrosyl radical cofactor (FeIII2-Y•) that is required to initiates nucleotide reduction in α. Cells have evolved multi-layered mechanisms to regulate RNR level and activity in order to maintain the adequate sizes and ratios of their dNTP pools to ensure high-fidelity DNA replication and repair. The central role of RNR in nucleotide metabolism also makes it a proven target of chemotherapeutics. In this review, we discuss recent progress in understanding the function and regulation of eukaryotic RNRs, with a focus on studies revealing the cellular machineries involved in RNR metallocofactor biosynthesis and its implication in RNR-targeting therapeutics. PMID:24899886

  11. Dynamics of trimethoprim bound to dihydrofolate reductase

    SciTech Connect

    Searle, M.S.; Forster, M.J.; Birdsall, B.; Roberts, G.C.K.; Feeney, J.; Cheung, H.T.A.; Kompis, I.; Geddes, A.J. )

    1988-06-01

    The conformation of a small molecule in its binding site on a protein is a major factor in the specificity of the interaction between them. In this paper, the authors report the use of {sup 1}H and {sup 13}C NMR spectroscopy to study the fluctuations in conformation of the anti-bacterial drug trimethoprim when it is bound to its target, dihydrofolate reductase. {sup 13}C relaxation measurements reveal dihedral angle changes of {plus minus}25{degree} to {plus minus}35{degree} on the subnanosecond time scale, while {sup 13}C line-shape analysis demonstrates dihedral angle changes of at least {plus minus}65{degree} on the millisecond time scale. {sup 1}H NMR shows that a specific hydrogen bond between the inhibitor and enzyme, which is believed to make an important contribution to binding, makes and breaks rapidly at room temperature.

  12. Nitrite Reductase Activity in Engineered Azurin Variants.

    PubMed

    Berry, Steven M; Strange, Jacob N; Bladholm, Erika L; Khatiwada, Balabhadra; Hedstrom, Christine G; Sauer, Alexandra M

    2016-05-01

    Nitrite reductase (NiR) activity was examined in a series of dicopper P.a. azurin variants in which a surface binding copper site was added through site-directed mutagenesis. Four variants were synthesized with copper binding motifs inspired by the catalytic type 2 copper binding sites found in the native noncoupled dinuclear copper enzymes nitrite reductase and peptidylglycine α-hydroxylating monooxygenase. The four azurin variants, denoted Az-NiR, Az-NiR3His, Az-PHM, and Az-PHM3His, maintained the azurin electron transfer copper center, with the second designed copper site located over 13 Å away and consisting of mutations Asn10His,Gln14Asp,Asn16His-azurin, Asn10His,Gln14His,Asn16His-azurin, Gln8Met,Gln14His,Asn16His-azurin, and Gln8His,Gln14His,Asn16His-azurin, respectively. UV-visible absorption spectroscopy, EPR spectroscopy, and electrochemistry of the sites demonstrate copper binding as well as interaction with small exogenous ligands. The nitrite reduction activity of the variants was determined, including the catalytic Michaelis-Menten parameters. The variants showed activity (0.34-0.59 min(-1)) that was slower than that of native NiRs but comparable to that of other model systems. There were small variations in activity of the four variants that correlated with the number of histidines in the added copper site. Catalysis was found to be reversible, with nitrite produced from NO. Reactions starting with reduced azurin variants demonstrated that electrons from both copper centers were used to reduce nitrite, although steady-state catalysis required the T2 copper center and did not require the T1 center. Finally, experiments separating rates of enzyme reduction from rates of reoxidation by nitrite demonstrated that the reaction with nitrite was rate limiting during catalysis. PMID:27055058

  13. Molecular evolution of nitrate reductase genes.

    PubMed

    Zhou, J; Kleinhofs, A

    1996-04-01

    To understand the evolutionary mechanisms and relationships of nitrate reductases (NRs), the nucleotide sequences encoding 19 nitrate reductase (NR) genes from 16 species of fungi, algae, and higher plants were analyzed. The NR genes examined show substantial sequence similarity, particularly within functional domains, and large variations in GC content at the third codon position and intron number. The intron positions were different between the fungi and plants, but conserved within these groups. The overall and nonsynonymous substitution rates among fungi, algae, and higher plants were estimated to be 4.33 x 10(-10) and 3.29 x 10(-10) substitutions per site per year. The three functional domains of NR genes evolved at about one-third of the rate of the N-terminal and the two hinge regions connecting the functional domains. Relative rate tests suggested that the nonsynonymous substitution rates were constant among different lineages, while the overall nucleotide substitution rates varied between some lineages. The phylogenetic trees based on NR genes correspond well with the phylogeny of the organisms determined from systematics and other molecular studies. Based on the nonsynonymous substitution rate, the divergence time of monocots and dicots was estimated to be about 340 Myr when the fungi-plant or algae-higher plant divergence times were used as reference points and 191 Myr when the rice-barley divergence time was used as a reference point. These two estimates are consistent with other estimates of divergence times based on these reference points. The lack of consistency between these two values appears to be due to the uncertainty of the reference times. PMID:8642612

  14. The cytochrome bd respiratory oxygen reductases

    PubMed Central

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

    2011-01-01

    Summary Cytochrome bd is a respiratory quinol:O2 oxidoreductase found in many prokaryotes, including a number of pathogens. The main bioenergetic function of the enzyme is the production of a proton motive force by the vectorial charge transfer of protons. The sequences of cytochromes bd are not homologous to those of the other respiratory oxygen reductases, i.e., the heme-copper oxygen reductases or alternative oxidases (AOX). Generally, cytochromes bd are noteworthy for their high affinity for O2 and resistance to inhibition by cyanide. In E. coli, for example, cytochrome bd (specifically, cytochrome bd-I) is expressed under O2-limited conditions. Among the members of the bd-family are the so-called cyanide-insensitive quinol oxidases (CIO) which often have a low content of the eponymous heme d but, instead, have heme b in place of heme d in at least a majority of the enzyme population. However, at this point, no sequence motif has been identified to distinguish cytochrome bd (with a stoichiometric complement of heme d) from an enzyme designated as CIO. Members of the bd-family can be subdivided into those which contain either a long or a short hydrophilic connection between transmembrane helices 6 and 7 in subunit I, designated as the Q-loop. However, it is not clear whether there is a functional consequence of this difference. This review summarizes current knowledge on the physiological functions, genetics, structural and catalytic properties of cytochromes bd. Included in this review are descriptions of the intermediates of the catalytic cycle, the proposed site for the reduction of O2, evidence for a proton channel connecting this active site to the bacterial cytoplasm, and the molecular mechanism by which a membrane potential is generated. PMID:21756872

  15. Vicenin 2 isolated from Artemisia capillaris exhibited potent anti-glycation properties.

    PubMed

    Islam, Md Nurul; Ishita, Ishrat Jahan; Jung, Hyun Ah; Choi, Jae Sue

    2014-07-01

    Vicenin 2, isolated from a traditionally used medicinal plant Artemisia capillaris, is a 6,8-di-C-glucoside of apigenin which has been previously reported to possess a wide variety of pharmacological activities including antioxidant, anti-inflammatory, anti-cancer, and hepatoprotective. However, there have not been any reports concerning its anti-diabetic potential until now. Therefore, in the present study, we evaluated the anti-diabetic potential of vicenin 2 via α-glucosidase, protein tyrosine phosphatase 1B (PTP1B), rat lens aldose reductase (RLAR), and advanced glycation end products (AGE) formation inhibitory assays. Vicenin 2 strongly inhibited α-glucosidase, PTP1B, and RLAR in the corresponding assays. In addition, vicenin 2 inhibited the formation of both fluorescent AGE and nonfluorescent AGE, e.g., CML, as well as the level of fructosamine in glucose-fructose-induced bovine serum albumin (BSA) glycation. In the test system, vicenin 2 suppressed glycation-induced protein oxidation by attenuating the formation of protein carbonyl groups as well as by inhibiting the modification of protein thiol groups. Moreover, vicenin 2 was found to be a potent inhibitor of glycation-induced formation of amyloid cross-β structures in BSA. Taken together, vicenin 2 might be a useful lead for the development of multiple target-oriented therapeutic modalities for the treatment of diabetes and diabetes-associated complications. PMID:24713265

  16. A comparison of glucose oxidase and aldose dehydrogenase as mediated anodes in printed glucose/oxygen enzymatic fuel cells using ABTS/laccase cathodes.

    PubMed

    Jenkins, Peter; Tuurala, Saara; Vaari, Anu; Valkiainen, Matti; Smolander, Maria; Leech, Dónal

    2012-10-01

    Current generation by mediated enzyme electron transfer at electrode surfaces can be harnessed to provide biosensors and redox reactions in enzymatic fuel cells. A glucose/oxygen enzymatic fuel cell can provide power for portable and implantable electronic devices. High volume production of enzymatic fuel cell prototypes will likely require printing of electrode and catalytic materials. Here we report on preparation and performance of, completely enzymatic, printed glucose/oxygen biofuel cells. The cells are based on filter paper coated with conducting carbon inks, enzyme and mediator. A comparison of cell performance using a range of mediators for either glucose oxidase (GOx) or aldose dehydrogenase (ALDH) oxidation of glucose at the anode and ABTS and a fungal laccase, for reduction of oxygen at the cathode, is reported. Highest power output, although of limited stability, is observed for ALDH anodes mediated by an osmium complex, providing a maximum power density of 3.5 μW cm(-2) at 0.34 V, when coupled to a laccase/ABTS cathode. The stability of cell voltage in a biobattery format, above a threshold of 200 mV under a moderate 75 kΩ load, is used to benchmark printed fuel cell performance. Highest stability is obtained for printed fuel cells using ALDH, providing cell voltages over the threshold for up to 74 h, compared to only 2 h for cells with anodes using GOx. These results provide promising directions for further development of mass-producible, completely enzymatic, printed biofuel cells. PMID:22200380

  17. Carboxylation mechanism and stereochemistry of crotonyl-CoA carboxylase/reductase, a carboxylating enoyl-thioester reductase

    PubMed Central

    Erb, Tobias J.; Brecht, Volker; Fuchs, Georg; Müller, Michael; Alber, Birgit E.

    2009-01-01

    Chemo- and stereoselective reductions are important reactions in chemistry and biology, and reductases from biological sources are increasingly applied in organic synthesis. In contrast, carboxylases are used only sporadically. We recently described crotonyl-CoA carboxylase/reductase, which catalyzes the reduction of (E)-crotonyl-CoA to butyryl-CoA but also the reductive carboxylation of (E)-crotonyl-CoA to ethylmalonyl-CoA. In this study, the complete stereochemical course of both reactions was investigated in detail. The pro-(4R) hydrogen of NADPH is transferred in both reactions to the re face of the C3 position of crotonyl-CoA. In the course of the carboxylation reaction, carbon dioxide is incorporated in anti fashion at the C2 atom of crotonyl-CoA. For the reduction reaction that yields butyryl-CoA, a solvent proton is added in anti fashion instead of the CO2. Amino acid sequence analysis showed that crotonyl-CoA carboxylase/reductase is a member of the medium-chain dehydrogenase/reductase superfamily and shares the same phylogenetic origin. The stereospecificity of the hydride transfer from NAD(P)H within this superfamily is highly conserved, although the substrates and reduction reactions catalyzed by its individual representatives differ quite considerably. Our findings led to a reassessment of the stereospecificity of enoyl(-thioester) reductases and related enzymes with respect to their amino acid sequence, revealing a general pattern of stereospecificity that allows the prediction of the stereochemistry of the hydride transfer for enoyl reductases of unknown specificity. Further considerations on the reaction mechanism indicated that crotonyl-CoA carboxylase/reductase may have evolved from enoyl-CoA reductases. This may be useful for protein engineering of enoyl reductases and their application in biocatalysis. PMID:19458256

  18. 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.

  19. COMPARISON OF THE METHYL REDUCTASE GENES AND GENE PRODUCTS

    EPA Science Inventory

    The DNA sequences encoding component C of methyl coenzyme M reductase (mcr genes) in Methanothermus fervidus, Methanobacterium thermoautotrophicum, Methanococcus vannielii, and Methanosarcina barkeri have been published. omparisons of transcription initiation and termination site...

  20. Structural features of the ribonucleotide reductase of Aujeszky's disease virus.

    PubMed

    Kaliman, A V; Boldogköi, Z; Fodor, I

    1994-01-01

    A gene construct of the Aujeszky's disease virus (ADV) genome was prepared and the DNA fragment encoding the ribonucleotide reductase was structurally characterized. We determined the entire DNA sequence of two adjacent open reading frames of the ribonucleotide reductase genes with the intergenic sequence of nine base pairs. From the sequence analysis we predict that Aujeszky's disease virus encodes a ribonucleotide reductase which comprises two polypeptides--large and small subunits, with sizes of 835 and 303 amino acids, respectively. Nucleotide and amino acid sequences of the large and small subunits of the Aujeszky's disease virus ribonucleotide reductase have been compared with that of other herpesviruses, and structural features of both proteins have been characterized. PMID:7810419

  1. Expression of bacterial mercuric ion reductase in Saccharomyces cerevisiae.

    PubMed Central

    Rensing, C; Kües, U; Stahl, U; Nies, D H; Friedrich, B

    1992-01-01

    The gene merA coding for bacterial mercuric ion reductase was cloned under the control of the yeast promoter for alcohol dehydrogenase I in the yeast-Escherichia coli shuttle plasmid pADH040-2 and transformed into Saccharomyces cerevisiae AH22. The resulting transformant harbored stable copies of the merA-containing hybrid plasmid, displayed a fivefold increase in the MIC of mercuric chloride, and synthesized mercuric ion reductase activity. Images PMID:1735719

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

    PubMed Central

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

    1996-01-01

    Nitrate assimilation in many plants, algae, yeasts and bacteria is mediated by two enzymes, nitrate reductase (EC 1.6.6.2) and nitrite reductase (EC 1.7.7.1). They catalyse the stepwise reduction of nitrate to nitrite and nitrite to ammonia respectively. The nitrite reductase from an industrially important yeast, Candida utilis, has been purified to homogeneity. Purified nitrite reductase is a heterodimer and the molecular masses of the two subunits are 58 and 66 kDa. The native enzyme exhibits a molecular mass of 126 kDa as analysed by gel filtration. The identify of the two subunits of nitrite reductase was confirmed by immunoblotting using antibody for Cucurbita pepo leaf nitrite reductase. The presence of two different sized transcripts coding for the two subunits was confirmed by (a) in vitro translation of mRNA from nitrate-induced C. utilis followed by immunoprecipitation of the in vitro translated products with heterologous nitrite reductase antibody and (b) Northern-blot analysis. The 66 kDa subunit is acidic in nature which is probably due to its phosphorylated status. The enzyme is stable over a range of temperatures. Both subunits can catalyse nitrite reduction, and the reconstituted enzyme, at a higher protein concentration, shows an activity similar to that of the purified enzyme. Each of these subunits has been shown to contain a few unique peptides in addition to a large number of common peptides. Reduced Methyl Viologen has been found to be as effective an electron donor as NADPH in the catalytic process, a phenomenon not commonly seen for nitrite reductases from other systems. PMID:8694757

  3. Reaction mechanism and regulation of mammalian thioredoxin/glutathione reductase.

    PubMed

    Sun, Qi-An; Su, Dan; Novoselov, Sergey V; Carlson, Bradley A; Hatfield, Dolph L; Gladyshev, Vadim N

    2005-11-01

    Thioredoxin/glutathione reductase (TGR) is a recently discovered member of the selenoprotein thioredoxin reductase family in mammals. In contrast to two other mammalian thioredoxin reductases, it contains an N-terminal glutaredoxin domain and exhibits a wide spectrum of enzyme activities. To elucidate the reaction mechanism and regulation of TGR, we prepared a recombinant mouse TGR in the selenoprotein form as well as various mutants and individual domains of this enzyme. Using these proteins, we showed that the glutaredoxin and thioredoxin reductase domains of TGR could independently catalyze reactions normally associated with each domain. The glutaredoxin domain is a monothiol glutaredoxin containing a CxxS motif at the active site, which could receive electrons from either the thioredoxin reductase domain of TGR or thioredoxin reductase 1. We also found that the C-terminal penultimate selenocysteine was required for transfer of reducing equivalents from the thiol/disulfide active site of TGR to the glutaredoxin domain. Thus, the physiologically relevant NADPH-dependent activities of TGR were dependent on this residue. In addition, we examined the effects of selenium levels in the diet and perturbations in selenocysteine tRNA function on TGR biosynthesis and found that expression of this protein was regulated by both selenium and tRNA status in liver, but was more resistant to this regulation in testes. PMID:16262253

  4. Effects of thioredoxin reductase-1 deletion on embryogenesis and transcriptome

    PubMed Central

    Bondareva, Alla A.; Capecchi, Mario R.; Iverson, Sonya V.; Li, Yan; Lopez, Nathan I.; Lucas, Olivier; Merrill, Gary F.; Prigge, Justin R.; Siders, Ashley M.; Wakamiya, Maki; Wallin, Stephanie L.; Schmidt, Edward E.

    2007-01-01

    Thioredoxin reductases (Txnrd)1 maintain intracellular redox homeostasis in most organisms. Metazoans Txnrds also participate in signal transduction. Mouse embryos homozygous for a targeted null mutation of the txnrd1 gene, encoding the cytosolic thioredoxin reductase, were viable at embryonic day 8.5 (E8.5) but not at E9.5. Histology revealed that txnrd1−/− cells were capable of proliferation and differentiation; however, mutant embryos were smaller than wild-type littermates and failed to gastrulate. In situ marker gene analyses indicated primitive streak mesoderm did not form. Microarray analyses on E7.5 txnrd−/− and txnrd+/+ littermates showed similar mRNA levels for peroxiredoxins, glutathione reductases, mitochondrial Txnrd2, and most markers of cell proliferation. Conversely, mRNAs encoding sulfiredoxin, IGF-binding protein 1, carbonyl reductase 3, glutamate cysteine ligase, glutathione S-transferases, and metallothioneins were more abundant in mutants. Many gene expression responses mirrored those in thioredoxin reductase 1-null yeast; however mice exhibited a novel response within the peroxiredoxin catalytic cycle. Thus, whereas yeast induce peroxiredoxin mRNAs in response to thioredoxin reductase disruption, mice induced sulfiredoxin mRNA. In summary, Txnrd1 was required for correct patterning of the early embryo and progression to later development. Conserved responses to Txnrd1 disruption likely allowed proliferation and limited differentiation of the mutant embryo cells. PMID:17697936

  5. An overview on 5alpha-reductase inhibitors.

    PubMed

    Aggarwal, Saurabh; Thareja, Suresh; Verma, Abhilasha; Bhardwaj, Tilak Raj; Kumar, Manoj

    2010-02-01

    Benign prostatic hyperplasia (BPH) is the noncancerous proliferation of the prostate gland associated with benign prostatic obstruction and lower urinary tract symptoms (LUTS) such as frequency, hesitancy, urgency, etc. Its prevalence increases with age affecting around 70% by the age of 70 years. High activity of 5alpha-reductase enzyme in humans results in excessive dihydrotestosterone levels in peripheral tissues and hence suppression of androgen action by 5alpha-reductase inhibitors is a logical treatment for BPH as they inhibit the conversion of testosterone to dihydrotestosterone. Finasteride (13) was the first steroidal 5alpha-reductase inhibitor approved by U.S. Food and Drug Administration (USFDA). In human it decreases the prostatic DHT level by 70-90% and reduces the prostatic size. Dutasteride (27) another related analogue has been approved in 2002. Unlike Finasteride, Dutasteride is a competitive inhibitor of both 5alpha-reductase type I and type II isozymes, reduced DHT levels >90% following 1 year of oral administration. A number of classes of non-steroidal inhibitors of 5alpha-reductase have also been synthesized generally by removing one or more rings from the azasteroidal structure or by an early non-steroidal lead (ONO-3805) (261). In this review all categories of inhibitors of 5alpha-reductase have been covered. PMID:19879888

  6. Regulation of the Neurospora crassa assimilatory nitrate reductase.

    PubMed Central

    Ketchum, P A; Zeeb, D D; Owens, M S

    1977-01-01

    Reduced nicotinamide adenine dinucleotide phosphate (NADPH)-nitrate reductase from Neurospora crassa was purified and found to be stimulated by certain amino acids, citrate, and ethylenediaminetetraacetic acid (EDTA). Stimulation by citrate and the amino acids was dependent upon the prior removal of EDTA from the enzyme preparations, since low quantities of EDTA resulted in maximal stimulation. Removal of EDTA from enzyme preparations by dialysis against Chelex-containing buffer resulted in a loss of nitrate reductase activity. Addition of alanine, arginine, glycine, glutamine, glutamate, histidine, tryptophan, and citrate restored and stimulated nitrate reductase activity from 29- to 46-fold. The amino acids tested altered the Km of NADPH-nitrate reductase for NADPH but did not significantly change that for nitrate. The Km of nitrate reductase for NADPH increased with increasing concentrations of histidine but decreased with increasing concentrations of glutamine. Amino acid modulation of NADPH-nitrate reductase activity is discussed in relation to the conservation of energy (NADPH) by Neurospora when nitrate is the nitrogen source. PMID:19423

  7. In vitro antidiabetic potential of the fruits of Crataegus pinnatifida

    PubMed Central

    Chowdhury, S.S.; Islam, M.N.; Jung, H.A.; Choi, J.S.

    2014-01-01

    In an attempt to develop alternative medicine for the treatment of diabetes and related complications, the antidiabetic potential of the fruits of Crataegus pinnatifida was evaluated. The antidiabetic potential of the methanol (MeOH) extract as well as different solvent soluble fractions of the fruits of C. pinnatifida was evaluated via α-glucosidase, protein tyrosine phosphatase 1B (PTP1B), rat lens aldose reductase (RLAR), and advanced glycation end products (AGEs) formation inhibitory assays. The MeOH extract showed potent inhibitory activity against α-glucosidase, PTP1B, and AGEs formation with IC50 values of 122.11, 3.66 and 65.83 μg/ml respectively, while it showed moderate inhibitory activity against RLAR with the IC50 value of 160.54 μg/ml. Among different fractions, the ethyl acetate (EtOAc) and the dichloromethane (CH2Cl2) fractions were found as active fractions exhibiting potential α-glucosidase, PTP1B, RLAR inhibitory, and AGEs formation inhibitory activities. Seven compounds including hyperoside, chlorogenic acid, ursolic acid, oleanolic acid, 3-epicorosolic acid, β-sitosterol, β-sitosterol glucoside were isolated from these two fractions. 3-Epicorosolic acid showed both potent α-glucosidase and PTP1B inhibitory activities with IC50 values of 30.18 and 4.08 μg/ml respectively. Moreover, kinetic study revealed that 3-epicorosolic acid showed mixed type inhibition against PTP1B, while it showed uncompetitive inhibition against α-glucosidase. Therefore, these results suggest that the fruits of C. pinnatifida and its constituents have potential antidiabetic activity which might be used as a functional food for the treatment of diabetes and associated complications. PMID:25598795

  8. 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

  9. Equine 5α-reductase activity and expression in epididymis.

    PubMed

    Corbin, C J; Legacki, E L; Ball, B A; Scoggin, K E; Stanley, S D; Conley, A J

    2016-10-01

    The 5α-reductase enzymes play an important role during male sexual differentiation, and in pregnant females, especially equine species where maintenance relies on 5α-reduced progesterone, 5α-dihydroprogesterone (DHP). Epididymis expresses 5α-reductases but was not studied elaborately in horses. Epididymis from younger and older postpubertal stallions was divided into caput, corpus and cauda and examined for 5α-reductase activity and expression of type 1 and 2 isoforms by quantitative real-time polymerase chain reaction (qPCR). Metabolism of progesterone and testosterone to DHP and dihydrotestosterone (DHT), respectively, by epididymal microsomal protein was examined by thin-layer chromatography and verified by liquid chromatography tandem mass spectrometry (LC-MS/MS). Relative inhibitory potencies of finasteride and dutasteride toward equine 5α-reductase activity were investigated. Pregnenolone was investigated as an additional potential substrate for 5α-reductase, suggested previously from in vivo studies in mares but never directly examined. No regional gradient of 5α-reductase expression was observed by either enzyme activity or transcript analysis. Results of PCR experiments suggested that type 1 isoform predominates in equine epididymis. Primers for the type 2 isoform were unable to amplify product from any samples examined. Progesterone and testosterone were readily reduced to DHP and DHT, and activity was effectively inhibited by both inhibitors. Using epididymis as an enzyme source, no experimental evidence was obtained supporting the notion that pregnenolone could be directly metabolized by equine 5α-reductases as has been suggested by previous investigators speculating on alternative metabolic pathways leading to DHP synthesis in placenta during equine pregnancies. PMID:27466384

  10. DNA damage induction of ribonucleotide reductase.

    PubMed Central

    Elledge, S J; Davis, R W

    1989-01-01

    RNR2 encodes the small subunit of ribonucleotide reductase, the enzyme that catalyzes the first step in the pathway for the production of deoxyribonucleotides needed for DNA synthesis. RNR2 is a member of a group of genes whose activities are cell cycle regulated and that are transcriptionally induced in response to the stress of DNA damage. An RNR2-lacZ fusion was used to further characterize the regulation of RNR2 and the pathway responsible for its response to DNA damage. beta-Galactosidase activity in yeast strains containing the RNR2-lacZ fusion was inducible in response to DNA-damaging agents (UV light, 4-nitroquinoline-1-oxide [4-NQO], and methyl methanesulfonate [MMS]) and agents that block DNA replication (hydroxyurea [HU] and methotrexate) but not heat shock. When MATa cells were arrested in G1 by alpha-factor, RNR2 mRNA was still inducible by DNA damage, indicating that the observed induction can occur outside of S phase. In addition, RNR2 induction was not blocked by the presence of cycloheximide and is therefore likely to be independent of protein synthesis. A mutation, rnr2-314, was found to confer hypersensitivity to HU and increased sensitivity to MMS. In rnr2-314 mutant strains, the DNA damage stress response was found to be partially constitutive as well as hypersensitive to induction by HU but not MMS. The induction properties of RNR2 were examined in a rad4-2 mutant background; in this genetic background, RNR2 was hypersensitive to induction by 4-NQO but not MMS. Induction of the RNR2-lacZ fusion in a RAD(+) strain in response to 4-NQO was not enhanced by the presence of an equal number of rad4-2 cells that lacked the fusion, implying that the DNA damage stress response in cell autonomous. Images PMID:2513480

  11. Sulfur Isotope Effects of Dissimilatory Sulfite Reductase

    PubMed Central

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

    2015-01-01

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

  12. Sulfur Isotope Effects of Dissimilatory Sulfite Reductase.

    PubMed

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

    2015-01-01

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

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

    PubMed

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

    2009-10-01

    Nitrate serves as a terminal electron acceptor under anaerobic conditions in Pseudomonas aeruginosa. Reduction of nitrate to nitrite generates a transmembrane proton motive force allowing ATP synthesis and anaerobic growth. The inner membrane-bound nitrate reductase NarGHI is encoded within the narK1K2GHJI operon, and the periplasmic nitrate reductase NapAB is encoded within the napEFDABC operon. The roles of the 2 dissimilatory nitrate reductases in anaerobic growth, and the regulation of their expressions, were examined by use of a set of deletion mutants in P. aeruginosa PAO1. NarGHI mutants were unable to grow anaerobically, but plate cultures remained viable up to 120 h. In contrast, the nitrate sensor-response regulator mutant DeltanarXL displayed growth arrest initially, but resumed growth after 72 h and reached the early stationary phase in liquid culture after 120 h. Genetic, transcriptional, and biochemical studies demonstrated that anaerobic growth recovery by the NarXL mutant was the result of NapAB periplasmic nitrate reductase expression. A novel transcriptional start site for napEFDABC expression was identified in the NarXL mutant grown anaerobically. Furthermore, mutagenesis of a consensus NarL-binding site monomer upstream of the novel transcriptional start site restored anaerobic growth recovery in the NarXL mutant. The data suggest that during anaerobic growth of wild-type P. aeruginosa PAO1, the nitrate response regulator NarL directly represses expression of periplasmic nitrate reductase, while inducing maximal expression of membrane nitrate reductase. PMID:19935885

  14. Comparative Studies on the Induction and Inactivation of Nitrate Reductase in Corn Roots and Leaves 1

    PubMed Central

    Aslam, Muhammad; Oaks, Ann

    1976-01-01

    A comparison of induction and inactivation of nitrate reductase and two of its component activities, namely FMNH2-nitrate reductase and NO3−-induced NADH-cytochrome c reductase, was made in roots and leaves of corn (Zea mays L. var. W64A × 182E). The three activities were induced in parallel in both tissues when NO3− was supplied. WO4= suppressed the induction of NADH- and FMNH2-nitrate reductase activities in root tips and leaves. The NO3−-induced NADH-cytochrome c reductase activity showed a normal increase in roots treated with WO4=. In leaves, on the other hand, there was a marked superinduction of the NO3−-induced NADH-cytochrome c reductase in the presence of WO4=. The half-life values of NADH-nitrate reductase and FMNH2-nitrate reductase measured by removing NO3− and adding WO4= to the medium, were 4 hours in root tips and 6 hours in excised leaves. Addition of NO3− in the induction medium together with WO4= gave partial protection of NADH-nitrate reductase and FMNH2-nitrate reductase activities in both root tips and leaves with a t0.5 of 6 and 8 hours, respectively. NO3− also reduced the loss of nitrate reductase activity from mature root sections. In the presence of cycloheximide, both NADH-nitrate reductase and NO3−-induced NADH-cytochrome c reductase activities were lost at similar rates in root tips. NO3− protected the loss of NO3−-induced NADH-cytochrome c reductase to the same extent as that of NADH-nitrate reductase. PMID:16659529

  15. 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.

  16. 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.

  17. 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)

  18. 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...

  19. 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...

  20. 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...

  1. 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...

  2. 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...

  3. 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.

  4. The arsenic hyperaccumulating Pteris vittata expresses two arsenate reductases

    PubMed Central

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

    2015-01-01

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

  5. Characterization of mitochondrial thioredoxin reductase from C. elegans

    SciTech Connect

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

    2006-08-04

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

  6. 5. cap alpha. -reductase activity in rat adipose tissue

    SciTech Connect

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

    1987-11-01

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

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

    PubMed

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

    2015-09-01

    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 of

  8. 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

  9. Measurement of nitrous oxide reductase activity in aquatic sediments

    SciTech Connect

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

    1986-01-01

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

  10. Nickel site of methane catalysis in the methyl reductase enzyme

    SciTech Connect

    Shelnutt, J.A.; Shiemke, A.K.; Scott, R.A.

    1988-01-01

    Methyl reductase is the enzyme of methanogenic bacteria that catalyzed the two-electron reduction of the methyl group of 2-(methylthio)ethanesulfonic acid (methyl-S-CoM) to methane and HS-CoM. The methyl group of methyl-S-CoM ultimately comes from the six-electron reduction of CO/sub 2/ by hydrogen, which also provides the reducing equivalents needed by methyl reductase. The nature of the catalytic site of methyl reductase is of current interest from the point of view of developing biomimetic C/sub 1/, chemistries directed toward methane synthesis and activation. In particular, Sandia is using molecular graphics and energy optimization techniques to design macromolecular catalysts that mimic the structure of sites of proteins that carry out C/sub 1/ chemistry. The goal is to produce catalysts whose function is the oxidation of low molecular weight hydrocarbon gases to generate liquid fuels or, alternatively, the reduction of abundant inorganic resources such as CO/sub 2/ to generate gaseous fuels. Unfortunately, the catalytic sites of many of the enzymes of interest, e.g., methyl reductase and methane monooxygenase, have not been characterized by X-ray crystallography and other structural techniques. With the goal of learning more about the structure of one of these naturally occurring sites of C/sub 1/ chemistry, we have obtained the first resonance Raman spectra of the nickel-macrocycle, called F/sub 430/, at the site of catalysis in methyl reductase. To help us structurally interpret the Raman spectra of the enzyme we have also obtained Raman spectra of solutions of the major forms of F/sub 430/ (salt-extracted and cytosol-free) at room temperature and at 77/degree/K and also, under similar solution conditions, spectra of a nickel-corphinoid derivative that is related to F/sub 430/.

  11. Characterization of recombinant glutathione reductase from the psychrophilic Antarctic bacterium Colwellia psychrerythraea.

    PubMed

    Ji, Mikyoung; Barnwell, Callie V; Grunden, Amy M

    2015-07-01

    Glutathione reductases catalyze the reduction of oxidized glutathione (glutathione disulfide, GSSG) using NADPH as the substrate to produce reduced glutathione (GSH), which is an important antioxidant molecule that helps maintain the proper reducing environment of the cell. A recombinant form of glutathione reductase from Colwellia psychrerythraea, a marine psychrophilic bacterium, has been biochemically characterized to determine its molecular and enzymatic properties. C. psychrerythraea glutathione reductase was shown to be a homodimer with a molecular weight of 48.7 kDa using SDS-PAGE, MALDI-TOF mass spectrometry and gel filtration. The C. psychrerythraea glutathione reductase sequence shows significant homology to that of Escherichia coli glutathione reductase (66 % identity), and it possesses the FAD and NADPH binding motifs, as well as absorption spectrum features which are characteristic of flavoenzymes such as glutathione reductase. The psychrophilic C. psychrerythraea glutathione reductase exhibits higher k cat and k cat/K m at lower temperatures (4 °C) compared to mesophilic Baker's yeast glutathione reductase. However, C. psychrerythraea glutathione reductase was able to complement an E. coli glutathione reductase deletion strain in oxidative stress growth assays, demonstrating the functionality of C. psychrerythraea glutathione reductase over a broad temperature range, which suggests its potential utility as an antioxidant enzyme in heterologous systems. PMID:26101017

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

    PubMed

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

    2003-12-12

    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. PMID:13129921

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

    NASA Technical Reports Server (NTRS)

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

    2003-01-01

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

  14. 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.

  15. 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.

  16. Tungstate, a Molybdate Analog Inactivating Nitrate Reductase, Deregulates the Expression of the Nitrate Reductase Structural Gene

    PubMed Central

    Deng, Mingde; Moureaux, Thérèse; Caboche, Michel

    1989-01-01

    Nitrate reductase (NR, EC 1.6.6.1) from higher plants is a homodimeric enzyme carrying a molybdenum cofactor at the catalytic site. Tungsten can be substituted for molybdenum in the cofactor structure, resulting in an inactive enzyme. When nitratefed Nicotiana tabacum plants were grown on a nutrient solution in which tungstate was substituted for molybdate, NR activity in the leaves decreased to a very low level within 24 hours while NR protein accumulated progressively to a level severalfold higher than the control after 6 days. NR mRNA level in molybdate-grown plants exhibited a considerable day-night fluctuation. However, when plants were treated with tungstate, NR mRNA level remained very high. NR activity and protein increased over a 24-hour period when nitrate was added back to N-starved molybdate-grown plants. NR mRNA level increased markedly during the first 2 hours and then decreased. In the presence of tungstate, however, the induction of NR activity by nitrate was totally abolished while high levels of NR protein and mRNA were both induced, and the high level of NR mRNA was maintained over a 10-hour period. These results suggest that the substitution of tungsten for molybdenum in NR complex leads to an overexpression of the NR structural gene. Possible mechanisms involved in this deregulation are discussed. Images Figure 2 Figure 3 Figure 5 PMID:16667015

  17. Purine nucleoside phosphorylase as a cytosolic arsenate reductase.

    PubMed

    Gregus, Zoltán; Németi, Balázs

    2002-11-01

    The findings of the accompanying paper (Németi and Gregus, Toxicol: Sci. 70, 4-12) indicate that the arsenate (AsV) reductase activity of rat liver cytosol is due to an SH enzyme that uses phosphate (or its analogue, arsenate, AsV) and a purine nucleoside (guanosine or inosine) as substrates. Purine nucleoside phosphorylase (PNP) is such an enzyme. It catalyzes the phosphorolytic cleavage of 6-oxopurine nucleosides according to the following scheme: guanosine (or inosine) + phosphate <--> guanine (or hypoxanthine) + ribose-1-phosphate. Therefore, we have tested the hypothesis that PNP is responsible for the thiol- and purine nucleoside-dependent reduction of AsV to AsIII by rat liver cytosol. AsIII formed from AsV was quantified by HPLC-hydride generation-atomic fluorescence spectrometry analysis of the deproteinized incubates. The following findings support the conclusion that PNP reduces AsV to AsIII, using AsV instead of phosphate in the reaction above: (1) Specific PNP inhibitors (CI-1000, BCX-1777) at a concentration of 1 microM completely inhibited cytosolic AsV reductase activity. (2) During anion-exchange chromatography of cytosolic proteins, PNP activity perfectly coeluted with the AsV reductase activity, suggesting that both activities belong to the same protein. (3) PNP purified from calf spleen catalyzed reduction of AsV to AsIII in the presence of dithiothreitol (DTT) and a 6-oxopurine nucleoside (guanosine or inosine). (4) AsV reductase activity of purified PNP, like the cytosolic AsV reductase activity, was inhibited by phosphate (a substrate of PNP alternative to AsV), guanine and hypoxanthine (products of PNP favoring the reverse reaction), mercurial thiol reagents (nonspecific inhibitors of PNP), as well as CI-1000 and BCX-1777 (specific PNP inhibitors). Thus, PNP appears to be responsible for the AsV reductase activity of rat liver cytosol in the presence of DTT. Further research should clarify the mechanism and the in vivo significance of PNP

  18. Involvement of nitrate reductase in auxin-induced NO synthesis

    PubMed Central

    Erdei, L

    2008-01-01

    It is well known for a long time, that nitric oxide (NO) functions in variable physiological and developmental processes in plants, however the source of this signaling molecule in the diverse plant responses is very obscure.1 Although existance of nitric oxide sythase (NOS) in plants is still questionable, LNMMA (NG-monomethyl-L-arginine)-sensitive NO generation was observed in different plant species.2,3 In addition, nitrate reductase (NR) is confirmed to have a major role as source of NO.4,5 This multifaced molecule acts also in auxin-induced lateral root (LR) formation, since exogenous auxin enhanced NO levels in regions of Arabidopsis LR initiatives. Our results pointed out the involvement of nitrate reductase enzyme in auxin-induced NO formation. In this addendum, we speculate on auxin-induced NO production in lateral root primordial formation. PMID:19704423

  19. Involvement of nitrate reductase in auxin-induced NO synthesis.

    PubMed

    Kolbert, Zsuzsanna; Erdei, L

    2008-11-01

    It is well known for a long time, that nitric oxide (NO) functions in variable physiological and developmental processes in plants, however the source of this signaling molecule in the diverse plant responses is very obscure.1 Although existance of nitric oxide sythase (NOS) in plants is still questionable, LNMMA (N(G)-monomethyl-L-arginine)-sensitive NO generation was observed in different plant species.2,3 In addition, nitrate reductase (NR) is confirmed to have a major role as source of NO.4,5 This multifaced molecule acts also in auxin-induced lateral root (LR) formation, since exogenous auxin enhanced NO levels in regions of Arabidopsis LR initiatives. Our results pointed out the involvement of nitrate reductase enzyme in auxin-induced NO formation. In this addendum, we speculate on auxin-induced NO production in lateral root primordial formation. PMID:19704423

  20. Methyltetrahydrofolate reductase polymorphism influences onset of Huntington's disease.

    PubMed

    Brune, N; Andrich, J; Gencik, M; Saft, C; Müller, Th; Valentin, S; Przuntek, H; Epplen, J T

    2004-01-01

    Onset of Huntington's disease (HD) negatively correlates with CAG repeat length of the HD gene, which encodes the protein huntingtin. This protein interacts with the homocysteine metabolizing enzyme cystathionine betasynthase (CBS). Objective of this study was to analyze the impact of CAG repeats, polymorphisms of various homocysteine metabolizing enzymes, like CBS, Methyltetrahydrofolate Reductase (MTHTR), Methionine Synthase Reductase (MSR) and methionine synthase (MS) on HD onset in 171 patients. The significant impact of CAG repeats on HD onset (chi2= 25.54, FG = 4, p<0.0001) with a significant correlation between both (R= -0.521, p=0.01) was obvious. HD patients with the homozygous MTHFR-1298-CC significantly (p = 0.024) earlier experienced HD symptoms. There was no influence demonstrable of CBS, MSR and MS. Determination of MTHFR polymorphisms and CAG repeats enables screening for subjects with putative early HD onset in order to study neuroprotective compounds in their efficacy to delay HD symptoms. PMID:15354395

  1. Structural Basis for Substrate Specificity in Human Monomeric Carbonyl Reductases

    PubMed Central

    El-Hawari, Yasser; Dunford, James E.; Kochan, Grazyna; Wsol, Vladimir; Martin, Hans-Joerg; Maser, Edmund; Oppermann, Udo

    2009-01-01

    Carbonyl reduction constitutes a phase I reaction for many xenobiotics and is carried out in mammals mainly by members of two protein families, namely aldo-keto reductases and short-chain dehydrogenases/reductases. In addition to their capacity to reduce xenobiotics, several of the enzymes act on endogenous compounds such as steroids or eicosanoids. One of the major carbonyl reducing enzymes found in humans is carbonyl reductase 1 (CBR1) with a very broad substrate spectrum. A paralog, carbonyl reductase 3 (CBR3) has about 70% sequence identity and has not been sufficiently characterized to date. Screening of a focused xenobiotic compound library revealed that CBR3 has narrower substrate specificity and acts on several orthoquinones, as well as isatin or the anticancer drug oracin. To further investigate structure-activity relationships between these enzymes we crystallized CBR3, performed substrate docking, site-directed mutagenesis and compared its kinetic features to CBR1. Despite high sequence similarities, the active sites differ in shape and surface properties. The data reveal that the differences in substrate specificity are largely due to a short segment of a substrate binding loop comprising critical residues Trp229/Pro230, Ala235/Asp236 as well as part of the active site formed by Met141/Gln142 in CBR1 and CBR3, respectively. The data suggest a minor role in xenobiotic metabolism for CBR3. Enhanced version This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1. PMID:19841672

  2. Two fatty acyl reductases involved in moth pheromone biosynthesis.

    PubMed

    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

  3. Using chemical approaches to study selenoproteins - focus on thioredoxin reductases

    PubMed Central

    Hondal, Robert J.

    2009-01-01

    The study of selenocysteine-containing proteins is difficult due to the problems associated with the heterologous production of these proteins. These problems are due to the intricate recoding mechanism used by cells to translate the UGA codon as a sense codon for selenocysteine. The process is further complicated by the fact that eukaryotes and prokaryotes have different UGA recoding machineries. This review focuses on chemical approaches to produce selenoproteins and study the mechanism of selenoenzymes. The use of intein-mediated peptide ligation is discussed with respect to the production of the mammalian selenoenzymes thioredoxin reductase and selenoprotein R, also known as methionine sulfoxide reductase B1. New methods for removing protecting groups from selenocysteine post-synthesis and methods for selenosulfide/diselenide formation are also reviewed. Chemical approaches have also been used to study the enzymatic mechanism of thioredoxin reductase. The approach divides the enzyme into two modules, a large protein module lacking selenocysteine and a small, synthetic selenocysteine-containing peptide. Study of this semisynthetic enzyme has revealed three distinct enzymatic pathways that depend on the properties of the substrate. The enzyme utilizes a macromolecular mechanism for protein substrates, a second mechanism for small molecule substrates and a third pathway for selenium-containing substrates such as selenocystine. PMID:19406205

  4. Perchlorate Reductase Is Distinguished by Active Site Aromatic Gate Residues.

    PubMed

    Youngblut, Matthew D; Tsai, Chi-Lin; Clark, Iain C; Carlson, Hans K; Maglaqui, Adrian P; Gau-Pan, Phonchien S; Redford, Steven A; Wong, Alan; Tainer, John A; Coates, John D

    2016-04-22

    Perchlorate is an important ion on both Earth and Mars. Perchlorate reductase (PcrAB), a specialized member of the dimethylsulfoxide reductase superfamily, catalyzes the first step of microbial perchlorate respiration, but little is known about the biochemistry, specificity, structure, and mechanism of PcrAB. Here we characterize the biophysics and phylogeny of this enzyme and report the 1.86-Å resolution PcrAB complex crystal structure. Biochemical analysis revealed a relatively high perchlorate affinity (Km = 6 μm) and a characteristic substrate inhibition compared with the highly similar respiratory nitrate reductase NarGHI, which has a relatively much lower affinity for perchlorate (Km = 1.1 mm) and no substrate inhibition. Structural analysis of oxidized and reduced PcrAB with and without the substrate analog SeO3 (2-) bound to the active site identified key residues in the positively charged and funnel-shaped substrate access tunnel that gated substrate entrance and product release while trapping transiently produced chlorate. The structures suggest gating was associated with shifts of a Phe residue between open and closed conformations plus an Asp residue carboxylate shift between monodentate and bidentate coordination to the active site molybdenum atom. Taken together, structural and mutational analyses of gate residues suggest key roles of these gate residues for substrate entrance and product release. Our combined results provide the first detailed structural insight into the mechanism of biological perchlorate reduction, a critical component of the chlorine redox cycle on Earth. PMID:26940877

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

    NASA Astrophysics Data System (ADS)

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

    2014-01-01

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

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

    SciTech Connect

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

    2009-05-28

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

  7. Differential Light Induction of Nitrate Reductases in Greening and Photobleached Soybean Seedlings 1

    PubMed Central

    Kakefuda, Genichi; Duke, Stanley H.; Duke, Stephen O.

    1983-01-01

    Soybean (Glycine max [L.] Merr.) seeds were imbibed and germinated with or without NO3−, tungstate, and norflurazon (San 9789). Norflurazon is a herbicide which causes photobleaching of chlorophyll by inhibiting carotenoid synthesis and which impairs normal chloroplast development. After 3 days in the dark, seedlings were placed in white light to induce extractable nitrate reductase activity. The induction of maximal nitrate reductase activity in greening cotyledons did not require NO3− and was not inhibited by tungstate. Induction of nitrate reductase activity in norflurazon-treated cotyledons had an absolute requirement for NO3− and was completely inhibited by tungstate. Nitrate was not detected in seeds or seedlings which had not been treated with NO3−. The optimum pH for cotyledon nitrate reductase activity from norflurazon-treated seedlings was at pH 7.5, and near that for root nitrate reductase activity, whereas the optimum pH for nitrate reductase activity from greening cotyledons was pH 6.5. Induction of root nitrate reductase activity was also inhibited by tungstate and was dependent on the presence of NO3−, further indicating that the isoform of nitrate reductase induced in norflurazon-treated cotyledons is the same or similar to that found in roots. Nitrate reductases with and without a NO3− requirement for light induction appear to be present in developing leaves. In vivo kinetics (light induction and dark decay rates) and in vitro kinetics (Arrhenius energies of activation and NADH:NADPH specificities) of nitrate reductases with and without a NO3− requirement for induction were quite different. Km values for NO3− were identical for both nitrate reductases. PMID:16663185

  8. The effect of copper and gallium compounds on ribonucleotide reductase

    SciTech Connect

    Narasimhan, J.

    1992-01-01

    The mode of action of copper complexes (CuL and CuKTS) and gallium compounds (gallium nitrate and citrate) in cytotoxicity was studied. The effects of these agents on the enzyme ribonucleotide reductase was investigated by monitoring the tyrosyl free radical present in the active site of the enzyme through electron spin resonance (ESR) spectroscopy. Ribonucleotide reductase, a key enzyme in cellular proliferation, consists of two subunits. M1, a dimer of molecular weight 170,000 contains the substrate and effector binding sites. M2, a dimer of molecular weight 88,000, contains non-heme iron and tyrosyl free radical essential for the activity of the enzyme. In studies using copper complexes, the cellular oxidative chemistry was examined by ESR studies on adduct formation with membranes, and oxidation of thiols. Membrane thiols were oxidized through the reduction of the ESR signal of the thiol adduct and the analysis of sulfhydryl content. Using the radiolabel [sup 59]Fe, the inhibitory action of copper thiosemicarbazones on cellular iron uptake was shown. The inhibitory action of CuL on ribonucleotide reductase was shown by the quenching of the tyrosyl free radical on the M2 subunit. The hypothesis that gallium directly interacts with the M2 subunit of the enzyme and displaces the iron from it was proven. The tyrosyl free radical signal from cell lysates was inhibited by the direct addition of gallium compounds. Gallium content in the cells was measured by a fluorimetric method, to ensure the presence of sufficient amounts of gallium to compete with the iron in the M2 subunit. The enzyme activity, measured by the conversion of [sup 14]C-CDP to the labeled deoxy CDP, was inhibited by the addition of gallium nitrate in a cell free assay system. The immunoprecipitation studies of the [sup 59]Fe labeled M2 protein using the monoclonal antibody directed against this subunit suggested that gallium releases iron from the M2 subunit.

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

    PubMed

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

    2015-10-01

    The incidence of congenital heart defects is higher in infants with mutation of methylenetetrahydrofolate reductase (MTHFR) gene. The MTHFR C677T gene decreases the bioavailability of folate and increases plasma homocysteine, a risk factor for thrombosis. There have been no reported cases in the literature on the clinical implications of this procoagulable state in the setting of cyanotic heart disease, which itself has prothrombotic predisposition. Two patients with hypoplastic left heart syndrome developed postoperative thrombotic complications, both were homozygous for MTHFR C677T. We present these cases and highlight the implications of MTHFR mutation in the management of complex congenital heart disease. PMID:26467879

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

    PubMed

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

    2016-03-01

    Two new ent-prenylaromadendrane-type diterpenoids, diplotaxifols A (1) and B (2), a new ent-eudesmol, ent-eudesma-4(15),11(13)-dien-6α,12-diol (3), eight new eudesmanolides enantiomers (4-11) of the corresponding compounds from higher plants along with four known ent-eudesmanolides (12-15) were isolated from the 95% EtOH extract of Chinese liverwort Diplophyllum taxifolium. Their structures were elucidated on the basis of MS, NMR and IR spectral data, and confirmed by single-crystal X-ray diffraction analysis. The quinone reductase-inducing activity of the compounds was evaluated. PMID:26656409

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

    PubMed

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

    2016-08-10

    Cytochromes P450 (P450s) require electron transfer partners to catalyze substrate conversions. With regard to biotechnological approaches, the elucidation of novel electron transfer proteins is of special interest, as they can influence the enzymatic activity and specificity of the P450s. In the current work we present the identification and characterization of a novel soluble NADPH-dependent diflavin reductase from Bacillus megaterium with activity towards a bacterial (CYP106A1) and a microsomal (CYP21A2) P450 and, therefore, we referred to it as B. megaterium cytochrome P450 reductase (BmCPR). Sequence analysis of the protein revealed besides the conserved FMN-, FAD- and NADPH-binding motifs, the presence of negatively charged cluster, which is thought to represent the interaction domain with P450s and/or cytochrome c. BmCPR was expressed and purified to homogeneity in Escherichia coli. The purified BmCPR exhibited a characteristic diflavin reductase spectrum, and showed a cytochrome c reducing activity. Furthermore, in an in vitro reconstituted system, the BmCPR was able to support the hydroxylation of testosterone and progesterone with CYP106A1 and CYP21A2, respectively. Moreover, in view of the biotechnological application, the BmCPR is very promising, as it could be successfully utilized to establish CYP106A1- and CYP21A2-based whole-cell biotransformation systems, which yielded 0.3g/L hydroxy-testosterone products within 8h and 0.16g/L 21-hydroxyprogesterone within 6h, respectively. In conclusion, the BmCPR reported herein owns a great potential for further applications and studies and should be taken into consideration for bacterial and/or microsomal CYP-dependent bioconversions. PMID:27238232

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  13. Evaluation of 5α-reductase inhibitory activity of certain herbs useful as antiandrogens.

    PubMed

    Nahata, A; Dixit, V K

    2014-08-01

    This study demonstrates 5α-reductase inhibitory activity of certain herbs useful in the management of androgenic disorders. Ganoderma lucidum (Curtis) P. Karst (GL), Urtica dioica Linn. (UD), Caesalpinia bonducella Fleming. (CB), Tribulus terrestris Linn. (TT), Pedalium murex Linn. (PM), Sphaeranthus indicus Linn. (SI), Cuscuta reflexa Roxb. (CR), Citrullus colocynthis Schrad. (CC), Benincasa hispida Cogn. (BH), Phyllanthus niruri Linn. (PN) and Echinops echinatus Linn. (EE) were included in the study. Petroleum ether, ethanol and aqueous extracts of these herbs were tested for their 5α-reductase inhibitory activity against the standard 5α-reductase inhibitor, finasteride. A biochemical method to determine the activity of 5α-reductase was used to evaluate the inhibition of different extracts to the enzyme. The optical density (OD) value of each sample was measured continuously with ultraviolet spectrophotometer for the reason that the substrate NADPH has a specific absorbance at 340 nm. As the enzyme 5α-reductase uses NADPH as a substrate, so in the presence of 5α-reductase inhibitor, the NADPH concentration will increase with the function of time. This method thus implicates the activity of 5α-reductase. The method proved to be extremely useful to screen the herbs for their 5α-reductase inhibitory potential. GL, UD, BH, SI and CR came out to be promising candidates for further exploring their antiandrogenic properties. PMID:23710567

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

    PubMed Central

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

    1996-01-01

    Pentaerythritol tetranitrate reductase, which reductively liberates nitrite from nitrate esters, is related to old yellow enzyme. Pentaerythritol tetranitrate reductase follows a ping-pong mechanism with competitive substrate inhibition by NADPH, is strongly inhibited by steroids, and is capable of reducing the unsaturated bond of 2-cyclohexen-1-one. PMID:8932320

  15. 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.

  16. The functional nitrite reductase activity of the heme-globins

    PubMed Central

    2008-01-01

    Hemoglobin and myoglobin are among the most extensively studied proteins, and nitrite is one of the most studied small molecules. Recently, multiple physiologic studies have surprisingly revealed that nitrite represents a biologic reservoir of NO that can regulate hypoxic vasodilation, cellular respiration, and signaling. These studies suggest a vital role for deoxyhemoglobin- and deoxymyoglobin-dependent nitrite reduction. Biophysical and chemical analysis of the nitrite-deoxyhemoglobin reaction has revealed unexpected chemistries between nitrite and deoxyhemoglobin that may contribute to and facilitate hypoxic NO generation and signaling. The first is that hemoglobin is an allosterically regulated nitrite reductase, such that oxygen binding increases the rate of nitrite conversion to NO, a process termed R-state catalysis. The second chemical property is oxidative denitrosylation, a process by which the NO formed in the deoxyhemoglobin-nitrite reaction that binds to other deoxyhemes can be released due to heme oxidation, releasing free NO. Third, the reaction undergoes a nitrite reductase/anhydrase redox cycle that catalyzes the anaerobic conversion of 2 molecules of nitrite into dinitrogen trioxide (N2O3), an uncharged molecule that may be exported from the erythrocyte. We will review these reactions in the biologic framework of hypoxic signaling in blood and the heart. PMID:18596228

  17. Life with too much polyprenol–polyprenol reductase deficiency

    PubMed Central

    Gründahl, J.E.H.; Guan, Z.; Rust, S.; Reunert, J.; Müller, B.; Du Chesne, I.; Zerres, K.; Rudnik-Schöneborn, S.; Ortiz-Brüchle, N.; Häusler, M.G.; Siedlecka, J.; Swiezewska, E.; Raetz, C.R.H.; Marquardt, T.

    2012-01-01

    Congenital disorders of glycosylation (CDG) are caused by a dysfunction of glycosylation, an essential step in the manufacturing process of glycoproteins. This paper focuses on a 6-year-old patient with a new type of CDG-I caused by a defect of the steroid 5α reductase type 3 gene (SRD5A3). The clinical features were psychomotor retardation, pathological nystagmus, slight muscular hypotonia and microcephaly. SRD5A3 was recently identified encoding the polyprenol reductase, an enzyme catalyzing the final step of the biosynthesis of dolichol, which is required for the assembly of the glycans needed for N-glycosylation. Although an early homozygous stop-codon (c.57G> A [W19X]) with no functional protein was found in the patient, about 70% of transferrin (Tf) was correctly glycosylated. Quantification of dolichol and unreduced polyprenol in the patient's fibroblasts demonstrated a high polyprenol/dolichol ratio with normal amounts of dolichol, indicating that high polyprenol levels might compete with dolichol for the initiation of N-glycan assembly but without supporting normal glycosylation and that there must be an alternative pathway for dolichol biosynthesis. PMID:22304929

  18. Optimisation of nitrate reductase enzyme activity to synthesise silver nanoparticles.

    PubMed

    Khodashenas, Bahareh; Ghorbani, Hamid Reza

    2016-06-01

    Today, the synthesis of silver nanoparticles (Ag NPs) is very common since it has many applications in different areas. The synthesis of these nanoparticles is done by means of physical, chemical, or biological methods. However, due to its inexpensive and environmentally friendly features, the biological method is more preferable. In the present study, using nitrate reductase enzyme available in the Escherichia coli (E. coli) bacterium, the biosynthesis of Ag NPs was investigated. In addition, the activity of the nitrate reductase enzyme was optimised by changing its cultural conditions, and the effects of silver nitrate (AgNO(3)) concentration and enzyme amount on nanoparticles synthesis were studied. Finally, the produced nanoparticles were studied using ultraviolet -visible (UV-Vis) spectrophotometer, dynamic light scattering technique, and transmission electron microscopy. UV-Visible spectrophotometric study showed the characteristic peak for Ag NPs at wavelength 405-420 nm for 1 mM metal precursor solution (AgNO(3)) with 1, 5, 10, and 20 cc supernatant and 435 nm for 0.01M AgNO(3) with 20 cc supernatant. In this study, it was found that there is a direct relationship between the AgNO(3) concentration and the size of produced Ag NPs. PMID:27256897

  19. New pteridine substrates for dihydropteridine reductase and horseradish peroxidase.

    PubMed Central

    Armarego, W L; Ohnishi, A; Taguchi, H

    1986-01-01

    The oxidation of 4,5-diaminopyrimidin-6(1H)-one, 5,6,7,8-tetrahydropteridin-4(3H)-one, its 6-methyl and cis-6,7-dimethyl derivatives, and 6-methyl- and cis-6-7-dimethyl-5,6,7,8-tetrahydropterins, by horseradish peroxidase/H2O2 is enzymic and follows Michaelis-Menten kinetics, and its Km and kcat. values were determined. This oxidation of 5,6,7,8-tetrahydropterins produces quinonoid dihydropterins of established structure, and they are known to be specific substrates for dihydropteridine reductase. By analogy the peroxidase/H2O2 oxidation of the 5,6,7,8-tetrahydropteridin-4(3H)-ones should produce similar quinonoid dihydro species. The quinonoid species derived from 5,6,7,8-tetrahydropteridin-4(3H)-one and its 6-methyl and cis-6,7-dimethyl derivatives are shown to be viable substrates for human brain dihydropteridine reductase, and apparent Km and Vmax. values are reported. PMID:3718470

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

    PubMed

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

    2015-12-01

    Primary nitrogen assimilation in plants includes the reduction of nitrite to ammonium in the chloroplasts by the enzyme nitrite reductase (NiR EC:1.7.7.1) or in the plastids of non-photosynthetic organs. Here we report on a study overexpressing the Arabidopsis thaliana NiR (AtNiR) gene in tobacco plants under the control of a constitutive promoter (CERV - Carnation Etched Ring Virus). The aim was to overexpress AtNiR in an attempt to alter the level of residual nitrite in the leaf which can act as precursor to the formation of nitrosamines. The impact of increasing the activity of AtNiR produced an increase in leaf protein and a stay-green phenotype in the primary transformed AtNiR population. Investigation of the T1 homozygous population demonstrated elevated nitrate reductase (NR) activity, reductions in leaf nitrite and nitrate and the amino acids proline, glutamine and glutamate. Chlorophyl content of the transgenic lines was increased, as evidenced by the stay-green phenotype. This reveals the importance of NiR in primary nitrogen assimilation and how modification of this key enzyme affects both the nitrogen and carbon metabolism of tobacco plants. PMID:26447683

  1. 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

  2. Evolution Alters the Enzymatic Reaction Coordinate of Dihydrofolate Reductase

    PubMed Central

    2015-01-01

    How evolution has affected enzyme function is a topic of great interest in the field of biophysical chemistry. Evolutionary changes from Escherichia coli dihydrofolate reductase (ecDHFR) to human dihydrofolate reductase (hsDHFR) have resulted in increased catalytic efficiency and an altered dynamic landscape in the human enzyme. Here, we show that a subpicosecond protein motion is dynamically coupled to hydride transfer catalyzed by hsDHFR but not ecDHFR. This motion propagates through residues that correspond to mutational events along the evolutionary path from ecDHFR to hsDHFR. We observe an increase in the variability of the transition states, reactive conformations, and times of barrier crossing in the human system. In the hsDHFR active site, we detect structural changes that have enabled the coupling of fast protein dynamics to the reaction coordinate. These results indicate a shift in the DHFR family to a form of catalysis that incorporates rapid protein dynamics and a concomitant shift to a more flexible path through reactive phase space. PMID:25369552

  3. Azotobacter vinelandii NADPH:ferredoxin reductase cloning, sequencing, and overexpression.

    PubMed

    Isas, J M; Yannone, S M; Burgess, B K

    1995-09-01

    Azotobacter vinelandii ferredoxin I (AvFdI) controls the expression of another protein that was originally designated Protein X. Recently we reported that Protein X is a NADPH-specific flavoprotein that binds specifically to FdI (Isas, J.M., and Burgess, B.K. (1994) J. Biol. Chem. 269, 19404-19409). The gene encoding this protein has now been cloned and sequenced. Protein X is 33% identical and has an overall 53% similarity with the fpr gene product from Escherichia coli that encodes NADPH:ferredoxin reductase. On the basis of this similarity and the similarity of the physical properties of the two proteins, we now designate Protein X as A. vinelandii NADPH:ferredoxin reductase and its gene as the fpr gene. The protein has been overexpressed in its native background in A. vinelandii by using the broad host range multicopy plasmid, pKT230. In addition to being regulated by FdI, the fpr gene product is overexpressed when A. vinelandii is grown under N2-fixing conditions even though the fpr gene is not preceded by a nif specific promoter. By analogy to what is known about fpr expression in E. coli, we propose that FdI may exert its regulatory effect on fpr by interacting with the SoxRS regulon. PMID:7673160

  4. SORGOdb: Superoxide Reductase Gene Ontology curated DataBase

    PubMed Central

    2011-01-01

    Background Superoxide reductases (SOR) catalyse the reduction of superoxide anions to hydrogen peroxide and are involved in the oxidative stress defences of anaerobic and facultative anaerobic organisms. Genes encoding SOR were discovered recently and suffer from annotation problems. These genes, named sor, are short and the transfer of annotations from previously characterized neelaredoxin, desulfoferrodoxin, superoxide reductase and rubredoxin oxidase has been heterogeneous. Consequently, many sor remain anonymous or mis-annotated. Description SORGOdb is an exhaustive database of SOR that proposes a new classification based on domain architecture. SORGOdb supplies a simple user-friendly web-based database for retrieving and exploring relevant information about the proposed SOR families. The database can be queried using an organism name, a locus tag or phylogenetic criteria, and also offers sequence similarity searches using BlastP. Genes encoding SOR have been re-annotated in all available genome sequences (prokaryotic and eukaryotic (complete and in draft) genomes, updated in May 2010). Conclusions SORGOdb contains 325 non-redundant and curated SOR, from 274 organisms. It proposes a new classification of SOR into seven different classes and allows biologists to explore and analyze sor in order to establish correlations between the class of SOR and organism phenotypes. SORGOdb is freely available at http://sorgo.genouest.org/index.php. PMID:21575179

  5. Recessive congenital methaemoglobinaemia: cytochrome b(5) reductase deficiency.

    PubMed

    Percy, Melanie J; Lappin, Terry R

    2008-05-01

    Some 60 years ago, Quentin Gibson reported the first hereditary disorder involving an enzyme when he deduced that familial methaemoglobinaemia was caused by an enzymatic lesion associated with the glycolysis pathway in red blood cells. This disorder, now known as recessive congenital methaemoglobinaemia (RCM), is caused by NADH-cytochrome b5 reductase (cb(5)r) deficiency. Two distinct clinical forms, types I and II, have been recognized, both characterized by cyanosis from birth. In type II, the cyanosis is accompanied by neurological impairment and reduced life expectancy. Cytochrome b(5) reductase is composed of one FAD and one NADH binding domain linked by a hinge region. It is encoded by the CYB5R3 (previously known as DIA1) gene and more than 40 mutations have been described, some of which are common to both types of RCM. Mutations associated with type II tend to cause incorrect splicing, disruption of the active site or truncation of the protein. At present the description of the sequence variants of cb(5)r in the literature is confusing, due to the use of two conventions which differ by one codon position. Herein we propose a new system for nomenclature of cb(5)r based on recommendations of the Human Genome Variation Society. The development of a heterologous expression system has allowed the impact of naturally occurring variants of cb(5)r to be assessed and has provided insight into the function of cb(5)r. PMID:18318771

  6. 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. PMID:21723457

  7. The Role of Thioredoxin Reductases in Brain Development

    PubMed Central

    Soerensen, Jonna; Jakupoglu, Cemile; Beck, Heike; Förster, Heidi; Schmidt, Jörg; Schmahl, Wolfgang; Schweizer, Ulrich

    2008-01-01

    The thioredoxin-dependent system is an essential regulator of cellular redox balance. Since oxidative stress has been linked with neurodegenerative disease, we studied the roles of thioredoxin reductases in brain using mice with nervous system (NS)-specific deletion of cytosolic (Txnrd1) and mitochondrial (Txnrd2) thioredoxin reductase. While NS-specific Txnrd2 null mice develop normally, mice lacking Txnrd1 in the NS were significantly smaller and displayed ataxia and tremor. A striking patterned cerebellar hypoplasia was observed. Proliferation of the external granular layer (EGL) was strongly reduced and fissure formation and laminar organisation of the cerebellar cortex was impaired in the rostral portion of the cerebellum. Purkinje cells were ectopically located and their dendrites stunted. The Bergmann glial network was disorganized and showed a pronounced reduction in fiber strength. Cerebellar hypoplasia did not result from increased apoptosis, but from decreased proliferation of granule cell precursors within the EGL. Of note, neuron-specific inactivation of Txnrd1 did not result in cerebellar hypoplasia, suggesting a vital role for Txnrd1 in Bergmann glia or neuronal precursor cells. PMID:18350150

  8. Synthesis and metabolism of inhibitors of ribonucleotide reductase

    SciTech Connect

    Smith, F.T.

    1985-01-01

    In an effort to prepare more effective inhibitors of ribo-nucleotide reductase a series of 2-substituted-4,6-dihydroxypyrimidines was prepared via the appropriately substituted benzamidine. None of the compounds exhibited in vivo activity against L1210 leukemia. No further testing was performed. In order to investigate the metabolism of 3,4-dihydroxybenzohydroxamic acid, a known inhibitor of ribonucleotide reductase, radiolabeled 3,4-dihydroxybenzohydroxamic acid was synthesized by a modification of the procedure of Pichat and Tostain. /sup 14/C-3,4-Dihydroxybenzoic acid was converted to the methyl ester and subsequently reacted with hydroxylamine to give the hydroxamic acid. /sup 14/C-3,4-Dihydroxybenzohydroxamic acid was given i.p. to Sprague-Dawley rats. Excretion occurred mainly (72%) via the urine. HPLC coupled with GC/MS analyses showed that the compound was excreted mainly unchanged. The compound was metabolized to 3,4-dihydroxybenzamide, 4-methoxy-3-hydroxybenzohydroxamic acid, and 4-hydroxy-3-methoxybenzohydroxamic acid. HPLC analysis also showed the lack of formation of any glucuronide or sulfate conjugates through either the hydroxamic acid or catechol functionalities.

  9. Interspecific variation for thermal dependence of glutathione reductase in sainfoin.

    PubMed

    Kidambi, S P; Mahan, J R; Matches, A G

    1990-05-01

    Understanding the biochemical and physiological consequences of species variation would expedite improvement in agronomically useful genotypes of sainfoin (Onobrychis spp.) Information on variation among sainfoin species is lacking on thermal dependence of glutathione reductase (B.C. 1.6.4.2.), which plays an important role in the protection of plants from both high and low temperature stresses by preventing harmful oxidation of enzymes and membranes. Our objective was to investigate the interspecific variation for thermal dependency of glutathione reductase in sainfoin. Large variation among species was found for: (i) the minimum apparent Km (0.4-2.5 μM NADPH), (ii) the temperature at which the minimum apparent Km was observed (15°-5°C), and (iii) the thermal kinetic windows (2°-30°C width) over a 15°-45°C temperature gradient. In general, tetraploid species had narrower (≤17°C) thermal kinetic windows than did diploid species (∼30°C), with one exception among the diploids. Within the tetraploid species, the cultivars of O. viciifolia had a broader thermal kinetic window (≥7°C) than the plant introduction (PI 212241, >2 °C) itself. PMID:24226572

  10. A mutant of barley lacking NADH-hydroxypyruvate reductase

    SciTech Connect

    Blackwell, R.; Lea, P. )

    1989-04-01

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

  11. 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. PMID:3511844

  12. An electron-bifurcating caffeyl-CoA reductase.

    PubMed

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

    2013-04-19

    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. Properties of seleno-methionine substituted assimilatory nitrate reductase

    SciTech Connect

    Solomonson, L.P.; Barber, M.J. )

    1991-03-11

    Assimilatory NADH:nitrate reductase contains FAD, heme and Mo-pterin arranged in an NADH{yields}FAD{yields}heme{yields}Mo-Pterin{yields}NO{sub 3} electron transfer sequence. A functional Mo-pterin center is essential for all nitrate-reducing activities. To assess the possible functional role of Met, a Se-Met substituted NR was obtained by addition of Se-Met to ammonia-grown Chlorella cells prior to induction of NR activity. Increase in NADH:dehydrogenase partial activities and nitrate reductase protein proceeded normally following induction but little or no nitrate-reducing activity was expressed. This effect was observed with as little as 10{sup {minus}5} Se-Met and was prevented by a 10-fold excess of Met. A less pronounced effect was observed with 10{sup {minus}4}M Se-Cys. The purified Se-Met substituted enzyme exhibited the same apparent physical size, spectral properties and NADH dehydrogenase activities as control NR but was devoid of nitrate-reducing activities. These results suggest that one or more Met residues are essential for the catalytic function of the molybdo-pterin center of assimilatory NR.

  14. Anti-diabetic and anti-Alzheimer's disease activities of Angelica decursiva.

    PubMed

    Yousof Ali, Md; Jung, Hyun Ah; Choi, Jae Sue

    2015-12-01

    Diabetes mellitus (DM) and Alzheimer's disease (AD) constitute two global health issues. DM is an ever-increasing epidemic affecting millions of elderly people worldwide, causing major repercussions on patients' daily lives, mostly due to chronic complications. Complications from DM can affect the brain, thereby characterizing DM as a risk factor for AD. In the present study, we examined the inhibitory activity of methanol extracts of different parts of 12 Angelica species against α-glucosidase, protein tyrosine phosphatase 1B (PTP1B), acetylcholinesterase (AChE), and butyrylcholinesterase (BChE). The methanol extract of Angelica decursiva exhibited the highest inhibitory activities against α-glucosidase, PTP1B, AChE, and BChE and so was selected for further investigation. Repeated column chromatography based on bioactivity-guided fractionation yielded seven compounds (1-7). Among these compounds, nodakenin (1), nodakenetin (2), umbelliferone (3), cis-3'-acetyl-4'-angeloylkhellactone (4), 3'(R)-O-acetyl-4'(S)-O-tigloylkhellactone (5), isorutarine (6), and para-hydroxybenzoic acid (7) exhibited potent inhibitory activities against α-glucosidase, PTP1B, rat lens aldose reductase (RLAR), AChE, BChE, and β-site amyloid precursor protein cleaving enzyme 1 (BACE1). Our results clearly indicate the potential inhibition of α-glucosidase, PTP1B, RLAR, AChE, BChE, and BACE1 by A. decursiva as well as its isolated constituents, which could be further explored to develop therapeutic modalities for the treatment of DM and AD. PMID:26152875

  15. Pinpointing a Mechanistic Switch Between Ketoreduction and "Ene" Reduction in Short-Chain Dehydrogenases/Reductases.

    PubMed

    Lygidakis, Antonios; Karuppiah, Vijaykumar; Hoeven, Robin; Ní Cheallaigh, Aisling; Leys, David; Gardiner, John M; Toogood, Helen S; Scrutton, Nigel S

    2016-08-01

    Three enzymes of the Mentha essential oil biosynthetic pathway are highly homologous, namely the ketoreductases (-)-menthone:(-)-menthol reductase and (-)-menthone:(+)-neomenthol reductase, and the "ene" reductase isopiperitenone reductase. We identified a rare catalytic residue substitution in the last two, and performed comparative crystal structure analyses and residue-swapping mutagenesis to investigate whether this determines the reaction outcome. The result was a complete loss of native activity and a switch between ene reduction and ketoreduction. This suggests the importance of a catalytic glutamate vs. tyrosine residue in determining the outcome of the reduction of α,β-unsaturated alkenes, due to the substrate occupying different binding conformations, and possibly also to the relative acidities of the two residues. This simple switch in mechanism by a single amino acid substitution could potentially generate a large number of de novo ene reductases. PMID:27411040

  16. Natural variation in arsenate tolerance identifies an arsenate reductase in Arabidopsis thaliana.

    PubMed

    Sánchez-Bermejo, Eduardo; Castrillo, Gabriel; del Llano, Bárbara; Navarro, Cristina; Zarco-Fernández, Sonia; Martinez-Herrera, Dannys Jorge; Leo-del Puerto, Yolanda; Muñoz, Riansares; Cámara, Carmen; Paz-Ares, Javier; Alonso-Blanco, Carlos; Leyva, Antonio

    2014-01-01

    The enormous amount of environmental arsenic was a major factor in determining the biochemistry of incipient life forms early in the Earth's history. The most abundant chemical form in the reducing atmosphere was arsenite, which forced organisms to evolve strategies to manage this chemical species. Following the great oxygenation event, arsenite oxidized to arsenate and the action of arsenate reductases became a central survival requirement. The identity of a biologically relevant arsenate reductase in plants nonetheless continues to be debated. Here we identify a quantitative trait locus that encodes a novel arsenate reductase critical for arsenic tolerance in plants. Functional analyses indicate that several non-additive polymorphisms affect protein structure and account for the natural variation in arsenate reductase activity in Arabidopsis thaliana accessions. This study shows that arsenate reductases are an essential component for natural plant variation in As(V) tolerance. PMID:25099865

  17. Inhibition of rat steroid 5 alpha-reductase (isozyme 1) by suramin.

    PubMed

    Taylor, M F; Bhattacharyya, A K; Collins, D C

    1995-07-01

    In this study, we show the inhibition of rat steroid 5 alpha-reductase (isozyme 1) by suramin. The enzyme activity decreased in a dose-dependent manner as suramin concentrations increased with the calculated drug dose required for 50% inhibition (at 5 microM testosterone and 200 microM NADPH) being 13 microM. Suramin showed non-competitive inhibition of 5 alpha-reductase with respect to testosterone (KT1 = 2.4 microM) and competitive inhibition with respect to NADPH (KiNADPH = 220 nM). Furthermore, suramin and NADP+, but not NAD+, protected 5 alpha-reductase from labeling by 2-azido-NADP+, a photoactive probe which has recently been used to identify the NADPH binding domain of 5 alpha-reductase. These results suggest that suramin inhibits rat steroid 5 alpha-reductase (isozyme 1) at the level of NADPH binding to the enzyme. PMID:7482629

  18. Immunocytochemical localization of short-chain family reductases involved in menthol biosynthesis in peppermint.

    PubMed

    Turner, Glenn W; Davis, Edward M; Croteau, Rodney B

    2012-06-01

    Biosynthesis of the p-menthane monoterpenes in peppermint occurs in the secretory cells of the peltate glandular trichomes and results in the accumulation of primarily menthone and menthol. cDNAs and recombinant enzymes are well characterized for eight of the nine enzymatic steps leading from the 5-carbon precursors to menthol, and subcellular localization of several key enzymes suggests a complex network of substrate and product movement is required during oil biosynthesis. In addition, studies concerning the regulation of oil biosynthesis have demonstrated a temporal partition of the pathway into an early, biosynthetic program that results in the accumulation of menthone and a later, oil maturation program that leads to menthone reduction and concomitant menthol accumulation. The menthone reductase responsible for the ultimate pathway reduction step, menthone-menthol reductase (MMR), has been characterized and found to share significant sequence similarity with its counterpart reductase, a menthone-neomenthol reductase, which catalyzes a minor enzymatic reaction associated with oil maturation. Further, the menthone reductases share significant sequence similarity with the temporally separate and mechanistically different isopiperitenone reductase (IPR). Here we present immunocytochemical localizations for these reductases using a polyclonal antibody raised against menthone-menthol reductase. The polyclonal antibody used for this study showed little specificity between these three reductases, but by using it for immunostaining of tissues of different ages we were able to provisionally separate staining of an early biosynthetic enzyme, IPR, found in young, immature leaves from that of the oil maturation enzyme, MMR, found in older, mature leaves. Both reductases were localized to the cytoplasm and nucleoplasm of the secretory cells of peltate glandular trichomes, and were absent from all other cell types examined. PMID:22170164

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

    PubMed Central

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

    1993-01-01

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

  20. Functional complementation of a nitrate reductase defective mutant of a green alga Dunaliella viridis by introducing the nitrate reductase gene.

    PubMed

    Sun, Yu; Gao, Xiaoshu; Li, Qiyun; Zhang, Qingqi; Xu, Zhengkai

    2006-08-01

    Nitrate reductase (NR) catalyzes NAD (P) H dependent reduction of nitrate to nitrite. Transformation systems have been established in several species of green algae by nitrate reductase gene functional complementation. In this report, an endogenous NR cDNA (3.4 kb) and a genomic fragment (14.6 kb) containing the NR gene (DvNIA1) were isolated from the D. viridis cDNA and genomic libraries respectively. Southern blot and Northern blot analyses showed that this gene exists as a single copy in D. viridis and is induced by nitrate. To obtain a NR defective mutant as a recipient strain, D. viridis cells were treated with a chemical mutagen and then cultured on a chlorate-containing plate to enrich chlorate tolerant mutants. Southern analysis showed that one isolate, B14, had a deletion in the DvNIA1 gene region. Using electroporation conditions determined in this laboratory, plasmid pDVNR containing the intact DvNIA1 gene has been electroporated into the defective mutant B14. Strains retaining a nitrate assimilation phenotype were obtained from nitrate plates after spreading the electroporated cells. In some individual strains, transcription of the introduced gene was detected. NR activity in these strains was slightly higher than that in the defective B14 cell, but excretion of nitrite into culture media was almost as high as that of the wild-type cell. Possible episomal presence of the introduced DNA in D. viridis is discussed. PMID:16797881

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

    PubMed

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

    2015-06-01

    Curcumin is a major component of the plant Curcuma longa L. It is traditionally used as a spice and coloring in foods and is an important ingredient in curry. Curcuminoids have anti-oxidant and anti-inflammatory properties and gained increasing attention as potential neuroprotective and cancer preventive compounds. In the present study, we report that curcumin is a potent tight-binding inhibitor of human carbonyl reductase 1 (CBR1, Ki=223 nM). Curcumin acts as a non-competitive inhibitor with respect to the substrate 2,3-hexandione as revealed by plotting IC50-values against various substrate concentrations and most likely as a competitive inhibitor with respect to NADPH. Molecular modeling supports the finding that curcumin occupies the cofactor binding site of CBR1. Interestingly, CBR1 is one of the most effective human reductases in converting the anthracycline anti-tumor drug daunorubicin to daunorubicinol. The secondary alcohol metabolite daunorubicinol has significantly reduced anti-tumor activity and shows increased cardiotoxicity, thereby limiting the clinical use of daunorubicin. Thus, inhibition of CBR1 may increase the efficacy of daunorubicin in cancer tissue and simultaneously decrease its cardiotoxicity. Western-blots demonstrated basal expression of CBR1 in several cell lines. Significantly less daunorubicin reduction was detected after incubating A549 cell lysates with increasing concentrations of curcumin (up to 60% less with 50 μM curcumin), suggesting a beneficial effect in the co-treatment of anthracycline anti-tumor drugs together with curcumin. PMID:25541467

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

    PubMed

    Zhu, L

    2015-01-01

    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. PMID:26345779

  3. 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

  4. The nitrate-sensing NasST system regulates nitrous oxide reductase and periplasmic nitrate reductase in Bradyrhizobium japonicum.

    PubMed

    Sánchez, Cristina; Itakura, Manabu; Okubo, Takashi; Matsumoto, Takashi; Yoshikawa, Hirofumi; Gotoh, Aina; Hidaka, Masafumi; Uchida, Takafumi; Minamisawa, Kiwamu

    2014-10-01

    The soybean endosymbiont Bradyrhizobium japonicum is able to scavenge the greenhouse gas N2O through the N2O reductase (Nos). In previous research, N2O emission from soybean rhizosphere was mitigated by B. japonicum Nos(++) strains (mutants with increased Nos activity). Here, we report the mechanism underlying the Nos(++) phenotype. Comparative analysis of Nos(++) mutant genomes showed that mutation of bll4572 resulted in Nos(++) phenotype. bll4572 encodes NasS, the nitrate (NO3(-))-sensor of the two-component NasST regulatory system. Transcriptional analyses of nosZ (encoding Nos) and other genes from the denitrification process in nasS and nasST mutants showed that, in the absence of NO3(-) , nasS mutation induces nosZ and nap (periplasmic nitrate reductase) via nasT. NO3(-) addition dissociated the NasS-NasT complex in vitro, suggesting the release of the activator NasT. Disruption of nasT led to a marked decrease in nosZ and nap transcription in cells incubated in the presence of NO3(-). Thus, although NasST is known to regulate the NO3(-)-mediated response of NO3(-) assimilation genes in bacteria, our results show that NasST regulates the NO3(-) -mediated response of nosZ and napE genes, from the dissimilatory denitrification pathway, in B. japonicum. PMID:24947409

  5. Vibrio harveyi Nitroreductase Is Also a Chromate Reductase

    PubMed Central

    Kwak, Young Hak; Lee, Dong Seok; Kim, Han Bok

    2003-01-01

    The chromate reductase purified from Pseudomonas ambigua was found to be homologous with several nitroreductases. Escherichia coli DH5α and Vibrio harveyi KCTC 2720 nitroreductases were chosen for the present study, and their chromate-reducing activities were determined. A fusion between glutathione S-transferase (GST) and E. coli DH5α NfsA (GST-EcNfsA), a fusion between GST and E. coli DH5α NfsB (GST-EcNfsB), and a fusion between GST and V. harveyi KCTC 2720 NfsA (GST-VhNfsA) were prepared for their overproduction and easy purification. GST-EcNfsA, GST-EcNFsB, and GST-VhNFsA efficiently reduced nitrofurazone and 2,4,6-trinitrotoluene (TNT) as their nitro substrates. The Km values for GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA for chromate reduction were 11.8, 23.5, and 5.4 μM, respectively. The Vmax values for GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA were 3.8, 3.9, and 10.7 nmol/min/mg of protein, respectively. GST-VhNfsA was the most effective of the three chromate reductases, as determined by each Vmax/Km value. The optimal temperatures of GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA for chromate reduction were 55, 30, and 30°C, respectively. Thus, it is confirmed that nitroreductase can also act as a chromate reductase. Nitroreductases may be used in chromate remediation. GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA have a molecular mass of 50 kDa and exist as a monomer in solution. Thin-layer chromatography showed that GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA contain FMN as a cofactor. GST-VhNfsA reduced Cr(VI) to Cr(III). Cr(III) was much less toxic to E. coli than Cr(VI). PMID:12902220

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

    PubMed Central

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

    1997-01-01

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

  7. Ascorbate free radical reductase mRNA levels are induced by wounding.

    PubMed Central

    Grantz, A A; Brummell, D A; Bennett, A B

    1995-01-01

    A cDNA clone encoding ascorbate free radical (AFR) reductase (EC 1.6.5.4) was isolated from tomato (Lycopersicon esculentum Mill.) and its mRNA levels were analyzed. The cDNA encoded a deduced protein of 433 amino acids and possessed amino acid domains characteristic of flavin adenine dinucleotide- and NAD(P)H-binding proteins but did not possess typical eukaryotic targeting sequences, suggesting that it encodes a cytosolic form of AFR reductase. Low-stringency genomic DNA gel blot analysis indicated that a single nuclear gene encoded this enzyme. Total ascorbate contents were greatest in leaves, with decreasing amounts in stems and roots and relatively constant levels in all stages of fruit. AFR reductase activity was inversely correlated with total ascorbate content, whereas the relative abundance of AFR reductase mRNA was directly correlated with enzyme activity in tissues examined. AFR reductase mRNA abundance increased dramatically in response to wounding, a treatment that is known to also induce ascorbate-dependent prolyl hydroxylation required for the accumulation of hydroxyproline-rich glycoproteins. In addition, AFR reductase may contribute to maintaining levels of ascorbic acid for protection against wound-induced free radical-mediated damage. Collectively, the results suggest that AFR reductase activity is regulated at the level of mRNA abundance by low ascorbate contents or by factors that promote ascorbate utilization. PMID:7784511

  8. Immunochemical characterization of NADPH-cytochrome P-450 reductase from Jerusalem artichoke and other higher plants.

    PubMed Central

    Benveniste, I; Lesot, A; Hasenfratz, M P; Durst, F

    1989-01-01

    Polyclonal antibodies were prepared against NADPH-cytochrome P-450 reductase purified from Jerusalem artichoke. These antibodies inhibited efficiently the NADPH-cytochrome c reductase activity of the purified enzyme, as well as of Jerusalem artichoke microsomes. Likewise, microsomal NADPH-dependent cytochrome P-450 mono-oxygenases (cinnamate and laurate hydroxylases) were efficiently inhibited. The antibodies were only slightly inhibitory toward microsomal NADH-cytochrome c reductase activity, but lowered NADH-dependent cytochrome P-450 mono-oxygenase activities. The Jerusalem artichoke NADPH-cytochrome P-450 reductase is characterized by its high Mr (82,000) as compared with the enzyme from animals (76,000-78,000). Western blot analysis revealed cross-reactivity of the Jerusalem artichoke reductase antibodies with microsomes from plants belonging to different families (monocotyledons and dicotyledons). All of the proteins recognized by the antibodies had an Mr of approx. 82,000. No cross-reaction was observed with microsomes from rat liver or Locusta migratoria midgut. The cross-reactivity generally paralleled well the inhibition of reductase activity: the enzyme from most higher plants tested was inhibited by the antibodies; whereas Gingko biloba, Euglena gracilis, yeast, rat liver and insect midgut activities were insensitive to the antibodies. These results point to structural differences, particularly at the active site, between the reductases from higher plants and the enzymes from phylogenetically distant plants and from animals. Images Fig. 5. PMID:2499315

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

    PubMed

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

    2015-11-25

    A steroid 5β-reductase gene corresponding to the hypothetical protein LOC100247199 from leaves of Vitis vinifera (var. 'Chardonnay') was cloned and overexpressed in Escherichia coli. The recombinant protein showed 5β-reductase activity when progesterone was used as a substrate. The reaction was stereoselective, producing only 5β-products such as 5β-pregnane-3,20-dione. Other small substrates (terpenoids and enones) were also accepted as substrates, indicating the highly promiscuous character of the enzyme class. Our results show that the steroid 5β-reductase gene, encoding an orthologous enzyme described as a key enzyme in cardenolide biosynthesis, is also expressed in leaves of the cardenolide-free plant V. vinifera. We emphasize the fact that, on some occasions, different reductases (e.g., progesterone 5β-reductase and monoterpenoid reductase) can also use molecules that are similar to the final products as a substrate. Therefore, in planta, the different reductases may contribute to the immense number of diverse small natural products finally leading to the flavor of wine. PMID:26537436

  10. Divergent evolution of protein conformational dynamics in dihydrofolate reductase.

    PubMed

    Bhabha, Gira; Ekiert, Damian C; Jennewein, Madeleine; Zmasek, Christian M; Tuttle, Lisa M; Kroon, Gerard; Dyson, H Jane; Godzik, Adam; Wilson, Ian A; Wright, Peter E

    2013-11-01

    Molecular evolution is driven by mutations, which may affect the fitness of an organism and are then subject to natural selection or genetic drift. Analysis of primary protein sequences and tertiary structures has yielded valuable insights into the evolution of protein function, but little is known about the evolution of functional mechanisms, protein dynamics and conformational plasticity essential for activity. We characterized the atomic-level motions across divergent members of the dihydrofolate reductase (DHFR) family. Despite structural similarity, Escherichia coli and human DHFRs use different dynamic mechanisms to perform the same function, and human DHFR cannot complement DHFR-deficient E. coli cells. Identification of the primary-sequence determinants of flexibility in DHFRs from several species allowed us to propose a likely scenario for the evolution of functionally important DHFR dynamics following a pattern of divergent evolution that is tuned by cellular environment. PMID:24077226

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

    SciTech Connect

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

    2010-03-19

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

  12. Go Green: The Anti-Inflammatory Effects of Biliverdin Reductase

    PubMed Central

    Wegiel, Barbara; Otterbein, Leo E.

    2012-01-01

    Biliverdin (BV) has emerged as a cytoprotective and important anti-inflammatory molecule. Conversion of BV to bilirubin (BR) is catalyzed by biliverdin reductase (BVR) and is required for the downstream signaling and nuclear localization of BVR. Recent data by others and us make clear that BVR is a critical regulator of innate immune responses resulting from acute insult and injury and moreover, that a lack of BVR results in an enhanced proinflammatory phenotype. In macrophages, BVR is regulated by its substrate BV which leads to activation of the PI3K–Akt-IL-10 axis and inhibition of TLR4 expression via direct binding of BVR to the TLR4 promoter. In this review, we will summarize recent findings on the role of BVR and the bile pigments in inflammation in context with its activity as an enzyme, receptor, and transcriptional regulator. PMID:22438844

  13. Genetic Evidence for a Molybdopterin-Containing Tellurate Reductase

    PubMed Central

    Theisen, Joanne; Zylstra, Gerben J.

    2013-01-01

    The genetic identity and cofactor composition of the bacterial tellurate reductase are currently unknown. In this study, we examined the requirement of molybdopterin biosynthesis and molybdate transporter genes for tellurate reduction in Escherichia coli K-12. The results show that mutants deleted of the moaA, moaB, moaE, or mog gene in the molybdopterin biosynthesis pathway lost the ability to reduce tellurate. Deletion of the modB or modC gene in the molybdate transport pathway also resulted in complete loss of tellurate reduction activity. Genetic complementation by the wild-type sequences restored tellurate reduction activity in the mutant strains. These findings provide genetic evidence that tellurate reduction in E. coli involves a molybdoenzyme. PMID:23475618

  14. Pulse radiolysis studies on superoxide reductase from Treponema pallidum.

    PubMed

    Nivière, V; Lombard, M; Fontecave, M; Houée-Levin, C

    2001-05-25

    Superoxide reductases (SORs) are small metalloenzymes, which catalyze reduction of O2*- to H2O2. The reaction of the enzyme from Treponema pallidum with superoxide was studied by pulse radiolysis methods. The first step is an extremely fast bi-molecular reaction of the ferrous center with O2, with a rate constant of 6 x 10 (8) M(-1) s(-1). A first intermediate is formed which is converted to a second one with a slower rate constant of 4800 s(-1). This latter value is 10 times higher than the corresponding one previously reported in the case of SOR from Desulfoarculus baarsii. The reconstituted spectra for the two intermediates are consistent with formation of transient iron-peroxide species. PMID:11377434

  15. Divergent evolution of protein conformational dynamics in dihydrofolate reductase

    PubMed Central

    Bhabha, Gira; Ekiert, Damian C.; Jennewein, Madeleine; Zmasek, Christian M.; Tuttle, Lisa M.; Kroon, Gerard; Dyson, H. Jane; Godzik, Adam; Wilson, Ian A.; Wright, Peter E.

    2013-01-01

    Molecular evolution is driven by mutations, which may affect the fitness of an organism and are then subject to natural selection or genetic drift. Analysis of primary protein sequences and tertiary structures has yielded valuable insights into the evolution of protein function, but little is known about evolution of functional mechanisms, protein dynamics and conformational plasticity essential for activity. We characterized the atomic-level motions across divergent members of the dihydrofolate reductase (DHFR) family. Despite structural similarity, E. coli and human DHFRs use different dynamic mechanisms to perform the same function, and human DHFR cannot complement DHFR-deficient E. coli cells. Identification of the primary sequence determinants of flexibility in DHFRs from several species allowed us to propose a likely scenario for the evolution of functionally important DHFR dynamics, following a pattern of divergent evolution that is tuned by the cellular environment. PMID:24077226

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

    SciTech Connect

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

    2009-02-13

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

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

    PubMed

    Wang, Xiaoqiang; He, Xianzhi; Lin, Jianqiao; Shao, Hui; Chang, Zhenzhan; Dixon, Richard A

    2006-05-19

    Isoflavonoids play important roles in plant defense and exhibit a range of mammalian health-promoting activities. Isoflavone reductase (IFR) specifically recognizes isoflavones and catalyzes a stereospecific NADPH-dependent reduction to (3R)-isoflavanone. The crystal structure of Medicago sativa IFR with deletion of residues 39-47 has been determined at 1.6A resolution. Structural analysis, molecular modeling and docking, and comparison with the structures of other NADPH-dependent enzymes, defined the putative binding sites for co-factor and substrate and potential key residues for enzyme activity and substrate specificity. Further mutagenesis has confirmed the role of Lys144 as a catalytic residue. This study provides a structural basis for understanding the enzymatic mechanism and substrate specificity of IFRs as well as the functions of IFR-like proteins. PMID:16600295

  18. Thioredoxin reductase 1 suppresses adipocyte differentiation and insulin responsiveness

    PubMed Central

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

    2016-01-01

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

  19. 5-Alpha-Reductase Inhibitors and Combination Therapy.

    PubMed

    Füllhase, Claudius; Schneider, Marc P

    2016-08-01

    By inhibiting the conversion from testosterone to dihydrotestosterone 5-Alpha reductase inhibitors (5ARIs) are able to hinder prostatic growth, shrink prostate volumes, and improve BPH-related LUTS. 5ARIs are particularly beneficial for patients with larger prostates (>30-40ml). Generally the side effects of 5ARI treatment are mild, and according to the FORTA classification 5ARIs are suitable for frail elderly. 5ARI / alpha-blocker (AB) combination therapy showed the best symptomatic outcome and risk reduction for clinical progression. Combining Phosphodieseterase type 5 inhbibitors (PDE5Is) with 5ARIs counteracts the negative androgenic sexual side effects of 5ARIs, and simultaneously combines their synergistic effects on LUTS. PMID:27476125

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

    PubMed Central

    Ellis, E M; Hayes, J D

    1995-01-01

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

  1. 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

  2. Evidence for a hexaheteromeric methylenetetrahydrofolate reductase in Moorella thermoacetica.

    PubMed

    Mock, Johanna; Wang, Shuning; Huang, Haiyan; Kahnt, Jörg; Thauer, Rudolf K

    2014-09-01

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

  3. 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

  4. The modulation of carbonyl reductase 1 by polyphenols.

    PubMed

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

    2015-01-01

    Carbonyl reductase 1 (CBR1), an enzyme belonging to the short-chain dehydrogenases/reductases family, has been detected in all human tissues. CBR1 catalyzes the reduction of many xenobiotics, including important drugs (e.g. anthracyclines, nabumetone, bupropion, dolasetron) and harmful carbonyls and quinones. Moreover, it participates in the metabolism of a number of endogenous compounds and it may play a role in certain pathologies. Plant polyphenols are not only present in many human food sources, but are also a component of many popular dietary supplements and herbal medicines. Many studies reviewed herein have demonstrated the potency of certain flavonoids, stilbenes and curcuminoids in the inhibition of the activity of CBR1. Interactions of these polyphenols with transcriptional factors, which regulate CBR1 expression, have also been reported in several studies. As CBR1 plays an important role in drug metabolism as well as in the protection of the organism against potentially harmful carbonyls, the modulation of its expression/activity may have significant pharmacological and/or toxicological consequences. Some polyphenols (e.g. luteolin, apigenin and curcumin) have been shown to be very potent CBR1 inhibitors. The inhibition of CBR1 seems useful regarding the increased efficacy of anthracycline therapy, but it may cause the worse detoxification of reactive carbonyls. Nevertheless, all known information about the interactions of polyphenols with CBR1 have only been based on the results of in vitro studies. With respect to the high importance of CBR1 and the frequent consumption of polyphenols, in vivo studies would be very helpful for the evaluation of risks/benefits of polyphenol interactions with CBR1. PMID:26415702

  5. Characterization of the nitric oxide reductase from Thermus thermophilus

    PubMed Central

    Schurig-Briccio, Lici A.; Venkatakrishnan, Padmaja; Hemp, James; Bricio, Carlos; Berenguer, José; Gennis, Robert B.

    2013-01-01

    Nitrous oxide (N2O) is a powerful greenhouse gas implicated in climate change. The dominant source of atmospheric N2O is incomplete biological dentrification, and the enzymes responsible for the release of N2O are NO reductases. It was recently reported that ambient emissions of N2O from the Great Boiling Spring in the United States Great Basin are high, and attributed to incomplete denitrification by Thermus thermophilus and related bacterial species [Hedlund BP, et al. (2011) Geobiology 9(6)471–480]. In the present work, we have isolated and characterized the NO reductase (NOR) from T. thermophilus. The enzyme is a member of the cNOR family of enzymes and belongs to a phylogenetic clade that is distinct from previously examined cNORs. Like other characterized cNORs, the T. thermophilus cNOR consists of two subunits, NorB and NorC, and contains a one heme c, one Ca2+, a low-spin heme b, and an active site consisting of a high-spin heme b and FeB. The roles of conserved residues within the cNOR family were investigated by site-directed mutagenesis. The most important and unexpected result is that the glutamic acid ligand to FeB is not essential for function. The E211A mutant retains 68% of wild-type activity. Mutagenesis data and the pattern of conserved residues suggest that there is probably not a single pathway for proton delivery from the periplasm to the active site that is shared by all cNORs, and that there may be multiple pathways within the T. thermophilus cNOR. PMID:23858452

  6. Isolation and characterization of nitric oxide reductase from Paracoccus halodenitrificans.

    PubMed

    Sakurai, N; Sakurai, T

    1997-11-11

    Nitric oxide reductase was isolated from the membrane fraction of a denitrifying bacterium, Paracoccus halodenitrificans, in the presence of n-dodecyl beta-D-maltoside. A relatively simple and effective procedure to purify NO reductase using DEAE-Toyopearl and hydroxyapatite (ceramic) chromatographies has been developed. The enzyme consisted of two subunits with molecular masses of 20 and 42 kDa associated with the c-type heme and two b-type hemes, respectively. The optical and magnetic circular dichroism (MCD) spectra of the oxidized (as isolated) and reduced enzymes indicated that the heme c is in the low-spin state and the hemes b are in the high- and low-spin states. The EPR spectrum also showed the presence of the split high-spin component (g perpendicular = 6.6, 6.0) and two low spin components (gz,y,x = 2.96, 2.26, 1.46, gz = 3.59). Although the presence of an extra iron was suggested from atomic absorption spectroscopy, a non-heme iron could not be detected by colorimetric titrations using ferene and 2-(5-nitro-2-pyridylazo)- 5-(N-propyl-N-sulfopropylamino)phenolate (PAPS). One of the extra signals at g = 4.3 and 2.00 might come from a non-heme iron, while they may originate from an adventitious iron and a certain nonmetallic radical, respectively. When CO acted on the reduced enzyme, both of the low-spin hemes were not affected, and when NO acted on the reduced enzyme, the optical and MCD spectra were of a mixture of the oxidized and reduced enzymes. Consequently, the reduction of NO was supposed to take place at the high-spin heme b. The heme c and the low-spin heme b centers were considered to function as electron mediators during the intermolecular and intramolecular processes. PMID:9374857

  7. Bacteriophage T4 Virion Baseplate Thymidylate Synthetase and Dihydrofolate Reductase

    PubMed Central

    Kozloff, L. M.; Lute, M.; Crosby, L. K.

    1977-01-01

    Additional evidence is presented that both the phage T4D-induced thymidylate synthetase (gp td) and the T4D-induced dihydrofolate reductase (gp frd) are baseplate structural components. With regard to phage td it has been found that: (i) low levels of thymidylate synthetase activity were present in highly purified preparations of T4D ghost particles produced after infection with td+, whereas particles produced after infection with td− had no measurable enzymatic activity; (ii) a mutation of the T4D td gene from tdts to td+ simultaneously produced a heat-stable thymidylate synthetase enzyme and heat-stable phage particles (it should be noted that the phage baseplate structure determines heat lability); (iii) a recombinant of two T4D mutants constructed containing both tdts and frdts genes produced particles whose physical properties indicate that these two molecules physically interact in the baseplate. With regard to phage frd it has been found that two spontaneous revertants each of two different T4D frdts mutants to frd+ not only produced altered dihydrofolate reductases but also formed phage particles with heat sensitivities different from their parents. Properties of T4D particles produced after infection with parental T4D mutants presumed to have a deletion of the td gene and/or the frd gene indicate that these particles still retain some characteristics associated with the presence of both the td and the frd molecules. Furthermore, the particles produced by the deletion mutants have been found to be physically different from the parent particles. PMID:894793

  8. Identification of imine reductase-specific sequence motifs.

    PubMed

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

    2016-05-01

    Chiral amines are valuable building blocks for the production of a variety of pharmaceuticals, agrochemicals and other specialty chemicals. Only recently, imine reductases (IREDs) were discovered which catalyze the stereoselective reduction of imines to chiral amines. Although several IREDs were biochemically characterized in the last few years, knowledge of the reaction mechanism and the molecular basis of substrate specificity and stereoselectivity is limited. To gain further insights into the sequence-function relationships, the Imine Reductase Engineering Database (www.IRED.BioCatNet.de) was established and a systematic analysis of 530 putative IREDs was performed. A standard numbering scheme based on R-IRED-Sk was introduced to facilitate the identification and communication of structurally equivalent positions in different proteins. A conservation analysis revealed a highly conserved cofactor binding region and a predominantly hydrophobic substrate binding cleft. Two IRED-specific motifs were identified, the cofactor binding motif GLGxMGx5 [ATS]x4 Gx4 [VIL]WNR[TS]x2 [KR] and the active site motif Gx[DE]x[GDA]x[APS]x3 {K}x[ASL]x[LMVIAG]. Our results indicate a preference toward NADPH for all IREDs and explain why, despite their sequence similarity to β-hydroxyacid dehydrogenases (β-HADs), no conversion of β-hydroxyacids has been observed. Superfamily-specific conservations were investigated to explore the molecular basis of their stereopreference. Based on our analysis and previous experimental results on IRED mutants, an exclusive role of standard position 187 for stereoselectivity is excluded. Alternatively, two standard positions 139 and 194 were identified which are superfamily-specifically conserved and differ in R- and S-selective enzymes. Proteins 2016; 84:600-610. © 2016 Wiley Periodicals, Inc. PMID:26857686

  9. Evidence for a Hexaheteromeric Methylenetetrahydrofolate Reductase in Moorella thermoacetica

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    1999-01-01

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

  11. Nitrite and nitrous oxide reductase regulation by nitrogen oxides in Rhodobacter sphaeroides f. sp. denitrificans IL106.

    PubMed

    Sabaty, M; Schwintner, C; Cahors, S; Richaud, P; Verméglio, A

    1999-10-01

    We have cloned the nap locus encoding the periplasmic nitrate reductase in Rhodobacter sphaeroides f. sp. denitrificans IL106. A mutant with this enzyme deleted is unable to grow under denitrifying conditions. Biochemical analysis of this mutant shows that in contrast to the wild-type strain, the level of synthesis of the nitrite and N(2)O reductases is not increased by the addition of nitrate. Growth under denitrifying conditions and induction of N oxide reductase synthesis are both restored by the presence of a plasmid containing the genes encoding the nitrate reductase. This demonstrates that R. sphaeroides f. sp. denitrificans IL106 does not possess an efficient membrane-bound nitrate reductase and that nitrate is not the direct inducer for the nitrite and N(2)O reductases in this species. In contrast, we show that nitrite induces the synthesis of the nitrate reductase. PMID:10498715

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

    PubMed

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

    2012-05-01

    The aromatase and quinone reductase-2 inhibition of synthesized heterocyclic pyrazole derivatives fused with steroidal structure for chemoprevention of cancer is reported herein. All compounds were interestingly less toxic than the reference drug (Cyproterone(®)). The aromatase inhibitory activities of these compounds were much more potent than the lead compound resveratrol, which has an IC(50) of 80 μM. In addition, all the compounds displayed potent quinone reductase-2 inhibition. Initially the acute toxicity of the compounds was assayed via the determination of their LD(50). The aromatase and quinone reductase-2 inhibitors resulting from this study have potential value in the treatment and prevention of cancer. PMID:22361454

  13. The use of 5-alpha reductase inhibitors for the prevention of prostate cancer.

    PubMed

    Yu, Eun-mi; El-Ayass, Walid; Aragon-Ching, Jeanny B

    2010-07-01

    The use of 5-alpha-reductase inhibitors has been studied not only in benign prostatic hyperplasia, but as a chemopreventive strategy in prostate cancer. Both finasteride and dutasteride, 5 alpha-reductase inhibitors (5ARI), have been shown to decrease the risk of prostate cancer. The results of the REDUCE trial using the dual alpha-reductase isoenzyme inhibitor dutasteride, has recently been published by Andriole et al. in the New England Journal of Medicine. Certain considerations regarding its use and applicability to men with high risk of developing prostate cancer are herein discussed. PMID:20574153

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

    PubMed Central

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

    1988-01-01

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

  15. An ethoxyquin-inducible aldehyde reductase from rat liver that metabolizes aflatoxin B1 defines a subfamily of aldo-keto reductases.

    PubMed Central

    Ellis, E M; Judah, D J; Neal, G E; Hayes, J D

    1993-01-01

    Protection of liver against the toxic and carcinogenic effects of aflatoxin B1 (AFB1) can be achieved through the induction of detoxification enzymes by chemoprotectors such as the phenolic antioxidant ethoxyquin. We have cloned and sequenced a cDNA encoding an aldehyde reductase (AFB1-AR), which is expressed in rat liver in response to dietary ethoxyquin. Expression of the cDNA in Escherichia coli and purification of the recombinant enzyme reveals that the protein exhibits aldehyde reductase activity and is capable of converting the protein-binding dialdehyde form of AFB1-dihydrodiol to the nonbinding dialcohol metabolite. We show that the mRNA encoding this enzyme is markedly elevated in the liver of rats fed an ethoxyquin-containing diet, correlating with acquisition of resistance to AFB1. AFB1-AR represents the only carcinogen-metabolizing aldehyde reductase identified to date that is induced by a chemoprotector. Alignment of the amino acid sequence of AFB1-AR with other known and putative aldehyde reductases shows that it defines a subfamily within the aldo-keto reductase superfamily. Images Fig. 2 Fig. 3 Fig. 4 PMID:8234296

  16. 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

  17. Methylenetetrahydrofolate reductase and methionine synthase reductase gene polymorphisms and protection from microvascular complications in adolescents with type 1 diabetes.

    PubMed

    Wiltshire, Esko J; Mohsin, Fauzia; Chan, Albert; Donaghue, Kim C

    2008-08-01

    Folate status has been associated with endothelial dysfunction in adolescents with type 1 diabetes, and elevated total plasma homoocyst(e)ine (tHcy) is a risk for vascular disease in the non-diabetic population. Polymorphisms in genes involved in folate and homocysteine metabolism are implicated in vascular disease. We aimed to determine whether polymorphisms in the methylenetetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR) genes are risk factors for early microvascular disease in a large group of adolescents with type 1 diabetes. Four hundred and eighty adolescents were screened annually for retinopathy and microalbuminuria for a median of 4 yr. Molecular analysis for the polymorphisms 677C-->T, 1298A-->C in MTHFR, and 66A-->G in MTRR was performed. The MTRR 66GG genotype reduced the risk for elevated albumin excretion rate (AER) (OR 0.47, CI 0.25, 0.88, p = 0.018) and showed a trend to reduced risk for microalbuminuria (OR 0.27, CI 0.06-1.21, p = 0.09). Survival without elevated AER was increased with the MTRR 66GG genotype (12.4 vs. 9.7 yr, p = 0.04) and with the MTHFR 1298CC genotype (15.2 vs. 10.2 yr, p = 0.007). Conversely, survival without retinopathy was reduced with the MTHFR 677TT and MTRR 66GG combined genotype (6.2 vs. 10.2 yr, p = 0.015). The MTRR 66GG and MTHFR 1298 CC genotypes may confer protection against early nephropathy, possibly because they are associated with lower tHcy. The MTHFR 677 TT was only related to earlier onset retinopathy in combination with MTRR 66GG. PMID:18774994

  18. Redesigning alcohol dehydrogenases/reductases for more efficient biosynthesis of enantiopure isomers.

    PubMed

    Zhang, Rongzhen; Xu, Yan; Xiao, Rong

    2015-12-01

    Alcohol dehydrogenases/reductases predominantly catalyze the asymmetric biosynthesis of optically pure stereoisomers because of their unique chiral constitutions. The enantioselectivities of alcohol dehydrogenases/reductases are substrate- and cofactor-dependent, and therefore they usually catalyze specific reactions with high enantioselectivity under physiological conditions; this may not be suitable for asymmetric biosynthesis with non-natural substrates or non-natural cofactors, and under nonphysiological conditions. It is therefore necessary to modify alcohol dehydrogenases/reductases using various redesigning tools such as directed evolution and rational design, and their combinations, as well as engineering enzyme modules for more efficient production of "non-natural" products. In this article, progress in these aspects of alcohol dehydrogenase/reductase design is reviewed, and future challenges are discussed. PMID:26320091

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

    SciTech Connect

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

    1981-12-01

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

  20. 5α-reductase inhibitors, antiviral and anti-tumor activities of some steroidal cyanopyridinone derivatives.

    PubMed

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

    2012-01-01

    We herein report the 5α-reductase inhibitors, antiviral and anti-tumor activities of some synthesized heterocyclic cyanopyridone and cyanothiopyridone derivatives fused with steroidal structure. Initially the acute toxicity of the compounds was assayed via the determination of their LD(50). All the compounds, except 3b, were interestingly less toxic than the reference drug (Prednisolone(®)). Seventeen heterocyclic derivatives containing a cyanopyridone or cyanothiopyridone rings fused to a steroidal moiety were synthesized and screened for their 5α-reductase inhibitors, antiviral and anti-tumor activities comparable to that of Anastrozole, Bicalutamide, Efavirenz, Capravirine, Ribavirin, Oseltamivir and Amantadine as the reference drugs. Some of the compounds exhibited better 5α-reductase inhibitors, antiviral and anti-tumor activities than the reference drugs. The detailed 5α-reductase inhibitors, antiviral and anti-tumor activities of the synthesized compounds were reported. PMID:22057085

  1. Design and synthesis of hepatoselective, pyrrole-based HMG-CoA reductase inhibitors.

    PubMed

    Pfefferkorn, Jeffrey A; Song, Yuntao; Sun, Kuai-Lin; Miller, Steven R; Trivedi, Bharat K; Choi, Chulho; Sorenson, Roderick J; Bratton, Larry D; Unangst, Paul C; Larsen, Scott D; Poel, Toni-Jo; Cheng, Xue-Min; Lee, Chitase; Erasga, Noe; Auerbach, Bruce; Askew, Valerie; Dillon, Lisa; Hanselman, Jeffrey C; Lin, Zhiwu; Lu, Gina; Robertson, Andrew; Olsen, Karl; Mertz, Thomas; Sekerke, Catherine; Pavlovsky, Alexander; Harris, Melissa S; Bainbridge, Graeme; Caspers, Nicole; Chen, Huifen; Eberstadt, Matthias

    2007-08-15

    This manuscript describes the design and synthesis of a series of pyrrole-based inhibitors of HMG-CoA reductase for the treatment of hypercholesterolemia. Analogs were optimized using structure-based design and physical property considerations resulting in the identification of 44, a hepatoselective HMG-CoA reductase inhibitor with excellent acute and chronic efficacy in a pre-clinical animal models. PMID:17574412

  2. 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

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

    PubMed Central

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

    2014-01-01

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

  4. Purification and partial characterization of NADPH-cytochrome c reductase from Petunia hybrida flowers.

    PubMed Central

    Menting, J G; Cornish, E; Scopes, R K

    1994-01-01

    NADPH-cytochrome c reductase was solubilized from the microsomal fraction of Petunia hybrida flowers by 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate detergent and purified by adenosine 2',5'-bisphosphate-Sepharose chromatography, followed by high-performance anion-exchange chromatography. Two proteins with molecular sizes of 75 and 81 kD were detected in the purified preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Western blot analysis showed that both purified proteins cross-reacted with two different monoclonal antibodies raised against P. hybrida NADPH-cytochrome c reductase and rabbit anti-Jerusalem artichoke NADPH-cytochrome P450 reductase antibodies. Only one 84-kD protein was detected by western blot analysis of fresh microsomal extracts. Amino acid sequence analysis of tryptic peptides revealed significant similarity to the NADPH binding region of plant and animal NADPH-cytochrome P450 reductases and Bacillus megaterium cytochrome P450:NADPH-cytochrome P450 reductase. The pH optimum for reduction of ferricytochrome c was 7.4 and the Km values for the binding of NADPH and ferricytochrome c were 9.2 and 2.8 microM, respectively. We believe that the purified enzyme is a P. hybrida NADPH-cytochrome P450 reductase (EC 1.6.2.4). PMID:7991686

  5. "Subversive" substrates for the enzyme trypanothione disulfide reductase: alternative approach to chemotherapy of Chagas disease.

    PubMed Central

    Henderson, G B; Ulrich, P; Fairlamb, A H; Rosenberg, I; Pereira, M; Sela, M; Cerami, A

    1988-01-01

    The trypanosomatid flavoprotein disulfide reductase, trypanothione reductase, is shown to catalyze one-electron reduction of suitably substituted naphthoquinone and nitrofuran derivatives. A number of such compounds have been chemically synthesized, and a structure-activity relationship has been established; the enzyme is most active with compounds that contain basic functional groups in side-chain residues. The reduced products are readily reoxidized by molecular oxygen and thus undergo classical enzyme-catalyzed redox cycling. In addition to their ability to act as substrates for trypanothione reductase, the compounds are also shown to effectively inhibit enzymatic reduction of the enzyme's physiological substrate, trypanothione disulfide. Under aerobic conditions, trypanothione reductase is not inactivated by these redox-cycling substrates, whereas under anaerobic conditions the nitrofuran compounds cause irreversible inactivation of the enzyme. When tested for biological activity against Trypanosoma cruzi trypomastigotes, many of the test compounds were trypanocidal, and this activity correlated with their relative ability to act as substrates for trypanothione reductase. The activity of the enzyme with these redox-cycling derivatives constitutes a subversion of its normal antioxidant role within the cell. For this reason these compounds may be termed "subversive" substrates for trypanothione reductase. PMID:3135548

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

    PubMed

    Couto, Narciso; Wood, Jennifer; Barber, Jill

    2016-06-01

    In this review article we examine the role of glutathione reductase in the regulation, modulation and maintenance of cellular redox homoeostasis. Glutathione reductase is responsible for maintaining the supply of reduced glutathione; one of the most abundant reducing thiols in the majority of cells. In its reduced form, glutathione plays key roles in the cellular control of reactive oxygen species. Reactive oxygen species act as intracellular and extracellular signalling molecules and complex cross talk between levels of reactive oxygen species, levels of oxidised and reduced glutathione and other thiols, and antioxidant enzymes such as glutathione reductase determine the most suitable conditions for redox control within a cell or for activation of programmed cell death. Additionally, we discuss the translation and expression of glutathione reductase in a number of organisms including yeast and humans. In yeast and human cells, a single gene expresses more than one form of glutathione reductase, destined for residence in the cytoplasm or for translocation to different organelles; in plants, however, two genes encoding this protein have been described. In general, insects and kinetoplastids (a group of protozoa, including Plasmodia and Trypanosoma) do not express glutathione reductase or glutathione biosynthetic enzymes. Instead, they express either the thioredoxin system or the trypanothione system. The thioredoxin system is also present in organisms that have the glutathione system and there may be overlapping functions with cross-talk between the two systems. Finally we evaluate therapeutic targets to overcome oxidative stress associated cellular disorders. PMID:26923386

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

    SciTech Connect

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

    1991-10-01

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

  8. Photoaffinity labelling of nuclear steroid 5 alpha-reductase of rat ventral prostate.

    PubMed

    Enderle-Schmitt, U; Seitz, J; Aumüller, G

    1989-09-01

    In order to get more information on the molecular structure of the rat prostatic 5 alpha-reductase (3-oxo-5 alpha-steroid: NADP+ 4-ene-oxidoreductase, EC 1.3:1.22) a systematic photoaffinity labelling study has been performed. To irreversibly freeze the status quo of interaction, either testosterone, the physiological ligand, or diazo-MAPD (21-diazo-4-methyl-4-aza-5 alpha-pregnane-3,20-dione), a specific 5 alpha-reductase inhibitor, was irradiated with isolated nuclei or with purified nuclear membranes or with solubilized nuclear membrane proteins and checked for optimal labelling conditions. The principal substances covalently labelled were phospholipids and at a minor ratio proteins. Analysis by SDS-PAGE and autoradiofluorography revealed two labelled polypeptides with molecular weights of 20 kDa and 26 kDa. The following evidence indicates that these polypeptides might be derived from the enzyme 5 alpha-reductase: both proteins are labelled only when specific ligands for 5 alpha-reductase are used; binding can be reduced by the addition of an excess of unlabelled ligand; enzyme activity is irreversibly suppressed when irradiated in the presence of these ligands; only subcellular fractions containing 5 alpha-reductase reveal the labelled proteins; in all 5 alpha-reductase containing preparations with increasing specific activity, independent of the polypeptide pattern, the same proteins are labelled. PMID:2779229

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

    SciTech Connect

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

    1989-06-01

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

  10. The Role of the 3-Hydroxy 3-Methylglutaryl Coenzyme A Reductase Cytosolic Domain in Karmellae Biogenesis

    PubMed Central

    Profant, Deborah A.; Roberts, Christopher J.; Koning, Ann J.; Wright, Robin L.

    1999-01-01

    In all cells examined, specific endoplasmic reticulum (ER) membrane arrays are induced in response to increased levels of the ER membrane protein 3-hydroxy 3-methylglutaryl coenzyme A (HMG-CoA) reductase. In yeast, expression of Hmg1p, one of two yeast HMG-CoA reductase isozymes, induces assembly of nuclear-associated ER stacks called karmellae. Understanding the features of HMG-CoA reductase that signal karmellae biogenesis would provide useful insights into the regulation of membrane biogenesis. The HMG-CoA reductase protein consists of two domains, a multitopic membrane domain and a cytosolic catalytic domain. Previous studies had indicated that the HMG-CoA reductase membrane domain was exclusively responsible for generation of ER membrane proliferations. Surprisingly, we discovered that this conclusion was incorrect: sequences at the carboxyl terminus of HMG-CoA reductase can profoundly affect karmellae biogenesis. Specifically, truncations of Hmg1p that removed or shortened the carboxyl terminus were unable to induce karmellae assembly. This result indicated that the membrane domain of Hmg1p was not sufficient to signal for karmellae assembly. Using β-galactosidase fusions, we demonstrated that the carboxyl terminus was unlikely to simply serve as an oligomerization domain. Our working hypothesis is that a truncated or misfolded cytosolic domain prevents proper signaling for karmellae by interfering with the required tertiary structure of the membrane domain. PMID:10512876

  11. Molecular cloning, expression and catalytic activity of a human AKR7 member of the aldo-keto reductase superfamily: evidence that the major 2-carboxybenzaldehyde reductase from human liver is a homologue of rat aflatoxin B1-aldehyde reductase.

    PubMed Central

    Ireland, L S; Harrison, D J; Neal, G E; Hayes, J D

    1998-01-01

    The masking of charged amino or carboxy groups by N-phthalidylation and O-phthalidylation has been used to improve the absorption of many drugs, including ampicillin and 5-fluorouracil. Following absorption of such prodrugs, the phthalidyl group is hydrolysed to release 2-carboxybenzaldehyde (2-CBA) and the pharmaceutically active compound; in humans, 2-CBA is further metabolized to 2-hydroxymethylbenzoic acid by reduction of the aldehyde group. In the present work, the enzyme responsible for the reduction of 2-CBA in humans is identified as a homologue of rat aflatoxin B1-aldehyde reductase (rAFAR). This novel human aldo-keto reductase (AKR) has been cloned from a liver cDNA library, and together with the rat protein, establishes the AKR7 family of the AKR superfamily. Unlike its rat homologue, human AFAR (hAFAR) appears to be constitutively expressed in human liver, and is widely expressed in extrahepatic tissues. The deduced human and rat protein sequences share 78% identity and 87% similarity. Although the two AKR7 proteins are predicted to possess distinct secondary structural features which distinguish them from the prototypic AKR1 family of AKRs, the catalytic- and NADPH-binding residues appear to be conserved in both families. Certain of the predicted structural features of the AKR7 family members are shared with the AKR6 beta-subunits of voltage-gated K+-channels. In addition to reducing the dialdehydic form of aflatoxin B1-8,9-dihydrodiol, hAFAR shows high affinity for the gamma-aminobutyric acid metabolite succinic semialdehyde (SSA) which is structurally related to 2-CBA, suggesting that hAFAR could function as both a SSA reductase and a 2-CBA reductase in vivo. This hypothesis is supported in part by the finding that the major peak of 2-CBA reductase activity in human liver co-purifies with hAFAR protein. PMID:9576847

  12. Characterization of two alkyl hydroperoxide reductase C homologs alkyl hydroperoxide reductase C_H1 and alkyl hydroperoxide reductase C_H2 in Bacillus subtilis

    PubMed Central

    Cha, Mee-Kyung; Bae, Yoo-Jeen; Kim, Kyu-Jeong; Park, Byung-Joon; Kim, Il-Han

    2015-01-01

    AIM: To identify alkyl hydroperoxide reductase subunit C (AhpC) homologs in Bacillus subtilis (B. subtilis) and to characterize their structural and biochemical properties. AhpC is responsible for the detoxification of reactive oxygen species in bacteria. METHODS: Two AhpC homologs (AhpC_H1 and AhpC_H2) were identified by searching the B. subtilis database; these were then cloned and expressed in Escherichia coli. AhpC mutants carrying substitutions of catalytically important Cys residues (C37S, C47S, C166S, C37/47S, C37/166S, C47/166S, and C37/47/166S for AhpC_H1; C52S, C169S, and C52/169S for AhpC_H2) were obtained by site-directed mutagenesis and purified, and their structure-function relationship was analyzed. The B. subtilis ahpC genes were disrupted by the short flanking homology method, and the phenotypes of the resulting AhpC-deficient bacteria were examined. RESULTS: Comparative characterization of AhpC homologs indicates that AhpC_H1 contains an extra C37, which forms a disulfide bond with the peroxidatic C47, and behaves like an atypical 2-Cys AhpC, while AhpC_H2 functions like a typical 2-Cys AhpC. Tryptic digestion analysis demonstrated the presence of intramolecular Cys37-Cys47 linkage, which could be reduced by thioredoxin, resulting in the association of the dimer into higher-molecular-mass complexes. Peroxidase activity analysis of Cys→Ser mutants indicated that three Cys residues were involved in the catalysis. AhpC_H1 was resistant to inactivation by peroxide substrates, but had lower activity at physiological H2O2 concentrations compared to AhpC_H2, suggesting that in B. subtilis, the enzymes may be physiologically functional at different substrate concentrations. The exposure to organic peroxides induced AhpC_H1 expression, while AhpC_H1-deficient mutants exhibited growth retardation in the stationary phase, suggesting the role of AhpC_H1 as an antioxidant scavenger of lipid hydroperoxides and a stress-response factor in B. subtilis

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

    PubMed

    Munoz, Tatiana; Patel, Jinesh; Badilla-Porras, Ramses; Kronick, Jonathan; Mercimek-Mahmutoglu, Saadet

    2015-01-01

    Severe methylenetetrahydrofolate reductase (MTHFR) deficiency is a rare autosomal recessively inherited inborn error of folate metabolism. We report a new patient with severe MTHFR deficiency who presented at age 4 months with early onset severe scoliosis associated with severe hypotonia. Markedly decreased MTHFR enzyme activity (0.3 nmoles CHO/mg protein/h; reference range>9) and compound heterozygous mutations (c. 1304T>C; p.Phe435Ser and c.1539dup; p.Glu514Argfs∗24) in the MTHFR gene confirmed the diagnosis. She was treated with vitamin B12, folic acid and betaine supplementation and showed improvements in her developmental milestones and hypotonia. To the best of our knowledge, this is the first patient with MTHFR deficiency reported with severe early onset scoliosis. Despite the late diagnosis and treatment initiation, she showed favorable short-term neurodevelopmental outcome. This case suggests that homocysteine measurement should be included in the investigations of patients with developmental delay, hypotonia and scoliosis within first year of life prior to organizing genetic investigations. PMID:24726568

  14. 5alpha-reductase: history and clinical importance.

    PubMed

    Marks, Leonard S

    2004-01-01

    The treatment of men with symptomatic benign prostatic hyperplasia (BPH) has shifted dramatically from surgery to drug therapy over the past decade. The revolution in BPH treatment began with the discovery of congenital 5alpha-reductase (5AR) deficiency, leading to the appreciation of 2 different androgenic hormones: testosterone, which mediates overt masculinization in the adult male, and dihydrotestosterone (DHT), which mediates prostatic growth, acne, facial beard, and male pattern baldness. Inhibition of DHT in adults results in prostatic shrinkage and symptomatic relief in many men, without the side effects seen with conventional androgen-deprivation therapy. The 5AR inhibitor drugs (finasteride and the dual inhibitor, dutasteride) are able to ablate the accumulation of intraprostatic DHT, the mechanism most responsible for prostate growth and maintenance. Not only may these drugs relieve symptoms, but they may also alter the natural history of the BPH process. Future indications for the 5ARI drugs could include chemoprevention of prostate cancer, prophylaxis of BPH-related complications, and treatment of BPH-associated hematuria. PMID:16985920

  15. Glyoxalase 1 and glutathione reductase 1 regulate anxiety in mice.

    PubMed

    Hovatta, Iiris; Tennant, Richard S; Helton, Robert; Marr, Robert A; Singer, Oded; Redwine, Jeffrey M; Ellison, Julie A; Schadt, Eric E; Verma, Inder M; Lockhart, David J; Barlow, Carrolee

    2005-12-01

    Anxiety and fear are normal emotional responses to threatening situations. In human anxiety disorders--such as panic disorder, obsessive-compulsive disorder, post-traumatic stress disorder, social phobia, specific phobias and generalized anxiety disorder--these responses are exaggerated. The molecular mechanisms involved in the regulation of normal and pathological anxiety are mostly unknown. However, the availability of different inbred strains of mice offers an excellent model system in which to study the genetics of certain behavioural phenotypes. Here we report, using a combination of behavioural analysis of six inbred mouse strains with quantitative gene expression profiling of several brain regions, the identification of 17 genes with expression patterns that correlate with anxiety-like behavioural phenotypes. To determine if two of the genes, glyoxalase 1 and glutathione reductase 1, have a causal role in the genesis of anxiety, we performed genetic manipulation using lentivirus-mediated gene transfer. Local overexpression of these genes in the mouse brain resulted in increased anxiety-like behaviour, while local inhibition of glyoxalase 1 expression by RNA interference decreased the anxiety-like behaviour. Both of these genes are involved in oxidative stress metabolism, linking this pathway with anxiety-related behaviour. PMID:16244648

  16. Catalytic Cycle of Human Glutathione Reductase Near 1 Å Resolution

    SciTech Connect

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

    2008-09-08

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

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

    PubMed

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

    2015-01-01

    Dihydroflavonol 4-reductase (DFR) genes from Rosa chinensis (Asn type) and Calibrachoa hybrida (Asp type), driven by a CaMV 35S promoter, were integrated into the petunia (Petunia hybrida) cultivar 9702. Exogenous DFR gene expression characteristics were similar to flower-color changes, and effects on anthocyanin concentration were observed in both types of DFR gene transformants. Expression analysis showed that exogenous DFR genes were expressed in all of the tissues, but the expression levels were significantly different. However, both of them exhibited a high expression level in petals that were starting to open. The introgression of DFR genes may significantly change DFR enzyme activity. Anthocyanin ultra-performance liquid chromatography results showed that anthocyanin concentrations changed according to DFR enzyme activity. Therefore, the change in flower color was probably the result of a DFR enzyme change. Pelargonidin 3-O-glucoside was found in two different transgenic petunias, indicating that both CaDFR and RoDFR could catalyze dihydrokaempferol. Our results also suggest that transgenic petunias with DFR gene of Asp type could biosynthesize pelargonidin 3-O-glucoside. PMID:25966276

  18. Optical observation of correlated motions in dihydrofolate reductase

    NASA Astrophysics Data System (ADS)

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

    2015-03-01

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

  19. Loop interactions during catalysis by dihydrofolate reductase from Moritella profunda.

    PubMed

    Behiry, Enas M; Evans, Rhiannon M; Guo, Jiannan; Loveridge, E Joel; Allemann, Rudolf K

    2014-07-29

    Dihydrofolate reductase (DHFR) is often used as a model system to study the relation between protein dynamics and catalysis. We have studied a number of variants of the cold-adapted DHFR from Moritella profunda (MpDHFR), in which the catalytically important M20 and FG loops have been altered, and present a comparison with the corresponding variants of the well-studied DHFR from Escherichia coli (EcDHFR). Mutations in the M20 loop do not affect the actual chemical step of transfer of hydride from reduced nicotinamide adenine dinucleotide phosphate to the substrate 7,8-dihydrofolate in the catalytic cycle in either enzyme; they affect the steady state turnover rate in EcDHFR but not in MpDHFR. Mutations in the FG loop also have different effects on catalysis by the two DHFRs. Despite the two enzymes most likely sharing a common catalytic cycle at pH 7, motions of these loops, known to be important for progression through the catalytic cycle in EcDHFR, appear not to play a significant role in MpDHFR. PMID:25014120

  20. Correlated Protein Motion Measurements of Dihydrofolate Reductase Crystals

    NASA Astrophysics Data System (ADS)

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

    2014-03-01

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

  1. Structural basis and mechanism of enoyl reductase inhibition by triclosan.

    PubMed

    Stewart, M J; Parikh, S; Xiao, G; Tonge, P J; Kisker, C

    1999-07-23

    The enoyl-acyl carrier protein reductase (ENR) is involved in bacterial fatty acid biosynthesis and is the target of the antibacterial diazaborine compounds and the front-line antituberculosis drug isoniazid. Recent studies suggest that ENR is also the target for the broad-spectrum biocide triclosan. The 1.75 A crystal structure of EnvM, the ENR from Escherichia coli, in complex with triclosan and NADH reveals that triclosan binds specifically to EnvM. These data provide a molecular mechanism for the antibacterial activity of triclosan and substantiate the hypothesis that its activity results from inhibition of a specific cellular target rather than non-specific disruption of the bacterial cell membrane. This has important implications for the emergence of drug-resistant bacteria, since triclosan is an additive in many personal care products such as toothpastes, mouthwashes and soaps. Based on this structure, rational design of triclosan derivatives is possible which might be effective against recently identified triclosan-resistant bacterial strains. PMID:10398587

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

    PubMed

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

    2012-11-01

    Dihydrofolate reductase (DHFR) is an essential enzyme involved in de novo purine and thymidine biosynthesis. For several decades, selective inhibition of DHFR has proven to be a potent therapeutic approach in the treatment of various cancers including acute lymphoblastic leukemia, non-Hodgkin's lymphoma, osteogenic sarcoma, carcinoma of the breast, and head and neck cancer. Therapeutic success with DHFR inhibitor methotrexate (MTX) has been compromised in the clinic, which limits the success of MTX treatment by both acquired and intrinsic resistance mechanisms. We report that benzamide riboside (BR), via anabolism to benzamide adenine dinucleotide (BAD) known to potently inhibit inosine monophosphate dehydrogenase (IMPDH), also inhibits cell growth through a mechanism involving downregulation of DHFR protein. Evidence to support this second site of action of BR includes the finding that CCRF-CEM/R human T-cell lymphoblasic leukemia cells, resistant to MTX as a consequence of gene amplification and overexpression of DHFR, are more resistant to BR than are parental cells. Studies of the mechanism by which BR lowers DHFR showed that BR, through its metabolite BAD, reduced NADP and NADPH cellular levels by inhibiting nicotinamide adenine dinucleotide kinase (NADK). As consequence of the lack of NADPH, DHFR was shown to be destabilized. We suggest that, inhibition of NADK is a new approach to downregulate DHFR and to inhibit cell growth. PMID:22954684

  3. Alkyl hydroperoxide reductase: a candidate Helicobacter pylori vaccine.

    PubMed

    O'Riordan, Avril A; Morales, Veronica Athie; Mulligan, Linda; Faheem, Nazia; Windle, Henry J; Kelleher, Dermot P

    2012-06-01

    Helicobacter pylori (H. pylori) is the most important etiological agent of chronic active gastritis, peptic ulcer disease and gastric cancer. The aim of this study was to evaluate the efficacy of alkyl hydroperoxide reductase (AhpC) and mannosylated AhpC (mAhpC) as candidate vaccines in the C57BL/6J mouse model of H. pylori infection. Recombinant AhpC was cloned, over-expressed and purified in an unmodified form and was also engineered to incorporate N and C-terminal mannose residues when expressed in the yeast Pichia pastoris. Mice were immunized systemically and mucosally with AhpC and systemically with mAhpC prior to challenge with H. pylori. Serum IgG responses to AhpC were determined and quantitative culture was used to determine the efficacy of vaccination strategies. Systemic prophylactic immunization with AhpC/alum and mAhpC/alum conferred protection against infection in 55% and 77.3% of mice, respectively. Mucosal immunization with AhpC/cholera toxin did not protect against infection and elicited low levels of serum IgG in comparison with systemic immunization. These data support the use of AhpC as a potential vaccine candidate against H. pylori infection. PMID:22512976

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

    PubMed

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

    2010-01-27

    Hydride transfer catalyzed by dihydrofolate reductase (DHFR) has been described previously within an environmentally coupled model of hydrogen tunneling, where protein motions control binding of substrate and cofactor to generate a tunneling ready conformation and modulate the width of the activation barrier and hence the reaction rate. Changes to the composition of the reaction medium are known to perturb protein motions. We have measured kinetic parameters of the reaction catalyzed by DHFR from Escherichia coli in the presence of various cosolvents and cosolutes and show that the dielectric constant, but not the viscosity, of the reaction medium affects the rate of reaction. Neither the primary kinetic isotope effect on the reaction nor its temperature dependence were affected by changes to the bulk solvent properties. These results are in agreement with our previous report on the effect of solvent composition on catalysis by DHFR from the hyperthermophile Thermotoga maritima. However, the effect of solvent on the temperature dependence of the kinetic isotope effect on hydride transfer catalyzed by E. coli DHFR is difficult to explain within a model, in which long-range motions couple to the chemical step of the reaction, but may indicate the existence of a short-range promoting vibration or the presence of multiple nearly isoenergetic conformational substates of enzymes with similar but distinct catalytic properties. PMID:20047317

  5. Molecular Cloning of Complementary DNA Encoding Maize Nitrite Reductase

    PubMed Central

    Lahners, Kristine; Kramer, Vance; Back, Eduard; Privalle, Laura; Rothstein, Steven

    1988-01-01

    Complementary DNA has been isolated that codes for maize nitrite reductase (NiR) by using the corresponding spinach gene (E Back et al. 1988 Mol Gen Genet 212:20-26) as a heterologous probe. The sequences of the complementary DNAs from the two species are 66% homologous while the deduced amino acid sequences are 86% similar when analogous amino acids are included. A high percentage of the differences in the DNA sequences is due to the extremely strong bias in the corn gene to have a G/C base in the third codon position with 559/569 codons ending in a G or C. Using a hydroponic system, maize seedlings grown in the absence of an exogenous nitrogen source were induced with nitrate or nitrite. Nitrate stimulated a rapid induction of the NiR mRNA in both roots and leaves. There is also a considerable induction of this gene in roots upon the addition of nitrite, although under the conditions used the final mRNA level was not as high as when nitrate was the inducer. There is a small but detectable level of NiR mRNA in leaves prior to induction, but no constitutive NiR mRNA can be seen in the roots. Analysis of genomic DNA supports the notion that there are at least two NiR genes in maize. Images Fig. 3 Fig. 4 Fig. 5 PMID:16666376

  6. Chromate reductase activity in Streptomyces sp. MC1.

    PubMed

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

    2010-02-01

    Biological transformation of Cr(VI) to Cr(III) by enzymatic reduction may provide a less costly and more environmentally friendly approach to remediation. In a previous report a Cr(VI) resistant actinomycete strain, Streptomyces sp. MC1, was able to reduce Cr(VI) present in a synthetic medium, soil extract and soil samples. This is the first time optimal conditions such as pH, temperature, growth phase and electron donor have been elucidated in vitro for Cr(VI) reduction by a streptomycete. Chromate reductase of Streptomyces sp. MC1 is a constitutive enzyme which was mainly associated with biomass and required NAD(P)H as an electron donor. It was active over a broad temperature (19-39 degrees C) and pH (5-8) range, and optimum conditions were 30 degrees C and pH 7. The enzyme was present in supernatant, pellet and cell free extract. Bioremediation with the enzyme was observed in non-compatible cell reproduction systems, conditions frequently found in contaminated environments. PMID:20339215

  7. Crystal structure studies on sulfur oxygenase reductase from Acidianus tengchongensis

    SciTech Connect

    Li Mei; Chen Zhiwei; Zhang Pingfeng; Pan Xiaowei; Jiang Chengying; An Xiaomin; Liu Shuangjiang; Chang Wenrui

    2008-05-09

    Sulfur oxygenase reductase (SOR) simultaneously catalyzes oxidation and reduction of elemental sulfur to produce sulfite, thiosulfate, and sulfide in the presence of molecular oxygen. In this study, crystal structures of wild type and mutants of SOR from Acidianus tengchongensis (SOR-AT) in two different crystal forms were determined and it was observed that 24 identical SOR monomers form a hollow sphere. Within the icosatetramer sphere, the tetramer and trimer channels were proposed as the paths for the substrate and products, respectively. Moreover, a comparison of SOR-AT with SOR-AA (SOR from Acidianus ambivalens) structures showed that significant differences existed at the active site. Firstly, Cys31 is not persulfurated in SOR-AT structures. Secondly, the iron atom is five-coordinated rather than six-coordinated, since one of the water molecules ligated to the iron atom in the SOR-AA structure is lost. Consequently, the binding sites of substrates and a hypothetical catalytic process of SOR were proposed.

  8. 5α-Reductase: History and Clinical Importance

    PubMed Central

    Marks, Leonard S

    2004-01-01

    The treatment of men with symptomatic benign prostatic hyperplasia (BPH) has shifted dramatically from surgery to drug therapy over the past decade. The revolution in BPH treatment began with the discovery of congenital 5α-reductase (5AR) deficiency, leading to the appreciation of 2 different androgenic hormones: testosterone, which mediates overt masculinization in the adult male, and dihydrotestosterone (DHT), which mediates prostatic growth, acne, facial beard, and male pattern baldness. Inhibition of DHT in adults results in prostatic shrinkage and symptomatic relief in many men, without the side effects seen with conventional androgen-deprivation therapy. The 5AR inhibitor drugs (finasteride and the dual inhibitor, dutasteride) are able to ablate the accumulation of intraprostatic DHT, the mechanism most responsible for prostate growth and maintenance. Not only may these drugs relieve symptoms, but they may also alter the natural history of the BPH process. Future indications for the 5ARI drugs could include chemoprevention of prostate cancer, prophylaxis of BPH-related complications, and treatment of BPH-associated hematuria. PMID:16985920

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

    PubMed

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

    2015-10-01

    Over the past decades, a number of authors have reported the presence of inactive species in as-prepared samples of members of the Mo/W-bisPGD enzyme family. This greatly complicated the spectroscopic studies of these enzymes, since it is impossible to discriminate between active and inactive species on the basis of the spectroscopic signatures alone. Escherichia coli nitrate reductase A (NarGHI) is a member of the Mo/W-bisPGD family that allows anaerobic respiration using nitrate as terminal electron acceptor. Here, using protein film voltammetry on NarGH films, we show that the enzyme is purified in a functionally heterogeneous form that contains between 20 and 40% of inactive species that activate the first time they are reduced. This activation proceeds in two steps: a non-redox reversible reaction followed by an irreversible reduction. By carefully correlating electrochemical and EPR spectroscopic data, we show that neither the two major Mo(V) signals nor those of the two FeS clusters that are the closest to the Mo center are associated with the two inactive species. We also conclusively exclude the possibility that the major "low-pH" and "high-pH" Mo(V) EPR signatures correspond to species in acid-base equilibrium. PMID:26073890

  10. Converting a Sulfenic Acid Reductase into a Disulfide Bond Isomerase

    PubMed Central

    Chatelle, Claire; Kraemer, Stéphanie; Ren, Guoping; Chmura, Hannah; Marechal, Nils; Boyd, Dana; Roggemans, Caroline; Ke, Na; Riggs, Paul; Bardwell, James

    2015-01-01

    Abstract Aims: Posttranslational formation of disulfide bonds is essential for the folding of many secreted proteins. Formation of disulfide bonds in a protein with more than two cysteines is inherently fraught with error and can result in incorrect disulfide bond pairing and, consequently, misfolded protein. Protein disulfide bond isomerases, such as DsbC of Escherichia coli, can recognize mis-oxidized proteins and shuffle the disulfide bonds of the substrate protein into their native folded state. Results: We have developed a simple blue/white screen that can detect disulfide bond isomerization in vivo, using a mutant alkaline phosphatase (PhoA*) in E. coli. We utilized this screen to isolate mutants of the sulfenic acid reductase (DsbG) that allowed this protein to act as a disulfide bond isomerase. Characterization of the isolated mutants in vivo and in vitro allowed us to identify key amino acid residues responsible for oxidoreductase properties of thioredoxin-like proteins such as DsbC or DsbG. Innovation and Conclusions: Using these key residues, we also identified and characterized interesting environmental homologs of DsbG with novel properties, thus demonstrating the capacity of this screen to discover and elucidate mechanistic details of in vivo disulfide bond isomerization. Antioxid. Redox Signal. 23, 945–957. PMID:26191605

  11. Imexon enhances gemcitabine cytotoxicity by inhibition of ribonucleotide reductase

    PubMed Central

    Roman, Nicholas O.; Samulitis, Betty K.; Wisner, Lee; Landowski, Terry H.; Dorr, Robert T.

    2010-01-01

    Purpose Gemcitabine (GEM) is currently the standard first line treatment for pancreatic cancer; however, the overall survival of patients with this disease remains poor. Imexon is a pro-oxidant small molecule which produced a high response rate in combination with GEM in a phase I trial in pancreatic cancer. In this study, we investigate the combination of GEM with a novel redox-active agent, imexon, in vitro and in vivo. Methods Median effect analysis was used for in vitro combination cytotoxicity. The effect of imexon on GEM metabolism and uptake into cells and into DNA and effects on ribonucleotide reductase (RNR) were examined in vitro. The pharmacokinetics and antitumor efficacy of the imexon/GEM combination was evaluated in mouse models. Results In three human pancreatic cancer lines, there was additivity for the imexon/GEM combination. There was significantly greater efficacy for the drug combination in Panc-1 xenograft tumors. A pharmacokinetic study in mice showed a near doubling in the AUC of imexon when GEM was co-administered, with no effect of imexon on GEM's pharmacokinetic disposition. In vitro, imexon did not alter GEM's metabolism or uptake into DNA, but significantly inhibited RNR, and this effect was greater when combined with GEM. Conclusions These results suggest that the interaction between imexon and GEM may be due to complimentary inhibition of RNR plus an enhanced exposure to imexon when the GEM is administered in vivo. This combination is currently being tested in a randomized phase II trial in pancreatic cancer. PMID:20339847

  12. Regulation and degradation of HMGCo-A reductase.

    PubMed

    Panda, T; Devi, V Amutha

    2004-12-01

    The enzyme, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) controls the biosynthesis of cholesterol. Hypercholesterolemia and atherosclerosis are critical health risk factors. One way of controlling these risk factors is to manipulate regulation as well as degradation of HMGR. At present, a class of compounds called statins, which are HMGR inhibitors, are used for the treatment of hypercholesterolemia. However, statins suffer major setbacks as their use produces more adverse reactions than the desirable one of inhibiting the enzyme. Genetically engineered forms of HMGR are also studied in primitive life forms like bacteria, but detailed investigation of this enzyme in human systems is certainly required. Extensive studies have been made on the regulatory aspects of this enzyme, but no breakthrough is conspicuous in the clinical background to find an alternative treatment for hypercholesterolemia. The immediate need is to find an alternate way of regulating degradation of the enzyme. This review presents the importance of regulation and degradation of the HMGR enzyme in different systems to gain possible insight into alternative schemes for regulating this enzyme and, if these exist, the feasibility of extending them same to studies in mammalian systems. A high degree of similarity exists between mammalian and yeast HMGR. Detailed studies reported on the regulation and degradation of the yeast enzyme also throw more light on the mammalian system, leading to a better understanding of ways of controlling hypercholesterolemia. PMID:15558272

  13. Mutation Update and Review of Severe Methylenetetrahydrofolate Reductase Deficiency.

    PubMed

    Froese, D Sean; Huemer, Martina; Suormala, Terttu; Burda, Patricie; Coelho, David; Guéant, Jean-Louis; Landolt, Markus A; Kožich, Viktor; Fowler, Brian; Baumgartner, Matthias R

    2016-05-01

    Severe 5,10-methylenetetrahydrofolate reductase (MTHFR) deficiency is caused by mutations in the MTHFR gene and results in hyperhomocysteinemia and varying severity of disease, ranging from neonatal lethal to adult onset. Including those described here, 109 MTHFR mutations have been reported in 171 families, consisting of 70 missense mutations, 17 that primarily affect splicing, 11 nonsense mutations, seven small deletions, two no-stop mutations, one small duplication, and one large duplication. Only 36% of mutations recur in unrelated families, indicating that most are "private." The most common mutation is c.1530A>G (numbered from NM_005957.4, p.Lys510 = ) causing a splicing defect, found in 13 families; the most common missense mutation is c.1129C>T (p.Arg377Cys) identified in 10 families. To increase disease understanding, we report enzymatic activity, detected mutations, and clinical onset information (early, <1 year; or late, >1 year) for all published patients available, demonstrating that patients with early onset have less residual enzyme activity than those presenting later. We also review animal models, diagnostic approaches, clinical presentations, and treatment options. This is the first large review of mutations in MTHFR, highlighting the wide spectrum of disease-causing mutations. PMID:26872964

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

    PubMed

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

    2010-09-22

    High levels of reactive oxygen species (ROS) are associated with deficits in learning and memory with age as well as in Alzheimer's disease. Using DNA microarray, we demonstrated the overexpression of quinone reductase 2 (QR2) in the hippocampus in two models of learning deficits, namely the aged memory impaired rats and the scopolamine-induced amnesia model. QR2 is a cytosolic flavoprotein that catalyzes the reduction of its substrate and enhances the production of damaging activated quinone and ROS. QR2-like immunostaining is enriched in cerebral structures associated with learning behaviors, such as the hippocampal formation and the temporofrontal cortex of rat, mouse, and human brains. In cultured rat embryonic hippocampal neurons, selective inhibitors of QR2, namely S26695 and S29434, protected against menadione-induced cell death by reversing its proapoptotic action. S26695 (8 mg/kg) also significantly inhibited scopolamine-induced amnesia. Interestingly, adult QR2 knock-out mice demonstrated enhanced learning abilities in various tasks, including Morris water maze, object recognition, and rotarod performance test. Other behaviors related to anxiety (elevated plus maze), depression (forced swim), and schizophrenia (prepulse inhibition) were not affected in QR2-deficient mice. Together, these data suggest a role for QR2 in cognitive behaviors with QR2 inhibitors possibly representing a novel therapeutic strategy toward the treatment of learning deficits especially observed in the aged brain. PMID:20861374

  15. Direct electrochemistry of nitrate reductase from the fungus Neurospora crassa.

    PubMed

    Kalimuthu, Palraj; Ringel, Phillip; Kruse, Tobias; Bernhardt, Paul V

    2016-09-01

    We report the first direct (unmediated) catalytic electrochemistry of a eukaryotic nitrate reductase (NR). NR from the filamentous fungus Neurospora crassa, is a member of the mononuclear molybdenum enzyme family and contains a Mo, heme and FAD cofactor which are involved in electron transfer from NAD(P)H to the (Mo) active site where reduction of nitrate to nitrite takes place. NR was adsorbed on an edge plane pyrolytic graphite (EPG) working electrode. Non-turnover redox responses were observed in the absence of nitrate from holo NR and three variants lacking the FAD, heme or Mo cofactor. The FAD response is due to dissociated cofactor in all cases. In the presence of nitrate, NR shows a pronounced cathodic catalytic wave with an apparent Michaelis constant (KM) of 39μM (pH7). The catalytic cathodic current increases with temperature from 5 to 35°C and an activation enthalpy of 26kJmol(-1) was determined. In spite of dissociation of the FAD cofactor, catalytically activity is maintained. PMID:27060250

  16. Constitutive nitrate reductase expression and inhibition in winged bean

    SciTech Connect

    Wu, Shenchuan; Harper, J.E. )

    1990-05-01

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

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

    PubMed

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

    2016-08-01

    Platyhelminth thioredoxin glutathione reductase (TGR) is a multifunctional enzyme that crosstalk between the conventional thioredoxin (Trx) and glutathione (GSH) system. It has been validated as a potential drug target in blood flukes. In the present study, we have performed a biochemical study on Fasciola gigantica TGR with substrates DTNB and GSSG. The Michaelis constant (Km) with DTNB was found to be 4.34±0.12μM while it was 61.15±1.50μM with GSSG. The kinetic results were compared with the TGR activities of other helminths. FgTGR showed typical hysteretic behavior with GSSG as other TGRs. We also described a homology-based structure of FgTGR. The cofactors (NADPH and FAD) and substrates (GSSG and DTNB) were docked, and two possible binding sites for substrates were identified in a single chain. The substrates were found to bind more favorably in the second site of TrxR domains. We also presented the first report on binding interaction of DTNB with a TGR. DTNB forms H-bond with His204 and Arg450 of chain A, Sec597, and Gly598 from chain B, salt-bridge with Lys124, and numerous other hydrophobic interactions. Helminth TGR represents an important enzyme in the redox and antioxidant system; hence, its inhibition can be used as an effective strategy against liver flukes. PMID:27112978

  18. Ligand-Dependent Conformational Dynamics of Dihydrofolate Reductase

    PubMed Central

    Reddish, Michael J.; Vaughn, Morgan B.; Fu, Rong; Dyer, R. Brian

    2016-01-01

    Enzymes are known to change among several conformational states during turnover. The role of such dynamic structural changes in catalysis is not fully understood. The influence of dynamics in catalysis can be inferred, but not proven, by comparison of equilibrium structures of protein variants and protein–ligand complexes. A more direct way to establish connections between protein dynamics and the catalytic cycle is to probe the kinetics of specific protein motions in comparison to progress along the reaction coordinate. We have examined the enzyme model system dihydrofolate reductase (DHFR) from Escherichia coli with tryptophan fluorescence-probed temperature-jump spectroscopy. We aimed to observe the kinetics of the ligand binding and ligand-induced conformational changes of three DHFR complexes to establish the relationship among these catalytic steps. Surprisingly, in all three complexes, the observed kinetics do not match a simple sequential two-step process. Through analysis of the relationship between ligand concentration and observed rate, we conclude that the observed kinetics correspond to the ligand binding step of the reaction and a noncoupled enzyme conformational change. The kinetics of the conformational change vary with the ligand's identity and presence but do not appear to be directly related to progress along the reaction coordinate. These results emphasize the need for kinetic studies of DHFR with highly specific spectroscopic probes to determine which dynamic events are coupled to the catalytic cycle and which are not. PMID:26901612

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

    SciTech Connect

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

    1998-08-01

    Enterobacter cloacae PB2 was originally isolated on the basis of its ability to utilize nitrate esters, such as pentaerythritol tetranitrate (PETN) and glycerol trinitrate, as the sole nitrogen source for growth. The enzyme responsible is an NADPH-dependent reductase designated PETN reductase. E. cloacae PB2 was found to be capable of slow aerobic growth with 2,4,6-trinitrotoluene (TNT) as the sole nitrogen source. Dinitrotoluenes were not produced and could not be used as nitrogen sources. Purified PETN reductase was found to reduce TNT to its hydride-Meisenheimer complex, which was further reduced to the dihydride-Meisenheimer complex. Purified PETN reductase and recombinant Escherichia coli expressing PETN reductase were able to liberate nitrogen as nitrite from TNT. The ability to remove nitrogen from TNT suggests that PB2 or recombinant organisms expressing PETN reductase may be useful for bioremediation of TNT-contaminated soil and water.

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

    SciTech Connect

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

    2006-12-01

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

  1. Posttranslational regulation of nitrogenase in Rhodospirillum rubrum strains overexpressing the regulatory enzymes dinitrogenase reductase ADP-ribosyltransferase and dinitrogenase reductase activating glycohydrolase.

    PubMed Central

    Grunwald, S K; Lies, D P; Roberts, G P; Ludden, P W

    1995-01-01

    Rhodospirillum rubrum strains that overexpress the enzymes involved in posttranslational nitrogenase regulation, dinitrogenase reductase ADP-ribosyltransferase (DRAT) and dinitrogenase reductase activating glycohydrolase (DRAG), were constructed, and the effect of this overexpression on in vivo DRAT and DRAG regulation was investigated. Broad-host-range plasmid constructs containing a fusion of the R. rubrum nifH promoter and translation initiation sequences to the second codon of draT, the first gene of the dra operon, were constructed. Overexpression plasmid constructs which overexpressed (i) only functional DRAT, (ii) only functional DRAG and presumably the putative downstream open reading frame (ORF)-encoded protein, or (iii) all three proteins were generated and introduced into wild-type R. rubrum. Overexpression of DRAT still allowed proper regulation of nitrogenase activity, with ADP-ribosylation of dinitrogenase reductase by DRAT occurring only upon dark or ammonium stimuli, suggesting that DRAT is still regulated upon overexpression. However, overexpression of DRAG and the downstream ORF altered nitrogenase regulation such that dinitrogenase reductase did not accumulate in the ADP-ribosylated form under inactivation conditions, suggesting that DRAG was constitutively active and that therefore DRAG regulation is altered upon overexpression. Proper DRAG regulation was observed in a strain overexpressing DRAT, DRAG, and the downstream ORF, suggesting that a proper balance of DRAT and DRAG levels is required for proper DRAG regulation. PMID:7836296

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

    SciTech Connect

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

    1995-05-01

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

  3. 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

  4. The Reaction Mechanism of Methyl-Coenzyme M Reductase

    PubMed Central

    Wongnate, Thanyaporn; Ragsdale, Stephen W.

    2015-01-01

    Methyl-coenzyme M reductase (MCR) is a nickel tetrahydrocorphinoid (coenzyme F430) containing enzyme involved in the biological synthesis and anaerobic oxidation of methane. MCR catalyzes the conversion of methyl-2-mercaptoethanesulfonate (methyl-SCoM) and N-7-mercaptoheptanoylthreonine phosphate (CoB7SH) to CH4 and the mixed disulfide CoBS-SCoM. In this study, the reaction of MCR from Methanothermobacter marburgensis, with its native substrates was investigated using static binding, chemical quench, and stopped-flow techniques. Rate constants were measured for each step in this strictly ordered ternary complex catalytic mechanism. Surprisingly, in the absence of the other substrate, MCR can bind either substrate; however, only one binary complex (MCR·methyl-SCoM) is productive whereas the other (MCR·CoB7SH) is inhibitory. Moreover, the kinetic data demonstrate that binding of methyl-SCoM to the inhibitory MCR·CoB7SH complex is highly disfavored (Kd = 56 mm). However, binding of CoB7SH to the productive MCR·methyl-SCoM complex to form the active ternary complex (CoB7SH·MCR(NiI)·CH3SCoM) is highly favored (Kd = 79 μm). Only then can the chemical reaction occur (kobs = 20 s−1 at 25 °C), leading to rapid formation and dissociation of CH4 leaving the binary product complex (MCR(NiII)·CoB7S−·SCoM), which undergoes electron transfer to regenerate Ni(I) and the final product CoBS-SCoM. This first rapid kinetics study of MCR with its natural substrates describes how an enzyme can enforce a strictly ordered ternary complex mechanism and serves as a template for identification of the reaction intermediates. PMID:25691570

  5. Short-chain dehydrogenases/reductases in cyanobacteria.

    PubMed

    Kramm, Anneke; Kisiela, Michael; Schulz, Rüdiger; Maser, Edmund

    2012-03-01

    The short-chain dehydrogenases/reductases (SDRs) represent a large superfamily of enzymes, most of which are NAD(H)-dependent or NADP(H)-dependent oxidoreductases. They display a wide substrate spectrum, including steroids, alcohols, sugars, aromatic compounds, and xenobiotics. On the basis of characteristic sequence motifs, the SDRs are subdivided into two main (classical and extended) and three smaller (divergent, intermediate, and complex) families. Despite low residue identities in pairwise comparisons, the three-dimensional structure among the SDRs is conserved and shows a typical Rossmann fold. Here, we used a bioinformatics approach to determine whether and which SDRs are present in cyanobacteria, microorganisms that played an important role in our ecosystem as the first oxygen producers. Cyanobacterial SDRs could indeed be identified, and were clustered according to the SDR classification system. Furthermore, because of the early availability of its genome sequence and the easy application of transformation methods, Synechocystis sp. PCC 6803, one of the most important cyanobacterial strains, was chosen as the model organism for this phylum. Synechocystis sp. SDRs were further analysed with bioinformatics tools, such as hidden Markov models (HMMs). It became evident that several cyanobacterial SDRs show remarkable sequence identities with SDRs in other organisms. These so-called 'homologous' proteins exist in plants, model organisms such as Drosophila melanogaster and Caenorhabditis  elegans, and even in humans. As sequence identities of up to 60% were found between Synechocystis and humans, it was concluded that SDRs seemed to have been well conserved during evolution, even after dramatic terrestrial changes such as the conversion of the early reducing atmosphere to an oxidizing one by cyanobacteria. PMID:22251568

  6. Differing views of the role of selenium in thioredoxin reductase

    PubMed Central

    Ruggles, Erik L.

    2010-01-01

    This review covers three different chemical explanations that could account for the requirement of selenium in the form of selenocysteine in the active site of mammalian thioredoxin reductase. These views are the following: (1) the traditional view of selenocysteine as a superior nucleophile relative to cysteine, (2) the superior leaving group ability of a selenol relative to a thiol due to its significantly lower pKa and, (3) the superior ability of selenium to accept electrons (electrophilicity) relative to sulfur. We term these chemical explanations as the “chemico-enzymatic” function of selenium in an enzyme. We formally define the chemico-enzymatic function of selenium as its specific chemical property that allows a selenoenzyme to catalyze its individual reaction. However we, and others, question whether selenocysteine is chemically necessary to catalyze an enzymatic reaction since cysteine-homologs of selenocysteine-containing enzymes catalyze their specific enzymatic reactions with high catalytic efficiency. There must be a unique chemical reason for the presence of selenocysteine in enzymes that explains the biological pressure on the genome to maintain the complex selenocysteine-insertion machinery. We term this biological pressure the “chemico-biological” function of selenocysteine. We discuss evidence that this chemico-biological function is the ability of selenoenzymes to resist inactivation by irreversible oxidation. The way in which selenocysteine confers resistance to oxidation could be due to the superior ability of the oxidized form of selenocysteine (Sec-SeO2−, seleninic acid) to be recycled back to its parent form (Sec-SeH, selenocysteine) in comparison to the same cycling of cysteine-sulfinic acid to cysteine (Cys-SO2− to Cys-SH). PMID:20397034

  7. Increased 5. cap alpha. -reductase activity in idiopathic hirsutism

    SciTech Connect

    Serafini, P.; Lobo, R.A.

    1985-01-01

    In vitro, genital skin 5..cap alpha..-reductase activity (5..cap alpha..-RA) was measured in ten hirsute women with normal androgen levels (idiopathic hirsutism (IH)) and in ten hirsute women with elevated androgen levels (polycystic ovary syndrome (PCO)) in order to determine the influence of secreted androgens on 5..cap alpha..-RA. In vitro 5..cap alpha..-RA was assessed by incubations of skin with /sup 14/C-testosterone (T) for 2 hours, after which steroids were separated and the radioactivity of dihydrotestosterone (DHT) and 5..cap alpha..-androstane 3..cap alpha..-17..beta..-estradiol (3..cap alpha..-diol) in specific eluates were determined. All androgens were normal in IH with the exception of higher levels of 3..cap alpha..-diol glucuronide which were similar to the levels of PCO. The conversion ratio (CR) of T to DHT in IH and PCO were similar, yet significantly greater than the CR of control subjects. The CR of T to 3..cap alpha..-diol in IH and PCO were similar, yet higher than in control subjects. Serum androgens showed no correlation with 5..cap alpha..-RA, while the CR of T to DHT showed a significant positive correlation with the Ferriman and Gallwey score. The increased 5..cap alpha..-RA in IH appears to be independent of serum androgen levels and is, therefore, an inherent abnormality. The term idiopathic is a misnomer, because hirsutism in these patients may be explained on the basis of increased skin 5..cap alpha..-RA.

  8. Characterization of thioredoxin glutathione reductase in Schiotosoma japonicum.

    PubMed

    Han, Yanhui; Zhang, Min; Hong, Yang; Zhu, Zhu; Li, Dong; Li, Xiangrui; Fu, Zhiqiang; Lin, Jiaojiao

    2012-09-01

    Schistosomiasis is one of the most prevalent and serious parasitic diseases in the world and remains an important public health problem in China. Screening and discovery of an effective vaccine candidate or new drug target is crucial for the control of this disease. In this study, we cloned a cDNA encoding Schistosoma japonicum (S. japonicum) thioredoxin glutathione reductase (SjTGR) from the cDNA of 42-day-old adult worms. The open reading frame (ORF) of the gene was 1791 base pairs (bp) encoding a protein of 596 amino acids. SjTGR was subcloned into pET-32a (+) and expressed in Escherichia coli (E. coli) BL21 (DE3). The recombinant protein rSjTGR exhibited enzymatic activity of 5.13U/mg with DTNB as the substrate, and showed strong immunogenecity. Real-time PCR results indicated that SjTGR was expressed at a higher level in 35-day-old schistosome worms in transcript. We vaccinated BALB/c mice with rSjTGR in combination with MONTANIDE™ ISA 206 VG (ISA 206) and observed a 33.50% to 36.51% (P<0.01) decrease in the adult worm burden and a 33.73%to 43.44% (P<0.01) decrease in the number of eggs counted compared to the ISA 206 or blank control groups in two independent vaccination tests. ELISA analysis demonstrated that rSjTGR induced a high level of SjTGR-specific IgG, IgG1, and IgG 2a antibodies and induced elevated production of IFN-γ. This study provides the basis for further investigations into the biological function of SjTGR and further evaluation of the potential use of this molecule as a vaccine candidate or new drug target is warranted. PMID:22484130

  9. Glutathione Reductase Is Essential for Host Defense against Bacterial Infection

    PubMed Central

    Yan, Jing; Ralston, Melissa M.; Meng, Xiaomei; Bongiovanni, Kathleen D.; Jones, Amanda L.; Benndorf, Rainer; Nelin, Leif D.; Frazier, W. Joshua; Rogers, Lynette K.; Smith, Charles V.; Liu, Yusen

    2013-01-01

    Glutathione reductase (Gsr)1 catalyzes the reduction of glutathione disulfide to glutathione, a major cellular antioxidant. We have recently shown that Gsr is essential for host defense against the Gram-negative bacteria Escherichia coli in a mouse model of sepsis. While we have demonstrated that Gsr is required for sustaining the oxidative burst and the development of neutrophil extracellular traps, the role of Gsr in other phagocytic functions remains unclear. It is also unclear whether Gsr-deficient mice exhibit host defense defects against Gram-positive bacteria. In the present study, we characterized the effects of Gsr deficiency on the innate immune responses to a Gram-positive bacterium, group B Streptococcus, and to the Gram-negative bacterial cell wall component lipopolysaccharide (LPS). We found that like, E. coli, group B Streptococcus resulted in a substantially more robust cytokine response and a markedly higher morbidity and mortality in Gsr-deficient mice than in wildtype mice. The increased morbidity and mortality were associated with greater bacterial burden in the Gsr-deficient mice. Interestingly, Gsr-deficient mice did not exhibit a greater sensitivity to LPS than did wildtype mice. Analysis of the neutrophils of Gsr-deficient mice revealed impaired phagocytosis. In response to thioglycollate stimulation, Gsr-deficient mice mobilized far fewer phagocytes, including neutrophils, macrophages, and eosinophils, into their peritoneal cavities than did wildtype mice. The defective phagocyte mobilization is associated with profound oxidation and aggregation of ascitic proteins, particularly albumin. Our results indicate that the oxidative defense mechanism mediated by Gsr is required for an effective innate immune response against bacteria, likely by preventing phagocyte dysfunction due to oxidative damage. PMID:23623936

  10. Comparative characterization of novel ene-reductases from cyanobacteria.

    PubMed

    Fu, Yilei; Castiglione, Kathrin; Weuster-Botz, Dirk

    2013-05-01

    The growing importance of biocatalysis in the syntheses of enantiopure molecules results from the benefits of enzymes regarding selectivity and specificity of the reaction and ecological issues of the process. Ene-reductases (ERs) from the old yellow enzyme family have received much attention in the last years. These flavo-enzymes catalyze the trans-specific reduction of activated C=C bonds, which is an important reaction in asymmetric synthesis, because up to two stereogenic centers can be created in one reaction. However, limitations of ERs described in the literature such as their moderate catalytic activity and their strong preference for NADPH promote the search for novel ERs with improved properties. In this study, we characterized nine novel ERs from cyanobacterial strains belonging to different taxonomic orders and habitats. ERs were identified with activities towards a broad spectrum of alkenes. The reduction of maleimide was catalyzed with activities of up to 35.5 U mg(-1) using NADPH. Ketoisophorone and (R)-carvone, which were converted to the highly valuable compounds (R)-levodione and (2R,5R)-dihydrocarvone, were reduced with reaction rates of up to 2.2 U mg(-1) with NADPH. In contrast to other homologous ERs from the literature, NADH was accepted at moderate to high rates as well: Enzyme activities of up to 16.7 U mg(-1) were obtained for maleimide and up to 1.3 U mg(-1) for ketoisophorone and (R)-carvone. Additionally, excellent stereoselectivities were achieved in the reduction of (R)-carvone (97-99% de). In particular, AnabaenaER3 from Anabaena variabilis ATCC 29413 and AcaryoER1 from Acaryochloris marina MBIC 11017 were identified as useful biocatalysts. Therefore, novel ERs from cyanobacteria with high catalytic efficiency were added to the toolbox for the asymmetric reduction of alkenes. PMID:23280373

  11. Aldo-keto reductase (AKR) superfamily: genomics and annotation.

    PubMed

    Mindnich, Rebekka D; Penning, Trevor M

    2009-07-01

    Aldo-keto reductases (AKRs) are phase I metabolising enzymes that catalyse the reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H)-dependent reduction of carbonyl groups to yield primary and secondary alcohols on a wide range of substrates, including aliphatic and aromatic aldehydes and ketones, ketoprostaglandins, ketosteroids and xenobiotics. In so doing they functionalise the carbonyl group for conjugation (phase II enzyme reactions). Although functionally diverse, AKRs form a protein superfamily based on their high sequence identity and common protein fold, the (alpha/beta) 8 -barrel structure. Well over 150 AKR enzymes, from diverse organisms, have been annotated so far and given systematic names according to a nomenclature that is based on multiple protein sequence alignment and degree of identity. Annotation of non-vertebrate AKRs at the National Center for Biotechnology Information or Vertebrate Genome Annotation (vega) database does not often include the systematic nomenclature name, so the most comprehensive overview of all annotated AKRs is found on the AKR website (http://www.med.upenn.edu/akr/). This site also hosts links to more detailed and specialised information (eg on crystal structures, gene expression and single nucleotide polymorphisms [SNPs]). The protein-based AKR nomenclature allows unambiguous identification of a given enzyme but does not reflect the wealth of genomic and transcriptomic variation that exists in the various databases. In this context, identification of putative new AKRs and their distinction from pseudogenes are challenging. This review provides a short summary of the characteristic features of AKR biochemistry and structure that have been reviewed in great detail elsewhere, and focuses mainly on nomenclature and database entries of human AKRs that so far have not been subject to systematic annotation. Recent developments in the annotation of SNP and transcript variance in AKRs are also summarised. PMID:19706366

  12. Diversity in Overall Activity Regulation of Ribonucleotide Reductase*

    PubMed Central

    Jonna, Venkateswara Rao; Crona, Mikael; Rofougaran, Reza; Lundin, Daniel; Johansson, Samuel; Brännström, Kristoffer; Sjöberg, Britt-Marie; Hofer, Anders

    2015-01-01

    Ribonucleotide reductase (RNR) catalyzes the reduction of ribonucleotides to the corresponding deoxyribonucleotides, which are used as building blocks for DNA replication and repair. This process is tightly regulated via two allosteric sites, the specificity site (s-site) and the overall activity site (a-site). The a-site resides in an N-terminal ATP cone domain that binds dATP or ATP and functions as an on/off switch, whereas the composite s-site binds ATP, dATP, dTTP, or dGTP and determines which substrate to reduce. There are three classes of RNRs, and class I RNRs consist of different combinations of α and β subunits. In eukaryotic and Escherichia coli class I RNRs, dATP inhibits enzyme activity through the formation of inactive α6 and α4β4 complexes, respectively. Here we show that the Pseudomonas aeruginosa class I RNR has a duplicated ATP cone domain and represents a third mechanism of overall activity regulation. Each α polypeptide binds three dATP molecules, and the N-terminal ATP cone is critical for binding two of the dATPs because a truncated protein lacking this cone could only bind dATP to its s-site. ATP activates the enzyme solely by preventing dATP from binding. The dATP-induced inactive form is an α4 complex, which can interact with β2 to form a non-productive α4β2 complex. Other allosteric effectors induce a mixture of α2 and α4 forms, with the former being able to interact with β2 to form active α2β2 complexes. The unique features of the P. aeruginosa RNR are interesting both from evolutionary and drug discovery perspectives. PMID:25971975

  13. Nitroimidazole Action in Entamoeba histolytica: A Central Role for Thioredoxin Reductase

    PubMed Central

    Leitsch, David; Kolarich, Daniel; Wilson, Iain B. H; Altmann, Friedrich; Duchêne, Michael

    2007-01-01

    Metronidazole, a 5-nitroimidazole drug, has been the gold standard for several decades in the treatment of infections with microaerophilic protist parasites, including Entamoeba histolytica. For activation, the drug must be chemically reduced, but little is known about the targets of the active metabolites. Applying two-dimensional gel electrophoresis and mass spectrometry, we searched for protein targets in E. histolytica. Of all proteins visualized, only five were found to form adducts with metronidazole metabolites: thioredoxin, thioredoxin reductase, superoxide dismutase, purine nucleoside phosphorylase, and a previously unknown protein. Recombinant thioredoxin reductase carrying the modification displayed reduced enzymatic activity. In treated cells, essential non-protein thiols such as free cysteine were also affected by covalent adduct formation, their levels being drastically reduced. Accordingly, addition of cysteine allowed E. histolytica to survive in the presence of otherwise lethal metronidazole concentrations and reduced protein adduct formation. Finally, we discovered that thioredoxin reductase reduces metronidazole and other nitro compounds, suggesting a new model of metronidazole activation in E. histolytica with a central role for thioredoxin reductase. By reducing metronidazole, the enzyme renders itself and associated thiol-containing proteins vulnerable to adduct formation. Because thioredoxin reductase is a ubiquitous enzyme, similar processes could occur in other eukaryotic or prokaryotic organisms. PMID:17676992

  14. Testosterone selectively affects aromatase and 5α-reductase activities in the green anole lizard brain

    PubMed Central

    Cohen, Rachel E.; Wade, Juli

    2011-01-01

    Testosterone (T) and its metabolites are important in the regulation of reproductive behavior in males of a variety of vertebrate species. Aromatase converts T to estradiol and 5α-reductase converts T to 5α-dihydrotestosterone (DHT). Male green anole reproduction depends on androgens, yet 5α-reductase in the brain is not sexually dimorphic and does not vary with season. In contrast, aromatase activity in the male brain is increased during the breeding compared to non-breeding season, and males have higher levels than females during the breeding season. Aromatase is important for female, but not male, sexual behaviors. The present experiment was conducted to determine whether 5α-reductase and aromatase are regulated by T. Enzyme activity was quantified in whole brain homogenates in both the breeding and non-breeding seasons in males and females that had been treated with either a T or blank implant. In males only, T increased 5α-reductase activity regardless of season and up-regulated aromatase during the breeding season specifically. Thus, regulation of both enzymes occurs in males, whereas females do not show parallel sensitivity to T. When considered with previous results, the data suggest that aromatase might influence a male function associated with the breeding season other than sexual behavior. 5α-Reductase can be mediated by T availability, but this regulation may not serve a sex- or season-specific purpose. PMID:19917285

  15. Characterization of the Reduction of Selenate and Tellurite by Nitrate Reductases

    PubMed Central

    Sabaty, Monique; Avazeri, Cécile; Pignol, David; Vermeglio, André

    2001-01-01

    Preliminary studies showed that the periplasmic nitrate reductase (Nap) of Rhodobacter sphaeroides and the membrane-bound nitrate reductases of Escherichia coli are able to reduce selenate and tellurite in vitro with benzyl viologen as an electron donor. In the present study, we found that this is a general feature of denitrifiers. Both the periplasmic and membrane-bound nitrate reductases of Ralstonia eutropha, Paracoccus denitrificans, and Paracoccus pantotrophus can utilize potassium selenate and potassium tellurite as electron acceptors. In order to characterize these reactions, the periplasmic nitrate reductase of R. sphaeroides f. sp. denitrificans IL106 was histidine tagged and purified. The Vmax and Km were determined for nitrate, tellurite, and selenate. For nitrate, values of 39 μmol · min−1 · mg−1 and 0.12 mM were obtained for Vmax and Km, respectively, whereas the Vmax values for tellurite and selenate were 40- and 140-fold lower, respectively. These low activities can explain the observation that depletion of the nitrate reductase in R. sphaeroides does not modify the MIC of tellurite for this organism. PMID:11679335

  16. Enzymatic removal of diacetyl from beer. II. Further studies on the use of diacetyl reductase.

    PubMed

    Tolls, T N; Shovers, J; Sandine, W E; Elliker, P R

    1970-04-01

    Diacetyl removal from beer was studied with whole cells and crude enzyme extracts of yeasts and bacteria. Cells of Streptococcus diacetilactis 18-16 destroyed diacetyl in solutions at a rate almost equal to that achieved by the addition of whole yeast cells. Yeast cells impregnated in a diatomaceous earth filter bed removed all diacetyl from solutions percolated through the bed. Undialyzed crude enzyme extracts from yeast cells removed diacetyl very slowly from beer at its normal pH (4.1); at a pH of 5.0 or higher, rapid diacetyl removal was achieved. Dialyzed crude enzyme extracts from yeast cells were found to destroy diacetyl in a manner quite similar to that of diacetyl reductase from Aerobacter aerogenes, and both the bacterial and the yeast extracts were stimulated significantly by the addition of reduced nicotinamide adenine dinucleotide (NADH). Diacetyl reductase activity of four strains of A. aerogenes was compared; three of the strains produced enzyme with approximately twice the specific activity of the other strain (8724). Gel electrophoresis results indicated that at least three different NADH-oxidizing enzymes were present in crude extracts of diacetyl reductase. Sephadex-gel chromotography separated NADH oxidase from diacetyl reductase. It was also noted that ethyl alcohol concentrations approximately equivalent to those found in beer were quite inhibitory to diacetyl reductase. PMID:4315861

  17. The archaeon Methanosarcina acetivorans contains a protein disulfide reductase with an iron-sulfur cluster.

    PubMed

    Lessner, Daniel J; Ferry, James G

    2007-10-01

    Methanosarcina acetivorans, a strictly anaerobic methane-producing species belonging to the domain Archaea, contains a gene cluster annotated with homologs encoding oxidative stress proteins. One of the genes (MA3736) is annotated as a gene encoding an uncharacterized carboxymuconolactone decarboxylase, an enzyme required for aerobic growth with aromatic compounds by species in the domain Bacteria. Methane-producing species are not known to utilize aromatic compounds, suggesting that MA3736 is incorrectly annotated. The product of MA3736, overproduced in Escherichia coli, had protein disulfide reductase activity dependent on a C(67)XXC(70) motif not found in carboxymuconolactone decarboxylase. We propose that MA3736 be renamed mdrA (methanosarcina disulfide reductase). Further, unlike carboxymuconolactone decarboxylase, MdrA contained an Fe-S cluster. Binding of the Fe-S cluster was dependent on essential cysteines C(67) and C(70), while cysteines C(39) and C(107) were not required. Loss of the Fe-S cluster resulted in conversion of MdrA from an inactive hexamer to a trimer with protein disulfide reductase activity. The data suggest that MdrA is the prototype of a previously unrecognized protein disulfide reductase family which contains an intermolecular Fe-S cluster that controls oligomerization as a mechanism to regulate protein disulfide reductase activity. PMID:17675382

  18. Purification and thermal dependence of glutathione reductase from two forage legume species.

    PubMed

    Kidambi, S P; Mahan, J R; Matches, A G

    1990-02-01

    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 degrees 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 degrees C), and (c) the thermal kinetic windows (6-19 degrees 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

  19. MEK2 regulates ribonucleotide reductase activity through functional interaction with ribonucleotide reductase small subunit p53R2.

    PubMed

    Piao, Chunmei; Youn, Cha-Kyung; Jin, Min; Yoon, Sang Pil; Chang, In-Youb; Lee, Jung Hee; You, Ho Jin

    2012-09-01

    The p53R2 protein, a newly identified member of the ribonucleotide reductase family that provides nucleotides for DNA damage repair, is directly regulated by p53. We show that p53R2 is also regulated by a MEK2 (ERK kinase 2/MAP kinase kinase 2)-dependent pathway. Increased MEK1/2 phosphorylation by serum stimulation coincided with an increase in the RNR activity in U2OS and H1299 cells. The inhibition of MEK2 activity, either by treatment with a MEK inhibitor or by transfection with MEK2 siRNA, dramatically decreased the serum-stimulated RNR activity. Moreover, p53R2 siRNA, but not R2 siRNA, significantly inhibits serum-stimulated RNR activity, indicating that p53R2 is specifically regulated by a MEK2-dependent pathway. Co-immunoprecipitation analyses revealed that the MEK2 segment comprising amino acids 65-171 is critical for p53R2-MEK2 interaction, and the binding domain of MEK2 is required for MEK2-mediated increased RNR activity. Phosphorylation of MEK1/2 was greatly augmented by ionizing radiation, and RNR activity was concurrently increased. Ionizing radiation-induced RNR activity was markedly attenuated by transfection of MEK2 or p53R2 siRNA, but not R2 siRNA. These data show that MEK2 is an endogenous regulator of p53R2 and suggest that MEK2 may associate with p53R2 and upregulate its activity. PMID:22895183

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

    PubMed Central

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

    1998-01-01

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

  1. Discovery and characterization of a xylose reductase from Zymomonas mobilis ZM4.

    PubMed

    Agrawal, Manoj; Chen, Rachel Ruizhen

    2011-11-01

    Formation of xylitol, a byproduct from xylose fermentation, is a major limiting factor in ethanol production from xylose in engineered Zymomonas strains, yet the postulated xylose reductase remains elusive. We report here the discovery of xylose reductase in Zymomonas mobilis and, for the first time, to associate the enzyme function with its gene. Besides xylose and xylulose, the enzyme was active towards benzaldehyde, furfural, 5-hydroxymethyl furfural, and acetaldehyde, exhibiting nearly 150-times higher affinity with benzaldehyde than xylose. The discovery of xylose reductase paves the way for further improvement of xylose fermentation in Z. mobilis. The enzyme may also be used to mitigate toxicity of furfural and other inhibitors from plant biomass. PMID:21720846

  2. HMG-CoA reductase inhibition causes neurite loss by interfering with geranylgeranylpyrophosphate synthesis.

    PubMed

    Schulz, Joachim G; Bösel, Julian; Stoeckel, Magali; Megow, Dirk; Dirnagl, Ulrich; Endres, Matthias

    2004-04-01

    To determine whether neurite outgrowth depends upon the mevalonate pathway, we blocked mevalonate synthesis in nerve growth factor-treated PC12 cells or primary cortical neurones with atorvastatin, a 3-hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, and substituted different intermediates of the mevalonate pathway. We show that HMG-CoA reductase inhibition causes a profound reduction of neurite length, neurite loss and ultimatively cell death in undifferentiated and pre-differentiated PC12 cells and also in rat primary cortical neurones. Geranylgeranylpyrophosphate, but not farnesylpyrophosphate, squalene or cholesterol, completely compensated for the lack of mevalonate. Our data indicate that, under HMG-CoA reductase inhibition, geranylgeranylpyrophosphate rather than farnesylpyrophosphate or cholesterol is critical for neurite outgrowth and/or maintenance. Loss of neurites is an early manifestation of various neurodegenerative disorders, and dysfunction of isoprenylation might play a role in their pathogenesis. PMID:15030386

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

    SciTech Connect

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

    1984-03-01

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

  4. Structural changes induced by catalytic turnover at the molybdenum site of arabidopsis nitrate reductase

    SciTech Connect

    George, G.N.; Mertens, J.A.; Campbell, W.H.

    1999-10-20

    Assimilatory nitrate reductases catalyze the reduction of nitrate to nitrite, which is the first and rate-limiting step of nitrogen assimilation in algae, fungi, and higher plants. The nitrate reductase from the thale cress, Arabidopsis thaliana, is a dimer, with each of the {approximately} 103,000 molecular weight monomers containing one molybdenum associated with a single pterin dithiolene cofactor, a flavin adenine dinucleotide cofactor, and a cytochrome b-type heme. During the catalytic cycle, reducing equivalents in the form of NADH enter the enzyme at the flavin site and are subsequently transferred by intramolecular electron transfer via the heme to the molybdenum center, where the two-electron reduction of nitrate takes place. In this paper the authors present the Mo K-edge EXAFS of Arabidopsis nitrate reductase in three different forms: oxidized as-isolated, reduced, and oxidized after catalytic turnover of excess nitrate (nitrate-oxidized).

  5. Overproduction of dihydrofolate reductase and gene amplification in methotrexate-resistant Chinese hamster ovary cells

    SciTech Connect

    Flintoff, W.F.; Weber, M.K.; Nagainis, C.R.; Essani, A.K.; Robertson, D.; Salser, W.

    1982-03-01

    Stable isolates of Chinese hamster ovary cells that are highly resistant to methotrexate have been selected in a multistep selection process. Quantitative immunoprecipitations have indicated that these isolates synthesize dihydrofolate reductase at an elevated rate over its synthesis in sensitive cells. Restriction enzyme and Southern blot analyses with a murine reductase cDNA probe indicate that the highly resistant isolates contain amplifications of the dihydrofolate reductase gene number. Depending upon the parental line used to select these resistant cells, they overproduce either a wild-type enzyme or a structurally altered enzyme. Karyotype analysis shows that some of these isolates contain chromosomes with homogeneously staining regions whereas others do not contain such chromosomes.

  6. Characterization of two dissimilatory sulfite reductases from sulfate-reducing bacteria

    NASA Astrophysics Data System (ADS)

    Huynh, B. H.; Moura, I.; Lino, A. R.; Moura, J. J. G.; Legall, J.

    1988-02-01

    Mössbauer, EPR, and biochemical techniques were used to characterize two dissimilatory sulfite reductases: desulforubidin from Desulfovibrio baculatus strain DSM 1743 and desulfoviridin from Desulfovibrio gigas. For each molecule of desulforubidin, there are two sirohemes and four [4Fe-4S] clusters. The [4Fe-4S] clusters are in the diamagnetic 2+ oxidation state. The sirohemes are high-spin ferric (S=5/2) and each siroheme is exchanged-coupled to a [4Fe-4S]2+ cluster. Such an exchange-coupled siroheme-[4Fe-4S] unit has also been found in the assimilatory sulfite reductase from Escherichia coli/1/ and in a low-molecular weight sulfite reductase from Desulfovibrio vulgaris/2/. For each molecule of defulfoviridin, there are two tetrahydroporphyrin groups and four [4Fe-4S]2+ clusters. To our surprise, we discovered that about 80% of the tetrahydroporphyrin groups, however, do not bind iron.

  7. Inactivation kinetics of dihydrofolate reductase from Chinese hamster during urea denaturation.

    PubMed Central

    Wu, J W; Wang, Z X; Zhou, J M

    1997-01-01

    The kinetic theory of substrate reaction during modification of enzyme activity has been applied to the study of inactivation kinetics of Chinese hamster dihydrofolate reductase by urea [Tsou (1988) Adv. Enzymol. Relat. Areas Mol. Biol. 61, 381-436]. On the basis of the kinetic equation of substrate reaction in the presence of urea, all microscopic kinetic constants for the free enzyme and enzyme-substrate binary and ternary complexes have been determined. The results of the present study indicate that the denaturation of dihydrofolate reductase by urea follows single-phase kinetics, and changes in enzyme activity and tertiary structure proceed simultaneously in the unfolding process. Both substrates, NADPH and 7,8-dihydrofolate, protect dihydrofolate reductase against inactivation, and enzyme-substrate complexes lose their activity less rapidly than the free enzyme. PMID:9182696

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

    SciTech Connect

    Beierlein, J.; Frey, K; Bolstad, D; Pelphrey, P; Joska, T; Smith, A; Priestley, N; Wright, D; Anderson, A

    2008-01-01

    Bacillus anthracis, the causative agent of anthrax, poses a significant biodefense danger. Serious limitations in approved therapeutics and the generation of resistance have produced a compelling need for new therapeutic agents against this organism. Bacillus anthracis is known to be insensitive to the clinically used antifolate, trimethoprim, because of a lack of potency against the dihydrofolate reductase enzyme. Herein, we describe a novel lead series of B. anthracis dihydrofolate reductase inhibitors characterized by an extended trimethoprim-like scaffold. The best lead compound adds only 22 Da to the molecular weight and is 82-fold more potent than trimethoprim. An X-ray crystal structure of this lead compound bound to B. anthracis dihydrofolate reductase in the presence of NADPH was determined to 2.25 A resolution. The structure reveals several features that can be exploited for further development of this lead series.

  9. Biochemical predetermination of the NO synthase and nitrite reductase components of the nitric oxide cycle.

    PubMed

    Reutov, V P

    1999-05-01

    This review presents some aspects of a concept of cellular evolution bearing a relationship to nitrate--nitrite respiration, the endosymbiosis theory, and the origin of NO synthase and nitrite reductase activity in heme-containing proteins. Analysis of structural and functional unity of the NO synthase and nitrite reductase systems suggests that these systems did not arise without any relation to evolutionarily ancient energetic systems of cells. The use of symmetry principles reveals commonalities among many electron transport chains which in the language of physics is called "invariance". This work also comparatively analyzes the nitric oxide cycle and the known nitrogen cycle. The ideas about evolution of the NO synthase and nitrite reductase systems developed here are clearly compatible with the endosymbiotic theory and the hypothesis that nitrate--nitrite respiration was a precursor of oxygen-dependent respiration. PMID:10381613

  10. Partial Purification and Characterization of d-Ribose-5-phosphate Reductase from Adonis vernalis L. Leaves

    PubMed Central

    Negm, Fayek B.; Marlow, Gary C.

    1985-01-01

    This study presents evidence for a new enzyme, d-ribose-5-P reductase, which catalyzes the reaction: d-ribose-5-P + NADPH + H+ → d-ribitol-5-P + NADP+. The enzyme was isolated from Adonis vernalis L. leaves in 38% yield and was purified 71-fold. The reductase was NADPH specific and had a pH optimum in the range of 5.5 to 6.0. The Michaelis constant value for d-ribose-5-P reduction was 1.35 millimolar. The enzyme also reduced d-erythrose-4-P, d-erythrose, dl-glyceraldehyde, and the aromatic aldehyde 3-pyridinecarboxaldehyde. Hexoses, hexose phosphates, pentoses, and dihydroxyacetone did not serve as substrates. d-Ribose-5-P reductase is distinct from the other known ribitol synthesizing enzymes detected in bacteria and yeast, and may be responsible for ribitol synthesis in Adonis vernalis. PMID:16664320

  11. Converting the NiFeS Carbon Monoxide Dehydrogenase to a Hydrogenase and a Hydroxylamine Reductase

    PubMed Central

    Heo, Jongyun; Wolfe, Marcus T.; Staples, Christopher R.; Ludden, Paul W.

    2002-01-01

    Substitution of one amino acid for another at the active site of an enzyme usually diminishes or eliminates the activity of the enzyme. In some cases, however, the specificity of the enzyme is changed. In this study, we report that the changing of a metal ligand at the active site of the NiFeS-containing carbon monoxide dehydrogenase (CODH) converts the enzyme to a hydrogenase or a hydroxylamine reductase. CODH with alanine substituted for Cys531 exhibits substantial uptake hydrogenase activity, and this activity is enhanced by treatment with CO. CODH with valine substituted for His265 exhibits hydroxylamine reductase activity. Both Cys531 and His265 are ligands to the active-site cluster of CODH. Further, CODH with Fe substituted for Ni at the active site acquires hydroxylamine reductase activity. PMID:12374822

  12. Serum homocysteine, folate level and methylenetetrahydrofolate reductase 677, 1298 gene polymorphism in Korean schizophrenic patients.

    PubMed

    Lee, Young Sik; Han, Doug Hyun; Jeon, Chang Moo; Lyoo, In Kyoon; Na, Chul; Chae, Seok Lae; Cho, Soo Churl

    2006-05-15

    High homocysteine serum level has been regarded as one of the important factors that influence the development of schizophrenia. Genetic variations of methylenetetrahydrofolate reductase, which is a main enzyme reducing homocysteine level, are reported in schizophrenic patients. We measured the serum level of homocysteine/folate and methylenetetrahydrofolate reductase C677T/A1298C gene polymorphism in 235 patients with schizophrenia. Plasma homocysteine levels were higher and folate levels were lower in patients than in comparison subjects. Variations of C677T were more frequent in patients than in comparison subjects. Patients with the 677TT genotype showed higher homocysteine levels than patients with the CC and CT genotypes. These findings suggest that folate supplement may be beneficial to some schizophrenic patients with homocysteinemia due to the genetic defect of methylenetetrahydrofolate reductase. PMID:16641680

  13. Regulation of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Activity in Human Fibroblasts by Lipoproteins

    PubMed Central

    Brown, Michael S.; Dana, Suzanna E.; Goldstein, Joseph L.

    1973-01-01

    The activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.34), the rate-limiting enzyme of hepatic cholesterol biosynthesis, is suppressed in human fibroblasts cultured in the presence of serum. This enzyme activity increases by more than 10-fold after the removal of serum from the medium. The rise in enzyme activity requires de novo protein synthesis and is not accompanied by changes in the activities of several other cellular enzymes. The factor responsible for the suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in cultured fibroblasts is present in the sera of at least four mammalian species, and in human serum it is found in the low-density lipoproteins. Human high-density lipoproteins, very low-density lipoproteins from chicken egg yolk, and the fraction of human serum containing no lipoproteins do not suppress the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase. PMID:4352976

  14. 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.

  15. Periplasmic Nitrate Reductase (NapABC Enzyme) Supports Anaerobic Respiration by Escherichia coli K-12

    PubMed Central

    Stewart, Valley; Lu, Yiran; Darwin, Andrew J.

    2002-01-01

    Periplasmic nitrate reductase (NapABC enzyme) has been characterized from a variety of proteobacteria, especially Paracoccus pantotrophus. Whole-genome sequencing of Escherichia coli revealed the structural genes napFDAGHBC, which encode NapABC enzyme and associated electron transfer components. E. coli also expresses two membrane-bound proton-translocating nitrate reductases, encoded by the narGHJI and narZYWV operons. We measured reduced viologen-dependent nitrate reductase activity in a series of strains with combinations of nar and nap null alleles. The napF operon-encoded nitrate reductase activity was not sensitive to azide, as shown previously for the P. pantotrophus NapA enzyme. A strain carrying null alleles of narG and narZ grew exponentially on glycerol with nitrate as the respiratory oxidant (anaerobic respiration), whereas a strain also carrying a null allele of napA did not. By contrast, the presence of napA+ had no influence on the more rapid growth of narG+ strains. These results indicate that periplasmic nitrate reductase, like fumarate reductase, can function in anaerobic respiration but does not constitute a site for generating proton motive force. The time course of Φ(napF-lacZ) expression during growth in batch culture displayed a complex pattern in response to the dynamic nitrate/nitrite ratio. Our results are consistent with the observation that Φ(napF-lacZ) is expressed preferentially at relatively low nitrate concentrations in continuous cultures (H. Wang, C.-P. Tseng, and R. P. Gunsalus, J. Bacteriol. 181:5303-5308, 1999). This finding and other considerations support the hypothesis that NapABC enzyme may function in E. coli when low nitrate concentrations limit the bioenergetic efficiency of nitrate respiration via NarGHI enzyme. PMID:11844760

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

    PubMed

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

    2016-05-01

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

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

    PubMed Central

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

    2012-01-01

    Several aldo–keto reductase (AKR) enzymes from subfamilies 1B and 1C show retinaldehyde reductase activity, having low Km and kcat values. Only AKR1B10 and 1B12, with all-trans-retinaldehyde, and AKR1C3, with 9-cis-retinaldehyde, display high catalytic efficiency. Major structural determinants for retinaldehyde isomer specificity are located in the external loops (A and C for AKR1B10, and B for AKR1C3), as assessed by site-directed mutagenesis and molecular dynamics. Cellular models have shown that AKR1B and 1C enzymes are well suited to work in vivo as retinaldehyde reductases and to regulate retinoic acid (RA) biosynthesis at hormone pre-receptor level. An additional physiological role for the retinaldehyde reductase activity of these enzymes, consistent with their tissue localization, is their participation in β-carotene absorption. Retinaldehyde metabolism may be subjected to subcellular compartmentalization, based on enzyme localization. While retinaldehyde oxidation to RA takes place in the cytosol, reduction to retinol could take place in the cytosol by AKRs or in the membranes of endoplasmic reticulum by microsomal retinaldehyde reductases. Upregulation of some AKR1 enzymes in different cancer types may be linked to their induction by oxidative stress and to their participation in different signaling pathways related to cell proliferation. AKR1B10 and AKR1C3, through their retinaldehyde reductase activity, trigger a decrease in the RA biosynthesis flow, resulting in RA deprivation and consequently lower differentiation, with an increased cancer risk in target tissues. Rational design of selective AKR inhibitors could lead to development of novel drugs for cancer treatment as well as reduction of chemotherapeutic drug resistance. PMID:22529810

  18. Endothelial human dihydrofolate reductase low activity limits vascular tetrahydrobiopterin recycling

    PubMed Central

    Whitsett, Jennifer; Filho, Artur Rangel; Sethumadhavan, Savitha; Celinska, Joanna; Widlansky, Michael; Vásquez-Vivar, Jeannette

    2013-01-01

    Tetrahydrobiopterin (BH4) is required for NO synthesis and inhibition of superoxide release from eNOS. Clinical trials using BH4 to treat endothelial dysfunction have produced mixed results. Poor outcomes may be explained by the rapid systemic and cellular oxidation of BH4. One of the oxidation products of BH4, 7,8-dihydrobiopterin (7,8-BH2), is recycled back to BH4 by dihydrofolate reductase (DHFR). This enzyme is ubiquitously distributed and shows a wide range of activity depending on species-specific factors and cell type. Information about the kinetics and efficiency of BH4 recycling in human endothelial cells receiving BH4 treatment is lacking. To characterize this reaction, we applied a novel multi-electrode coulometric HPLC method that enabled the direct quantification of 7,8-BH2 and BH4 which is not possible with fluorescent-based methodologies. We found that basal untreated BH4 and 7,8-BH2 concentrations in human ECs is lower than bovine and murine endothelioma cells. Treatment of human ECs with BH4 transiently increased intracellular BH4 while accumulating the more stable 7,8-BH2. This was different from bovine or murine ECs that resulted in preferential BH4 increase. Using BH4 diastereomers, 6S-BH4 and 6R-BH4, the narrow contribution of enzymatic DHFR recycling to total intracellular BH4 was demonstrated. Reduction of 7,8-BH2 to BH4 occurs at very slow rates in cells and needs supra-physiological levels of 7,8-BH2, indicating this reaction is kinetically limited. Activity assays verified that hDHFR has very low affinity for 7,8-BH2 (DHF7,8-BH2) and folic acid inhibits 7,8-BH2 recycling. We conclude that low activity of endothelial DHFR is an important factor limiting the benefits of BH4 therapies which may be further aggravated by folate supplements. PMID:23707606

  19. Expression and purification of spinach nitrite reductase in E. coli

    SciTech Connect

    Bellissimo, D.; Privalle, L. )

    1991-03-11

    The study of structure-function relationships in nitrite reductase (NiR) by site-directed mutagenesis requires an expression system from which suitable quantities of active enzyme can be purified. Spinach NiR cDNA was cloned into pUC18 and expressed in E.coli JM109 as a beta-galactosidase fusion protein. The IPTG-induced fusion protein contains five additional amino acids at the N-terminus. The expressed NiR in aerobic cultures was mostly insoluble and inactive indicating the presence of inclusion bodies. By altering growth conditions, active NiR could represent 0.5-1.0% of the total E.coli protein, Effects of the addition of delta-aminolevulinic acid, a heme precursor, and anaerobic growth were also examined. Spinach NiR was purified approximately 200 fold to homogeneity. When subjected to electrophoresis on SDS polyacrylamide gels, the NiR migrated as a single band with similar mobility to pure spinach enzyme. The expressed enzyme also reacted with rabbit anti-spinach NiR antibody as visualized by Western blot analysis. The absorption spectrum of the E.coli-expressed enzyme was identical to spinach enzyme with a Soret and alpha band a 386 and 573 nm, respectively, and an A{sub 278}/A{sub 386} = 1.9. The addition of nitrite produced the characteristic shifts in the spectrum. The E. coli-expressed NiR catalyzed the methylviologen-dependent reduction of nitrite. The specific activity was 100 U/mg. The K{sub m} determined for nitrite was 0.3 mM which is in agreement with values reported for the enzyme. These results indicate that the E.coli-expressed NiR is fully comparable to spinach NiR in purity, catalytic activity and physical state. Site-directed mutants have been made using PCR to examine structure-function relationships in this enzyme.

  20. Methylenetetrahydrofolate reductase, diet, and risk of colon cancer.

    PubMed

    Slattery, M L; Potter, J D; Samowitz, W; Schaffer, D; Leppert, M

    1999-06-01

    Individuals with different forms of the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene, carriers of the C677T mutation versus wild type, show differences in enzyme levels; these differences have been hypothesized to be related to DNA methylation and, perhaps, to the nucleotide pool size. Using data from an incident case-control study, we evaluated the combined effect of dietary intake of folate, methionine, vitamin B6, vitamin B12, and alcohol and various forms of the MTHFR gene on risk of colon cancer. Individuals homozygous for the variant form of the MTHFR gene (TT) had a slightly lower risk of colon cancer than did individuals who were wild type [CC, odds ratio (OR) = 0.8, 95% confidence interval (CI) = 0.6-1.1 for men; and OR = 0.9, 95% CI = 0.6-1.2 for women]. High levels of intake of folate, vitamin B6, and vitamin B12 were associated with a 30-40% reduction in risk of colon cancer among those with the TT relative to those with low levels of intake who were CC genotype. Associations were stronger for proximal tumors, in which high levels of intake of these nutrients were associated with a halving of risk among those with the TT genotype. The inverse association with high levels of these nutrients in those with the TT genotype was stronger among those diagnosed at an older age. Although imprecise, the inverse association with the low-risk diet that was high in folate and methionine and without alcohol was observed for both the TT genotype (OR = 0.4 95% CI = 0.1-0.9) and the CC/CT genotype (OR = 0.6, 95% CI = 0.4-1.0), but this association was not seen with the high-risk diet for either the TT or CC/CT genotype. Although associations were generally weak, these findings suggest that those with differing MTHFR genotypes may have different susceptibilities to colon cancer, based on dietary consumption of folate, vitamin B6, and vitamin B12. PMID:10385141

  1. Importance of P450 reductase activity in determining sensitivity of breast tumour cells to the bioreductive drug, tirapazamine (SR 4233).

    PubMed Central

    Patterson, A. V.; Barham, H. M.; Chinje, E. C.; Adams, G. E.; Harris, A. L.; Stratford, I. J.

    1995-01-01

    P450 reductase (NADPH:cytochrome P450 reductase, EC 1.6.2.4) is known to be important in the reductive activation of the benzotriazene-di-N-oxide tirapazamine (SR 4233). Using a panel of six human breast adenocarcinoma cell lines we have examined the relationship between P450 reductase activity and sensitivity to tirapazamine. The toxicity of tirapazamine was found to correlate strongly with P450 reductase activity following an acute (3 h) exposure under hypoxic conditions, the drug being most toxic in the cell lines with the highest P450 reductase activity. A similar correlation was also observed following a chronic (96 h) exposure to the drug in air but not following acute (3 h) exposure in air. We have also determined the ability of lysates prepared from the cell lines to metabolise tirapazamine to its two-electron reduced product, SR 4317, under hypoxic conditions using NADPH as an electron donor. The rate of SR 4317 formation was found to correlate both with P450 reductase activity and with sensitivity to tirapazamine, the highest rates of SR 4317 formation being associated with the highest levels of P450 reductase activity and the greatest sensitivity to the drug. These findings indicate a major role for P450 reductase in determining the hypoxic toxicity of tirapazamine in breast tumour cell lines. Images Figure 4 PMID:7577460

  2. MYC Phosphorylation, Activation, and Tumorigenic Potential in Hepatocellular Carcinoma are Regulated by HMG-CoA Reductase

    PubMed Central

    Cao, Zhongwei; Fan-Minogue, Hua; Bellovin, David I.; Yevtodiyenko, Aleksey; Arzeno, Julia; Yang, Qiwei; Gambhir, Sanjiv Sam; Felsher, Dean W.

    2011-01-01

    MYC is a potential target for many cancers but is not amenable to existing pharmacological approaches. Inhibition of HMG-CoA reductase by statins has shown potential efficacy against a number of cancers. Here, we demonstrate that inhibition of HMG-CoA reductase by atorvastatin blocks both MYC phosphorylation and activation, suppressing tumor initiation and growth in vivo in a transgenic model of MYC-induced HCC as well as in human HCC-derived cell lines. To confirm specificity, we show that the anti-tumor effects of atorvastatin are blocked by co-treatment with the HMG-CoA reductase product, Mevalonate. Moreover, by using a novel molecular imaging sensor, we confirm that inhibition of HMG-CoA reductase blocks MYC phosphorylation in vivo. Importantly, the introduction of phosphorylation mutants of MYC at Ser62 or Thr58 into tumors blocks their sensitivity to inhibition of HMG-CoA reductase. Finally, we demonstrate that inhibition of HMG-CoA reductase suppresses MYC phosphorylation through Rac GTPase. Therefore, HMG-CoA reductase is a critical regulator of MYC phosphorylation, activation, and tumorigenic properties. The inhibition of HMG-CoA reductase may be a useful target for the treatment of MYC-associated HCC as well as other tumors. PMID:21262914

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  4. The role of 5'-adenylylsulfate reductase in the sulfur assimilation pathway of soybean: molecular cloning, kinetic characterization, and gene expression

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Soybean seeds are a major source of protein, but contain low levels of sulfur-containing amino acids. With the objective of studying the sulfur assimilation pathway of soybean, a full-length cDNA clone for 5’-adenylylsulfate reductase (APS reductase) was isolated and characterized. The cDNA clone ...

  5. Molecular cloning and transcription expression of 3-dehydroecdysone 3α-reductase (3de 3α-reductase) in the different tissues and the developing stage from the silkworm, Bombyx mori L.

    PubMed

    Yang, Hua-jun; Xin, Hu-hu; Lu, Yan; Cai, Zi-zheng; Wang, Mei-xian; Chen, Rui-Ting; Liang, Shuang; Singh, Chabungbam Orville; Kim, Jong-nam; Miao, Yun-gen

    2013-10-01

    Molting in insects is regulated by molting hormones (ecdysteroids), which are also crucial to insect growth, development, and reproduction etc. The decreased ecdysteroid in titre results from enhanced ecdysteroid inactivation reactions including the formation of 3-epiecdyson under ecdysone oxidase and 3-dehydroecdysone 3α-reductase (3DE 3α-reductase). In this paper, we cloned and characterized 3-dehydroecdysone 3α-reductase (3DE 3α-reductase) in different tissues and developing stage of the silkworm, Bombyx mori L. The B. mori 3DE 3α-reductase cDNA contains an ORF 783 bp and the deduced protein sequence containing 260 amino acid residues. Analysis showed the deduced 3DE 3α-reductase belongs to SDR family, which has the NAD(P)-binding domain. Using the Escherichia coli, a high level expression of a fusion polypeptide band of approx. 33 kDa was observed. High transcription of 3DE 3α-reductase was mainly presented in the midgut and hemolymph in the third day of fifth instar larvae in silkworm. The expression of 3DE 3α-reductase at different stages of larval showed that the activity in the early instar was high, and then reduced in late instar. This is parallel to the changes of molting hormone titer in larval. 3DE 3α-reductase is key enzyme in inactivation path of ecdysteroid. The data elucidate the regulation of 3DE 3α-reductase in ecdyteroid titer of its targeting organs and the relationship between the enzyme and metamorphosis. PMID:24038161

  6. Isolation of xylose reductase gene of Pichia stipitis and its expression in Saccharomyces cerevisiae

    SciTech Connect

    Takuma, Shinya; Nakashima, Noriyuki; Tantirungkij, Manee

    1991-12-31

    A NADPH/NADH-dependent xylose reductase gene was isolated from the xylose-assimilating yeast, Pichia stipitis. DNA sequence analysis showed that the gene consists of 951 bp. The gene introduced in Saccharomyces cerevisiae was transcribed to mRNA, and a considerable amount of enzyme activity was observed constitutively, whereas transcription and translation in P steps were inducible. S. cerevisiae carrying the xylose reductase gene could not, however, grow on xylose medium, and could not produce ethanol from xylose. Since xylose uptake and accumulation of xylitol by S. cerevisiae were observed, the conversion of xylitol to xylulose seemed to be limited.

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

    PubMed

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

    1996-12-15

    The genes that encode the hc-type nitric-oxide reductase from Paracoccus denitrificans have been identified. They are part of a cluster of six genes (norCBQDEF) and are found near the gene cluster that encodes the cd1-type nitrite reductase, which was identified earlier [de Boer, A. P. N., Reijnders, W. N. M., Kuenen, J. G., Stouthamer, A. H. & van Spanning, R. J. M. (1994) Isolation, sequencing and mutational analysis of a gene cluster involved in nitrite reduction in Paracoccus denitrificans, Antonie Leeu wenhoek 66, 111-127]. norC and norB encode the cytochrome-c-containing subunit II and cytochrome b-containing subunit I of nitric-oxide reductase (NO reductase), respectively. norQ encodes a protein with an ATP-binding motif and has high similarity to NirQ from Pseudomonas stutzeri and Pseudomonas aeruginosa and CbbQ from Pseudomonas hydrogenothermophila. norE encodes a protein with five putative transmembrane alpha-helices and has similarity to CoxIII, the third subunit of the aa3-type cytochrome-c oxidases. norF encodes a small protein with two putative transmembrane alpha-helices. Mutagenesis of norC, norB, norQ and norD resulted in cells unable to grow anaerobically. Nitrite reductase and NO reductase (with succinate or ascorbate as substrates) and nitrous oxide reductase (with succinate as substrate) activities were not detected in these mutant strains. Nitrite extrusion was detected in the medium, indicating that nitrate reductase was active. The norQ and norD mutant strains retained about 16% and 23% of the wild-type level of NorC, respectively. The norE and norF mutant strains had specific growth rates and NorC contents similar to those of the wild-type strain, but had reduced NOR and NIR activities, indicating that their gene products are involved in regulation of enzyme activity. Mutant strains containing the norCBQDEF region on the broad-host-range vector pEG400 were able to grow anaerobically, although at a lower specific growth rate and with lower

  8. 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.

  9. Interaction of Product Analogues With the Active Site of Rhodobacter Sphaeroides Dimethyl Sulfoxide Reductase

    SciTech Connect

    George, G.N.; Nelson, K.J.; Harris, H.H.; Doonan, C.J.; Rajagopalan, K.V.; /Saskatchewan U. /Duke U. /Sydney U.

    2007-07-09

    We report a structural characterization using X-ray absorption spectroscopy of Rhodobacter sphaeroides dimethylsulfoxide (DMSO) reductase reduced with trimethylarsine, and show that this is structurally analogous to the physiologically relevant dimethylsulfide-reduced DMSO reductase. Our data unambiguously indicate that these species should be regarded as formal MoIV species, and indicate a classical coordination complex of trimethylarsine oxide, with no special structural distortions. The similarity of the trimethylarsine and dimethylsulfide complexes suggests in turn that the dimethylsulfide reduced enzyme possesses a classical coordination of DMSO with no special elongation of the S-O bond, as previously suggested.

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

    SciTech Connect

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

    2006-07-01

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

  11. Imaging the activity of nitrate reductase by means of a scanning electrochemical microscope.

    PubMed

    Zaumseil, J; Wittstock, G; Bahrs, S; Steinrücke, P

    2000-06-01

    Scanning electrochemical microscopy (SECM) was used to characterize immobilized nitrate reductase (NaR) from Pseudonomonas stutzeri (E.C. 1.7.99.4). Nitrate reductase with membrane fragment was embedded in a polyurethane hydrogel in a capillary and solubilized NaR without membrane fragment was covalently coupled to a diaminoethyl-cellulose-carbamitate film on glass. After systematic studies of possible mediators, SECM feedback imaging of both forms of immobilized NaR was accomplished with methylviologen as redox mediator. PMID:11225859

  12. Interaction of Product Analogs with the Active Site of Rhodobacter sphaeroides Dimethylsulfoxide Reductase

    PubMed Central

    George, Graham N.; Nelson, Kimberly Johnson; Harris, Hugh H.; Doonan, Christian J.; Rajagopalan, K.V.

    2007-01-01

    We report a structural characterization using X-ray absorption spectroscopy of Rhodobacter sphaeroides dimethylsulfoxide (DMSO) reductase reduced with trimethylarsine, and show that this is structurally analogous to the physiologically relevant dimethylsulfide-reduced DMSO reductase. Our data unambiguously indicate that these species should be regarded as formal MoIV species, and indicate a classical coordination complex of trimethylarsine oxide, with no special structural distortions. The similarity of the trimethylarsine and dimethylsulfide complexes suggests in turn that the dimethylsulfide reduced enzyme possesses a classical coordination of DMSO with no special elongation of the S—O bond, as previously suggested. PMID:17361996

  13. Crystallization and preliminary X-ray diffraction studies of a ferredoxin reductase from Rhodopseudomonas palustris CGA009

    SciTech Connect

    Peng, Ying; Xu, Feng; Bell, Stephen G.; Wong, Luet-Lok; Rao, Zihe

    2007-05-01

    Palustrisredoxin reductase (RPA3782, PuR), a flavin-dependent ferredoxin reductase, is an essential component of the Class I cytochrome P450 systems in Rhodopseudomonas palustris CGA009. Crystals of PuR that diffract to 2.2 Å resolution have been obtained. Palustrisredoxin reductase from Rhodopseudomonas palustris CGA009, a member of the oxygenase-coupled NADH-dependent ferredoxin reductase (ONFR) family, catalyzes electron transfer from NADH to ferredoxins. It is an essential component of the cytochrome P450 systems in R. palustris CGA009, a model organism with diverse metabolic pathways. Here, the crystallization of palustrisredoxin reductase is reported. The crystals belong to the trigonal space group P3{sub 2}21, with unit-cell parameters a = 107.5, b = 107.5, c = 69.9 Å, and diffract to 2.2 Å resolution on a synchrotron source.

  14. Pinpointing a Mechanistic Switch Between Ketoreduction and “Ene” Reduction in Short‐Chain Dehydrogenases/Reductases

    PubMed Central

    Lygidakis, Antonios; Karuppiah, Vijaykumar; Hoeven, Robin; Ní Cheallaigh, Aisling; Leys, David; Gardiner, John M.; Toogood, Helen S.

    2016-01-01

    Abstract Three enzymes of the Mentha essential oil biosynthetic pathway are highly homologous, namely the ketoreductases (−)‐menthone:(−)‐menthol reductase and (−)‐menthone:(+)‐neomenthol reductase, and the “ene” reductase isopiperitenone reductase. We identified a rare catalytic residue substitution in the last two, and performed comparative crystal structure analyses and residue‐swapping mutagenesis to investigate whether this determines the reaction outcome. The result was a complete loss of native activity and a switch between ene reduction and ketoreduction. This suggests the importance of a catalytic glutamate vs. tyrosine residue in determining the outcome of the reduction of α,β‐unsaturated alkenes, due to the substrate occupying different binding conformations, and possibly also to the relative acidities of the two residues. This simple switch in mechanism by a single amino acid substitution could potentially generate a large number of de novo ene reductases. PMID:27587903

  15. Pinpointing a Mechanistic Switch Between Ketoreduction and “Ene” Reduction in Short‐Chain Dehydrogenases/Reductases

    PubMed Central

    Lygidakis, Antonios; Karuppiah, Vijaykumar; Hoeven, Robin; Ní Cheallaigh, Aisling; Leys, David; Gardiner, John M.; Toogood, Helen S.

    2016-01-01

    Abstract Three enzymes of the Mentha essential oil biosynthetic pathway are highly homologous, namely the ketoreductases (−)‐menthone:(−)‐menthol reductase and (−)‐menthone:(+)‐neomenthol reductase, and the “ene” reductase isopiperitenone reductase. We identified a rare catalytic residue substitution in the last two, and performed comparative crystal structure analyses and residue‐swapping mutagenesis to investigate whether this determines the reaction outcome. The result was a complete loss of native activity and a switch between ene reduction and ketoreduction. This suggests the importance of a catalytic glutamate vs. tyrosine residue in determining the outcome of the reduction of α,β‐unsaturated alkenes, due to the substrate occupying different binding conformations, and possibly also to the relative acidities of the two residues. This simple switch in mechanism by a single amino acid substitution could potentially generate a large number of de novo ene reductases. PMID:27411040

  16. Diabetic neuropathy: structural analysis of nerve hydration by Magnetic Resonance Spectroscopy

    SciTech Connect

    Griffey, R.H.; Eaton, P.; Sibbitt, R.R.; Sibbitt, W.L. Jr.; Bicknell, J.M.

    1988-11-18

    The water content of the sural nerve of diabetic patients was quantitatively defined by magnetic resonance proton imaging as a putative reflection of activity of the aldose-reductase pathway. Thirty-nine patients were evaluated, comparing group A, symptomatic diabetic men with sensory neuropathy; group B, similarly symptomatic diabetic men treated aldose-reductase inhibition; group C, neurologically asymptomatic diabetic men; and group D, control nondiabetic men. Marked increase in hydration of the sural nerve was seen in more than half of the symptomatic diabetic patients. Two of 11 neurologically asymptomatic diabetics had increased nerve hydration, suggesting a presymptomatic alteration of the nerve. Symptomatic diabetics treated with aldose-reductase inhibitors had normal nerve water levels. Increased level of peripheral nerve water represents a new finding in diabetes mellitus. It seems to be related to aldose-reductase activity, involved in the development of neuropathy, and similar to events that occur in other target tissue in human diabetes.

  17. The isolation of a hexaheme cytochrome from Desulfovibrio desulfuricans and its identification as a new type of nitrite reductase

    SciTech Connect

    Liu, M.-C.; Peck, H.D., Jr.

    1981-12-01

    Desulfovibrio desulfuricans (ATCC 27774), a strictly anaerobic sulfate-reducing bacteria, is able to perform anaerobic nitrate respiration in which nitrate is first reduced to nitrite by the action of nitrate reductase, and nitrite reductase then catalyzes the six-electron reduction of nitrite to ammonia. The nitrite reductase was found to be a membrane-bound enzyme and has been purified to electrophoretic homogeneity. The purified enzyme has a minimal M/sub r/=66,000 as judged by sodium dodecyl sulfate gel electrophoresis and contains 6 c-type heme groups/molecule. Pure nitrite reductase exhibits a typical c-type cytochrome absorption spectrum with reduced..cap alpha..-band at 552.5 nm. NADH and NADPH do not function as direct electron donors for the nitrite reductase. Desulfovibrio vulgaris hydrogenase,however, is able to transfer electrons from H/sub 2/ to the nitrite reductase using FAD as the electron transfer mediator. The dithionite-reduced nitrite reductase was demonstrated to be auto-oxidizable even in the presence of potassium cyanide. On addition of nitrite, the dithionite-reduced enzyme is re-oxidized immediately. Hydroxylamine, however, can only partially reoxidize the reduced enzyme. Ascorbate reduces the enzyme to a limited extent and the partially reduced enzyme is neither auto-oxidizable by nitrite or hydroxylamine. Purified nitrite reductase has a pH optimum in the range of 8.0-9.5 and optimal activity at 57/sup o/C. Purified nitrite reductase also has hydroxylamine reductase activity, and the K/sub m/ for nitrite was determined to be 1.14 mM.

  18. Three spinach leaf nitrate reductase-3-hydroxy-3-methylglutaryl-CoA reductase kinases that are required by reversible phosphorylation and/or Ca2+ ions.

    PubMed Central

    Douglas, P; Pigaglio, E; Ferrer, A; Halfords, N G; MacKintosh, C

    1997-01-01

    In spinach (Spinacea oleracea L.) leaf extracts, three protein kinases (PKI, PKII and PKIII) were identified each of which phosphorylated spinach nitrate reductase on serine-543, and inactivated the enzyme in the presence of nitrate reductase inhibitor, 14-3-3. PKIII was also very active in phosphorylating and inactivating Arabidopsis (Landsberg erecta) 3-hydroxy-3-methylglutaryl-coenzyme A reductase 1 (HMGR1). PKI and PKII phosphorylated HMGR1 more slowly than PKIII, compared with their relative rates of phosphorylation of nitrate reductase. HMGR1 identical with those that are seen after phosphorylation of serine-577 by the sucrose non-fermenting (SNF1)-like PK, 3-hydroxy-3-methylglutaryl-Co A reductase kinase A (HRK-A), from cauliflower [Dale, Arró, Becerra, Morrice, Boronat, Hardie and Ferrer (1995) Eur. J. Biochem. 233, 506-513]. PKI was Ca2+-dependent when prepared in the absence of protein phosphatase (PP) inhibitors, and largely Ca2+-dependent when prepared in the presence of PP inhibitors (NaF and EGTA). The Ca2+-independent portion of PKI was inactivated by either PP2A or PP2C, while the Ca2+-dependent portion of PKI became increasingly activated during storage, which we presume was mimicking the effect of an unidentified PP. These findings indicate that PK1 is regulated by two functionally distinct phosphorylations. PKI had a molecular mass of 45 kDa on gel filtration and was active towards substrate peptides that terminated at the +2 residue from the phosphorylation site, whereas PKIII was inactive towards these peptides. PKII was Ca2+-stimulated under all conditions tested. PKIII was Ca2+-indepdented, inactivated by PP2A or PP2C, had a requirement for a hydrophobic residue in the +4 position of peptide substrates, had a molecular mass by gel filtration of approximately 140 kDa, and an antibody against the rye SNF1-related PK (RKIN1) recognized a 58 kDa subunit in fractions containing PKIII. These properties of PKIII are identical with those reported

  19. Protective role of glutathione reductase in paraquat induced neurotoxicity.

    PubMed

    Djukic, Mirjana M; Jovanovic, Marina D; Ninkovic, Milica; Stevanovic, Ivana; Ilic, Katarina; Curcic, Marijana; Vekic, Jelena

    2012-08-30

    Paraquat (PQ), a widely used herbicide is a well-known free radical producing agent. The mechanistic pathways of PQ neurotoxicity were examined by assessing oxidative/nitrosative stress markers. Focus was on the role of glutathione (GSH) cycle and to examine whether the pre-treatment with enzyme glutathione reductase (GR) could protect the vulnerable brain regions (VBRs) against harmful oxidative effect of PQ. The study was conducted on Wistar rats, randomly divided in five groups: intact-control group, (n = 8) and four experimental groups (n = 24). All tested compounds were administered intrastriatally (i.s.) in one single dose. The following parameters of oxidative status were measured in the striatum, hippocampus and cortex, at 30 min, 24 h and 7 days post treatment: superoxide anion radical (O₂·⁻), nitrate (NO₃⁻), malondialdehyde (MDA), superoxide dismutase (SOD), total GSH (tGSH) and its oxidized, disulfide form (GSSG) and glutathione peroxidase (GPx). Results obtained from the intact and the sham operated groups were not statistically different, confirming that invasive i.s. route of administration would not influence the reliability of results. Also, similar pattern of changes were observed between ipsi- and contra- lateral side of examined VBRs, indicating rapid spatial spreading of oxidative stress. Mortality of the animals (10%), within 24h, along with symptoms of Parkinsonism, after awakening from anesthesia for 2-3 h, were observed in the PQ group, only. Increased levels of O₂·⁻, NO₃⁻ and MDA, increased ratio of GSSG/GSH and considerably high activity of GPx were measured at 30 min after the treatment. Cytotoxic effect of PQ was documented by drastic drop of all measured parameters and extremely high peak of the ratio GSSG/GSH at 24th hrs after the PQ i.s. injection. In the GR+PQ group, markedly low activity of GPx and low content of NO₃⁻ (in striatum and cortex) were measured during whole experiment, while increase value was

  20. The dihydrolipoamide dehydrogenase of Aeromonas caviae ST exhibits NADH-dependent tellurite reductase activity.

    PubMed

    Castro, Miguel E; Molina, Roberto; Díaz, Waldo; Pichuantes, Sergio E; Vásquez, Claudio C

    2008-10-10

    Potassium tellurite (K(2)TeO(3)) is extremely toxic for most forms of life and only a limited number of organisms are naturally resistant to the toxic effects of this compound. Crude extracts prepared from the environmental isolate Aeromonas caviae ST catalize the in vitro reduction of TeO32- in a NADH-dependent reaction. Upon fractionation by ionic exchange column chromatography three major polypeptides identified as the E1, E2, and E3 components of the pyruvate dehydrogenase (PDH) complex were identified in fractions exhibiting tellurite-reducing activity. Tellurite reductase and pyruvate dehydrogenase activities co-eluted from a Sephadex gel filtration column. To determine which component(s) of the PDH complex has tellurite reductase activity, the A. caviae ST structural genes encoding for E1 (aceE), E2 (aceF), and E3 (lpdA) were independently cloned and expressed in Escherichia coli and their gene products purified. Results indicated that tellurite reductase activity lies almost exclusively in the E3 component, dihydrolipoamide dehydrogenase. The E3 component of the PDH complex from E. coli, Zymomonas mobilis, Streptococcus pneumoniae, and Geobacillus stearothermophilus also showed NADH-dependent tellurite reductase in vitro suggesting that this enzymatic activity is widely distributed among microorganisms. PMID:18675788

  1. Structure of Physarum polycephalum cytochrome b{sub 5} reductase at 1.56 Å resolution

    SciTech Connect

    Kim, Sangwoo; Suga, Michihiro; Ogasahara, Kyoko; Ikegami, Terumi; Minami, Yoshiko; Yubisui, Toshitsugu; Tsukihara, Tomitake

    2007-04-01

    The structure of P. polycephalum cytochrome b{sub 5} reductase, an enzyme which catalyzes the reduction of cytochrome b{sub 5} by NADH, was determined at a resolution of 1.56 Å. Physarum polycephalum cytochrome b{sub 5} reductase catalyzes the reduction of cytochrome b{sub 5} by NADH. The structure of P. polycephalum cytochrome b{sub 5} reductase was determined at a resolution of 1.56 Å. The molecular structure was compared with that of human cytochrome b{sub 5} reductase, which had previously been determined at 1.75 Å resolution [Bando et al. (2004 ▶), Acta Cryst. D60, 1929–1934]. The high-resolution structure revealed conformational differences between the two enzymes in the adenosine moiety of the FAD, the lid region and the linker region. The structural properties of both proteins were inspected in terms of hydrogen bonding, ion pairs, accessible surface area and cavity volume. The differences in these structural properties between the two proteins were consistent with estimates of their thermostabilities obtained from differential scanning calorimetry data.

  2. Ferric reductase activity and PsFRO1 sequence variation in pisum sps

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Physiological studies in pea (Pisum sativum) suggest that the reduction of iron (Fe) is the rate-limiting physiological process in Fe acquisition by dicotyledonous plants. Previous molecular work suggests that ferric reductase activity is regulated at both the transcriptional and post-translational ...

  3. Design and synthesis of novel, conformationally restricted HMG-CoA reductase inhibitors.

    PubMed

    Pfefferkorn, Jeffrey A; Choi, Chulho; Song, Yuntao; Trivedi, Bharat K; Larsen, Scott D; Askew, Valerie; Dillon, Lisa; Hanselman, Jeffrey C; Lin, Zhiwu; Lu, Gina; Robertson, Andrew; Sekerke, Catherine; Auerbach, Bruce; Pavlovsky, Alexander; Harris, Melissa S; Bainbridge, Graeme; Caspers, Nicole

    2007-08-15

    Using structure-based design, a novel series of conformationally restricted, pyrrole-based inhibitors of HMG-CoA reductase were discovered. Leading analogs demonstrated potent inhibition of cholesterol synthesis in both in vitro and in vivo models and may be useful for the treatment of hypercholesterolemia and related lipid disorders. PMID:17574411

  4. A phase II trial of sequential ribonucleotide reductase inhibition in aggressive myeloproliferative neoplasms

    PubMed Central

    Zeidner, Joshua F.; Karp, Judith E.; Blackford, Amanda L.; Smith, B. Douglas; Gojo, Ivana; Gore, Steven D.; Levis, Mark J.; Carraway, Hetty E.; Greer, Jacqueline M.; Ivy, S. Percy; Pratz, Keith W.; McDevitt, Michael A.

    2014-01-01

    Myeloproliferative neoplasms are a varied group of disorders that can have prolonged chronic phases, but eventually accelerate and can transform into a secondary acute myeloid leukemia that is ultimately fatal. Triapine is a novel inhibitor of the M2 subunit of ribonucleotide reductase. Sequential inhibition of ribonucleotide reductase with triapine and an M1 ribonucleotide reductase inhibitor (fludarabine) was noted to be safe, and led to a 29% complete plus partial response rate in myeloproliferative neoplasms. This article reports the findings of a phase II trial of triapine (105 mg/m2/day) followed by fludarabine (30 mg/m2/day) daily for 5 consecutive days in 37 patients with accelerated myeloproliferative neoplasms and secondary acute myeloid leukemia. The overall response rate was 49% (18/37), with a complete remission rate of 24% (9/37). Overall response rates and complete remissions were seen in all disease subsets, including secondary acute myeloid leukemia, in which the overall response rate and complete remission rate were 48% and 33%, respectively. All patients with known JAK2 V617F mutations (6/6) responded. The median overall survival of the entire cohort was 6.9 months, with a median overall survival of both overall responders and complete responders of 10.6 months. These data further demonstrate the promise of sequential inhibition of ribonucleotide reductase in patients with accelerated myeloproliferative neoplasms and secondary acute myeloid leukemia. This study was registered with clinicaltrials.gov (NCT00381550). PMID:24362550

  5. 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. PMID:25193000

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  7. Glyphosate Effect on Shikimate, Nitrate Reductase Activity, Yield, and Seed Composition in Corn

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A 2-yr field study investigated the effects of glyphosate drift rate on plant injury, shikimate accumulation, nitrate reductase activity, leaf nitrogen, yield, and seed composition in non-glyphosate-resistant (non-GR) corn (Zea mays L.) and the effects of glyphosate at label rates on nitrate reducta...

  8. CONTROL OF NITRATE REDUCTASE BY CIRCADIAN AND DURRAL RHYTHM IN TOMATO

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Nitrate reductase (NR) (E.C. 1.6.6.1) is a key, regulatory step in the assimilation of nitrate into amino acids in plant leaves. NR activity is intricately controlled by multifarious regulatory mechanisms acting at different levels ranging from transcription to protein degradation. It is one of a ...

  9. Cloning and expression of Candida guilliermondii xylose reductase gene (xyl1) in Pichia pastoris.

    PubMed

    Handumrongkul, C; Ma, D P; Silva, J L

    1998-04-01

    A xylose reductase gene (xyl1) of Candida guilliermondii ATCC 20118 was cloned and characterized. The open reading frame of xyl1 contained 954 nucleotides encoding a protein of 317 amino acids with a predicted molecular mass of 36 kDa. The derived amino acid sequence of C. guilliermondii xylose reductase was 70.4% homologous to that of Pichia stipitis. The gene was placed under the control of an alcohol oxidase promoter (AOX1) and integrated into the genome of a methylotrophic yeast, Pichia pastoris. Methanol induced the expression of the 36-kDa xylose reductase in both intracellular and secreted expression systems. The expressed enzyme preferentially utilized NADPH as a cofactor and was functional both in vitro and in vivo. The different cofactor specificity between P. pastoris and C. guilliermondii xylose reductases might be due to the difference in the numbers of histidine residues and their locations between the two proteins. The recombinant was able to ferment xylose, and the maximum xylitol accumulation (7.8 g/l) was observed when the organism was grown under aerobic conditions. PMID:9615481

  10. Loss and stabilization of amplified dihydrofolate reductase genes in mouse sarcoma S-180 cell lines

    SciTech Connect

    Kaufman, R.J.; Brown, P.C.; Schimke, R.T.

    1981-12-01

    The authors studied the loss and stabilization of dihydrofolate reductase genes in clones of a methotrexate-resistant murine S-180 cell line. These cells contained multiple copies of the dihydrofolate reductase gene which were associated with double minute chromosomes. The growth rate of these cells in the absence of methotrexate was inversely related to the degree of gene amplification (number of double minute chromosomes). Cells could both gain and lose genes as a result of an unequal distribution of double minute chromosomes into daughter cells at mitosis. The loss of amplified dihydrofolate reductase genes during growth in the absence of methotrexate resulted from the continual generation of cells containing lower numbers of double minute chromosomes. Because of the growth advantage of these cells, they became dominant in the population. They also studied an unstably resistant S-180 cell line (clone) that, after 3 years of continuous growth in methotrexate, generated cells containing stably amplified dihydrofolate reductase genes. These genes were present on one or more chromosomes, and they were retained in a stable state.

  11. Purification and characterization of the periplasmic nitrate reductase from Thiosphaera pantotropha.

    PubMed

    Berks, B C; Richardson, D J; Robinson, C; Reilly, A; Aplin, R T; Ferguson, S J

    1994-02-15

    The periplasmic nitrate reductase of Thiosphaera pantotropha has been purified from a mutant strain (M-6) that overproduces the enzyme activity under anaerobic growth conditions. The enzyme is a complex of a 93-kDa polypeptide and a 16-kDa nitrate-oxidizable cytochrome c552. The complex contains molybdenum; a fluorescent compound with spectral features of a pterin derivative can be extracted. In contrast to the dissimilatory membrane-bound nitrate reductases, the periplasmic nitrate reductase shows high specificity for nitrate as a substrate and is insensitive to inhibition by azide. The 93-kDa subunit exhibits immunological cross-reactivity with the catalytic subunit of Rhodobacter capsulatus N22DNAR+ periplasmic nitrate reductase. Mass spectrometric comparisons of holo-cytochrome c552 and apo-cytochrome c552 demonstrated that the polypeptide bound two haem groups. Mediated redox potentiometry of the cytochrome indicated that the haem groups have reduction potentials (pH = 7.0) of approximately -15 mV and + 80 mV. The functional significance of these potentials is discussed in relation to the proposed physiological role of the enzyme as a redox valve. PMID:8119278

  12. Roles of rat and human aldo-keto reductases in metabolism of farnesol and geranylgeraniol

    PubMed Central

    Endo, Satoshi; Matsunaga, Toshiyuki; Ohta, Chisato; Soda, Midori; Kanamori, Ayano; Kitade, Yukio; Ohno, Satoshi; Tajima, Kazuo; El-Kabbani, Ossama; Hara, Akira

    2011-01-01

    Farnesol (FOH) and geranylgeraniol (GGOH) with multiple biological actions are produced from the mevalonate pathway, and catabolized into farnesoic acid and geranylgeranoic acid, respectively, via the aldehyde intermediates (farnesal and geranylgeranial). We investigated the intracellular distribution, sequences and properties of the oxidoreductases responsible for the metabolic steps in rat tissues. The oxidation of FOH and GGOH into their aldehyde intermediates were mainly mediated by alcohol dehydrogenases 1 (in the liver and colon) and 7 (in the stomach and lung), and the subsequent step into the carboxylic acids was catalyzed by a microsomal aldehyde dehydrogenase. In addition, high reductase activity catalyzing the aldehyde intermediates into FOH (or GGOH) was detected in the cytosols of the extra-hepatic tissues, where the major reductase was identified as aldo-keto reductase (AKR) 1C15. Human reductases with similar specificity were identified as AKR1B10 and AKR1C3, which most efficiently reduced farnesal and geranylgeranial among seven enzymes in the AKR1A-1C subfamilies. The overall metabolism from FOH to farnesoic acid in cultured cells was significantly decreased by overexpression of AKR1C15, and increased by addition of AKR1C3 inhibitors, tolfenamic acid and R-flurbiprofen. Thus, AKRs (1C15 in rats, and 1B10 and 1C3 in humans) may play an important role in controlling the bioavailability of FOH and GGOH. PMID:21187079

  13. Purification and Characterization of the Bifunctional Enzyme Lysine-Ketoglutarate Reductase-Saccharopine Dehydrogenase from Maize.

    PubMed Central

    Goncalves-Butruille, M.; Szajner, P.; Torigoi, E.; Leite, A.; Arruda, P.

    1996-01-01

    The first enzyme of the lysine degradation pathway in maize (Zea mays L.), lysine-ketoglutarate reductase, condenses lysine and [alpha]-ketoglutarate into saccharopine using NADPH as a cofactor, whereas the second, saccharopine dehydrogenase, converts saccharopine to [alpha]-aminoadipic-[delta]-semialdehyde and glutamic acid using NAD+ or NADP+ as a cofactor. The reductase and dehydrogenase activities are optimal at pH 7.0 and 9.0, respectively. Both enzyme activities, co-purified on diethylaminoethyl-cellulose and gel filtration columns, were detected on nondenaturing polyacrylamide gels as single bands with identical electrophoretic mobilities and share tissue specificity for the endosperm. The highly purified preparation containing the reductase and dehydrogenase activities showed a single polypeptide band of 125 kD on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native form of the enzyme is a dimer of 260 kD. Limited proteolysis with elastase indicated that lysine-ketoglutarate reductase and saccharopine dehydrogenase from maize endosperm are located in two functionally independent domains of a bifunctional polypeptide. PMID:12226216

  14. Purification to homogeneity and characterization of a novel Pseudomonas putida chromate reductase

    SciTech Connect

    Park, C.H.; Keyhan, M.; Wielinga, B.; Fendorf, S.; Matin, A.

    2000-05-01

    Cr(VI) (chromate) is a widespread environmental contaminant. Bacterial chromate reductases can convert soluble and toxic chromate to the insoluble and less toxic Cr(III). Bioremediation can therefore be effective in removing chromate from the environment, especially if the bacterial propensity for such removal is enhanced by genetic and biochemical engineering. To clone the chromate reductase-encoding gene, the authors purified to homogeneity and characterized a novel soluble chromate reductase from Pseudomonas putida, using ammonium sulfate precipitation, anion-exchange chromatography, chromatofocusing, and gel filtration. The enzyme activity was dependent on NADH or NADPH; the temperature and pH optima for chromate reduction were 80 C and 5, respectively; and the K{sub m} was 374 {micro}M, with a V{sub max} of 1.72 {micro}mol/min/mg of protein. Sulfate inhibited the enzyme activity noncompetitively. The reductase activity remained virtually unaltered after 30 min of exposure to 50 C; even exposure to higher temperatures did not immediately inactivate the enzyme. X-ray absorption near-edge-structure spectra showed quantitative conversion of chromate to Cr(III) during the enzyme reaction.

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

    PubMed

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

    2004-09-28

    Soluble methane monooxygenase (sMMO) catalyzes the hydroxylation of methane by dioxygen to methanol, the first step in carbon assimilation by methanotrophs. This multicomponent system transfers electrons from NADH through a reductase component to the non-heme diiron center in the hydroxylase where O(2) is activated. The reductase component comprises three distinct domains, a [2Fe-2S] ferredoxin domain along with FAD- and NADH-binding domains. We report the solution structure of the reduced 27.6 kDa FAD- and NADH-binding domains (MMOR-FAD) of the reductase from Methylococcus capsulatus (Bath). The FAD-binding domain consists of a six-stranded antiparallel beta-barrel and one alpha-helix, with the first 10 N-terminal residues unstructured. In the interface between the two domains, the FAD cofactor is tightly bound in an unprecedented extended conformation. The NADH-binding domain consists of a five-stranded parallel beta-sheet with four alpha-helices packing closely around this sheet. MMOR-FAD is structurally homologous to other FAD-containing oxidoreductases, and we expect similar structures for the FAD/NADH-binding domains of reductases that occur in other multicomponent monooxygenases. PMID:15379538

  16. 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

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

    SciTech Connect

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

    1985-04-25

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

  18. Cloning and functional characterization of MtFRO1, a root iron reductase from Medicago truncatula

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Iron is an essential micronutrient, and although it is abundant in the soil, it can be poorly available under certain soil conditions. The activity of the Fe(III) reductase enzyme, an integral plasma membrane protein belonging to the super-family of the flavocytochromes (1), is the rate-limiting phy...

  19. X-Ray Absorption Spectroscopic Characterization of the Molybdenum Site of 'Escherichia Coli' Dimethyl Sulfoxide Reductase

    SciTech Connect

    George, G.N.; Doonan, C.J.; Rothery, R.A.; Boroumand, N.; Weiner, J.H.; /Saskatchewan U. /Alberta U.

    2007-07-09

    Structural studies of dimethyl sulfoxide (DMSO) reductases were hampered by modification of the active site during purification. We report an X-ray absorption spectroscopic analysis of the molybdenum active site of Escherichia coli DMSO reductase contained within its native membranes. The enzyme in these preparations is expected to be very close to the form found in vivo. The oxidized active site was found to have four Mo-S ligands at 2.43 angstroms, one Mo=O at 1.71 angstroms, and a longer Mo-O at 1.90 angstroms. We conclude that the oxidized enzyme is a monooxomolybdenum(VI) species coordinated by two molybdopterin dithiolenes and a serine. The bond lengths determined for E. coli DMSO reductase are very similar to those determined for the well-characterized Rhodobacter sphaeroides DMSO reductase, suggesting similar active site structures for the two enzymes. Furthermore, our results suggest that the form found in vivo is the monooxobis(molybdopterin) species.

  20. Use of a Simple, Colorimetric Assay to Demonstrate Conditions for Induction of Nitrate Reductase in Plants.

    ERIC Educational Resources Information Center

    Harley, Suzanne M.

    1993-01-01

    Nitrate assimilation by plants provides an excellent system for demonstrating control of gene expression in a eukaryotic organism. Describes an assay method that allows students to complete experiments designed around the measurement of nitrate reductase within a three-hour laboratory experiment. (PR)

  1. The anaerobic ribonucleoside triphosphate reductase from Escherichia coli requires S-adenosylmethionine as a cofactor.

    PubMed Central

    Eliasson, R; Fontecave, M; Jörnvall, H; Krook, M; Pontis, E; Reichard, P

    1990-01-01

    Extracts from anaerobically grown Escherichia coli contain an oxygen-sensitive activity that reduces CTP to dCTP in the presence of NADPH, dithiothreitol, Mg2+ ions, and ATP, different from the aerobic ribonucleoside diphosphate reductase (2'-deoxyribonucleoside-diphosphate: oxidized-thioredoxin 2'-oxidoreductase, EC 1.17.4.1) present in aerobically grown E. coli. After fractionation, the activity required at least five components, two heat-labile protein fractions and several low molecular weight fractions. One protein fraction, suggested to represent the actual ribonucleoside triphosphate reductase was purified extensively and on denaturing gel electrophoresis gave rise to several defined protein bands, all of which were stained by a polyclonal antibody against one of the two subunits (protein B1) of the aerobic reductase but not by monoclonal anti-B1 antibodies. Peptide mapping and sequence analyses revealed partly common structures between two types of protein bands but also suggested the presence of an additional component. Obviously, the preparations are heterogeneous and the structure of the reductase is not yet established. The second, crude protein fraction is believed to contain several ancillary enzymes required for the reaction. One of the low molecular weight components is S-adenosylmethionine; a second component is a loosely bound metal. We propose that S-adenosylmethionine together with a metal participates in the generation of the radical required for the reduction of carbon 2' of the ribosyl moiety of CTP. Images PMID:2185465

  2. 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. PMID:26773812

  3. Regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity in mouse peritoneal macrophages.

    PubMed Central

    Angelin, B

    1988-01-01

    The lipoprotein-mediated regulation of 3-hydroxy-3-methylglutaryl-(HMG-) CoA reductase in cultured mouse peritoneal macrophages has been investigated. In contrast to what has been reported for other cells, HMG-CoA reductase activity is not suppressed by normal serum or by normal low density lipoproteins (LDL) from humans or dogs. Suppression of reductase activity occurred when cells were cultured in the presence of beta-migrating very low density lipoproteins (beta-VLDL) or LDL from hypercholesterolaemic dogs, or LDL modified by acetoacetylation. Human beta-VLDL from an atypical type III hyperlipoproteinaemic patient was also effective, as was apolipoprotein (apo) E-containing high density lipoproteins (HDL) from cholesterol-fed dogs (apo-E HDLc). The results indicate that cholesterol biosynthesis in mouse peritoneal macrophages is regulated by lipoprotein cholesterol entering via receptor-mediated endocytosis. Normal LDL were not effective because of the poor binding and uptake of these lipoproteins by the apo-B, E (LDL) receptor. Only beta-VLDL, apo-E HDLc, and hypercholesterolaemic LDL were avidly taken up by this receptor and were able to suppress HMG-CoA reductase. Acetoacetylated LDL were internalized via the acetyl-LDL (scavenger) receptor. Thus, mouse macrophages differ from human fibroblasts and smooth muscle cells in their physiological regulation of cholesterogenesis. PMID:3202831

  4. Differential antioxidant and quinone reductase inducing activity of American, Asian, and Siberian ginseng

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The antioxidant and quinone reductase (QR) inducing activities of American, Asian, and Siberian ginseng have been reported using various plant materials, solvents, and assays. To directly establish their comparative bioactivity, the effects of extracts obtained from acidified methanol (MeOH), a gas...

  5. Extraction methods determine the antioxidant capacity and induction of quinone reductase by soy products in vitro

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Gastrointestinal mimic (GI) and organic solvent extracts of whole soybean powder (WSP), soy protein concentrate (SPC), and soy protein isolate (SPI) as well as soy isoflavone concentrate (SIC) were analyzed for total phenols; quinone reductase (QR) induction in hepa1c1c7 cells; antioxidant scavengi...

  6. EFFECT OF LINDANE ON INTESTINAL NITROREDUCTASE, AZO REDUCTASE, B-GLUCURONIDASE, DECHLORINASE AND DEHYDROCHLORINASE ACTIVITY

    EPA Science Inventory

    The effect of daily p.o. injections of 20 mg/kg lindane on nitroreductase, azo reductase, B-glucuronidase, dechlorinase and dehydrochlorinase enzyme activity in the rat intestinal tract vas investigated after 2 weeks and 5 weeks of treatment. Antibiotics were administered to half...

  7. Structural and transcriptional analysis of plant genes encoding the bifunctional lysine ketoglutarate reductase saccharopine dehydrogenase enzyme

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The analysis of a wheat lysine ketoglutarate reductase – saccharopine dehydrogenase (LKR/SDH) gene and comparative structural and functional analyses among available plant genes provides new information on this important gene. Both the structure of the LKR/SDH gene and the immediately adjacent genes...

  8. Monoterpene Metabolism. Cloning, Expression, and Characterization of Menthone Reductases from Peppermint1

    PubMed Central

    Davis, Edward M.; Ringer, Kerry L.; McConkey, Marie E.; Croteau, Rodney

    2005-01-01

    (−)-Menthone is the predominant monoterpene produced in the essential oil of maturing peppermint (Mentha x piperita) leaves during the filling of epidermal oil glands. This early biosynthetic process is followed by a second, later oil maturation program (approximately coincident with flower initiation) in which the C3-carbonyl of menthone is reduced to yield (−)-(3R)-menthol and (+)-(3S)-neomenthol by two distinct NADPH-dependent ketoreductases. An activity-based in situ screen, by expression in Escherichia coli of 23 putative redox enzymes from an immature peppermint oil gland expressed sequence tag library, was used to isolate a cDNA encoding the latter menthone:(+)-(3S)-neomenthol reductase. Reverse transcription-PCR amplification and RACE were used to acquire the former menthone:(−)-(3R)-menthol reductase directly from mRNA isolated from the oil gland secretory cells of mature leaves. The deduced amino acid sequences of these two reductases share 73% identity, provide no apparent subcellular targeting information, and predict inclusion in the short-chain dehydrogenase/reductase family of enzymes. The menthone:(+)-(3S)-neomenthol reductase cDNA encodes a 35,722-D protein, and the recombinant enzyme yields 94% (+)-(3S)-neomenthol and 6% (−)-(3R)-menthol from (−)-menthone as substrate, and 86% (+)-(3S)-isomenthol and 14% (+)-(3R)-neoisomenthol from (+)-isomenthone as substrate, has a pH optimum of 9.3, and Km values of 674 μm, > 1 mm, and 10 μm for menthone, isomenthone, and NADPH, respectively, with a kcat of 0.06 s−1. The recombinant menthone:(−)-(3R)-menthol reductase has a deduced size of 34,070 D and converts (−)-menthone to 95% (−)-(3R)-menthol and 5% (+)-(3S)-neomenthol, and (+)-isomenthone to 87% (+)-(3R)-neoisomenthol and 13% (+)-(3S)-isomenthol, displays optimum activity at neutral pH, and has Km values of 3.0 μm, 41 μm, and 0.12 μm for menthone, isomenthone, and NADPH, respectively, with a kcat of 0.6 s−1. The respective

  9. Enhanced seed phytosterol accumulation through expression of a modified HMG-CoA reductase.

    PubMed

    Hey, Sandra J; Powers, Stephen J; Beale, Michael H; Hawkins, Nathaniel D; Ward, Jane L; Halford, Nigel G

    2006-03-01

    The regulation of phytosterol biosynthesis in seeds is of interest to biotechnologists because of the efficacy of dietary phytosterols in reducing blood cholesterol in humans. Mevalonate synthesis via 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase) is a key step in phytosterol biosynthesis. HMG-CoA reductase is inactivated by phosphorylation by SNF1-related protein kinase 1 (SnRK1). With the aim of increasing seed phytosterol levels, transgenic tobacco plants were produced expressing a full-length Arabidopsis (Arabidopsis thaliana) HMG-CoA reductase gene (HMG1) coding sequence, a modified HMG1 sequence encoding a protein lacking the target serine residue for phosphorylation by SnRK1, or a chimaeric sequence encoding the N-terminal domain of the Arabidopsis HMG1 enzyme fused with the catalytic domain of yeast HMG-CoA reductase, which lacks an SnRK1 target site. All three transgenes (35S-AtHMG1, 35S-AtHMG1m and 35S-AtScHMG1) were under the control of a cauliflower mosaic virus 35S RNA promoter. Levels of seed phytosterols were up to 2.44-fold higher in plants transformed with the 35S-AtHMG1m gene than in the wild-type, and were significantly higher than in plants expressing 35S-AtHMG1 or 35S-AtScHMG1. In contrast, levels of phytosterols in leaves of plants transformed with the 35S-AtHMG1m gene were unchanged, suggesting that regulation of HMG-CoA reductase by SnRK1 is an important factor in seeds but not in leaves. A total of 11 independent transgenic lines expressing 35S-AtHMG1m or 35S-AtScHMG1 also showed an altered flower phenotype, comprising a compact floret, prolonged flowering, short, pale petals, a protruding style, short stamens, late anther development, little or no pollen production, premature flower abscission and poor seed set. Because of this phenotype, the modified HMG-CoA reductase gene would have to be expressed seed specifically if it were to be engineered into a crop plant for biotechnological purposes. PMID:17177798

  10. The metabolism of nitrosothiols in the Mycobacteria: identification and characterization of S-nitrosomycothiol reductase.

    PubMed Central

    Vogt, Ryan N; Steenkamp, Daniel J; Zheng, Renjian; Blanchard, John S

    2003-01-01

    When grown in culture Mycobacterium smegmatis metabolized S-nitrosoglutathione to oxidized glutathione and nitrate, which suggested a possible involvement of an S-nitrosothiol reductase and mycobacterial haemoglobin. The mycothiol-dependent formaldehyde dehydrogenase from M. smegmatis was purified by a combination of Ni2+-IMAC (immobilized metal ion affinity chromatography), hydrophobic interaction, anion-exchange and affinity chromatography. The enzyme had a subunit molecular mass of 38263 kDa. Steady-state kinetic studies indicated that the enzyme catalyses the NAD+-dependent conversion of S-hydroxymethylmycothiol into formic acid and mycothiol by a rapid-equilibrium ordered mechanism. The enzyme also catalysed an NADH-dependent decomposition of S-nitrosomycothiol (MSNO) by a sequential mechanism and with an equimolar stoichiometry of NADH:MSNO, which indicated that the enzyme reduces the nitroso group to the oxidation level of nitroxyl. Vmax for the MSNO reductase reaction indicated a turnover per subunit of approx. 116700 min(-1), which was 76-fold faster than the formaldehyde dehydrogenase activity. A gene, Rv2259, annotated as a class III alcohol dehydrogenase in the Mycobacterium tuberculosis genome was cloned and expressed in M. smegmatis as the C-terminally His6-tagged product. The purified recombinant enzyme from M. tuberculosis also catalysed both activities. M. smegmatis S-nitrosomycothiol reductase converted MSNO into the N -hydroxysulphenamide, which readily rearranged to mycothiolsulphinamide. In the presence of MSNO reductase, M. tuberculosis HbN (haemoglobin N) was converted with low efficiency into metHbN [HbN(Fe3+)] and this conversion was dependent on turnover of MSNO reductase. These observations suggest a possible route in vivo for the dissimilation of S-nitrosoglutathione. PMID:12809551

  11. Biochemical and antitumor activity of trimidox, a new inhibitor of ribonucleotide reductase.

    PubMed

    Szekeres, T; Gharehbaghi, K; Fritzer, M; Woody, M; Srivastava, A; van't Riet, B; Jayaram, H N; Elford, H L

    1994-01-01

    Trimidox (3,4,5-trihydroxybenzamidoxime), a newly synthesized analog of didox (N,3,4-trihydroxybenzamide) reduced the activity of ribonucleotide reductase (EC 1.17.4.1) in extracts of L1210 cells by 50% (50% growth-inhibitory concentration, IC50) at 5 microM, whereas hydroxyurea, the only ribonucleotide reductase inhibitor in clinical use, exhibited an IC50 of 500 microM. Ribonucleotide reductase activity was also measured in situ by incubating L1210 cells for 24 h with trimidox at 7.5 microM, a concentration that inhibits cell proliferation by 50% (IC50) or at 100 microM for 2 h; these concentrations resulted in a decrease in enzyme activity to 22% and 50% of the control value, respectively. Trimidox and hydroxyurea were cytotoxic to L1210 cells with IC50 values of 7.5 and 50 microM, respectively. Versus ribonucleotide reductase, trimidox and hydroxyurea yielded IC50 values of 12 and 87 microM, respectively. A dose-dependent increase in life span was observed in mice bearing intraperitoneally transplanted L1210 tumors. Trimidox treatment (200 mg/kg; q1dx9) significantly increased the life span of mice bearing L1210 leukemia (by 82% in male mice and 112% in female mice). The anti-tumor activity appeared more pronounced in female mice than in male mice. Viewed in concert, these findings suggest that trimidox is a new and potent inhibitor of ribonucleotide reductase and that it is a promising candidate for the chemotherapy of cancer in humans. PMID:8174204

  12. Conversion of NfsA, the Major Escherichia coli Nitroreductase, to a Flavin Reductase with an Activity Similar to That of Frp, a Flavin Reductase in Vibrio harveyi, by a Single Amino Acid Substitution

    PubMed Central

    Zenno, Shuhei; Kobori, Toshiro; Tanokura, Masaru; Saigo, Kaoru

    1998-01-01

    NfsA is the major oxygen-insensitive nitroreductase of Escherichia coli, similar in amino acid sequence to Frp, a flavin reductase of Vibrio harveyi. Here, we show that a single amino acid substitution at position 99, which may destroy three hydrogen bonds in the putative active center, transforms NfsA from a nitroreductase into a flavin reductase that is as active as the authentic Frp and a tartrazine reductase that is 30-fold more active than wild-type NfsA. PMID:9440535

  13. Deletion of the varicella-zoster virus large subunit of ribonucleotide reductase impairs growth of virus in vitro.

    PubMed Central

    Heineman, T C; Cohen, J I

    1994-01-01

    Cells infected with varicella-zoster virus (VZV) express a viral ribonucleotide reductase which is distinct from that present in uninfected cells. VZV open reading frames 18 and 19 (ORF18 and ORF19) are homologous to the herpes simplex virus type 1 genes encoding the small and large subunits of ribonucleotide reductase, respectively. We generated recombinant VZV by transfecting cultured cells with four overlapping cosmid DNAs. To construct a virus lacking ribonucleotide reductase, we deleted 97% of VZV ORF19 from one of the cosmids. Transfection of this cosmid with the other parental cosmids yielded a VZV mutant with a 2.3-kbp deletion confirmed by Southern blot analysis. Virus-specific ribonucleotide reductase activity was not detected in cells infected with VZV lacking ORF19. Infection of melanoma cells with ORF19-deleted VZV resulted in plaques smaller than those produced by infection with the parental VZV. The mutant virus also exhibited a growth rate slightly slower than that of the parental virus. Chemical inhibition of the VZV ribonucleotide reductase has been shown to potentiate the anti-VZV activity of acyclovir. Similarly, the concentration of acyclovir required to inhibit plaque formation by 50% was threefold lower for the VZV ribonucleotide reductase deletion mutants than for parental virus. We conclude that the VZV ribonucleotide reductase large subunit is not essential for virus infection in vitro; however, deletion of the gene impairs the growth of VZV in cell culture and renders the virus more susceptible to inhibition by acyclovir. Images PMID:8151792

  14. Involvement of nitrate reductase and pyoverdine in competitiveness of Pseudomonas fluorescens strain C7R12 in soil.

    PubMed

    Mirleau, P; Philippot, L; Corberand, T; Lemanceau, P

    2001-06-01

    Involvement of nitrate reductase and pyoverdine in the competitiveness of the biocontrol strain Pseudomonas fluorescens C7R12 was determined, under gnotobiotic conditions, in two soil compartments (bulk and rhizosphere soil), with the soil being kept at two different values of matric potential (-1 and -10 kPa). Three mutants affected in the synthesis of either the nitrate reductase (Nar(-)), the pyoverdine (Pvd(-)), or both (Nar(-) Pvd(-)) were used. The Nar(-) and Nar(-) Pvd(-) mutants were obtained by site-directed mutagenesis of the wild-type strain and of the Pvd(-) mutant, respectively. The selective advantage given by nitrate reductase and pyoverdine to the wild-type strain was assessed by measuring the dynamic of each mutant-to-total-inoculant (wild-type strain plus mutant) ratio. All three mutants showed a lower competitiveness than the wild-type strain, indicating that both nitrate reductase and pyoverdine are involved in the fitness of P. fluorescens C7R12. The double mutant presented the lowest competitiveness. Overall, the competitive advantages given to C7R12 by nitrate reductase and pyoverdine were similar. However, the selective advantage given by nitrate reductase was more strongly expressed under conditions of lower aeration (-1 kPa). In contrast, the selective advantage given by nitrate reductase and pyoverdine did not differ in bulk and rhizosphere soil, indicating that these bacterial traits are not specifically involved in the rhizosphere competence but rather in the saprophytic ability of C7R12 in soil environments. PMID:11375173

  15. Distinguishing two groups of flavin reductases by analyzing the protonation state of an active site carboxylic acid.

    PubMed

    Dumit, Verónica I; Cortez, Néstor; Matthias Ullmann, G

    2011-07-01

    Flavin-containing reductases are involved in a wide variety of physiological reactions such as photosynthesis, nitric oxide synthesis, and detoxification of foreign compounds, including therapeutic drugs. Ferredoxin-NADP(H)-reductase (FNR) is the prototypical enzyme of this family. The fold of this protein is highly conserved and occurs as one domain of several multidomain enzymes such as the members of the diflavin reductase family. The enzymes of this family have emerged as fusion of a FNR and a flavodoxin. Although the active sites of these enzymes are very similar, different enzymes function in opposite directions, that is, some reduce oxidized nicotinamide adenine dinucleotide phosphate (NADP(+)) and some oxidize reduced nicotinamide adenine dinucleotide phosphate (NADPH). In this work, we analyze the protonation behavior of titratable residues of these enzymes through electrostatic calculations. We find that a highly conserved carboxylic acid in the active site shows a different titration behavior in different flavin reductases. This residue is deprotonated in flavin reductases present in plastids, but protonated in bacterial counterparts and in diflavin reductases. The protonation state of the carboxylic acid may also influence substrate binding. The physiological substrate for plastidic enzymes is NADP(+), but it is NADPH for the other mentioned reductases. In this article, we discuss the relevance of the environment of this residue for its protonation and its importance in catalysis. Our results allow to reinterpret and explain experimental data. PMID:21538544

  16. Farnesol is not the nonsterol regulator mediating degradation of HMG-CoA reductase in rat liver.

    PubMed

    Keller, R K; Zhao, Z; Chambers, C; Ness, G C

    1996-04-15

    A recent report, in which cultured tumor cells were used, identified farnesol as the nonsterol mevalonate-derived metabolite required for the accelerated degradation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (C. C. Correll, L. Ng, and P. A. Edwards, 1994, J. Biol. Chem. 269, 17390-17393). We examined this proposed linkage in animals by measuring hepatic farnesol levels and rates of HMG-CoA reductase degradation under conditions previously shown to alter the stability of the reductase. In normal rats, the hepatic farnesol level, quantified by high-pressure liquid chromatography, was 0.10 +/- 0.08 microgram/g and the half-life of HMG-CoA reductase was 2.5 h. Administration of mevalonolactone at 1 g/kg body wt to provide all nonsterol metabolites in addition to cholesterol increased farnesol levels 6-fold without significantly affecting the half-life of the reductase. Treatment of rats with zaragozic acid A, an inhibitor of squalene synthase, raised hepatic farnesol levels 10-fold and decreased the half-life of HMG-CoA reductase to 0.25 h. However, feeding lovastatin to rats did not lower hepatic farnesol levels despite a marked stabilization of HMB-CoA reductase protein. Moreover, intubation of rats with 500 mg/kg body wt of farnesol failed to decrease the half-life of HMG-CoA reductase protein, alter the levels of enzyme activity, or change of the levels of immunoreactive protein despite an increase of 1000-fold in hepatic farnesol levels. These observations indicate that farnesol per se does not induce accelerated degradation of HMG-CoA reductase in rat liver. PMID:8645011

  17. Biochemical Characterization of Inducible 'Reductase' Component of Benzoate Dioxygenase and Phthalate Isomer Dioxygenases from Pseudomonas aeruginosa strain PP4.

    PubMed

    Karandikar, Rohini; Badri, Abinaya; Phale, Prashant S

    2015-09-01

    The first step involved in the degradation of phthalate isomers (phthalate, isophthalate and terephthalate) is the double hydroxylation by respective aromatic-ring hydroxylating dioxygenases. These are two component enzymes consisting of 'oxygenase' and 'reductase' components. Soil isolate Pseudomonas aeruginosa strain PP4 degrades phthalate isomers via protocatechuate and benzoate via catechol 'ortho' ring cleavage pathway. Metabolic studies suggest that strain PP4 has carbon source-specific inducible phthalate isomer dioxygenase and benzoate dioxygenase. Thus, it was of interest to study the properties of reductase components of these enzymes. Reductase activity from phthalate isomer-grown cells was 3-5-folds higher than benzoate grown cells. In-gel activity staining profile showed a reductase activity band of R f 0.56 for phthalate isomer-grown cells as compared to R f 0.73 from benzoate-grown cells. Partially purified reductase components from phthalate isomer grown cells showed K m in the range of 30-40 μM and V max = 34-48 μmol min(-1) mg(-1). However, reductase from benzoate grown cells showed K m = 49 μM and V max = 10 μmol min(-1) mg(-1). Strikingly similar molecular and kinetic properties of reductase component from phthalate isomer-grown cells suggest that probably the same reductase component is employed in three phthalate isomer dioxygenases. However, reductase component is different, with respect to kinetic properties and zymogram analysis, from benzoate-grown cells when compared to that from phthalate isomer grown cells of PP4. PMID:26201480

  18. Establishment of type II 5alpha-reductase over-expressing cell line as an inhibitor screening model.

    PubMed

    Jang, Sunhyae; Lee, Young; Hwang, Seong-Lok; Lee, Min-Ho; Park, Su Jin; Lee, In Ho; Kang, Sangjin; Roh, Seok-Seon; Seo, Young-Joon; Park, Jang-Kyu; Lee, Jeung-Hoon; Kim, Chang Deok

    2007-01-01

    Dihydrotestosterone (DHT) is the most potent male hormone that causes androgenetic alopecia. The type II 5alpha-reductase is an enzyme that catalyzes the conversion of testosterone (T) to DHT, therefore it can be expected that specific inhibitors for type II 5alpha-reductase may improve the pathophysiologic status of androgenetic alopecia. In this study, we attempted to establish the reliable and convenient screening model for type II 5alpha-reductase inhibitors. After transfection of human cDNA for type II 5alpha-reductase into HEK293 cells, the type II 5alpha-reductase over-expressing stable cells were selected by G418 treatment. RT-PCR and Western blot analyses confirmed that type II 5alpha-reductase gene was expressed in the stable cells. In in vitro enzymatic assay, 10 microg of stable cell extract completely converted 1 microCi (approximately 0.015 nmol) of T into DHT. The type II 5alpha-reductase activity was inhibited by finasteride in a dose-dependent manner, confirming the reliability of screening system. In cell culture condition, 2 x 10(5) of stable cells completely converted all the input T (approximately 0.03 nmol) into DHT by 4h incubation, demonstrating that the stable cell line can be used as a cell-based assay system. Using this system, we selected the extracts of Curcumae longae rhizoma and Mori ramulus as the potential inhibitors for type II 5alpha-reductase. These results demonstrate that the type II 5alpha-reductase over-expressing stable cell line is a convenient and reliable model for screening and evaluation of inhibitors. PMID:17646096

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

    PubMed

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

    1994-06-30

    Two fused genes were constructed which encode for two chimeric proteins in which either 10 or 191 N-terminal amino acids of mature mitochondrial aspartate aminotransferase had been attached to the entire polypeptide chain of cytosolic dihydrofolate reductase. The precursor and mature form of mitochondrial aspartate aminotransferase, dihydrofolate reductase and both chimeric proteins were synthesized in vitro and their import into isolated mitochondria was studied. Both chimeric proteins were taken up by isolated organelles, where they became protease resistant, thus indicating the ability of the N-terminal portion of the mature moiety of the precursor of mitochondrial aspartate aminotransferase to direct cytosolic dihydrofolate reductase into mitochondria. PMID:8024546

  20. Structure of xylose reductase bound to NAD+ and the basis for single and dual co-substrate specificity in family 2 aldo-keto reductases.

    PubMed Central

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

    2003-01-01

    The co-ordinates reported have been submitted to the Protein Data Bank under accession number 1MI3. Xylose reductase (XR; AKR2B5) is an unusual member of aldo-keto reductase superfamily, because it is one of the few able to efficiently utilize both NADPH and NADH as co-substrates in converting xylose into xylitol. In order to better understand the basis for this dual specificity, we have determined the crystal structure of XR from the yeast Candida tenuis in complex with NAD(+) to 1.80 A resolution (where 1 A=0.1 nm) with a crystallographic R -factor of 18.3%. A comparison of the NAD(+)- and the previously determined NADP(+)-bound forms of XR reveals that XR has the ability to change the conformation of two loops. To accommodate both the presence and absence of the 2'-phosphate, the enzyme is able to adopt different conformations for several different side chains on these loops, including Asn(276), which makes alternative hydrogen-bonding interactions with the adenosine ribose. Also critical is the presence of Glu(227) on a short rigid helix, which makes hydrogen bonds to both the 2'- and 3'-hydroxy groups of the adenosine ribose. In addition to changes in hydrogen-bonding of the adenosine, the ribose unmistakably adopts a 3'- endo conformation rather than the 2'- endo conformation seen in the NADP(+)-bound form. These results underscore the importance of tight adenosine binding for efficient use of either NADH or NADPH as a co-substrate in aldo-keto reductases. The dual specificity found in XR is also an important consideration in designing a high-flux xylose metabolic pathway, which may be improved with an enzyme specific for NADH. PMID:12733986

  1. Coenzyme A disulfide reductase, the primary low molecular weight disulfide reductase from Staphylococcus aureus. Purification and characterization of the native enzyme.

    PubMed

    delCardayre, S B; Stock, K P; Newton, G L; Fahey, R C; Davies, J E

    1998-03-01

    The human pathogen Staphylococcus aureus does not utilize the glutathione thiol/disulfide redox system employed by eukaryotes and many bacteria. Instead, this organism produces CoA as its major low molecular weight thiol. We report the identification and purification of the disulfide reductase component of this thiol/disulfide redox system. Coenzyme A disulfide reductase (CoADR) catalyzes the specific reduction of CoA disulfide by NADPH. CoADR has a pH optimum of 7.5-8.0 and is a dimer of identical subunits of Mr 49,000 each. The visible absorbance spectrum is indicative of a flavoprotein with a lambdamax = 452 nm. The liberated flavin from thermally denatured enzyme was identified as flavin adenine dinucleotide. Steady-state kinetic analysis revealed that CoADR catalyzes the reduction of CoA disulfide by NADPH at pH 7.8 with a Km for NADPH of 2 muM and for CoA disulfide of 11 muM. In addition to CoA disulfide CoADR reduces 4,4'-diphosphopantethine but has no measurable ability to reduce oxidized glutathione, cystine, pantethine, or H2O2. CoADR demonstrates a sequential kinetic mechanism and employs a single active site cysteine residue that forms a stable mixed disulfide with CoA during catalysis. These data suggest that S. aureus employs a thiol/disulfide redox system based on CoA/CoA-disulfide and CoADR, an unorthodox new member of the pyridine nucleotide-disulfide reductase superfamily. PMID:9488707

  2. Crystal structure and catalytic mechanism of leucoanthocyanidin reductase from Vitis vinifera.

    PubMed

    Maugé, Chloé; Granier, Thierry; d'Estaintot, Béatrice Langlois; Gargouri, Mahmoud; Manigand, Claude; Schmitter, Jean-Marie; Chaudière, Jean; Gallois, Bernard

    2010-04-01

    Leucoanthocyanidin reductase (LAR) catalyzes the NADPH-dependent reduction of 2R,3S,4S-flavan-3,4-diols into 2R,3S-flavan-3-ols, a subfamily of flavonoids that is important for plant survival and for human nutrition. LAR1 from Vitis vinifera has been co-crystallized with or without NADPH and one of its natural products, (+)-catechin. Crystals diffract to a resolution between 1.75 and 2.72 A. The coenzyme and substrate binding pocket is preformed in the apoprotein and not markedly altered upon NADPH binding. The structure of the abortive ternary complex, determined at a resolution of 2.28 A, indicates the ordering of a short 3(10) helix associated with substrate binding and suggests that His122 and Lys140 act as acid-base catalysts. Based on our 3D structures, a two-step catalytic mechanism is proposed, in which a concerted dehydration precedes an NADPH-mediated hydride transfer at C4. The dehydration step involves a Lys-catalyzed deprotonation of the phenolic OH7 through a bridging water molecule and a His-catalyzed protonation of the benzylic hydroxyl at C4. The resulting quinone methide serves as an electrophilic target for hydride transfer at C4. LAR belongs to the short-chain dehydrogenase/reductase superfamily and to the PIP (pinoresinol-lariciresinol reductase, isoflavone reductase, and phenylcoumaran benzylic ether reductase) family. Our data support the concept that all PIP enzymes reduce a quinone methide intermediate and that the major role of the only residue that has been conserved from the short-chain dehydrogenase/reductase catalytic triad (Ser...TyrXXXLys), that is, lysine, is to promote the formation of this intermediate by catalyzing the deprotonation of a phenolic hydroxyl. For some PIP enzymes, this lysine-catalyzed proton abstraction may be sufficient to trigger the extrusion of the leaving group, whereas in LAR, the extrusion of a hydroxide group requires a more sophisticated mechanism of concerted acid-base catalysis that involves histidine

  3. Expression and characterization of a functional canine variant of cytochrome b5 reductase.

    PubMed

    Roma, Glenn W; Crowley, Louis J; Barber, Michael J

    2006-08-01

    Cytochrome b5 reductase (cb5r), a member of the flavoprotein transhydrogenase family of oxidoreductase enzymes, catalyzes the transfer of reducing equivalents from the physiological electron donor, NADH, to two molecules of cytochrome b5. We have determined the correct nucleotide sequence for the putative full-length, membrane-associated enzyme from Canis familiaris, and have generated a heterologous expression system for production of a histidine-tagged variant of the soluble, catalytic diaphorase domain, comprising residues I33 to F300. Using a simple two-step chromatographic procedure, the recombinant diaphorase domain has been purified to homogeneity and demonstrated to be a simple flavoprotein with a molecular mass of 31,364 (m/z) that retained both NADH:ferricyanide reductase and NADH:cytochrome b5 reductase activities. The recombinant protein contained a full complement of FAD and exhibited absorption and CD spectra comparable to those of a recombinant form of the rat cytochrome b5 reductase diaphorase domain generated using an identical expression system, suggesting similar protein folding. Oxidation-reduction potentiometric titrations yielded a standard midpoint potential (Eo') for the FAD/FADH2 couple of -273+/-5 mV which was identical to the value obtained for the corresponding rat domain. Thermal denaturation studies revealed that the canine domain exhibited stability comparable to that of the rat protein, confirming similar protein conformations. Initial-rate kinetic studies revealed the canine diaphorase domain retained a marked preference for NADH versus NADPH as reducing substrate and exhibited kcat's of 767 and 600 s(-1) for NADH:ferricyanide reductase and NADH:cytochrome b5 reductase activities, respectively, with Km's of 7, 8, and 12 microM for NADH, K3Fe(CN)6, and cytochrome b5, respectively. Spectral-binding constants (Ks) determined for a variety of NAD+ analogs indicated the highest and lowest affinities were observed for APAD+ (Ks=71 micro

  4. Human monomethylarsonic acid (MMA(V)) reductase is a member of the glutathione-S-transferase superfamily.

    PubMed

    Zakharyan, R A; Sampayo-Reyes, A; Healy, S M; Tsaprailis, G; Board, P G; Liebler, D C; Aposhian, H V

    2001-08-01

    The drinking of water containing large amounts of inorganic arsenic is a worldwide major public health problem because of arsenic carcinogenicity. Yet an understanding of the specific mechanism(s) of inorganic arsenic toxicity has been elusive. We have now partially purified the rate-limiting enzyme of inorganic arsenic metabolism, human liver MMA(V) reductase, using ion exchange, molecular exclusion, and hydroxyapatite chromatography. When SDS-beta-mercaptoethanol-PAGE was performed on the most purified fraction, seven protein bands were obtained. Each band was excised from the gel, sequenced by LC-MS/MS and identified according to the SWISS-PROT and TrEMBL Protein Sequence databases. Human liver MMA(V) reductase is 100% identical, over 92% of sequence that we analyzed, with the recently discovered human glutathione-S-transferase Omega class hGSTO 1-1. Recombinant human GSTO1-1 had MMA(V) reductase activity with K(m) and V(max) values comparable to those of human liver MMA(V) reductase. The partially purified human liver MMA(V) reductase had glutathione S-transferase (GST) activity. MMA(V) reductase activity was competitively inhibited by the GST substrate, 1-chloro 2,4-dinitrobenzene and also by the GST inhibitor, deoxycholate. Western blot analysis of the most purified human liver MMA(V) reductase showed one band when probed with hGSTO1-1 antiserum. We propose that MMA(V) reductase and hGSTO 1-1 are identical proteins. PMID:11511179

  5. The association between methylenetetrahydrofolate reductase gene C677T polymorphisms and breast cancer risk in Chinese population.

    PubMed

    Wang, Yadong; Yang, Haiyan; Gao, Huiyan; Wang, Haiyu

    2015-12-01

    With great interest, we read the recent article entitled "Methylenetetrahydrofolate reductase polymorphisms and breast cancer risk in Chinese population: a meta-analysis of 22 case-control studies" published online in Tumor Biology, 2014, 35: 1695-1701. This article suggests that methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism was significantly associated with breast cancer risk in Chinese population. The result is encouraging. Nevertheless, three key issues in this meta-analysis are worth noticing. PMID:26537580

  6. Crystal Structure of Saccharomyces Cerevisiae 3'-Phosphoadenosine-5'-Phosphosulfate Reductase Complexed With Adenosine 3',5'-Bisphosphate

    SciTech Connect

    Yu, Z.; Lemongello, D.; Segel, I.H.; Fisher, A.J.

    2009-05-28

    Most assimilatory bacteria, fungi, and plants species reduce sulfate (in the activated form of APS or PAPS) to produce reduced sulfur. In yeast, PAPS reductase reduces PAPS to sulfite and PAP. Despite the difference in substrate specificity and catalytic cofactor, PAPS reductase is homologous to APS reductase in both sequence and structure, and they are suggested to share the same catalytic mechanism. Metazoans do not possess the sulfate reduction pathway, which makes APS/PAPS reductases potential drug targets for human pathogens. Here, we present the 2.05 A resolution crystal structure of the yeast PAPS reductase binary complex with product PAP bound. The N-terminal region mediates dimeric interactions resulting in a unique homodimer assembly not seen in previous APS/PAPS reductase structures. The 'pyrophosphate-binding' sequence (47)TTAFGLTG(54) defines the substrate 3'-phosphate binding pocket. In yeast, Gly54 replaces a conserved aspartate found in APS reductases vacating space and charge to accommodate the 3'-phosphate of PAPS, thus regulating substrate specificity. Also, for the first time, the complete C-terminal catalytic motif (244)ECGIH(248) is revealed in the active site. The catalytic residue Cys245 is ideally positioned for an in-line attack on the beta-sulfate of PAPS. In addition, the side chain of His248 is only 4.2 A from the Sgamma of Cys245 and may serve as a catalytic base to deprotonate the active site cysteine. A hydrophobic sequence (252)RFAQFL(257) at the end of the C-terminus may provide anchoring interactions preventing the tail from swinging away from the active site as seen in other APS/PAPS reductases.

  7. A qualitative and quantitative cytochemical assay of dihydrofolate reductase in erythroid cells.

    PubMed

    Nano, R; Gerzeli, G; Invernizzi, R; Supino, R

    1989-01-01

    The distribution and intensity of dihydrofolate reductase (DHFR) cytochemically demonstrable was studied in erythroid cells. Cells of normal human bone marrow, of human erythroleukaemia (M6), and cells of the Friend (MEL) clone 745A murine erythroleukaemia (also after differentiation with dimethylsulphoxide, DMSO) were stained according to Gerzeli and de Piceis Polver (1969) technique; quantification of the reaction product was made using a Vickers M86 microdensitometer. The enzyme activity progressively decreased during the normal differentiation of the erythropoietic series while persisted at high levels in erythroleukaemia cells. It can be suggested that in the 1st case, the cytochemical pattern of dihydrofolate reductase may be a useful added tool for studying the erythroid differentiation. In the 2nd case, the increased level of this enzyme may be related to an amplification of the gene of DHFR in the malignant transformation. PMID:2496572

  8. Discovery of pyrrole-based hepatoselective ligands as potent inhibitors of HMG-CoA reductase.

    PubMed

    Bratton, Larry D; Auerbach, Bruce; Choi, Chulho; Dillon, Lisa; Hanselman, Jeffrey C; Larsen, Scott D; Lu, Gina; Olsen, Karl; Pfefferkorn, Jeffrey A; Robertson, Andrew; Sekerke, Catherine; Trivedi, Bharat K; Unangst, Paul C

    2007-08-15

    In an effort to identify hepatoselective inhibitors of HMG-CoA reductase, two series of pyrroles were synthesized and evaluated. Efforts were made to modify (3R,5R)-7-[3-(4-fluorophenyl)-1-isopropyl-4-phenyl-5-phenylcarbamoyl-1H-pyrrol-2-yl]-3,5-dihydroxy-heptanoic acid sodium salt 30 in order to reduce its lipophilicity and therefore increase hepatoselectivity. Two strategies that were explored were replacement of the lipophilic 3-phenyl substituent with either a polar function (pyridyl series) or with lower alkyl substituents (lower alkyl series) and attachment of additional polar moieties at the 2-position of the pyrrole ring. One compound was identified to be both highly hepatoselective and active in vivo. We report the discovery, synthesis, and optimization of substituted pyrrole-based hepatoselective ligands as potent inhibitors of HMG-CoA reductase for reducing low density lipoprotein cholesterol (LDL-c) in the treatment of hypercholesterolemia. PMID:17560788

  9. 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

  10. The properties of monoclonal antibody against sepiapterin reductase from fat body of the silkworm, Bombyx mori.

    PubMed

    Iino, T; Sawada, H; Gyure, W L; Tsusué, M

    1992-12-01

    A specific monoclonal antibody prepared for the 29-kDa a subunit of silkworm fat body sepiapterin reductase (SPR) was able to recognize the subunit in crude extract of fat body after SDS treatment. Although SPR from the silkworm fat body has biochemical properties similar to those reported for SPR from mammalian sources, especially rat erythrocytes, the antibody failed to recognize the 28-kDa subunit of rat erythrocyte SPR. This result indicates that SPR from silkworm fat body has a different amino-acid sequence from that of the rat erythrocyte enzyme. Sepiapterin reductase activity has not been found in crude extract of fat body from the silkworm mutant lemon. Although the antibody recognized only 29-kDa protein in the crude extract of silkworm fat body from normal strain after SDS-treatment, the antibody recognized only an approximately 80-kDa protein in the crude extract of the lemon mutant after SDS-treatment. PMID:1292509

  11. The class III ribonucleotide reductase from Neisseria bacilliformis can utilize thioredoxin as a reductant

    PubMed Central

    Wei, Yifeng; Funk, Michael A.; Rosado, Leonardo A.; Baek, Jiyeon; Drennan, Catherine L.; Stubbe, JoAnne

    2014-01-01

    The class III anaerobic ribonucleotide reductases (RNRs) studied to date couple the reduction of ribonucleotides to deoxynucleotides with the oxidation of formate to CO2. Here we report the cloning and heterologous expression of the Neisseria bacilliformis class III RNR and show that it can catalyze nucleotide reduction using the ubiquitous thioredoxin/thioredoxin reductase/NADPH system. We present a structural model based on a crystal structure of the homologous Thermotoga maritima class III RNR, showing its architecture and the position of conserved residues in the active site. Phylogenetic studies suggest that this form of class III RNR is present in bacteria and archaea that carry out diverse types of anaerobic metabolism. PMID:25157154

  12. Crystal Structure of ChrR -- A Quinone Reductase with the Capacity to Reduce Chromate

    SciTech Connect

    Eswaramoorthy S.; Poulain, S.; Hienerwadel, R.; Bremond, N.; Sylvester, M. D.; Zhang, Y.-B.; Berthomieu, C.; van der Lelie, D.; Matin, A.

    2012-04-01

    The Escherichia coli ChrR enzyme is an obligatory two-electron quinone reductase that has many applications, such as in chromate bioremediation. Its crystal structure, solved at 2.2 {angstrom} resolution, shows that it belongs to the flavodoxin superfamily in which flavin mononucleotide (FMN) is firmly anchored to the protein. ChrR crystallized as a tetramer, and size exclusion chromatography showed that this is the oligomeric form that catalyzes chromate reduction. Within the tetramer, the dimers interact by a pair of two hydrogen bond networks, each involving Tyr128 and Glu146 of one dimer and Arg125 and Tyr85 of the other; the latter extends to one of the redox FMN cofactors. Changes in each of these amino acids enhanced chromate reductase activity of the enzyme, showing that this network is centrally involved in chromate reduction.

  13. Immobilization of mercuric reductase from a pseudomonas putida strain on different activated carriers

    SciTech Connect

    Anspach, F.B.; Hueckel, M.; Brunke, M.

    1994-02-01

    Mercuric reductase was isolated from Pseudomonas putida KT2442::mer-73 and immobilized on chromatographic carriers activated by various methods. The immobilization methods for covalent coupling were compared with regard to preservation of enzymatic activity and coupling yields. Highest yields were obtained with carriers bearing the most reactive functional groups. Best results were achieved with tresyl chloride-activated carriers. The optimum binding conditions were found at pH 8. Application of the immobilized mercuric reductase for continuous treatment of Hg(II)-containing water was examined in a fixed bed reactor. Space-time yields up to 510 nmol/min{center_dot}mL were attained. The kinetics of immobilized enzyme systems were not diffusion-controlled. 22 refs., 7 figs., 2 tabs.

  14. Crystal Structure of ChrR—A Quinone Reductase with the Capacity to Reduce Chromate

    PubMed Central

    Hienerwadel, Rainer; Bremond, Nicolas; Sylvester, Matthew D.; Zhang, Yian-Biao; Berthomieu, Catherine; Van Der Lelie, Daniel; Matin, A.

    2012-01-01

    The Escherichia coli ChrR enzyme is an obligatory two-electron quinone reductase that has many applications, such as in chromate bioremediation. Its crystal structure, solved at 2.2 Å resolution, shows that it belongs to the flavodoxin superfamily in which flavin mononucleotide (FMN) is firmly anchored to the protein. ChrR crystallized as a tetramer, and size exclusion chromatography showed that this is the oligomeric form that catalyzes chromate reduction. Within the tetramer, the dimers interact by a pair of two hydrogen bond networks, each involving Tyr128 and Glu146 of one dimer and Arg125 and Tyr85 of the other; the latter extends to one of the redox FMN cofactors. Changes in each of these amino acids enhanced chromate reductase activity of the enzyme, showing that this network is centrally involved in chromate reduction. PMID:22558308

  15. The dual function of flavodiiron proteins: oxygen and/or nitric oxide reductases.

    PubMed

    Romão, Célia V; Vicente, João B; Borges, Patrícia T; Frazão, Carlos; Teixeira, Miguel

    2016-03-01

    Flavodiiron proteins have emerged in the last two decades as a newly discovered family of oxygen and/or nitric oxide reductases widespread in the three life domains, and present in both aerobic and anaerobic organisms. Herein we present the main features of these fascinating enzymes, with a particular emphasis on the metal sites, as more appropriate for this special issue in memory of the exceptional bioinorganic scientist R. J. P. Williams who pioneered the notion of (metal) element availability-driven evolution. We also compare the flavodiiron proteins with the other oxygen and nitric oxide reductases known until now, highlighting how throughout evolution Nature arrived at different solutions for similar functions, in some cases adding extra features, such as energy conservation. These enzymes are an example of the (bioinorganic) unpredictable diversity of the living world. PMID:26767750

  16. Structure-Based Design of Pteridine Reductase Inhibitors Targeting African Sleeping Sickness and the Leishmaniases†

    PubMed Central

    2009-01-01

    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. PMID:19916554

  17. Effects of environmental conditions on xylose reductase and xylitol dehydrogenase production by Candida guilliermondii.

    PubMed

    Sene, L; Vitolo, M; Felipe, M G; Silva, S S

    2000-01-01

    The effects of environmental conditions, namely initial pH (2.5-7.0) and temperature (25 and 35 degrees C), on xylose reductase and xylitol dehydrogenase levels, as well as on xylitol production, were evaluated. Although the fermentative parameter values increased with an increase in pH and temperature (the maximum Yp/s and Qp were 0.75 g/g and 0.95 g/[L.h], respectively, both attained at pH 6.0, 35 degrees C), the highest xylose reductase activities (nearly 900 IU/mg of protein) were observed at an initial pH varying from 4.0 to 6.0. Xylitol dehydrogenase was favored by an increase in both initial pH and temperature of the medium. The highest xylitol dehydrogenase specific activity was attained at pH 6.5 and 35 degrees C (577 IU/mg of protein). PMID:10849803

  18. 5alpha-Reductase inhibitor treatment of prostatic diseases: background and practical implications.

    PubMed

    Dörsam, J; Altwein, J

    2009-01-01

    This literature review discusses the theoretical background of 5alpha-reductase inhibitor (5ARI) treatment and the resulting clinical implications. A Medline-based search for peer-reviewed articles addressing 5ARIs, benign prostatic hyperplasia and prostate cancer was performed. The 5ARIs Finasteride and Dutasteride, which specifically inhibit the production of dihydrotestosterone by acting as competitive inhibitors of 5alpha-reductase, are clinically well tolerated and represent an effective treatment option for benign prostatic obstruction. Finasteride is the first compound which has a proven efficacy in chemoprevention of prostate cancer. The aim of this review was to elucidate, if there are sufficient data available to point out clinically relevant differences between the drugs. Both compounds achieve a significant reduction of prostate volume, an improvement of symptoms and a lower risk of acute urinary retention. Whether the different pharmacokinetic and pharmacodynamic properties of Finasteride and Dutasteride are of clinical importance cannot be judged at this time. PMID:19030020

  19. Mineral supplementation increases erythrose reductase activity in erythritol biosynthesis from glycerol by Yarrowia lipolytica.

    PubMed

    Tomaszewska, Ludwika; Rymowicz, Waldemar; Rywińska, Anita

    2014-03-01

    The aim of this study was to examine the impact of divalent copper, iron, manganese, and zinc ions on the production of erythritol from glycerol by Yarrowia lipolytica and their effect on the activity of erythrose reductase. No inhibitory effect of the examined minerals on yeast growth was observed in the study. Supplementation with MnSO4 · 7H2O (25 mg l(-1)) increased erythritol production by Y. lipolytica by 14.5%. In the bioreactor culture with manganese ion addition, 47.1 g l(-1) of erythritol was produced from 100.0 g l(-1) of glycerol, which corresponded to volumetric productivity of 0.87 g l(-1) h(-1). The addition of Mn(2+) enhanced the intracellular activity of erythrose reductase up to 24.9 U g(-1) of dry weight of biomass (DW), hence, about 1.3 times more than in the control. PMID:24488778

  20. Defining the Role of the NADH-Cytochrome-b5 Reductase 3 in the Mitochondrial Amidoxime Reducing Component Enzyme System.

    PubMed

    Plitzko, Birte; Havemeyer, Antje; Bork, Bettina; Bittner, Florian; Mendel, Ralf; Clement, Bernd

    2016-10-01

    The importance of the mitochondrial amidoxime reducing component (mARC)-containing enzyme system in N-reductive metabolism has been studied extensively. It catalyzes the reduction of various N-hydroxylated compounds and therefore acts as the counterpart of cytochrome P450- and flavin-containing monooxygenase-catalyzed oxidations at nitrogen centers. This enzyme system was found to be responsible for the activation of amidoxime and N-hydroxyguanidine prodrugs in drug metabolism. The synergy of three components (mARC, cytochrome b5, and the appropriate reductase) is crucial to exert the N-reductive catalytic effect. Previous studies have demonstrated the involvement of the specific isoforms of the molybdoenzyme mARC and the electron transport protein cytochrome b5 in N-reductive metabolism. To date, the corresponding reductase involved in N-reductive metabolism has yet to be defined because previous investigations have presented ambiguous results. Using small interfering RNA-mediated knockdown in human cells and assessing the stoichiometry of the enzyme system reconstituted in vitro, we provide evidence that NADH-cytochrome-b5 reductase 3 is the principal reductase involved in the mARC enzyme system and is an essential component of N-reductive metabolism in human cells. In addition, only minimal levels of cytochrome-b5 reductase 3 protein are sufficient for catalysis, which impeded previous attempts to identify the reductase. PMID:27469001

  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. PMID:26906096

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

    PubMed Central

    Hartsock, Angela; Shapleigh, James P.

    2011-01-01

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

  3. 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

  4. The Cellular Thioredoxin-1/Thioredoxin Reductase-1 Driven Oxidoreduction Represents a Chemotherapeutic Target for HIV-1 Entry Inhibition

    PubMed Central

    Curbo, Sophie; Pannecouque, Christophe; Noppen, Sam; Liekens, Sandra; Engman, Lars; Lundberg, Mathias; Balzarini, Jan; Karlsson, Anna

    2016-01-01

    Background The entry of HIV into its host cell is an interesting target for chemotherapeutic intervention in the life-cycle of the virus. During entry, reduction of disulfide bridges in the viral envelope glycoprotein gp120 by cellular oxidoreductases is crucial. The cellular thioredoxin reductase-1 plays an important role in this oxidoreduction process by recycling electrons to thioredoxin-1. Therefore, thioredoxin reductase-1 inhibitors may inhibit gp120 reduction during HIV-1 entry. In this present study, tellurium-based thioredoxin reductase-1 inhibitors were investigated as potential inhibitors of HIV entry. Results The organotellurium compounds inhibited HIV-1 and HIV-2 replication in cell culture at low micromolar concentrations by targeting an early event in the viral infection cycle. Time-of-drug-addition studies pointed to virus entry as the drug target, more specifically: the organotellurium compound TE-2 showed a profile similar or close to that of the fusion inhibitor enfuvirtide (T-20). Surface plasmon resonance-based interaction studies revealed that the compounds do not directly interact with the HIV envelope glycoproteins gp120 and gp41, nor with soluble CD4, but instead, dose-dependently bind to thioredoxin reductase-1. By inhibiting the thioredoxin-1/thioredoxin reductase-1-directed oxidoreduction of gp120, the organotellurium compounds prevent conformational changes in the viral glycoprotein which are necessary during viral entry. Conclusion Our findings revealed that thioredoxin-1/thioredoxin reductase-1 acts as a cellular target for the inhibition of HIV entry. PMID:26816344

  5. 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

  6. Nitrate reductase, nitrite reductase, glutamine synthetase, and glutamate synthase expression and activity in response to different nitrogen sources in nitrogen-starved wheat seedlings.

    PubMed

    Balotf, Sadegh; Kavoosi, Gholamreza; Kholdebarin, Bahman

    2016-03-01

    The objective of this study was to examine the expression and activity of nitrate reductase (NR, EC 1.7.1.1), nitrite reductase (NiR, EC 1.7.2.2), glutamine synthetase (GS, EC 6.3.1.2), and glutamate synthase (GOGAT, EC 1.4.7.1) in response to potassium nitrate, ammonium chloride, and ammonium nitrate in nitrogen-starved wheat seedlings. Plants were grown in standard nutrient solution for 17 days and then subjected to nitrogen starvation for 7 days. The starved plants were supplied with potassium nitrate ammonium nitrate and ammonium chloride (50 mM) for 4 days and the leaves were harvested. The relative expression of NR, NiR, GS, and GOGAT as well as the enzyme activities were investigated. Nitrogen starvation caused a significant decrease both in transcript levels and in NR, NiR, GS, and GOGAT activities. Potassium nitrate and ammonium nitrate treatments restored NR, NiR, GS, and GOGAT expressions and activities. Ammonium chloride increased only the expressions and activities of GS and GOGAT in a dose-dependent manner. The results of our study highlight the differential effects between the type and the amount of nitrogen salts on NR, NiR, GS, and GOGAT activities in wheat seedlings while potassium nitrate being more effective. PMID:25676153

  7. Metal complexes with 2-acetylpyridine-N(4)-orthochlorophenylthiosemicarbazone: cytotoxicity and effect on the enzymatic activity of thioredoxin reductase and glutathione reductase.

    PubMed

    Parrilha, Gabrieli L; Ferraz, Karina S O; Lessa, Josane A; de Oliveira, Kely Navakoski; Rodrigues, Bernardo L; Ramos, Jonas P; Souza-Fagundes, Elaine M; Ott, Ingo; Beraldo, Heloisa

    2014-09-12

    Metal complexes with 2-acetylpyridine-N(4)-orthochlorophenylthiosemicarbazone (H2Ac4oClPh) were assayed for their cytotoxicity against MCF-7 breast adenocarcinoma and HT-29 colon carcinoma cells. The thiosemicarbazone and most of the complexes were highly cytotoxic. H2Ac4oClPh and its gallium(III) and tin(IV) complexes did not show any inhibitory activity against thioredoxin reductase (TrxR) and glutathione reductase (GR). The palladium(II), platinum(II) and bismuth(III) complexes inhibited TrxR at micromolar concentrations but not GR. The antimony(III) and gold(III) complexes strongly inhibited TrxR at submicromolar doses with GR inhibition at higher concentrations. The selectivity of these complexes for TrxR suggests metal binding to a selenol residue in the active site of the enzyme. TrxR inhibition is likely a contributing factor to the mode of action of the gold and antimony derivatives. PMID:25058344

  8. Gene expression of monodehydroascorbate reductase and dehydroascorbate reductase during fruit ripening and in response to environmental stresses in acerola (Malpighia glabra).

    PubMed

    Eltelib, Hani A; Badejo, Adebanjo A; Fujikawa, Yukichi; Esaka, Muneharu

    2011-04-15

    Acerola (Malpighia glabra) is an exotic fruit cultivated primarily for its abundant ascorbic acid (AsA) content. The molecular mechanisms that regulate the metabolism of AsA in acerola have yet to be defined. Monodehydroascorbate reductase (MDHAR) and dehydroascorbate reductase (DHAR) are key enzymes of the ascorbate-glutathione cycle that maintain reduced pools of ascorbic acid and serve as important antioxidants. cDNAs encoding MDHAR and DHAR were isolated from acerola using RT-PCR and RACE. Phylogenetic trees associated acerola MDHAR and DHAR with other plant cytosolic MDHARs and DHARs. Expressions of the two genes correlated with their enzymatic activities and were differentially regulated during fruit ripening. Interestingly, MDHAR expression was only detected in overripe fruits, whereas the transcript level of DHAR was highest at the intermediate stage of fruit ripening. Under dark conditions, there was a sharp and significant decline in the total and reduced ascorbate contents, accompanied by a decrease in the level of transcripts and enzyme activities of the two genes in acerola leaves. MDHAR and DHAR transcripts and enzyme activities were significantly up-regulated in the leaves of acerola under cold and salt stress conditions, indicating that expression of both genes are transcriptionally regulated under these stresses. PMID:20933298

  9. Cloned and expressed nitric oxide synthase structurally resembles cytochrome P-450 reductase

    NASA Astrophysics Data System (ADS)

    Bredt, David S.; Hwang, Paul M.; Glatt, Charles E.; Lowenstein, Charles; Reed, Randall R.; Snyder, Solomon H.

    1991-06-01

    Nitric oxide is a messenger molecule, mediating the effect of endothelium-derived relaxing factor in blood vessels and the cytotoxic actions of macrophages, and playing a part in neuronal communication in the brain. Cloning of a complementary DNA for brain nitric oxide synthase reveals recognition sites for NADPH, FAD, flavin mononucleotide and calmodulin as well as phosphorylation sites, indicating that the synthase is regulated by many different factors. The only known mammalian enzyme with close homology is cytochrome P-450 reductase.

  10. Regulation of schistosome egg production by HMG CoA reductase

    SciTech Connect

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

    1986-03-05

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

  11. Identification of the reactive cysteine residue (Cys227) in human carbonyl reductase.

    PubMed

    Tinguely, J N; Wermuth, B

    1999-02-01

    Carbonyl reductase is highly susceptible to inactivation by organomercurials suggesting the presence of a reactive cysteine residue in, or close to, the active site. This residue is also close to a site which binds glutathione. Structurally, carbonyl reductase belongs to the short-chain dehydrogenase/reductase family and contains five cysteine residues, none of which is conserved within the family. In order to identify the reactive residue and investigate its possible role in glutathione binding, alanine was substituted for each cysteine residue of human carbonyl reductase by site-directed mutagenesis. The mutant enzymes were expressed in Escherichia coli and purified to homogeneity. Four of the five mutants (C26A, C122A C150A and C226A) exhibited wild-type-like enzyme activity, although K(m) values of C226A for three structurally different substrates were increased threefold to 10-fold. The fifth mutant, C227A, showed a 10-15-fold decrease in kcat and a threefold to 40-fold increase in K(m), resulting in a 30-500-fold drop in kcat/K(m). NaCl (300 mM) increased the activity of C227A 16-fold, whereas the activity of the wild-type enzyme was only doubled. Substitution of serine rather than alanine for Cys227 similarly affected the kinetic constants with the exception that NaCl did not activate the enzyme. Both C227A and C227S mutants were insensitive to inactivation by 4-hydroxymercuribenzoate. Unlike the parent carbonyl compounds, the glutathione adducts of menadione and prostaglandin A1 were better substrates for the C227A and C227S mutants than the wild-type enzyme. Conversely, the binding of free glutathione to both mutants was reduced. Our findings indicate that Cys227 is the reactive residue and suggest that it is involved in the binding of both substrate and glutathione. PMID:10091578

  12. Distinct roles of cytochrome P450 reductase in mitomycin c redox cycling and cytotoxicity

    PubMed Central

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

    2010-01-01

    Mitomycin c (MMC), a quinone-containing anticancer drug, is known to redox cycle and generate reactive oxygen species. A key enzyme mediating MMC redox cycling is cytochrome P450 reductase, a microsomal NADPH-dependent flavoenzyme. In the present studies, CHO cells overexpressing this enzyme (CHO-OR cells) and corresponding control cells (CHO-WT cells) were used to investigate the role of cytochrome P450 reductase in the actions of MMC. In lysates from both cell types, MMC was found to redox cycle and generate H2O2; this activity was greater in CHO-OR cells (Vmax = 1.2 ± 0.1 nmol H2O2/min/mg protein in CHO-WT cells vs. 32.4 ± 3.9 nmol H2O2/min/mg protein in CHO-OR cells). MMC was also more effective in generating superoxide anion and hydroxyl radicals in CHO-OR cells, relative to CHO-WT cells. Despite these differences in MMC redox cycling, MMC-induced cytotoxicity, as measured by growth inhibition, was similar in the two cell types (IC50 = 72 ± 20 nM for CHO-WT and 75 ± 23 nM for CHO-OR cells), as was its ability to induce G2/M and S phase arrest. Additionally, in 9 different tumor cell lines, although a strong correlation was observed between MMC-induced H2O2 generation and cytochrome P450 reductase activity, there was no relationship between redox cycling and cytotoxicity. Hypoxia, which stabilizes MMC radicals generated by redox cycling, also had no effect on the sensitivity of tumor cells to MMC-induced cytotoxicity. These data indicate that NADPH cytochrome P450 reductase-mediated MMC redox cycling is not involved in cytotoxicity of this chemotherapeutic agent. PMID:20501808

  13. Roles of thioredoxin and thioredoxin reductase in the resistance to oxidative stress in Lactobacillus casei.

    PubMed

    Serata, Masaki; Iino, Tohru; Yasuda, Emi; Sako, Tomoyuki

    2012-04-01

    The Lactobacillus casei strain Shirota used in this study has in the genome four putative thioredoxin genes designated trxA1, trxA2, trxA3 and trxA4, and one putative thioredoxin reductase gene designated trxB. To elucidate the roles of the thioredoxins and the thioredoxin reductase against oxidative stress in L. casei, we constructed gene disruption mutants, in which each of the genes trxA1, trxA2 and trxB, or both trxA1 and trxA2 were disrupted, and we characterized their growth and response to oxidative stresses. In aerobic conditions, the trxA1 (MS108) and the trxA2 (MS109) mutants had moderate growth defects, and the trxA1 trxA2 double mutant (MS110) had a severe growth defect, which was characterized by elongation of doubling time and a lower final turbidity level. Furthermore, the trxB mutant (MS111), which is defective in thioredoxin reductase, lost the ability to grow under aerobic conditions, although it grew partially under anaerobic conditions. The growth of these mutants, however, could be substantially restored by the addition of dithiothreitol or reduced glutathione. In addition, MS110 and MS111 were more sensitive to hydrogen peroxide and disulfide stress than the wild-type. In particular, the stress sensitivity of MS111 was significantly increased. On the other hand, transcription of all these genes was only weakly affected by these oxidative stresses. Taken together, these results suggest that the thioredoxin-thioredoxin reductase system is the major thiol/disulfide redox system and is essential to allow the facultative anaerobe L. casei to grow under aerobic conditions. PMID:22301908

  14. Structural Basis of Free Reduced Flavin Generation by Flavin Reductase from Thermus thermophilus HB8*

    PubMed Central

    Imagawa, Takahito; Tsurumura, Toshiharu; Sugimoto, Yasushi; Aki, Kenji; Ishidoh, Kazumi; Kuramitsu, Seiki; Tsuge, Hideaki

    2011-01-01

    Free reduced flavins are involved in a variety of biological functions. They are generated from NAD(P)H by flavin reductase via co-factor flavin bound to the enzyme. Although recent findings on the structure and function of flavin reductase provide new information about co-factor FAD and substrate NAD, there have been no reports on the substrate flavin binding site. Here we report the structure of TTHA0420 from Thermus thermophilus HB8, which belongs to flavin reductase, and describe the dual binding mode of the substrate and co-factor flavins. We also report that TTHA0420 has not only the flavin reductase motif GDH but also a specific motif YGG in C terminus as well as Phe-41 and Arg-11, which are conserved in its subclass. From the structure, these motifs are important for the substrate flavin binding. On the contrary, the C terminus is stacked on the NADH binding site, apparently to block NADH binding to the active site. To identify the function of the C-terminal region, we designed and expressed a mutant TTHA0420 enzyme in which the C-terminal five residues were deleted (TTHA0420-ΔC5). Notably, the activity of TTHA0420-ΔC5 was about 10 times higher than that of the wild-type enzyme at 20–40 °C. Our findings suggest that the C-terminal region of TTHA0420 may regulate the alternative binding of NADH and substrate flavin to the enzyme. PMID:22052907

  15. Phenotypic classification of male pseudohermaphroditism due to steroid 5{alpha}-reductase 2 deficiency

    SciTech Connect

    Sinnecker, G.H.G; Hiort, O.; Kruse, K.; Dibbelt, L.

    1996-05-03

    Conversion of testosterone (T) to dihydrotestosterone (DHT) in genital tissue is catalysed by the enzyme 5{alpha}-reductase 2, which is encoded by the SRD5A2 gene. The potent androgen DHT is required for full masculinization of the external genitalia. Mutations of the SRD5A2 gene inhibit enzyme activity, diminish DHT formation, and hence cause masculinization defects of varying degree. The classical syndrome, formerly described as pseudovaginal perineoscrotal hypospadias, is characterized by a predominantly female phenotype at birth and significant virilization without gynecomastia at puberty. We investigated nine patients with steroid 5{alpha}-reductase 2 deficiency (SRD). T/DHT-ratios were highly increased in the classical syndrome, but variable in the less severe affected patients. Mutations in the SRD5A2 gene had been characterized using PCR-SSCP analysis and direct DNA sequencing. A small deletion was encountered in two patients, while all other patients had single base mutations which result in amino acid substitutions. We conclude that phenotypes may vary widely in patients with SRD5A2 gene mutations spanning the whole range from completely female to normal male without distinctive clinical signs of the disease. Hence, steroid 5{alpha}-reductase deficiency should be considered not only in sex reversed patients with female or ambiguous phenotypes, but also in those with mild symptoms of undermasculinization as encountered in patients with hypospadias and/or micropenis. A classification based on the severity of the masculinization defect may be used for correlation of phenotypes with enzyme activities and genotypes, and for comparisons of phenotypes between different patients as the basis for clinical decisions to be made in patients with pseudohermaphroditism due to steroid 5{alpha}-reductase 2 deficiency. 22 refs., 2 figs., 2 tabs.

  16. Dihydrofolate reductase as a model for studies of enzyme dynamics and catalysis

    PubMed Central

    Kohen, Amnon

    2015-01-01

    Dihydrofolate reductase from Escherichia coli (ecDHFR) serves as a model system for investigating the role of protein dynamics in enzyme catalysis. We discuss calculations predicting a network of dynamic motions that is coupled to the chemical step catalyzed by this enzyme. Kinetic studies testing these predictions are presented, and their potential use in better understanding the role of these dynamics in enzyme catalysis is considered. The cumulative results implicate motions across the entire protein in catalysis. PMID:26918149

  17. Anxiety and Methylenetetrahydrofolate Reductase Mutation Treated With S-Adenosyl Methionine and Methylated B Vitamins.

    PubMed

    Anderson, Shanna; Panka, Jacob; Rakobitsch, Robin; Tyre, Kaitlin; Pulliam, Kerry

    2016-04-01

    This case report highlights challenges faced in the clinical management of patients with methylenetetrahydrofolate reductase (MTHFR) gene mutations and the importance of precise dosage when recommending methylated B vitamins to compensate for deficiencies caused by the polymorphism or symptoms related to the polymorphism. It also underscores the importance of obtaining ongoing objective assessments of anxiety (eg, Patient Reported Outcomes Measurement Information System, or PROMIS) to help gauge patient response. PMID:27330489

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

    PubMed

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

    1985-04-25

    21-Diazo-4-methyl-4-aza-5 alpha-pregnane-3,20-dione (Diazo-MAPD) inhibits steroid 5 alpha-reductase in liver microsomes of female rats with a Ki value of 8.7 +/- 1.7 nM, and the inhibition is competitive with testosterone. It also inhibits the binding of a 5 alpha-reductase inhibitor, [3H] 17 beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one ([3H]4-MA), to the enzyme in liver microsomes. The inhibition of 5 alpha-reductase activity and of inhibitor binding activity by diazo-MAPD becomes irreversible upon UV irradiation. [1,2-3H]Diazo-MAPD binds to a single high affinity site (Kd 8 nM, 125 pmol binding sites/mg of protein) in liver microsomes of female rats, and this binding requires NADPH. Without UV irradiation, this binding is reversible, and it becomes irreversible upon UV irradiation. Both the initial reversible binding and the subsequent irreversible conjugation after UV irradiation are inhibited by inhibitors (diazo-MAPD and 4-MA) and substrates (progesterone and testosterone) of 5 alpha-reductase, but they are not inhibited by 5 alpha-reduced steroids (5 alpha-dihydrotestosterone and 5 alpha-androstan-3 alpha, 17 beta-diol). NADPH stimulates the binding of [3H] diazo-MAPD to microsomes of male rat liver and prostate. UV irradiation also induces conjugation of [3H] diazo-MAPD to these microsomes. Photoaffinity labeled liver microsomes of female rats were solubilized and fractionated by high performance gel filtration. The radioactive conjugate eluted in one major peak at Mr 50,000. PMID:3988737

  19. Disruption of the plr1+ gene encoding pyridoxal reductase of Schizosaccharomyces pombe.

    PubMed

    Morita, Tomotake; Takegawa, Kaoru; Yagi, Toshiharu

    2004-02-01

    Pyridoxal (PL) reductase encoded by the plr1(+) gene practically catalyzes the irreversible reduction of PL by NADPH to form pyridoxine (PN). The enzyme has been suggested to be involved in the salvage synthesis of pyridoxal 5'-phosphate (PLP), a coenzyme form of vitamin B(6), or the excretion of PL as PN from yeast cells. In this study, a PL reductase-disrupted (plr1 Delta) strain was constructed and its phenotype was examined. The plr1 Delta cells showed almost the same growth curve as that of wild-type cells in YNB and EMM media. In EMM, the plr1 Delta strain became flocculent at the late stationary phase for an unknown reason. The plr1 Delta cells showed low but measurable PL reductase activity catalyzed by some other protein(s) than the enzyme encoded by the plr1(+) gene, which maintained the flow of "PL --> PN --> PNP --> PLP" in the salvage synthesis of PLP. The total vitamin B(6) and pyridoxamine 5'-phosphate contents in the plr1 Delta cells were significantly lower than those in the wild-type ones. The percentages of the PLP amount as to the other vitamin B(6) compounds were similar in the two cell types. The amount of PL in the culture medium of the disruptant was significantly higher than that in the wild-type. In contrast, PN was much higher in the latter than the former. The plr1 Delta cells accumulated a 6.1-fold higher amount of PL than the wild-type ones when they were incubated with PL. The results showed that PL reductase encoded by the plr1(+ )gene is involved in the excretion of PL after reducing it to PN, and may not participate in the salvage pathway for PLP synthesis. PMID:15047724

  20. Molecular modeling toward selective inhibitors of dihydrofolate reductase from the biological warfare agent Bacillus anthracis.

    PubMed

    Giacoppo, Juliana O S; Mancini, Daiana T; Guimarães, Ana P; Gonçalves, Arlan S; da Cunha, Elaine F F; França, Tanos C C; Ramalho, Teodorico C

    2015-02-16

    In the present work, we applied docking and molecular dynamics techniques to study 11 compounds inside the enzymes dihydrofolate reductase (DHFR) from the biological warfare agent Bacillus anthracis (BaDHFR) and Homo sapiens sapiens (HssDHFR). Six of these compounds were selected for a study with the mutant BaF96IDHFR. Our results corroborated with experimental data and allowed the proposition of a new molecule with potential activity and better selectivity for BaDHFR. PMID:24985033

  1. Direct electrochemistry of Shewanella oneidensis cytochrome c nitrite reductase: evidence for interactions across the dimeric interface

    PubMed Central

    Judd, Evan T.; Youngblut, Matthew; Pacheco, A. Andrew; Elliott, Sean J.

    2013-01-01

    Shewanella oneidensis cytochrome c nitrite reductase (soNrfA), a dimeric enzyme that houses five c-type hemes per protomer, carries out the six-electron reduction of nitrite and the two-electron reduction of hydroxylamine. Protein film voltammetry (PFV) has been used to study the cytochrome c nitrite reductase from Escherichia coli (ecNrfA) previously, revealing catalytic reduction of both nitrite and hydroxylamine substrates by ecNrfA adsorbed to a graphite electrode that is characterized by ‘boosts’ and attenuations in activity depending on the applied potential. Here, we use PFV to investigate the catalytic properties of soNrfA during both nitrite and hydroxylamine turnover and compare those properties to ecNrfA. Distinct differences in both the electrochemical and kinetic characteristics of soNrfA are observed, e.g., all detected electron transfer steps are one-electron in nature, contrary to what has been observed in ecNrfA (Angove, H. C., Cole, J. A., Richardson, D. J., and Butt, J. N. (2002) Protein film voltammetry reveals distinctive fingerprints of nitrite and hydroxylamine reduction by a cytochrome C nitrite reductase, J Biol Chem 277, 23374-23381). Additionally, we find evidence of substrate inhibition during nitrite turnover and negative cooperativity during hydroxylamine turnover, neither of which have previously been observed in any cytochrome c nitrite reductase. Collectively these data provide evidence that during catalysis, potential pathways of communication exist between the individual soNrfA monomers comprising the native homodimer. PMID:23210513

  2. Ferric reductase activity in Azotobacter vinelandii and its inhibition by Zn2+.

    PubMed

    Huyer, M; Page, W J

    1989-07-01

    Ferric reductase activity was examined in Azotobacter vinelandii and was found to be located in the cytoplasm. The specific activities of soluble cell extracts were not affected by the iron concentration of the growth medium; however, activity was inhibited by the presence of Zn2+ during cell growth and also by the addition of Zn2+ to the enzyme assays. Intracellular Fe2+ levels were lower and siderophore production was increased in Zn2+-grown cells. The ferric reductase was active under aerobic conditions, had an optimal pH of approximately 7.5, and required flavin mononucleotide and Mg2+ for maximum activity. The enzyme utilized NADH to reduce iron supplied as a variety of iron chelates, including the ferrisiderophores of A. vinelandii. The enzyme was purified by conventional protein purification techniques, and the final preparation consisted of two major proteins with molecular weights of 44,600 and 69,000. The apparent Km values of the ferric reductase for Fe3+ (supplied as ferric citrate) and NADH were 10 and 15.8 microM, respectively, and the data for the enzyme reaction were consistent with Ping Pong Bi Bi kinetics. The approximate Ki values resulting from inhibition of the enzyme by Zn2+, which was a hyperbolic (partial) mixed-type inhibitor, were 25 microM with respect to iron and 1.7 microM with respect to NADH. These results suggested that ferric reductase activity may have a regulatory role in the processes of iron assimilation in A. vinelandii. PMID:2525550

  3. Induction and inhibition of NAD(P)H: quinone reductase in murine and human skin.

    PubMed

    Merk, H; Jugert, F; Bonnekoh, B; Mahrle, G

    1991-01-01

    The purpose of this study was to characterize the human cutaneous NAD(P)H: quinone reductase (NQR) activity by known inhibitors of different reductases and to compare it with the murine skin and liver NQR activity. This enzyme plays a major role in the defence of cells against oxygen stress because it inhibits the 1-electron reduction of quinones to semiquinones and their subsequent oxidation to quinones termed as quinone redox cycle. It belongs to the aromatic hydrocarbon-responsive (Ah) battery. This gene battery includes Cyp1a1 (cytochrome P-450 IA1), Cyp1a2 (cytochrome P-450 IA2) and Nmo-1 [NAD(P)H: quinone reductase]. In the skin cytochrome P-450 IA1-dependent activity is about 1-5% compared to the corresponding activity in the liver, whereas NQR has the same activity in skin and liver. NQR was determined in the cytoplasm of murine skin, liver, and human keratinocytes using 2,6-dichlorophenolindophenol as the substrate. The Ah-receptor binding compounds, such as coal tar constituents, or 3-methylcholanthrene induce cytochrome P-450-dependent activities such as aryl hydrocarbon hydroxylase or 7-ethoxyresorufin-O-de-ethylase and NQR, whereas butyl hydroxytoluol, which does not bind to the Ah receptor, induces only NQR. For inhibition studies several known inhibitors of dihydrodiol dehydrogenase, aldo-keto and carbonyl reductase activities were used. There was a similar pattern of inhibition of the basal and induced activity in all tissues investigated. Pyrazole, progesterone and phenobarbital did not inhibit, whereas dicoumarol, rutin and indomethacin inhibited NQR activity in murine skin and liver as well as in human keratinocytes.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1768430

  4. Testosterone and Adult Male Bone: Actions Independent of 5α-Reductase and Aromatase.

    PubMed

    Yarrow, Joshua F; Wronski, Thomas J; Borst, Stephen E

    2015-10-01

    Androgens and estrogens influence skeletal development and maintenance in males. However, the relative contributions of the circulating sex steroid hormones that originate from testicular/adrenal secretion versus those produced locally in bone via intracrine action require further elucidation. Our novel hypothesis is that testosterone exerts direct protective effects on the adult male skeleton independently of the actions of 5α-reductase or aromatase. PMID:26196865

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

    NASA Astrophysics Data System (ADS)

    Wu, Sangwook

    2016-04-01

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

  6. 5-alpha reductase inhibitors in patients on active surveillance: do the benefits outweigh the risk?

    PubMed

    Al Edwan, Ghazi; Fleshner, Neil

    2013-06-01

    Prostate cancer (PCa) is a slow, progressive disease. Prostate specific antigen testing, screening, and aggressive case identification has made PCa the most frequently diagnosed cancer. Concerns regarding overdiagnosis and overtreatment flourish on a large scale. In order to avoid overtreatment for those in whom therapeutic intervention is not required, active surveillance for eligible patients with the use of 5-alpha reductase can be considered a safe and a promising approach to delay the progression of the disease with minimal side effects. PMID:23579402

  7. 5alpha-reductase inhibitors in benign prostatic hyperplasia and prostate cancer risk reduction.

    PubMed

    Rittmaster, Roger S

    2008-04-01

    Androgens play an essential role in prostatic development and function, but are also involved in prostate disease pathogenesis. The primary prostatic androgen, dihydrotestosterone (DHT), is synthesized from testosterone by 5alpha-reductase types 1 and 2. Inhibition of the 5alpha-reductase isoenzymes therefore has potential therapeutic benefit in prostate disease. The two currently approved 5alpha-reductase inhibitors (5ARIs), finasteride and dutasteride, have demonstrated long-term efficacy and safety in the treatment of benign prostatic hyperplasia. Finasteride, a type-2 5ARI, has also been studied for its ability to reduce the incidence of biopsy-detectable prostate cancer in the Prostate Cancer Prevention Trial. Treatment with dutasteride, a dual 5ARI, has been shown to result in a greater degree and consistency of DHT suppression compared with finasteride. Two large-scale studies of dutasteride are currently investigating the role of near-maximal DHT suppression in the settings of prostate cancer risk reduction and expectant management of localized prostate cancer. PMID:18471794

  8. Identification of inhibitors of bacterial enoyl-acyl carrier protein reductase.

    PubMed

    Moir, Donald T

    2005-09-01

    The FabI-related enoyl-ACP reductase enzymes of bacteria meet many of the criteria for antibacterial targets. These enzymes are essential for the growth of several pathogenic species, have no significant mammalian homologs, catalyze a rate-limiting step in a vital macromolecular biosynthetic pathway, and are already the targets of antibacterials used in the clinic (isoniazid) and in consumer products (triclosan). The suitability of FabI as an antibiotic target is diminished somewhat by the discovery that many pathogens carry an alternate unrelated enoyl-ACP reductase (FabK) or both reductases. However, a key human pathogen, Staphylococcus aureus and its increasingly common drug-resistant derivative MRSA are sensitive to FabI inhibitors. Screening for inhibitors of this target has resulted in the identification of five chemical classes of potent inhibitors. In addition, analogs of triclosan with increased potency and with pro-drug features have been engineered. At least one of these classes of inhibitors has been optimized and tested in animals for pharmacokinetic properties and efficacy. Further development of one or more of these classes and further screening are expected to generate new FabI inhibitors for application in the clinic against drug-resistant S. aureus. PMID:16181147

  9. Increased Reactive Oxygen Species Production During Reductive Stress: The Roles of Mitochondrial Glutathione and Thioredoxin Reductases

    PubMed Central

    Korge, Paavo; Calmettes, Guillaume; Weiss, James N.

    2015-01-01

    Both extremes of redox balance are known to cause cardiac injury, with mounting evidence revealing that the injury induced by both oxidative and reductive stress is oxidative in nature. During reductive stress, when electron acceptors are expected to be mostly reduced, some redox proteins can donate electrons to O2 instead, which increases reactive oxygen species (ROS) production. However, the high level of reducing equivalents also concomitantly enhances ROS scavenging systems involving redox couples such as NADPH/NADP+ and GSH/GSSG. Here our objective was to explore how reductive stress paradoxically increases net mitochondrial ROS production despite the concomitant enhancement of ROS scavenging systems. Using recombinant enzymes and isolated permeabilized cardiac mitochondria, we show that two normally antioxidant matrix NADPH reductases, glutathione reductase and thioredoxin reductase, generate H2O2 by leaking electrons from their reduced flavoprotein to O2 when electron flow is impaired by inhibitors or because of limited availability of their natural electron acceptors, GSSG and oxidized thioredoxin. The spillover of H2O2 under these conditions depends on H2O2 reduction by peroxiredoxin activity, which may regulate redox signaling in response to endogenous or exogenous factors. These findings may explain how ROS production during reductive stress overwhelms ROS scavenging capability, generating the net mitochondrial ROS spillover causing oxidative injury. These enzymes could potentially targeted to increase cancer cell death or modulate H2O2-induced redox signaling to protect the heart against ischemia/reperfusion damage. PMID:25701705

  10. Cellular localization of 5α-reductase in the rat cerebellum.

    PubMed

    Kiyokage, Emi; Toida, Kazunori; Suzuki-Yamamoto, Toshiko; Ishimura, Kazunori

    2014-09-01

    The enzyme 5α-reductase catalyzes the transformation of progesterone, testosterone, and deoxycorticosterone into 5α-reduced metabolites, which are recognized as neurosteroids in the brain with variable potential neuroactivity. Two isoforms of 5α-reductase were identified in rodents, and, of these, 5α-reductase type 1 (5α-R1) is abundantly expressed in the brain. To understand the multiple influences of neurosteroids in the central nervous system, we need to know their region-specific synthesis. The present study reports the detailed localization of 5α-R1 in the adult rat cerebellum. The occurrence of 5α-R1 was detected by reverse transcription-polymerase chain reaction. The enzyme activity was also detected by thin layer chromatography. Immunocytochemistry showed 5α-R1 immunoreactive cells in all cerebellar layers. Multiple immunolabeling revealed that 5α-R1 was mainly localized in glia, such as astrocytes and oligodendrocytes. The most intense immunoreactivity for 5α-R1 was found in Bergmann glia, and the processes of these glia were associated with dendrites of both Purkinje cells and interneurons in the molecular layer. The 5α-R1 in the cerebellum was expressed consistently throughout different ages and sexes, in both gonadectomized and hypophysectomized rats. Thus, 5α-R1 may contribute to the formation and maintenance of the cerebellar neurons through 5α-reduced metabolites, which are synthesized through a complex interaction between neurons and glia. PMID:24810015

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

    PubMed

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

    2015-11-01

    This study aimed to define progesterone 5β-reductases (P5βR, EC 1.3.99.6, enone 1,4-reductases) as function-associated molecular markers at the plant family level. Therefore cDNAs were isolated from 25 Brassicaceae species, including two species, Erysimum crepidifolium and Draba aizoides, known to produce cardiac glycosides. The sequences were used in a molecular phylogeny study. The cladogram created is congruent to the existing molecular analyses. Recombinant His-tagged forms of the P5βR cDNAs from Aethionema grandiflorum, Draba aizoides, Nasturtium officinale, Raphanus sativus and Sisymbrium officinale were expressed in E. coli. Enone 1,4-reductase activity was demonstrated in vitro using progesterone and 2-cyclohexen-1-one as substrates. Evidence is provided that functional P5βRs are ubiquitous in the Brassicaceae. The recombinant P5βR enzymes showed different substrate preferences towards progesterone and 2-cyclohexen-1-one. Sequence comparison of the catalytic pocket of the P5βR enzymes and homology modelling using Digitalis lanata P5βR (PDB ID: 2V6G) as template highlighted the importance of the hydrophobicity of the binding pocket for substrate discrimination. It is concluded that P5βR genes or P5βR proteins can be used as valuable function-associated molecular markers to infer taxonomic relationship and evolutionary diversification from a metabolic/catalytic perspective. PMID:26108256

  12. Major peptides from amaranth (Amaranthus cruentus) protein inhibit HMG-CoA reductase activity.

    PubMed

    Soares, Rosana Aparecida Manólio; Mendonça, Simone; de Castro, Luíla Ívini Andrade; Menezes, Amanda Caroline Cardoso Corrêa Carlos; Arêas, José Alfredo Gomes

    2015-01-01

    The objective of this study was to identify the major peptides generated by the in vitro hydrolysis of Amaranthus cruentus protein and to verify the effect of these peptides on the activity of 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG-CoA reductase), a key enzyme in cholesterol biosynthesis. A protein isolate was prepared, and an enzymatic hydrolysis that simulated the in vivo digestion of the protein was performed. After hydrolysis, the peptide mixture was filtered through a 3 kDa membrane. The peptide profile of this mixture was determined by reversed phase high performance chromatography (RP-HPLC), and the peptide identification was performed by LC-ESI MS/MS. Three major peptides under 3 kDa were detected, corresponding to more than 90% of the peptides of similar size produced by enzymatic hydrolysis. The sequences identified were GGV, IVG or LVG and VGVI or VGVL. These peptides had not yet been described for amaranth protein nor are they present in known sequences of amaranth grain protein, except LVG, which can be found in amaranth α‑amylase. Their ability to inhibit the activity of HMG-CoA reductase was determined, and we found that the sequences GGV, IVG, and VGVL, significantly inhibited this enzyme, suggesting a possible hypocholesterolemic effect. PMID:25690031

  13. 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. PMID:23266822

  14. 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. PMID:27117030

  15. 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

  16. 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). PMID:23131110

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

    PubMed

    Vázquez-Torres, Andrés; Bäumler, Andreas J

    2016-02-01

    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 c oxidases 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

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

    SciTech Connect

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

    1984-03-01

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

  19. Discovery of epoxyqueuosine (oQ) reductase reveals parallels between halorespiration and tRNA modification

    PubMed Central

    Miles, Zachary D.; McCarty, Reid M.; Molnar, Gabriella; Bandarian, Vahe

    2011-01-01

    Transfer RNA is one of the most richly modified biological molecules. Biosynthetic pathways that introduce these modifications are underexplored, largely because their absence does not lead to obvious phenotypes under normal growth conditions. Queuosine (Q) is a hypermodified base found in the wobble positions of tRNA Asp, Asn, His, and Tyr from bacteria to mankind. Using liquid chromatography MS methods, we have screened 1,755 single gene knockouts of Escherichia coli and have identified the key final step in the biosynthesis of Q. The protein is homologous to B12-dependent iron-sulfur proteins involved in halorespiration. The recombinant Bacillus subtilis epoxyqueuosine (oQ) reductase catalyzes the conversion of oQ to Q in a synthetic substrate, as well as undermodified RNA isolated from an oQ reductase knockout strain. The activity requires inclusion of a reductant and a redox mediator. Finally, exogenously supplied cobalamin stimulates the activity. This work provides the framework for studies of the biosynthesis of other modified RNA components, where lack of accessible phenotype or obvious gene clustering has impeded discovery. Moreover, discovery of the elusive oQ reductase protein completes the biosynthetic pathway of Q. PMID:21502530

  20. Human leukemia and normal leukocytes contain a species of immunoreactive but nonfunctional dihydrofolate reductase

    SciTech Connect

    Rothenbery, S.P.; Iqbal, M.P.

    1982-01-01

    A quantitative radioimmunoassay has been developed for human dihydrofolate reductase (tetrahydrofolate dehydrogenase; 5,6,7,8-tetrahdrofolate:NADP/sup +/ oxidoreductase, EC 1.5.1.3) by using antiserum raised in rabbits against the active enzyme purified from calf liver. An immunoreactive protein could be identified in the cytoplasm of chronic myelogenous leukemia cells, which contained no functional dihydrofolate reductase activity. Its concentration was stoichiometric to the volume of cytoplasm assayed and paralleled the standard curve obtained with purified enzyme, indicating that this protein in the human cells is antigenically similar to the homologous antigen. The concentration of this immunoreactive protein in the cytoplasm of human leukemia and normal leukocytes in all instances greatly exceeded the concentration of functional dihydrofolate reductase, which was measured by the binding of (/sup 3/H)methotrexate. This nonfunctional immunoreactive protein in the cytoplasm and cytosol from two different samples of chronic myelogenous leukemia cells analyzed by gel filtration had an apparent molecular weight of 41,000, which is twice the molecular weight of the functional enzyme.