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Sample records for aldose reductase akr1b1

  1. 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

  2. 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

  3. Aldose reductases influence prostaglandin F2α levels and adipocyte differentiation in male mouse and human species.

    PubMed

    Pastel, Emilie; Pointud, Jean-Christophe; Loubeau, Gaëlle; Dani, Christian; Slim, Karem; Martin, Gwenaëlle; Volat, Fanny; Sahut-Barnola, Isabelle; Val, Pierre; Martinez, Antoine; Lefrançois-Martinez, Anne-Marie

    2015-05-01

    Aldose reductases (AKR1B) are widely expressed oxidoreductases whose physiological function remains elusive. Some isoforms are genuine prostaglandin F2α (PGF2α) synthases, suggesting they might influence adipose homeostasis because PGF2α inhibits adipogenesis. This was shown by Akr1b7 gene ablation in the mouse, which resulted in increased adiposity related to a lower PGF2α content in fat. Yet humans have no ortholog gene for Akr1b7, so the role of aldose reductases in human adipose homeostasis remains to be explored. We analyzed expression of genes encoding human and mouse aldose reductase isoforms in adipose tissues and differentiating adipocytes to assess conserved mechanisms regulating PGF2α synthesis and adipogenesis. The Akr1b3 gene encoded the most abundant isoform in mouse adipose tissue, whereas Akr1b7 encoded the only isoform enriched in the stromal vascular fraction. Most mouse aldose reductase gene expression peaked in early adipogenesis of 3T3-L1 cells and diminished with differentiation. In contrast with its mouse ortholog Akr1b3, AKR1B1 expression increased throughout differentiation of human multipotent adipose-derived stem cells, paralleling PGF2α release, whereas PGF2α receptor (FP) levels collapsed in early differentiation. Pharmacological inhibition of aldose reductase using Statil altered PGF2α production and enhanced human multipotent adipose-derived stem adipocyte differentiation. As expected, the adipogenic effects of Statil were counteracted by an FP agonist (cloprostenol). Thus, in both species aldose reductase-dependent PGF2α production could be important in early differentiation to restrict adipogenesis. PGF2α antiadipogenic signaling could then be toned down through the FP receptor or aldose reductases down-regulation in human and mouse cells, respectively. Our data suggest that aldose reductase inhibitors could have obesogenic potential.

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

    PubMed

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

    2015-06-01

    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

  5. 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

  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. 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

  8. 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

  9. 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

  10. Aldose Reductase, Oxidative Stress, and Diabetic Mellitus

    PubMed Central

    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

  11. 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

  12. 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

  13. 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.

  14. 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.

  15. 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

  16. 20(S)-Ginsenoside Rh2 as aldose reductase inhibitor from Panax ginseng.

    PubMed

    Fatmawati, Sri; Ersam, Taslim; Yu, Hongshan; Zhang, Chunzhi; Jin, Fengxie; Shimizu, Kuniyoshi

    2014-09-15

    The root of Panax ginseng C. A. Meyer (Araliaceae) is a well-known herbal medicine in East Asia. The major bioactive metabolites in this root are commonly identified as ginsenosides. A series of ginsenosides were determined for in vitro human recombinant aldose reductase. This Letter aims to clarify the structural requirement for aldose reductase inhibition. We discovered that only ginsenoside 20(S)-Rh2 showed potent against aldose reductase, with an IC50 of 147.3 μM. These results implied that the stereochemistry of the hydroxyl group at C-20 may play an important role in aldose reductase inhibition. An understanding of these requirements is considered necessary in order to develop a new type of aldose reductase inhibitor. Furthermore, P. ginseng might be an important herbal medicine in preventing diabetic complications.

  17. Aldose reductase catalysis and crystallography. Insights from recent advances in enzyme structure and function.

    PubMed

    Petrash, J M; Tarle, I; Wilson, D K; Quiocho, F A

    1994-08-01

    Enhanced metabolism of glucose via the polyol pathway may play an important role in the pathogenesis of diabetic retinopathy, neuropathy, and nephropathy. Aldose reductase catalyzes the NADPH-dependent conversion of glucose to sorbitol, the first step in the polyol pathway. Interruption of the polyol pathway by inhibition of aldose reductase holds considerable promise as a therapeutic measure to prevent or delay the onset and severity of these late complications of diabetes. Dramatic advances in our understanding of the molecular biology, enzymology, and three-dimensional structure of aldose reductase have occurred in recent years, providing new and challenging insights into the enzyme's catalytic mechanism. Recent developments in structure determination of aldose reductase and the implications for evaluation and development of aldose reductase inhibitors are summarized. PMID:8039602

  18. X-ray structure of the V301L aldo-keto reductase 1B10 complexed with NADP(+) and the potent aldose reductase inhibitor fidarestat: implications for inhibitor binding and selectivity.

    PubMed

    Ruiz, Francesc Xavier; Cousido-Siah, Alexandra; Mitschler, André; Farrés, Jaume; Parés, Xavier; Podjarny, Alberto

    2013-02-25

    Only one crystal structure is currently available for tumor marker AKR1B10, complexed with NADP(+) and tolrestat, which is an aldose reductase inhibitor (ARI) of the carboxylic acid type. Here, the X-ray structure of the complex of the V301L substituted AKR1B10 holoenzyme with fidarestat, an ARI of the cyclic imide type, was obtained at 1.60Å resolution by replacement soaking of crystals containing tolrestat. Previously, fidarestat was found to be safe in phase III trials for diabetic neuropathy and, consistent with its low in vivo side effects, was highly selective for aldose reductase (AR or AKR1B1) versus aldehyde reductase (AKR1A1). Now, inhibition studies showed that fidarestat was indeed 1300-fold more selective for AR as compared to AKR1B10, while the change of Val to Leu (found in AR) caused a 20-fold decrease in the IC50 value with fidarestat. Structural analysis of the V301L AKR1B10-fidarestat complex displayed enzyme-inhibitor interactions similar to those of the AR-fidarestat complex. However, a close inspection of both the new crystal structure and a computer model of the wild-type AKR1B10 complex with fidarestat revealed subtle changes that could affect fidarestat binding. In the crystal structure, a significant motion of loop A was observed between AR and V301L AKR1B10, linked to a Phe-122/Phe-123 side chain displacement. This was due to the presence of the more voluminous Gln-303 side chain (Ser-302 in AR) and of a water molecule buried in a subpocket located at the base of flexible loop A. In the wild-type AKR1B10 model, a short contact was predicted between the Val-301 side chain and fidarestat, but would not be present in AR or in V301L AKR1B10. Overall, these changes could contribute to the difference in inhibitory potency of fidarestat between AR and AKR1B10.

  19. 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.

  20. Metabolism of the aldose reductase inhibitor ALO1567 in man.

    PubMed Central

    Park, Y H; Hudson, J E; Barker, R C; York, B M; Brazzell, R K

    1991-01-01

    1. The metabolism of the aldose reductase inhibitor, ALO1567, was studied in man. The major biotransformation pathway was aromatic hydroxylation followed by glucuronide conjugation. 2. Hydroxylation occurred at several positions on the fluorene ring. The major metabolite was identified as the 7-hydroxy analogue of ALO1567 and three minor metabolites were characterized as positional isomers of the 7-hydroxy metabolite. 3. Oxidative defluorination and metabolism on the hydantoin ring were also indicated as minor pathways. 4. The capacity of normal subjects to oxidize ALO1567 was indicated by the urinary ratio of the parent drug to the 7-hydroxy metabolite after daily oral administration of 100 mg and 200 mg of ALO1567. Most subjects having higher ALO1567 plasma concentrations showed higher ratios. PMID:1931471

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

    PubMed

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

    2014-01-01

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

  2. Diuretic and natriuretic effects of sorbinil, an aldose reductase inhibitor.

    PubMed

    Springate, J E; Feld, L G; Van Liew, J B; Fildes, R D; Acara, M A

    1991-04-01

    The renal effects of sorbinil, an aldose reductase inhibitor that interferes with the conversion of glucose to sorbitol, were studied in rats and rabbits before and after fluid deprivation. The intracellular osmolar solute, sorbitol, is found in increasing concentrations from cortex to medulla in the kidney and may be involved in the urinary concentrating mechanism. Oral administration of sorbinil in the rabbit resulted in significant increases in urine flow rate and sodium excretion with a tendency toward decreased urine osmolality and increased potassium excretion both before and after water deprivation. When fluid intake was controlled in the rat study, significant increases in urine flow rate and sodium and potassium excretion and a significant decrease in urine osmolality occurred only in response to fluid deprivation. Thus, sorbinil has diuretic and natriuretic properties and may prevent the normal concentration of urine in the antidiuretic animal.

  3. Effect of Polygonum hydropiper sulfated flavonoids on lens aldose reductase and related enzymes.

    PubMed

    Haraguchi, H; Ohmi, I; Sakai, S; Fukuda, A; Toihara, Y; Fujimoto, T; Okamura, N; Yagi, A

    1996-04-01

    The sulfated flavonoids in Polygonum hydropiper showed potent inhibiton against lens aldose reductase. Among these flavonoids isorhamnetin 3,7-disulfate (5) was most potent. Kinetic analysis showed that 5 exhibited noncompetitive inhibition against both dl-glyceraldehyde and NADPH.

  4. 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

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

    PubMed

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

    1991-12-01

    The hot water extracts of Chrysanthemum morifolium, Bixa orellana and Ipomoea batatas, were found to have potent inhibitory activity towards lens aldose reductase (AR). Ellagic acid (4) was isolated from C. morifolium and I. batatas, isoscutellarein (7) from B. orellana and 3,5-dicaffeoylquinic acid (10) from I. batatas, respectively, as potent inhibitors. PMID:1814628

  6. 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

  7. Inhibition of Aldose Reductase by Gentiana lutea Extracts

    PubMed Central

    Akileshwari, Chandrasekhar; Muthenna, Puppala; Nastasijević, Branislav; Joksić, Gordana; Petrash, J. Mark; Reddy, Geereddy Bhanuprakash

    2012-01-01

    Accumulation of intracellular sorbitol due to increased aldose reductase (ALR2) activity has been implicated in the development of various secondary complications of diabetes. Thus, ALR2 inhibition could be an effective strategy in the prevention or delay of certain diabetic complications. Gentiana lutea grows naturally in the central and southern areas of Europe. Its roots are commonly consumed as a beverage in some European countries and are also known to have medicinal properties. The water, ethanol, methanol, and ether extracts of the roots of G. lutea were subjected to in vitro bioassay to evaluate their inhibitory activity on the ALR2. While the ether and methanol extracts showed greater inhibitory activities against both rat lens and human ALR2, the water and ethanol extracts showed moderate inhibitory activities. Moreover, the ether and methanol extracts of G. lutea roots significantly and dose-dependently inhibited sorbitol accumulation in human erythrocytes under high glucose conditions. Molecular docking studies with the constituents commonly present in the roots of G. lutea indicate that a secoiridoid glycoside, amarogentin, may be a potential inhibitor of ALR2. This is the first paper that shows G. lutea extracts exhibit inhibitory activity towards ALR2 and these results suggest that Gentiana or its constituents might be useful to prevent or treat diabetic complications. PMID:22844269

  8. Inhibition of aldose reductase by Gentiana lutea extracts.

    PubMed

    Akileshwari, Chandrasekhar; Muthenna, Puppala; Nastasijević, Branislav; Joksić, Gordana; Petrash, J Mark; Reddy, Geereddy Bhanuprakash

    2012-01-01

    Accumulation of intracellular sorbitol due to increased aldose reductase (ALR2) activity has been implicated in the development of various secondary complications of diabetes. Thus, ALR2 inhibition could be an effective strategy in the prevention or delay of certain diabetic complications. Gentiana lutea grows naturally in the central and southern areas of Europe. Its roots are commonly consumed as a beverage in some European countries and are also known to have medicinal properties. The water, ethanol, methanol, and ether extracts of the roots of G. lutea were subjected to in vitro bioassay to evaluate their inhibitory activity on the ALR2. While the ether and methanol extracts showed greater inhibitory activities against both rat lens and human ALR2, the water and ethanol extracts showed moderate inhibitory activities. Moreover, the ether and methanol extracts of G. lutea roots significantly and dose-dependently inhibited sorbitol accumulation in human erythrocytes under high glucose conditions. Molecular docking studies with the constituents commonly present in the roots of G. lutea indicate that a secoiridoid glycoside, amarogentin, may be a potential inhibitor of ALR2. This is the first paper that shows G. lutea extracts exhibit inhibitory activity towards ALR2 and these results suggest that Gentiana or its constituents might be useful to prevent or treat diabetic complications.

  9. 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

  10. 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

  11. Comprehensive analysis of prostaglandin metabolic enzyme expression during pregnancy and the characterization of AKR1B1 as a prostaglandin F synthase at the maternal-conceptus interface in pigs.

    PubMed

    Seo, Heewon; Choi, Yohan; Shim, Jangsoo; Yoo, Inkyu; Ka, Hakhyun

    2014-05-01

    Prostaglandins (PGs) are important lipid mediators regulating various reproductive processes in many species. In pigs, the expression pattern of PGE2 and PGF2α metabolic enzymes and the regulatory mechanism controlling PGE2 and PGF2α levels in the uterus during pregnancy are not completely understood. This study determined endometrial expression of the genes (PLA2G4A, PTGS1, PTGS2, PTGES, PTGES2, PTGES3, AKR1B1, CBR1, and HPGD) involved in PGE2 and PGF2α metabolism during the estrous cycle and pregnancy and measured levels of PGE2 and PGF2α in uterine endometrial tissues and uterine flushings at the time of conceptus implantation in pigs. Except PTGES3, expression of the genes studied changed in a pregnancy-stage-specific manner, and localization of PTGES, AKR1B1, CBR1, and HPGD mRNAs were cell-type specific in the uterine endometrium. Levels of both PGE2 and PGF2α in uterine endometrial tissues and uterine lumen were higher on Day 12 of pregnancy than those of the estrous cycle and affected by different morphology of spherical and filamentous conceptuses. Furthermore, we determined that endometrial expression of AKR1B1, known to encode a PGF2α synthase in other species, was increased by estrogen and interleukin-1beta and that AKR1B1 exhibited PGF2α synthase activity in the porcine uterine endometrium. These results in pigs indicate that the PGE2 and PGF2α metabolic enzymes are expressed stage specifically in the endometrium during pregnancy and regulate the abundance of PGE2 and PGF2α in the uterus at the time of implantation and that AKR1B1 may act as a major PGF synthase in the endometrium during early pregnancy.

  12. Comprehensive analysis of prostaglandin metabolic enzyme expression during pregnancy and the characterization of AKR1B1 as a prostaglandin F synthase at the maternal-conceptus interface in pigs.

    PubMed

    Seo, Heewon; Choi, Yohan; Shim, Jangsoo; Yoo, Inkyu; Ka, Hakhyun

    2014-05-01

    Prostaglandins (PGs) are important lipid mediators regulating various reproductive processes in many species. In pigs, the expression pattern of PGE2 and PGF2α metabolic enzymes and the regulatory mechanism controlling PGE2 and PGF2α levels in the uterus during pregnancy are not completely understood. This study determined endometrial expression of the genes (PLA2G4A, PTGS1, PTGS2, PTGES, PTGES2, PTGES3, AKR1B1, CBR1, and HPGD) involved in PGE2 and PGF2α metabolism during the estrous cycle and pregnancy and measured levels of PGE2 and PGF2α in uterine endometrial tissues and uterine flushings at the time of conceptus implantation in pigs. Except PTGES3, expression of the genes studied changed in a pregnancy-stage-specific manner, and localization of PTGES, AKR1B1, CBR1, and HPGD mRNAs were cell-type specific in the uterine endometrium. Levels of both PGE2 and PGF2α in uterine endometrial tissues and uterine lumen were higher on Day 12 of pregnancy than those of the estrous cycle and affected by different morphology of spherical and filamentous conceptuses. Furthermore, we determined that endometrial expression of AKR1B1, known to encode a PGF2α synthase in other species, was increased by estrogen and interleukin-1beta and that AKR1B1 exhibited PGF2α synthase activity in the porcine uterine endometrium. These results in pigs indicate that the PGE2 and PGF2α metabolic enzymes are expressed stage specifically in the endometrium during pregnancy and regulate the abundance of PGE2 and PGF2α in the uterus at the time of implantation and that AKR1B1 may act as a major PGF synthase in the endometrium during early pregnancy. PMID:24695626

  13. Cuminaldehyde: Aldose Reductase and alpha-Glucosidase Inhibitor Derived from Cuminum cyminum L. Seeds.

    PubMed

    Lee, Hoi-Seon

    2005-04-01

    The inhibitory activity of Cuminum cyminum seed-isolated component was evaluated against lens aldose reductase and alpha-glucosidase isolated from Sprague-Dawley male rats and compared to that of 11 commercially available components derived from C. cyminum seed oil, as well as quercitrin as an aldose reductase inhibitor and acarbose as an alpha-glucosidase inhibitor. The biologically active constituent of C. cyminum seed oil was characterized as cuminaldehyde by various spectral analyses. The IC(50) value of cuminaldehyde is 0.00085 mg/mL against aldose reductase and 0.5 mg/mL against alpha-glucosidase, respectively. Cuminaldehyde was about 1.8 and 1.6 times less in inhibitory activity than acarbose and quercitin, respectively. Nonetheless, cuminaldehyde may be useful as a lead compound and a new agent for antidiabetic therapeutics.

  14. [Progress in research of aldose reductase inhibitors in traditional medicinal herbs].

    PubMed

    Feng, Chang-Gen; Zhang, Lin-Xia; Liu, Xia

    2005-10-01

    The traditional medicinal herbs are natural product, and have no obviously toxic action and side effect, and their resources are extensive. The adverse effects produced by aldose reductase inhibitors in traditional medicinal herbs are less than those from chemical synthesis and micro-organism, they can effectively prevent and delay diabetic complication, such as diabetic nephropathy, vasculopathy, retinopathy, peripheral neuropathy, and so on. They will have a wonderful respect. Flavonoid compounds and their derivates from traditional medicinal herbs are active inhibitors to aldose reductase, such as quercetin, silymarin, puerarin, baicalim, berberine and so on. In addition, some compound preparations show more strongly activity in inhibiting aldose reductase and degrading sorbitol contents, such as Shendan in traditional medicinal herbs being active inhibitors and Jianyi capsule, Jinmaitong composita, Liuwei Di-huang pill, et al. The progresses definite functions of treating diabetes complications have been reviewed.

  15. 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

  16. Rapid Identification of Aldose Reductase Inhibitory Compounds from Perilla frutescens

    PubMed Central

    Paek, Ji Hun; Shin, Kuk Hyun; Kang, Young-Hee; Lee, Jae-Yong; Lim, Soon Sung

    2013-01-01

    The ethyl acetate (EtOAc) soluble fraction of methanol extracts of Perilla frutescens (P. frutescens) inhibits aldose reductase (AR), the key enzyme in the polyol pathway. Our investigation of inhibitory compounds from the EtOAc soluble fraction of P. frutescens was followed by identification of the inhibitory compounds by a combination of HPLC microfractionation and a 96-well enzyme assay. This allowed the biological activities to be efficiently matched with selected HPLC peaks. Structural analyses of the active compounds were performed by LC-MSn. The main AR inhibiting compounds were tentatively identified as chlorogenic acid and rosmarinic acid by LC-MSn. A two-step high speed counter current chromatography (HSCCC) isolation method was developed with a solvent system of n-hexane-ethyl acetate-methanol-water at 1.5 : 5 : 1 : 5, v/v and 3 : 7 : 5 : 5, v/v. The chemical structures of the isolated compounds were determined by 1H- and 13C-nuclear magnetic resonance spectrometry (NMR). The main compounds inhibiting AR in the EtOAc fraction of methanol extracts of P. frutescens were identified as chlorogenic acid (2) (IC50 = 3.16 μM), rosmarinic acid (4) (IC50 = 2.77 μM), luteolin (5) (IC50 = 6.34 μM), and methyl rosmarinic acid (6) (IC50 = 4.03 μM). PMID:24308003

  17. 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.

  18. Expression of AKR1B1, AKR1C3 and other genes of prostaglandin F2α biosynthesis and action in ovarian endometriosis tissue and in model cell lines.

    PubMed

    Sinreih, Maša; Anko, Maja; Kene, Neli Hevir; Kocbek, Vida; Rižner, Tea Lanišnik

    2015-06-01

    Endometriosis is a frequent benign gynecological disease characterized by endometrial tissue outside the uterine cavity. The estimated prevalence in the general population is 6-10%, but this reaches 30-50% in women with infertility and/or pain. As ectopic tissue within the pelvic cavity provokes inflammation, endometriosis is also considered a chronic inflammatory disease, and is characterized by increased peritoneal fluid levels of prostaglandin (PG)E2 and PGF2α. The AKR1B1 and AKR1C3 enzymes act as PG synthases and catalyze reduction of PGH2 to PGF2α, and PGD2 to 9α,11β-PGF2α, respectively. AKR1B1 and AKR1C3 may thus be associated with increased PGF2α production in endometriosis patients, as supported by our previous report of increased AKR1C1-AKR1C3 mRNA levels in endometriotic tissue, compared to control endometrium. Here, we initially evaluated PGF2α concentrations in peritoneal fluid from endometriosis patients and healthy women. We also examined expression of AKR1B1, AKR1C3 and other genes involved in PGF2α biosynthesis, metabolism, and action in ovarian endometriosis tissue versus healthy endometrium, and in peritoneal endometriosis and control endometrium model cell lines. Compared to controls, increased PGF2α concentrations in peritoneal fluid of patients were supported by endometriotic tissue showing increased AKR1B1 mRNA and protein levels, but unchanged AKR1C3 protein levels. Among genes involved in PGF2α biosynthesis, metabolism and action PLA2G2A, PTGS2/COX-2, ABCC4 and PTGFR were up-regulated, mRNA levels of SLCO2A, PTGDS and HPGDS were unchanged, and genes PLA2G4A and HPGD were down-regulated in diseased tissue. All of these PGF2α-associated genes were also expressed in control endometrial HIEEC epithelial and HIESC stromal cell lines, and in peritoneal endometriosis 12-Z epithelial and 22-B stromal cell lines. Higher expression of PLA2G2A, PTGS2, AKR1B1, AKR1C3 and ABCC4 was seen in 22-B endometriosis cells compared to HIESC control

  19. 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

  20. 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.

  1. Structure of constituents isolated from the flower buds of Cananga odorata and their inhibitory effects on aldose reductase.

    PubMed

    Matsumoto, Takahiro; Nakamura, Seikou; Fujimoto, Katsuyoshi; Ohta, Tomoe; Ogawa, Keiko; Yoshikawa, Masayuki; Matsuda, Hisashi

    2014-10-01

    Three new terpenoid derivatives, canangaterpenes IV-VI, were isolated from the flower buds of Cananga odorata, cultivated in Thailand, together with eight known flavonoids. The chemical structures of the new compounds were elucidated on the basis of chemical and physicochemical evidence. The inhibitory effects of the isolated compounds on aldose reductase were also investigated. Several terpenoid derivatives and flavonoids were shown to inhibit aldose reductase. PMID:24816646

  2. 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

  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. 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.

  5. 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

  6. Human and rodent aldo-keto reductases from the AKR1B subfamily and their specificity with retinaldehyde

    PubMed Central

    Ruiz, F. Xavier; Moro, Armando; Gallego, Oriol; Ardèvol, Albert; Rovira, Carme; Petrash, J. Mark; Parés, Xavier; Farrés, Jaume

    2011-01-01

    NADP(H)-dependent cytosolic aldo-keto reductases (AKR) are mostly monomeric enzymes which fold into a typical (α/β)8-barrel structure. Substrate specificity and inhibitor selectivity are determined by interaction with residues located in three highly variable loops (A, B, and C). Based on sequence identity, AKR have been grouped into families, namely AKR1–AKR15, containing multiple subfamilies. Two human enzymes from the AKR1B subfamily (AKR1B1 and AKR1B10) are of special interest. AKR1B1 (aldose reductase) is related to secondary diabetic complications, while AKR1B10 is induced in cancer cells and is highly active with all-trans-retinaldehyde. Residues interacting with all-trans-retinaldehyde and differing between AKR1B1 and AKR1B10 are Leu125Lys and Val131Ala (loop A), Leu301Val, Ser303Gln, and Cys304Ser (loop C). Recently, we demonstrated the importance of Lys125 as a determinant of AKR1B10 specificity for retinoids. Residues 301 and 304 are also involved in interactions with substrates or inhibitors, and thus we checked their contribution to retinoid specificity. We also extended our study with retinoids to rodent members of the AKR1B subfamily: AKR1B3 (aldose reductase), AKR1B7 (mouse vas deferens protein), AKR1B8 (fibroblast-growth factor 1-regulated protein), and AKR1B9 (Chinese hamster ovary reductase), which were tested against all-trans isomers of retinaldehyde and retinol. All enzymes were active with retinaldehyde, but with kcat values (0.02–0.52 min−1) much lower than that of AKR1B10 (27 min−1). None of the enzymes showed oxidizing activity with retinol. Since these enzymes (except AKR1B3) have Lys125, other residues should account for retinaldehyde specificity. Here, by using site-directed mutagenesis and molecular modeling, we further delineate the contribution of residues 301 and 304. We demonstrate that besides Lys125, Ser304 is a major structural determinant for all-trans-retinaldehyde specificity of AKR1B10. PMID:21329680

  7. 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

  8. 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

  9. Inhibition of aldose reductase and anti-cataract action of trans-anethole isolated from Foeniculum vulgare Mill. fruits.

    PubMed

    Dongare, Vandana; Kulkarni, Chaitanya; Kondawar, Manish; Magdum, Chandrakant; Haldavnekar, Vivek; Arvindekar, Akalpita

    2012-05-01

    Foeniculum vulgare fruits are routinely consumed for their carminative and mouth freshening effect. The plant was evaluated for aldose reductase inhibition and anti-diabetic action. Bioguided fractionation using silica gel column chromatography, HPLC, and GC-MS analysis revealed trans-anethole as the bioactive constituent possessing potent aldose reductase inhibitory action, with an IC50 value of 3.8μg/ml. Prolonged treatment with the pet ether fraction of the F. vulgare distillate demonstrated improvement in blood glucose, lipid profile, glycated haemoglobin and other parameters in streptozotocin-induced diabetic rats. Trans-anethole could effectively show anti-cataract activity through the increase in soluble lens protein, reduced glutathione, catalase and SOD activity on in vitro incubation of the eye lens with 55mM glucose. Trans-anethole demonstrated noncompetitive to mixed type of inhibition of lens aldose reductase using Lineweaver Burk plot.

  10. Aldose reductase inhibitors from the leaves of Myrciaria dubia (H. B. & K.) McVaugh.

    PubMed

    Ueda, H; Kuroiwa, E; Tachibana, Y; Kawanishi, K; Ayala, F; Moriyasu, M

    2004-11-01

    Ellagic acid (1) and its two derivatives, 4-O-methylellagic acid (2) and 4-(alpha-rhamnopyranosyl)ellagic acid (3) were isolated as inhibitors of aldose reductase (AR) from Myrciaria dubia (H. B. & K.) McVaugh. Compound 2 was the first isolated from the nature. Compound 3 showed the strongest inhibition against human recombinant AR (HRAR) and rat lens AR (RLAR). Inhibitory activity of compound 3 against HRAR (IC50 value = 4.1 x 10(-8) M) was 60 times more than that of quercetin (2.5 x 10(-6) M). The type of inhibition against HRAR was uncompetitive. PMID:15636180

  11. Substrate specificity and catalytic efficiency of aldo-keto reductases with phospholipid aldehydes.

    PubMed

    Spite, Matthew; Baba, Shahid P; Ahmed, Yonis; Barski, Oleg A; Nijhawan, Kanchan; Petrash, J Mark; Bhatnagar, Aruni; Srivastava, Sanjay

    2007-07-01

    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 C(16:0-20:4) phosphatidylcholine with acyl, plasmenyl or alkyl linkage at the sn-1 position or C(16:0-20:4) 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

  12. Inhibition of aldose reductase by phenylethanoid glycoside isolated from the seeds of Paulownia coreana.

    PubMed

    Kim, Jin Kyu; Lee, Yeon Sil; Kim, Seon Ha; Bae, Young Soo; Lim, Soon Sung

    2011-01-01

    Aldose reductase (AR) inhibitors have considerable therapeutic potential against diabetic complications and do not increase the risk of hypoglycemia. Through bioassay-guided fractionation of the 70% acetone extract obtained from Paulownia coreana seeds, phenylpropanoid glycosides (compounds 1-4) and 5 phenolic compounds were isolated (compounds 5-9). Their structures were determined on the basis of spectroscopic analysis and comparison with reported data. All the isolates were subjected to in vitro bioassays to evaluate their inhibitory activities against recombinant human aldose reductase (rhAR) and sorbitol formation in human erythrocytes. Phenylethanoid glycosides showed more effective than the phenolic compounds in inhibiting rhAR. Among the compounds, isocampneoside II (3) was found to significantly inhibit rhAR with an IC(50) value of 9.72 µM. In kinetic analyses performed using Lineweaver-Burk plots of 1/velocity and 1/concentration of substrate, isocampneoside II (3) showed uncompetitive inhibition against rhAR. Furthermore, it inhibited sorbitol formation in a rat lens incubated with a high concentration of glucose; this finding indicated that isocampneoside II (3) may effectively prevent osmotic stress in hyperglycemia. Thus, the P. coreana-derived phenylethanoid glycoside isocampneoside II (3) may have a potential therapeutics against diabetic complications. PMID:21212537

  13. 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

  14. Synthesis of organic nitrates of luteolin as a novel class of potent aldose reductase inhibitors.

    PubMed

    Wang, Qi-Qin; Cheng, Ning; Zheng, Xiao-Wei; Peng, Sheng-Ming; Zou, Xiao-Qing

    2013-07-15

    Aldose reductase (AR) plays an important role in the design of drugs that prevent and treat diabetic complications. Aldose reductase inhibitors (ARIs) have received significant attentions as potent therapeutic drugs. Based on combination principles, three series of luteolin derivatives were synthesised and evaluated for their AR inhibitory activity and nitric oxide (NO)-releasing capacity in vitro. Eighteen compounds were found to be potent ARIs with IC50 values ranging from (0.099±0.008) μM to (2.833±0.102) μM. O(7)-Nitrooxyethyl-O(3'),O(4')-ethylidene luteolin (La1) showed the most potent AR inhibitory activity [IC50=(0.099±0.008) μM]. All organic nitrate derivatives released low concentrations of NO in the presence of l-cysteine. Structure-activity relationship studies suggested that introduction of an NO donor, protection of the catechol structure, and the ether chain of a 2-carbon spacer as a coupling chain on the luteolin scaffold all help increase the AR inhibitory activity of the resulting compound. This class of NO-donor luteolin derivatives as efficient ARIs offer a new concept for the development and design of new drug for preventive and therapeutic drugs for diabetic complications.

  15. 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).

  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. Characterization of WY 14,643 and its Complex with Aldose Reductase

    PubMed Central

    Sawaya, Michael R.; Verma, Malkhey; Balendiran, Vaishnavi; Rath, Nigam P.; Cascio, Duilio; Balendiran, Ganesaratnam K.

    2016-01-01

    The peroxisome proliferator, WY 14,643 exhibits a pure non-competitive inhibition pattern in the aldehyde reduction and in alcohol oxidation activities of human Aldose reductase (hAR). Fluorescence emission measurements of the equilibrium dissociation constants, Kd, of oxidized (hAR•NADP+) and reduced (hAR•NADPH) holoenzyme complexes display a 2-fold difference between them. Kd values for the dissociation of WY 14,643 from the oxidized (hAR•NADP+•WY 14,643) and reduced (hAR•NADPH•WY 14,643) ternary complexes are comparable to each other. The ternary complex structure of hAR•NADP+•WY 14,643 reveals the first structural evidence of a fibrate class drug binding to hAR. These observations demonstrate how fibrate molecules such as WY 14,643, besides being valued as agonists for PPAR, also inhibit hAR. PMID:27721416

  19. 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.

  20. Advance in dietary polyphenols as aldose reductases inhibitors: structure-activity relationship aspect.

    PubMed

    Xiao, Jianbo; Ni, Xiaoling; Kai, Guoyin; Chen, Xiaoqing

    2015-01-01

    The dietary polyphenols as aldose reductases inhibitors (ARIs) have attracted great interest among researchers. The aim of this review is to give an overview of the research reports on the structure-activity relationship of dietary polyphenols inhibiting aldose reductases (AR). The molecular structures influence the inhibition of the following: (1) The methylation and methoxylation of the hydroxyl group at C3, C3', and C4' of flavonoids decreased or little affected the inhibitory potency. However, the methylation and methoxylation of the hydroxyl group at C5, C6, and C8 significantly enhanced the inhibition. Moreover, the methylation and methoxylation of C7-OH influence the inhibitory activity depending on the substitutes on rings A and B of flavonoids. (2) The glycosylation on 3-OH of flavonoids significantly increased or little affected the inhibition. However, the glycosylation on 7-OH and 4'-OH of flavonoids significantly decreased the inhibition. (3) The hydroxylation on A-ring of flavones and isoflavones, especially at positions 5 and 7, significantly improved the inhibition and the hydroxylation on C3' and C4' of B-ring of flavonoids remarkably enhanced the inhibition; however, the hydroxylation on the ring C of flavones significantly weakened the inhibition. (4) The hydrogenation of the C2=C3 double bond of flavones reduced the inhibition. (5) The hydrogenation of α=β double bond of stilbenes hardly affected the inhibition and the hydroxylation on C3' of stilbenes decreased the inhibition. Moreover, the methylation of the hydroxyl group of stilbenes obviously reduced the activity. (6) The hydroxylation on C4 of chalcone significantly increased the inhibition and the methylation on C4 of chalcone remarkably weakened the inhibition.

  1. Identification of Novel Aldose Reductase Inhibitors from Spices: A Molecular Docking and Simulation Study.

    PubMed

    Antony, Priya; Vijayan, Ranjit

    2015-01-01

    Hyperglycemia in diabetic patients results in a diverse range of complications such as diabetic retinopathy, neuropathy, nephropathy and cardiovascular diseases. The role of aldose reductase (AR), the key enzyme in the polyol pathway, in these complications is well established. Due to notable side-effects of several drugs, phytochemicals as an alternative has gained considerable importance for the treatment of several ailments. In order to evaluate the inhibitory effects of dietary spices on AR, a collection of phytochemicals were identified from Zingiber officinale (ginger), Curcuma longa (turmeric) Allium sativum (garlic) and Trigonella foenum graecum (fenugreek). Molecular docking was performed for lead identification and molecular dynamics simulations were performed to study the dynamic behaviour of these protein-ligand interactions. Gingerenones A, B and C, lariciresinol, quercetin and calebin A from these spices exhibited high docking score, binding affinity and sustained protein-ligand interactions. Rescoring of protein ligand interactions at the end of MD simulations produced binding scores that were better than the initially docked conformations. Docking results, ligand interactions and ADMET properties of these molecules were significantly better than commercially available AR inhibitors like epalrestat, sorbinil and ranirestat. Thus, these natural molecules could be potent AR inhibitors. PMID:26384019

  2. Novel Role for Aldose Reductase in Mediating Acute Inflammatory Responses in the Lung1

    PubMed Central

    Ravindranath, Thyyar M.; Mong, Phyllus Y.; Ananthakrishnan, Radha; Li, Qing; Quadri, Nosirudeen; Schmidt, Ann Marie; Ramasamy, Ravichandran; Wang, Qin

    2011-01-01

    Exaggerated inflammatory responses and the resultant increases in alveolar-capillary permeability underlie the pathogenesis of acute lung injury during sepsis. This study examined the functions of aldose reductase (AR) in mediating acute lung inflammation. Transgenic mice expressing human AR (ARTg) were used to study the functions of AR since mice have low intrinsic AR activity. In a mild cecal ligation and puncture model, ARTg mice demonstrated an enhanced AR activity and a greater inflammatory response as evaluated by circulating cytokine levels, neutrophil accumulation in the lungs, and activation of Rho kinase in lung endothelial cells (ECs). Compared with WT lung cells, ARTg lung cells produced more IL-6 and showed augmented JNK activation in response to LPS stimulation ex vivo. In human neutrophils, AR activity was required for fMLP-included CD11b activation and up-regulation, respiratory burst, and shape changes. In human pulmonary microvascular ECs, AR activity was required for TNF-α-induced activation of the Rho kinase/MKK4/JNK pathway and IL-6 production, but not p38 activation or ICAM-1 expression. Importantly, AR activity in both human neutrophils and ECs was required for neutrophil adhesion to TNF-α-stimulated ECs. These data demonstrate a novel role for AR in regulating the signaling pathways leading to neutrophil-EC adhesion during acute lung inflammation. PMID:20007578

  3. Identification of Novel Aldose Reductase Inhibitors from Spices: A Molecular Docking and Simulation Study

    PubMed Central

    Antony, Priya; Vijayan, Ranjit

    2015-01-01

    Hyperglycemia in diabetic patients results in a diverse range of complications such as diabetic retinopathy, neuropathy, nephropathy and cardiovascular diseases. The role of aldose reductase (AR), the key enzyme in the polyol pathway, in these complications is well established. Due to notable side-effects of several drugs, phytochemicals as an alternative has gained considerable importance for the treatment of several ailments. In order to evaluate the inhibitory effects of dietary spices on AR, a collection of phytochemicals were identified from Zingiber officinale (ginger), Curcuma longa (turmeric) Allium sativum (garlic) and Trigonella foenum graecum (fenugreek). Molecular docking was performed for lead identification and molecular dynamics simulations were performed to study the dynamic behaviour of these protein-ligand interactions. Gingerenones A, B and C, lariciresinol, quercetin and calebin A from these spices exhibited high docking score, binding affinity and sustained protein-ligand interactions. Rescoring of protein ligand interactions at the end of MD simulations produced binding scores that were better than the initially docked conformations. Docking results, ligand interactions and ADMET properties of these molecules were significantly better than commercially available AR inhibitors like epalrestat, sorbinil and ranirestat. Thus, these natural molecules could be potent AR inhibitors. PMID:26384019

  4. 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

  5. Aldose Reductase Inhibition Alleviates Hyperglycemic Effects on Human Retinal Pigment Epithelial Cells

    PubMed Central

    Chang, Kun-Che; Snow, Anson; LaBarbera, Daniel V.; Petrash, J. Mark

    2014-01-01

    Chronic hyperglycemia is an important risk factor involved in the onset and progression of diabetic retinopathy (DR). Among other effectors, aldose reductase (AR) has been linked to the pathogenesis of this degenerative disease. The purpose of this study was to investigate whether the novel AR inhibitor, beta-glucogallin (BGG), can offer protection against various hyperglycemia-induced abnormalities in human adult retinal pigment epithelia (ARPE-19) cells. AR is an enzyme that contributes to cellular stress by production of reactive oxygen species (ROS) under high glucose conditions. A marked decrease in cell viability (from 100% to 78%) following long-term exposure (4 days) of RPE cells to high glucose (HG) was largely prevented by siRNA-mediated knockdown of AR gene expression (from 79% to 97%) or inhibition using sorbinil (from 66% to 86%). In HG, BGG decreased sorbitol accumulation (44%), ROS production (27%) as well as ER stress (22%). Additionally, we demonstrated that BGG prevented loss of mitochondrial membrane potential (MMP) under HG exposure. We also showed that AR inhibitor pretreatment reduced retinal microglia-induced apoptosis in APRE-19 cells. These results suggest that BGG may be useful as a therapeutic agent against retinal degeneration in the diabetic eye by preventing RPE cell death. PMID:25451566

  6. 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

  7. 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

  8. 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

  9. Identification of Novel Aldose Reductase Inhibitors from Spices: A Molecular Docking and Simulation Study.

    PubMed

    Antony, Priya; Vijayan, Ranjit

    2015-01-01

    Hyperglycemia in diabetic patients results in a diverse range of complications such as diabetic retinopathy, neuropathy, nephropathy and cardiovascular diseases. The role of aldose reductase (AR), the key enzyme in the polyol pathway, in these complications is well established. Due to notable side-effects of several drugs, phytochemicals as an alternative has gained considerable importance for the treatment of several ailments. In order to evaluate the inhibitory effects of dietary spices on AR, a collection of phytochemicals were identified from Zingiber officinale (ginger), Curcuma longa (turmeric) Allium sativum (garlic) and Trigonella foenum graecum (fenugreek). Molecular docking was performed for lead identification and molecular dynamics simulations were performed to study the dynamic behaviour of these protein-ligand interactions. Gingerenones A, B and C, lariciresinol, quercetin and calebin A from these spices exhibited high docking score, binding affinity and sustained protein-ligand interactions. Rescoring of protein ligand interactions at the end of MD simulations produced binding scores that were better than the initially docked conformations. Docking results, ligand interactions and ADMET properties of these molecules were significantly better than commercially available AR inhibitors like epalrestat, sorbinil and ranirestat. Thus, these natural molecules could be potent AR inhibitors.

  10. 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

  11. 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

  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. Nrf2 regulates curcumin-induced aldose reductase expression indirectly via nuclear factor-kappaB.

    PubMed

    Kang, Eun Sil; Kim, Gil Hyeong; Kim, Hyo Jung; Woo, Im Sun; Ham, Sun Ah; Jin, Hana; Kim, Min Young; Kim, Hye Jung; Lee, Jae Heun; Chang, Ki Churl; Seo, Han Geuk; Hwang, Jin-Yong

    2008-07-01

    The osmotic response element (ORE) differs from the nuclear factor-kappaB (NF-kappaB) binding sequence by a single base pair; therefore, we investigated the involvement of NF-kappaB in the induction of aldose reductase (AR) by curcumin. Curcumin, an herb-derived polyphenolic compound, elicited an increase in the expression and promoter activity of the AR gene in a nuclear factor-erythroid 2-related factor 2 (Nrf2)-dependent manner. Small interfering RNA (siRNA) against p65 or BAY11-7082, an inhibitor of NF-kappaB, significantly suppressed the curcumin and/or Nrf2-induced increase in expression levels and promoter activity of the AR gene. BAY11-7082 or siRNA against p65 also attenuated the curcumin-induced increase in the promoter activity of the wild type AR-ORE(wt) gene, but not that of the mutated AR-ORE(mt), indicating that the ORE is essential for the response to NF-kappaB. The expression of p65, the promoter activity and DNA binding activity of NF-kappaB were enhanced in the presence of curcumin in cells that were transfected with Nrf2 compared to those treated with curcumin alone. Cells that had been preincubated with curcumin demonstrated resistance to reactive oxygen species-induced cell damage through the suppressive effects in the generation of reactive aldehydes. These effects were significantly attenuated in the presence of BAY11-7082, indicating the involvement of NF-kappaB in the cellular response of AR to oxidative stress and toxic aldehydes.

  15. Role of Aldose Reductase in the Metabolism and Detoxification of Carnosine-Acrolein Conjugates*

    PubMed Central

    Baba, Shahid P.; Hoetker, Joseph David; Merchant, Michael; Klein, Jon B.; Cai, Jian; Barski, Oleg A.; Conklin, Daniel J.; Bhatnagar, Aruni

    2013-01-01

    Oxidation of unsaturated lipids generates reactive aldehydes that accumulate in tissues during inflammation, ischemia, or aging. These aldehydes form covalent adducts with histidine-containing dipeptides such as carnosine and anserine, which are present in high concentration in skeletal muscle, heart, and brain. The metabolic pathways involved in the detoxification and elimination of these conjugates are, however, poorly defined, and their significance in regulating oxidative stress is unclear. Here we report that conjugates of carnosine with aldehydes such as acrolein are produced during normal metabolism and excreted in the urine of mice and adult human non-smokers as carnosine-propanols. Our studies show that the reduction of carnosine-propanals is catalyzed by the enzyme aldose reductase (AR). Carnosine-propanals were converted to carnosine-propanols in the lysates of heart, skeletal muscle, and brain tissue from wild-type (WT) but not AR-null mice. In comparison with WT mice, the urinary excretion of carnosine-propanols was decreased in AR-null mice. Carnosine-propanals formed covalent adducts with nucleophilic amino acids leading to the generation of carnosinylated proteins. Deletion of AR increased the abundance of proteins bound to carnosine in skeletal muscle, brain, and heart of aged mice and promoted the accumulation of carnosinylated proteins in hearts subjected to global ischemia ex vivo. Perfusion with carnosine promoted post-ischemic functional recovery in WT but not in AR-null mouse hearts. Collectively, these findings reveal a previously unknown metabolic pathway for the removal of carnosine-propanal conjugates and suggest a new role of AR as a critical regulator of protein carnosinylation and carnosine-mediated tissue protection. PMID:23928303

  16. Probenecid Treatment Enhances Retinal and Brain Delivery of N-4-Benzoylaminophenylsulfonylglycine, An Anionic Aldose Reductase Inhibitor

    PubMed Central

    Sunkara, Gangadhar; Ayalasomayajula, Surya P.; DeRuiter, Jack; Kompella, Uday B.

    2009-01-01

    Anion efflux transporters are expected to minimize target tissue delivery of N-[4-(benzoylaminophenyl)sulfonyl]glycine (BAPSG), a novel carboxylic acid aldose reductase inhibitor, which exists as a monocarboxylate anion at physiological conditions. Therefore, the objective of this study was to determine whether BAPSG delivery to various eye tissues including the retina and the brain can be enhanced by probenecid, a competitive inhibitor of anion transporters. To determine the influence of probenecid on eye and brain distribution of BAPSG, probenecid was administered intraperitoneally (120 mg/kg body weight; i.p.) 20 minutes prior to BAPSG (50 mg/kg; i.p.) administration. Drug disposition in various eye tissues including the retina and the brain was determined at 15 min, 1, 2 and 4 hr after BAPSG dose in male Sprauge-Dawley rats. To determine whether probenecid alters plasma clearance of BAPSG, influence of probenecid (120 mg/kg; i.p.) on the plasma pharmacokinetics of intravenously administered BAPSG (15 mg/kg) was studied as well. Finally, the effect of probenecid co-administration on the ocular tissue distribution of BAPSG was assessed in rabbits following topical (eye drop) administration. Following pretreatment with probenecid in the rat study, retinal delivery at 1 hr was increased by about 11 fold (2580 vs 244 ng/gm; p<0.05). Further, following probenecid pretreatment, significant BAPSG levels were detectable in the brain (45 ± 20 ng/gm) at 1 hr, unlike controls where the drug was not detectable. Plasma concentrations, plasma elimination half-life, and total body clearance of intravenously administered BAPSG were not altered by i.p. probenecid pretreatment. In the topical dosing study, a significant decline in BAPSG delivery was observed in the iris-ciliary body but no significant changes were observed in other tissues of the anterior segment of the eye including tears. Thus, inhibition of anion transporters is a useful approach to elevate retinal and brain

  17. Aldose Reductase Mediates Endotoxin-Induced Production of Nitric oxide and Cytotoxicity in Murine Macrophages.

    PubMed Central

    Ramana, Kota V; Reddy, Aramati BM.; Tammali, Ravinder; Srivastava, Satish K.

    2007-01-01

    Aldose reductase (AR) is a ubiquitously expressed protein with pleiotrophic roles as an efficient catalyst for the reduction of toxic lipid aldehydes and mediator of hyperglycemia, cytokine and growth factor –induced redox sensitive signals that cause secondary diabetic complications. Although AR inhibition has been shown to be protective against oxidative stress signals, the role of AR in regulating nitric oxide (NO) synthesis and NO-mediated apoptosis has not been elucidated to date. We therefore investigated the role of AR in regulating lipopolysaccharide (LPS)-induced NO synthesis and apoptosis in RAW 264.7 macrophages. Inhibition or RNA interference ablation of AR suppressed LPS-stimulated production of NO and over-expression of iNOS mRNA. Inhibition or ablation of AR also prevented the LPS-induced apoptosis, cell cycle arrest, activation of caspase-3, p38-MAPK, JNK, NF-κB and AP1. In addition, AR inhibition prevented the LPS-induced down-regulation of Bcl-xl and up-regulation of Bax and Bak in macrophages. L-arginine increased and L-NAME decreased the severity of cell death caused by LPS and AR inhibitors prevented it. Furthermore, inhibition of AR prevents cell death caused by HNE and GS-HNE, but not GS-DHN. Our findings for the first time suggest that AR catalyzed lipid aldehyde-glutathione conjugates regulates the LPS-induced production of inflammatory marker NO and cytotoxicity in RAW 264.7 cells. Inhibition or ablation of AR activity may be potential therapeutic target in endotoximia and other inflammatory diseases. PMID:17382209

  18. Aldose reductase regulates acrolein-induced cytotoxicity in human small airway epithelial cells.

    PubMed

    Yadav, Umesh C S; Ramana, K V; Srivastava, Satish K

    2013-12-01

    Aldose reductase (AR), a glucose-metabolizing enzyme, reduces lipid aldehydes and their glutathione conjugates with more than 1000-fold efficiency (Km aldehydes 5-30 µM) relative to glucose. Acrolein, a major endogenous lipid peroxidation product as well as a component of environmental pollutants and cigarette smoke, is known to be involved in various pathologies including atherosclerosis, airway inflammation, COPD, and age-related disorders, but the mechanism of acrolein-induced cytotoxicity is not clearly understood. We have investigated the role of AR in acrolein-induced cytotoxicity in primary human small airway epithelial cells (SAECs). Exposure of SAECs to varying concentrations of acrolein caused cell death in a concentration- and time-dependent manner. AR inhibition by fidarestat prevented the low-dose (5-10 µM) but not the high-dose (>10 µM) acrolein-induced SAEC death. AR inhibition protected SAECs from low-dose (5 µM) acrolein-induced cellular reactive oxygen species (ROS). Inhibition of acrolein-induced apoptosis by fidarestat was confirmed by decreased condensation of nuclear chromatin, DNA fragmentation, comet tail moment, and annexin V fluorescence. Further, fidarestat inhibited acrolein-induced translocation of the proapoptotic proteins Bax and Bad from the cytosol to the mitochondria and that of Bcl2 and BclXL from the mitochondria to the cytosol. Acrolein-induced cytochrome c release from mitochondria was also prevented by AR inhibition. The mitogen-activated protein kinases (MAPKs), such as extracellular signal-regulated kinases 1 and 2, stress-activated protein kinase/c-Jun NH2-terminal kinase, and p38MAPK, and c-Jun were transiently activated in airway epithelial cells by acrolein in a concentration- and time-dependent fashion, which was significantly prevented by AR inhibition. These results suggest that AR inhibitors could prevent acrolein-induced cytotoxicity in the lung epithelial cells.

  19. 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.

  20. Chemical constituents from the aerial parts of Aster koraiensis with protein glycation and aldose reductase inhibitory activities.

    PubMed

    Lee, Jun; Lee, Yun Mi; Lee, Byong Won; Kim, Joo-Hwan; Kim, Jin Sook

    2012-02-24

    Two new eudesmane-type sesquiterpene glucosides, 9β-O-(E-p-hydroxycinnamoyl)-1β,6β-dihydroxy-trans-eudesm-3-en-6-O-β-D-glucopyranoside (1) and 9α-O-(E-p-hydroxycinnamoyl)-1α,6α-11-trihydroxy-trans-eudesm-3-en-6-O-β-D-glucopyranoside (2), were isolated by the activity-guidedfractionation of an EtOAc-soluble fraction from the aerial parts of Aster koraiensis. A new dihydrobenzofuran glucoside, (2R,3S)-6-acetyl-2-[1-O-(β-D-glucopyranosyl)-2-propenyl]-5-hydroxy-3-methoxy-2,3-dihydrobenzofuran (3), was also isolated, in addition to 15 known compounds. The structures of 1-3 were determined by spectroscopic data interpretation. All of the isolates were evaluated for in vitro inhibitory activity against the formation of advanced glycation end-products and rat lens aldose reductase.

  1. 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

  2. Coumarin-thiazole and -oxadiazole derivatives: Synthesis, bioactivity and docking studies for aldose/aldehyde reductase inhibitors.

    PubMed

    Ibrar, Aliya; Tehseen, Yildiz; Khan, Imtiaz; Hameed, Abdul; Saeed, Aamer; Furtmann, Norbert; Bajorath, Jürgen; Iqbal, Jamshed

    2016-10-01

    In continuation of our previous efforts directed towards the development of potent and selective inhibitors of aldose reductase (ALR2), and to control the diabetes mellitus (DM), a chronic metabolic disease, we synthesized novel coumarin-thiazole 6(a-o) and coumarin-oxadiazole 11(a-h) hybrids and screened for their inhibitory activity against aldose reductase (ALR2), for the selectivity against aldehyde reductase (ALR1). Compounds were also screened against ALR1. Among the newly designed compounds, 6c, 11d, and 11g were selective inhibitors of ALR2. Whereas, (E)-3-(2-(2-(2-bromobenzylidene)hydrazinyl)thiazol-4-yl)-2H-chromen-2-one 6c yielded the lowest IC50 value of 0.16±0.06μM for ALR2. Moreover, compounds (E)-3-(2-(2-benzylidenehydrazinyl)thiazol-4-yl)-2H-chromen-2-one (6a; IC50=2.94±1.23μM for ARL1 and 0.12±0.05μM for ARL2) and (E)-3-(2-(2-(1-(4-bromophenyl)ethylidene)hydrazinyl)thiazol-4-yl)-2H-chromen-2-one (6e; IC50=1.71±0.01μM for ARL1 and 0.11±0.001μM for ARL2) were confirmed as dual inhibitors. Furthermore, compounds 6i, 6k, 6m, and 11b were found to be selective inhibitors for ALR1, among which (E)-3-(2-(2-((2-amino-4-chlorophenyl)(phenyl)methylene)hydrazinyl)thiazol-4-yl)-2H-chromen-2-one (6m) was most potent (IC50=0.459±0.001μM). Docking studies performed using X-ray structures of ALR1 and ALR2 with the given synthesized inhibitors showed that coumarinyl thiazole series lacks the carboxylate function that could interact with the anionic binding site being a common ALR1/ALR2 inhibitors trait. Molecular docking study with dual inhibitor 6e also suggested plausible binding modes for the ALR1 and ALR2 enzymes. Hence, the results of this study revealed that coumarinyl thiazole and oxadiazole derivatives could act as potential ALR1/ALR2 inhibitors.

  3. 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

  4. 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.

  5. A novel zwitterionic inhibitor of aldose reductase interferes with polyol pathway in ex vivo and in vivo models of diabetic complications.

    PubMed

    Karasova, M Juskova; Prnova, M Soltesova; Stefek, M

    2014-10-01

    Recently a zwitterionic principle has been suggested as an alternative to bioisosteric replacement for increasing low bioavailability of aldose reductase inhibitors bearing an acidic function. In the present work we studied the effect of a novel zwitterionic inhibitor of aldose reductase [(2-benzyl-2,3,4,5-tetrahydro-1 H-pyrido[4,3-b]indole-8-yl)-acetic acid, compound 1] on sorbitol accumulation in ex vivo and in vivo models of diabetic complications. The effect of 1 on sorbitol accumulation in isolated rat eye lenses incubated with high glucose and in selected organs of streptozotocin-induced diabetic rats was evaluated. Significantly increased sorbitol levels were recorded in the lenses incubated with 50 mM glucose in comparison with controls. Sorbitol production was inhibited by 1 at concentrations of 25 and 100 μM. Under in vivo conditions in diabetic rats, significant elevation of sorbitol levels in selected organs was recorded. Compound 1 administered i.g. for five consecutive days (twice a day 25 mg/kg) inhibited sorbitol accumulation in erythrocytes and the sciatic nerve, yet it was without effect in eye lenses. A similar picture of inhibition was observed after i.p. administration of 1. To conclude, the results suggest that the zwitterionic principle may represent a practicable way of improving bioavailability of aldose reductase inhibitors bearing an acidic function.

  6. Galloyl glucoses from the seeds of Cornus officinalis with inhibitory activity against protein glycation, aldose reductase, and cataractogenesis ex vivo.

    PubMed

    Lee, Jun; Jang, Dae Sik; Kim, Nan Hee; Lee, Yun Mi; Kim, Junghyun; Kim, Jin Sook

    2011-01-01

    In an ongoing project directed toward the discovery of novel treatments for diabetic complications from traditional herbal medicines, six galloyl glucoses, 1,2,3-tri-O-galloyl-β-D-glucose (1), 1,2,6-tri-O-galloyl-β-D-glucose (2), 1,2,3,6-tetra-O-galloyl-β-D-glucose (3), 1,2,4,6-tetra-O-galloyl-β-D-glucose (4), 1,2,3,4,6-penta-O-galloyl-β-D-glucose (5), and tellimagrandin II (6), and two phenolic acids, gallic acid 4-O-β-D-glucoside (7) and gallic acid 4-O-β-D-(6'-O-galloyl)-glucoside (8), were isolated from an EtOAc-soluble fraction of the seeds of Cornus officinalis (Cornaceae). The structures of the compounds were identified using physical and spectroscopic methods, as well as by comparison of their data with values reported in the literature. All the isolates were evaluated in vitro for inhibitory activity against the formation of advanced glycation end-products (AGEs) and rat lens aldose reductase (RLAR). Compounds 1-6 were subjected to further bioassay to examine their inhibitory effects on AGE cross-linking. The opacity of lenses was significantly prevented when treated with 3 in an ex vivo experiment.

  7. Scopoletin from the flower buds of Magnolia fargesii inhibits protein glycation, aldose reductase, and cataractogenesis ex vivo.

    PubMed

    Lee, Jun; Kim, Nan Hee; Nam, Joo Won; Lee, Yun Mi; Jang, Dae Sik; Kim, Young Sook; Nam, Sang Hae; Seo, Eun-Kyoung; Yang, Min Suk; Kim, Jin Sook

    2010-09-01

    Five compounds previously known structures, scopoletin (1), northalifoline (2), stigmast-4-en-3-one (3), tiliroside (4), and oplopanone (5) were obtained from the flower buds of Magnolia fargesii using chromatographic separation methods. The structures of 1-5 were identified by the interpretation of their spectroscopic data including 1D- and 2D-NMR as well as by comparison with reported values. Three compounds 1-3 were found from M. fargesii for the first time in this study. All the isolates (1-5) were subjected to in vitro bioassays to evaluate the inhibitory activity on advanced glycation end products formation and rat lens aldose reductase (RLAR). Compound 1 showed a remarkable inhibitory activity on advanced glycation end products formation with IC(50) value of 2.93 μM (aminoguanidine: 961 μM), and showed a significant RLAR inhibitory activity with IC(50) value of 22.5 μM (3.3-tetramethyleneglutaric acid: 28.7 μM). Compound 4 exhibited potent inhibitory activity against RLAR (IC(50) = 14.9 μM). In the further experiment ex vivo, cataractogenesis of rat lenses induced with xylose was significantly inhibited by compound 1 treatment.

  8. 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.

  9. Aldose reductase (-106) C/T gene polymorphism and possibility of macrovascular complications in Egyptian type 2 diabetic patients

    PubMed Central

    Nomair, Azhar Mohamed; ElDeeb, Mona Mohamed Kamal; Maharem, Dalia Aly

    2016-01-01

    Introduction: Over the past three decades, the number of people with diabetes mellitus (DM) has more than doubled globally, making it one of the most important public health challenges to all nations. Aldose reductase (AR) is a rate-limiting enzyme in the polyol pathway, which has been implicated in the pathogenesis of diabetic microvascular complications; however, the association of the AR gene with diabetic macrovascular complications has rarely been investigated. Aim: The study aimed to identify the possible association between C(-106) T polymorphism of the AR gene and diabetic macroangiopathy in a cohort of Egyptian patients with type 2 DM. Settings and Design: This study was conducted on 100 Egyptian subjects, the control group (n = 20) and the patient group (n = 80) with type 2 diabetes which were further subdivided into two subgroups with (n = 48) and without macroangiopathic complications (n = 32) as evidenced by carotid intima-media thickness, electrocardiography (ECG) ischemic changes, cerebrovascular insufficiency, and peripheral vascular insufficiency. Subjects and Methods: All studied subjects were subjected to detailed history taking, clinical examination, ECG, carotid ultrasonography, routine laboratory investigations, and molecular studies including the detection of AR C(-106) T gene polymorphisms using the polymerase chain reaction (PCR)/restriction fragment length polymorphism technique. Results: The genotype distribution and allele frequency of AR C(-106) T showed no statistical significance also the genotypes were not associated with any of the different studied parameters. Conclusions: The results suggest that the C(-106) T polymorphism in the AR gene is not involved in the pathogenesis of macroangiopathy in type 2 diabetes. PMID:27730075

  10. 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

  11. Evaluation of in vitro aldose reductase inhibitory potential of different fraction of Hybanthus enneaspermus Linn F. Muell

    PubMed Central

    Patel, DK; Kumar, R; Kumar, M; Sairam, K; Hemalatha, S

    2012-01-01

    Objective To evaluate the aldose reductase inhibitory (ARI) activity of different fractions of Hybanthus enneaspermus for potential use in diabetic cataract. Methods Total phenol and flavonoid content of different fractions was determined. ARI activity of different fractions in rat lens was investigated in vitro. Results The results showed significant level of phenolic and flavonoid content in ethyl acetate fraction [total phenol (212.15±0.79 mg/g), total flavonoid (39.11±2.27 mg/g)] and aqueous fraction [total phenol (140.62±0.57 mg/g), total flavonoid (26.07±1.49 mg/g)] as compared with the chloroform fraction [total phenol (68.56±0.51 mg/g), total flavonoid (13.41±0.82 mg/g)] and petrolium ether fraction [total phenol (36.68±0.43 mg/g), total flavonoid (11.55±1.06 mg/g)]. There was a significant difference in the ARI activity of each fraction, and it was found to be the highest in ethyl acetate fraction [IC50 (49.26±1.76 µg/mL)] followed by aqueous extract [IC50 (70.83±2.82 µg/mL)] and it was least in the petroleum ether fraction [IC50 (118.89±0.71 µg/mL)]. Chloroform fraction showed moderate activity [IC50 (98.52±1.80 µg/mL)]. Conclusions Different fractions showed significanct amount of ARI activity, where in ethyl acetate fraction it was found to be maximum which may be due to its high phenolic and flavonoid content. The extract after further evaluation may be used in the treatment of diabetic cataract. PMID:23569883

  12. [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

  13. 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.

  14. 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

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

    PubMed

    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.

  16. Design and synthesis of pyridazinone-substituted benzenesulphonylurea derivatives as anti-hyperglycaemic agents and inhibitors of aldose reductase - an enzyme embroiled in diabetic complications.

    PubMed

    Yaseen, Raed; Pushpalatha, H; Reddy, G Bhanuprakash; Ismael, Ameer; Ahmed, Ayad; Dheyaa, Alhamza; Ovais, Syed; Rathore, Pooja; Samim, Mohammed; Akthar, Mymoona; Sharma, Kalicharan; Shafi, Syed; Singh, Surender; Javed, Kalim

    2016-12-01

    Thirty new aryl-pyridazinone-substituted benzenesulphonylurea derivatives (I-XXX) were synthesized and evaluated for their anti-hyperglycaemic activity in glucose-fed hyperglycaemic normal rats. Twenty-three compounds (III-XI, XIV-XVII, XIX-XXIV, XXVI and XXVIII-XXX) showed more or comparable area under the curve (AUC) reduction percentage (ranging from 21.9% to 35.5%) as compared to the standard drug gliclazide (22.0%). On the basis of docking results, 18 compounds were screened for their in vitro ability to inhibit rat lens aldose reductase. Ten compounds (III-VI, XII, XVI-XVIII, XXI and XXVII) showed ARI activity with IC50 ranging from 34 to 242 μM. Out of these, two compounds IV and V showed best ARI activity which is comparable with that of quercetin. As a result, two compounds (IV and V) possessing significant dual action (anti-hyperglycaemic and aldose reductase inhibition) were identified and may be used as lead compounds for developing new drugs. PMID:26879420

  17. 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

  18. Long-term clinical effects of epalrestat, an aldose reductase inhibitor, on progression of diabetic neuropathy and other microvascular complications: multivariate epidemiological analysis based on patient background factors and severity of diabetic neuropathy

    PubMed Central

    Hotta, N; Kawamori, R; Fukuda, M; Shigeta, Y

    2012-01-01

    Abstract Aims The goal of the study was to evaluate the efficacy of epalrestat, an aldose reductase inhibitor, on diabetic retinopathy and diabetic nephropathy, based on analysis of the results of the Aldose Reductase Inhibitor–Diabetes Complications Trial, a 3-year multicentre comparative clinical trial of conventional therapy (control group) and epalrestat therapy (epalrestat group) in Japanese patients with mild diabetic neuropathy. Methods The subjects of the study were patients enrolled in the Aldose Reductase Inhibitor–Diabetes Complications Trial for whom data for major patient characteristics, severity of diabetic neuropathy at the end of the study and time-courses of diabetic retinopathy and diabetic nephropathy were available (57 and 52 patients from the control and epalrestat groups, respectively). Progression of diabetic retinopathy/nephropathy (a primary endpoint) in relation to major patient characteristics, severity of diabetic neuropathy at the end of the study (assessed from the mean of z-scores in four neurological function tests) and epalrestat treatment were analysed using univariate analysis and multiple logistic regression analysis. Results Progression of diabetic retinopathy/nephropathy was significantly inhibited in the epalrestat group compared with the control group (odds ratio = 0.323, P = 0.014) and was dependent on the severity of diabetic neuropathy at the end of the study (odds ratio = 2.131, P = 0.025). Conclusions Epalrestat prevented progression of diabetic neuropathy and retinopathy/nephropathy. The effect on diabetic retinopathy/nephropathy may have occurred indirectly because of the prevention of progression of diabetic neuropathy, in addition to the inhibitory action of epalrestat on aldose reductase. PMID:22507139

  19. Construction of an Indonesian herbal constituents database and its use in Random Forest modelling in a search for inhibitors of aldose reductase.

    PubMed

    Naeem, Sadaf; Hylands, Peter; Barlow, David

    2012-02-01

    Data on phytochemical constituents of plants commonly used in traditional Indonesian medicine have been compiled as a computer database. This database (the Indonesian Herbal constituents database, IHD) currently contains details on ∼1,000 compounds found in 33 different plants. For each entry, the IHD gives details of chemical structure, trivial and systematic name, CAS registry number, pharmacology (where known), toxicology (LD(50)), botanical species, the part(s) of the plant(s) where the compounds are found, typical dosage(s) and reference(s). A second database has been also been compiled for plant-derived compounds with known activity against the enzyme, aldose reductase (AR). This database (the aldose reductase inhibitors database, ARID) contains the same details as the IHD, and currently comprises information on 120 different AR inhibitors. Virtual screening of all compounds in the IHD has been performed using Random Forest (RF) modelling, in a search for novel leads active against AR-to provide for new forms of symptomatic relief in diabetic patients. For the RF modelling, a set of simple 2D chemical descriptors were employed to classify all compounds in the combined ARID and IHD databases as either active or inactive as AR inhibitors. The resulting RF models (which gave misclassification rates of 21%) were used to identify putative new AR inhibitors in the IHD, with such compounds being identified as those giving RF scores >0.5 (in each of the three different RF models developed). In vitro assays were subsequently performed for four of the compounds obtained as hits in this in silico screening, to determine their inhibitory activity against human recombinant AR. The two compounds having the highest RF scores (prunetin and ononin) were shown to have the highest activities experimentally (giving ∼58% and ∼52% inhibition at a concentration of 15μM, respectively), while the compounds with lowest RF scores (vanillic acid and cinnamic acid) showed the

  20. Comparative functional analysis of human medium-chain dehydrogenases, short-chain dehydrogenases/reductases and aldo-keto reductases with retinoids

    PubMed Central

    Gallego, Oriol; Belyaeva, Olga V.; Porté, Sergio; Ruiz, F. Xavier; Stetsenko, Anton V.; Shabrova, Elena V.; Kostereva, Natalia V.; Farrés, Jaume; Parés, Xavier; Kedishvili, Natalia Y.

    2006-01-01

    Retinoic acid biosynthesis in vertebrates occurs in two consecutive steps: the oxidation of retinol to retinaldehyde followed by the oxidation of retinaldehyde to retinoic acid. Enzymes of the MDR (medium-chain dehydrogenase/reductase), SDR (short-chain dehydrogenase/reductase) and AKR (aldo-keto reductase) superfamilies have been reported to catalyse the conversion between retinol and retinaldehyde. Estimation of the relative contribution of enzymes of each type was difficult since kinetics were performed with different methodologies, but SDRs would supposedly play a major role because of their low Km values, and because they were found to be active with retinol bound to CRBPI (cellular retinol binding protein type I). In the present study we employed detergent-free assays and HPLC-based methodology to characterize side-by-side the retinoid-converting activities of human MDR [ADH (alcohol dehydrogenase) 1B2 and ADH4), SDR (RoDH (retinol dehydrogenase)-4 and RDH11] and AKR (AKR1B1 and AKR1B10) enzymes. Our results demonstrate that none of the enzymes, including the SDR members, are active with CRBPI-bound retinoids, which questions the previously suggested role of CRBPI as a retinol supplier in the retinoic acid synthesis pathway. The members of all three superfamilies exhibit similar and low Km values for retinoids (0.12–1.1 μM), whilst they strongly differ in their kcat values, which range from 0.35 min−1 for AKR1B1 to 302 min−1 for ADH4. ADHs appear to be more effective retinol dehydrogenases than SDRs because of their higher kcat values, whereas RDH11 and AKR1B10 are efficient retinaldehyde reductases. Cell culture studies support a role for RoDH-4 as a retinol dehydrogenase and for AKR1B1 as a retinaldehyde reductase in vivo. PMID:16787387

  1. 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

  2. Structure-activity relationships and molecular modelling of new 5-arylidene-4-thiazolidinone derivatives as aldose reductase inhibitors and potential anti-inflammatory agents.

    PubMed

    Maccari, Rosanna; Vitale, Rosa Maria; Ottanà, Rosaria; Rocchiccioli, Marco; Marrazzo, Agostino; Cardile, Venera; Graziano, Adriana Carol Eleonora; Amodeo, Pietro; Mura, Umberto; Del Corso, Antonella

    2014-06-23

    A series of 5-(carbamoylmethoxy)benzylidene-2-oxo/thioxo-4-thiazolidinone derivatives (6-9) were synthesized as inhibitors of aldose reductase (AR), enzyme which plays a crucial role in the development of diabetes complications as well as in the inflammatory processes associated both to diabetes mellitus and to other pathologies. In vitro inhibitory activity indicated that compounds 6-9a-d were generally good AR inhibitors. Acetic acid derivatives 8a-d and 9a-d were shown to be the best enzyme inhibitors among the tested compounds endowed with significant inhibitory ability levels reaching submicromolar IC50 values. Moreover, some representative AR inhibitors (7a, 7c, 9a, 9c, 9d) were assayed in cultures of human keratinocytes in order to evaluate their capability to reduce NF-kB activation and iNOS expression. Compound 9c proved to be the best derivative endowed with both interesting AR inhibitory effectiveness and ability to reduce NF-kB activation and iNOS expression. Molecular docking and molecular dynamics simulations were undertaken to investigate the binding modes of selected compounds into the active site of AR in order to rationalize the inhibitory effectiveness of these derivatives.

  3. Tissue-specific effects of aldose reductase inhibition on fluorescence and cross-linking of extracellular matrix in chronic galactosemia. Relationship to pentosidine cross-links.

    PubMed

    Richard, S; Tamas, C; Sell, D R; Monnier, V M

    1991-08-01

    Chronic experimental hyperglycemia mediated by galactose has been shown to induce browning and cross-linking of rat tail tendon collagen that could be duplicated in vitro by nonenzymatic galactosylation. To investigate the nature of these changes, Sprague-Dawley rats were placed on a 33% galactose diet without and with sorbinil for 6 and 12 mo. Collagen-linked fluorescence and pentosidine cross-links increased with age and galactosemia in tail tendons (P less than 0.001) and skin but were essentially unresponsive to aldose reductase inhibition (ARI). In contrast, tendon breaking time in urea, a likely parameter of cross-linking, was markedly improved (P less than 0.001) by ARI. Fluorescence that was inhibited by sorbinil treatment was increased in pepsin and proteinase K digest of aortic tissue from galactosemic rats (P less than 0.001), but impaired enzymatic digestibility was not observed. Systolic blood pressure as potential consequence of aortic stiffening was not increased in galactosemia. These data suggest that fluorescence in skin and tendon might be in part due to advanced glycosylation and pentosidine formation because these were not decreased by ARI. However, they also suggest that nonfluorescent cross-links may also be forming because, in contrast to fluorescence, tail tendon breaking time was partly corrected by ARI. Thus, it appears that extracellular matrix changes in chronic galactosemia are complex, being partly attributable to advanced glycosylation and partly to polyol-pathway activation.

  4. Prospecting for Novel Plant-Derived Molecules of Rauvolfia serpentina as Inhibitors of Aldose Reductase, a Potent Drug Target for Diabetes and Its Complications

    PubMed Central

    Pathania, Shivalika; Randhawa, Vinay; Bagler, Ganesh

    2013-01-01

    Aldose Reductase (AR) is implicated in the development of secondary complications of diabetes, providing an interesting target for therapeutic intervention. Extracts of Rauvolfia serpentina, a medicinal plant endemic to the Himalayan mountain range, have been known to be effective in alleviating diabetes and its complications. In this study, we aim to prospect for novel plant-derived inhibitors from R. serpentina and to understand structural basis of their interactions. An extensive library of R. serpentina molecules was compiled and computationally screened for inhibitory action against AR. The stability of complexes, with docked leads, was verified using molecular dynamics simulations. Two structurally distinct plant-derived leads were identified as inhibitors: indobine and indobinine. Further, using these two leads as templates, 16 more leads were identified through ligand-based screening of their structural analogs, from a small molecules database. Thus, we obtained plant-derived indole alkaloids, and their structural analogs, as potential AR inhibitors from a manually curated dataset of R. serpentina molecules. Indole alkaloids reported herein, as a novel structural class unreported hitherto, may provide better insights for designing potential AR inhibitors with improved efficacy and fewer side effects. PMID:23613832

  5. Pressure-induced inhibition of fast axonal transport of proteins in the rabbit vagus nerve in galactose neuropathy: prevention by an aldose reductase inhibitor.

    PubMed

    McLean, W G

    1988-07-01

    Fast and slow anterograde axonal transport and retrograde axonal transport of proteins were studied in the mainly non-myelinated sensory fibres of the vagus nerve of rabbits fed a diet of 50% galactose over a period of 29 days. Galactose feeding had no effect on the rate or protein composition of slow transport nor on the amount of retrogradely transported proteins. There was a slight retardation of fast transported proteins although their composition was unchanged. The galactose feeding led to a significant increase (p less than 0.005) in nerve water content and nerve galactitol but no significant change in myo-inositol. When 20 mm Hg pressure was applied locally to the cervical vagus nerve, fast transported proteins accumulated proximal to the compression zone in the galactose-fed but not in control rabbits. Administration of the aldose reductase inhibitor Statil (ICI 128436) throughout the experiment prevented the increased susceptibility to pressure and the increase in nerve galactitol and water content. The effects of pressure are similar to those found in the streptozotocin-diabetic rat although the underlying mechanisms may differ.

  6. Effects of Long-Term Treatment with Ranirestat, a Potent Aldose Reductase Inhibitor, on Diabetic Cataract and Neuropathy in Spontaneously Diabetic Torii Rats

    PubMed Central

    Ota, Ayumi; Kakehashi, Akihiro; Toyoda, Fumihiko; Kinoshita, Nozomi; Shinmura, Machiko; Takano, Hiroko; Obata, Hiroto; Matsumoto, Takafumi; Tsuji, Junichi; Dobashi, Yoh; Fujimoto, Wilfred Y.; Kawakami, Masanobu; Kanazawa, Yasunori

    2013-01-01

    We evaluated ranirestat, an aldose reductase inhibitor, in diabetic cataract and neuropathy (DN) in spontaneously diabetic Torii (SDT) rats compared with epalrestat, the positive control. Animals were divided into groups and treated once daily with oral ranirestat (0.1, 1.0, 10 mg/kg) or epalrestat (100 mg/kg) for 40 weeks, normal Sprague-Dawley rats, and untreated SDT rats. Lens opacification was scored from 0 (normal) to 3 (mature cataract). The combined scores (0–6) from both lenses represented the total for each animal. DN was assessed by measuring the motor nerve conduction velocity (MNCV) in the sciatic nerve. Sorbitol and fructose levels were measured in the lens and sciatic nerve 40 weeks after diabetes onset. Cataracts developed more in untreated rats than normal rats (P < 0.01). Ranirestat significantly (P < 0.01) inhibited rapid cataract development; epalrestat did not. Ranirestat significantly reversed the MNCV decrease (40.7 ± 0.6 m/s) in SDT rats dose-dependently (P < 0.01). Epalrestat also reversed the prevented MNCV decrease (P < 0.05). Sorbitol levels in the sciatic nerve increased significantly in SDT rats (2.05 ± 0.10 nmol/g), which ranirestat significantly suppressed dose-dependently, (P < 0.05, <0.01, and <0.01); epalrestat did not. Ranirestat prevents DN and cataract; epalrestat prevents DN only. PMID:23671855

  7. Relation of Na+, K(+)-ATPase to delayed motor nerve conduction velocity: effect of aldose reductase inhibitor, ADN-138, on Na+, K(+)-ATPase activity.

    PubMed

    Hirata, Y; Okada, K

    1990-06-01

    The role of sorbitol, myo-inositol, and Na+, K(+)-adenosine triphosphatase (ATPase) activity on motor nerve conduction velocity (MNCV) in streptozotocin (STZ)-diabetic rats was studied. Reduction of MNCV and Na+, K(+)-ATPase in caudal nerves appeared after 3 weeks of diabetes, and at this time treatment with aldose reductase inhibitor (ARI), ADN-138 and 1% myo-inositol supplement was begun. One percent myo-inositol supplement for 3 weeks resulted in a significant increase in myo-inositol levels in diabetic nerves, but left MNCV and sorbitol levels unchanged. In contrast, treatment with ADN-138 for 3 weeks reduced sorbitol levels in diabetic nerves and resulted in significant increases in MNCV and Na+, K(+)-ATPase in the nerves. Since ADN-138 did not restore myo-inositol levels, the increase in Na+, K(+)-ATPase levels by ADN-138 treatment was independent of myo-inositol levels. Also, nerve Na+ levels in ADN-138-treated rats were reduced and the ratio of K+ to Na+ was raised, while 1% myo-inositol supplement did not affect them. These results suggest that treatment with ADN-138 elevates MNCV through a series of processes: ARI----reduction of sorbitol level----increase in Na+, K(+)-ATPase activity----correction of K+, Na+ imbalance----increase in MNCV.

  8. Catalytic mechanism of the primary human prostaglandin F2α synthase, aldo-keto reductase 1B1--prostaglandin D2 synthase activity in the absence of NADP(H).

    PubMed

    Nagata, Nanae; Kusakari, Yukiko; Fukunishi, Yoshifumi; Inoue, Tsuyoshi; Urade, Yoshihiro

    2011-04-01

    Aldo-keto reductase 1B1 and 1B3 (AKR1B1 and AKR1B3) are the primary human and mouse prostaglandin F(2α) (PGF(2α)) synthases, respectively, which catalyze the NADPH-dependent reduction of PGH(2), a common intermediate of various prostanoids, to form PGF(2α). In this study, we found that AKR1B1 and AKR1B3, but not AKR1B7 and AKR1C3, also catalyzed the isomerization of PGH(2) to PGD(2) in the absence of NADPH or NADP(+). Both PGD(2) and PGF(2α) synthase activities of AKR1B1 and AKR1B3 completely disappeared in the presence of NADP(+) or after heat treatment of these enzymes at 100 °C for 5 min. The K(m), V(max), pK and optimum pH values of the PGD(2) synthase activities of AKR1B1 and AKR1B3 were 23 and 18 μM, 151 and 57 nmol·min(-1)·(mg protein)(-1), 7.9 and 7.6, and pH 8.5 for both AKRs, respectively, and those of PGF(2α) synthase activity were 29 and 33 μM, 169 and 240 nmol·min(-1)·(mg protein)(-1), 6.2 and 5.4, and pH 5.5 and pH 5.0, respectively, in the presence of 0.5 mm NADPH. Site-directed mutagenesis of the catalytic tetrad of AKR1B1, composed of Tyr, Lys, His and Asp, revealed that the triad of Asp43, Lys77 and His110, but not Tyr48, acts as a proton donor in most AKR activities, and is crucial for PGD(2) and PGF(2α) synthase activities. These results, together with molecular docking simulation of PGH(2) to the crystallographic structure of AKR1B1, indicate that His110 acts as a base in concert with Asp43 and Lys77 and as an acid to generate PGD(2) and PGF(2α) in the absence of NADPH or NADP(+) and in the presence of NADPH, respectively.

  9. Triple aldose reductase/α-glucosidase/radical scavenging high-resolution profiling combined with high-performance liquid chromatography-high-resolution mass spectrometry-solid-phase extraction-nuclear magnetic resonance spectroscopy for identification of antidiabetic constituents in crude extract of Radix Scutellariae.

    PubMed

    Tahtah, Yousof; Kongstad, Kenneth T; Wubshet, Sileshi G; Nyberg, Nils T; Jønsson, Louise H; Jäger, Anna K; Qinglei, Sun; Staerk, Dan

    2015-08-21

    In this work, development of a new microplate-based high-resolution profiling assay using recombinant human aldose reductase is presented. Used together with high-resolution radical scavenging and high-resolution α-glucosidase assays, it provided the first report of a triple aldose reductase/α-glucosidase/radical scavenging high-resolution inhibition profile - allowing proof of concept with Radix Scutellariae crude extract as a polypharmacological herbal drug. The triple bioactivity high-resolution profiles were used to pinpoint bioactive compounds, and subsequent structure elucidation was performed with hyphenated high-performance liquid chromatography-high-resolution mass spectrometry-solid-phase extraction-nuclear magnetic resonance spectroscopy. The only α-glucosidase inhibitor was baicalein, whereas main aldose reductase inhibitors in the crude extract were baicalein and skullcapflavone II, and main radical scavengers were ganhuangemin, viscidulin III, baicalin, oroxylin A 7-O-glucuronide, wogonoside, baicalein, wogonin, and skullcapflavone II.

  10. 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.

  11. 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

  12. 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

  13. Diabetic neuropathy in db/db mice develops independently of changes in ATPase and aldose reductase. A biochemical and immunohistochemical study.

    PubMed

    Bianchi, R; Marelli, C; Marini, P; Fabris, M; Triban, C; Fiori, M G

    1990-03-01

    ATPase activity was investigated in sciatic and optic nerves of female mutant diabetic C57Bl/Ks (db/db) mice and age-matched control mice (db/m and m/m). Nerves from animals aged 50, 70, 125, 180 and 280 days were assayed in vitro for ATPase activity in the presence or absence of ouabain: the ouabain-sensitive fraction contained Na+,K(+)-ATPase. Enzymatic activity was compared within and between age-matched groups. No significant difference in Na+,K(+)-ATPase activity was detected between the diabetic and control mice, whether expressed as mumol Pi/h-1 formed per gramme wet weight or per nerve (protein content). The activity decreased by about 25% in both the sciatic and optic nerves of the oldest animals. These results were strikingly similar in all groups, regardless of the type of nerve examined, confirming that the development of neuropathy in this animal model is unrelated to the postulated derangement of Na+,K(+)-ATPase activity. Among possible explanations, a lack of polyol pathway activation was investigated by staining the sciatic nerves of animals from all groups with the peroxidase-antiperoxidase procedure using a polyclonal antiserum raised against the enzyme aldose reductase. Histological sections of all nerves were consistently negative, suggesting that these animals actually lack the enzyme involved in activating the self-perpetuating metabolic cycle leading to deranged nerve function. The db/db mouse appears to present particular biochemical changes which merit attention with a view to clarifying the pathogenesis of diabetic neuropathy.

  14. Effects of the New Aldose Reductase Inhibitor Benzofuroxane Derivative BF-5m on High Glucose Induced Prolongation of Cardiac QT Interval and Increase of Coronary Perfusion Pressure

    PubMed Central

    Di Filippo, C.; Ferraro, B.; Maisto, R.; Trotta, M. C.; Di Carluccio, N.; Sartini, S.; La Motta, C.; Ferraraccio, F.; Rossi, F.; D'Amico, M.

    2016-01-01

    This study investigated the effects of the new aldose reductase inhibitor benzofuroxane derivative 5(6)-(benzo[d]thiazol-2-ylmethoxy)benzofuroxane (BF-5m) on the prolongation of cardiac QT interval and increase of coronary perfusion pressure (CPP) in isolated, high glucose (33.3 mM D-glucose) perfused rat hearts. BF-5m was dissolved in the Krebs solution at a final concentration of 0.01 μM, 0.05 μM, and 0.1 μM. 33.3 mM D-glucose caused a prolongation of the QT interval and increase of CPP up to values of 190 ± 12 ms and 110 ± 8 mmHg with respect to the values of hearts perfused with standard Krebs solution (11.1 mM D-glucose). The QT prolongation was reduced by 10%, 32%, and 41%, respectively, for the concentration of BF-5m 0.01 μM, 0.05 μM, and 0.1 μM. Similarly, the CPP was reduced by 20% for BF-5m 0.05 μM and by 32% for BF-5m 0.1 μM. BF-5m also increased the expression levels of sirtuin 1, MnSOD, eNOS, and FOXO-1, into the heart. The beneficial actions of BF-5m were partly abolished by the pretreatment of the rats with the inhibitor of the sirtuin 1 activity EX527 (10 mg/kg/day/7 days i.p.) prior to perfusion of the hearts with high glucose + BF-5m (0.1 μM). Therefore, BF-5m supplies cardioprotection from the high glucose induced QT prolongation and increase of CPP. PMID:26839893

  15. 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.

  16. A meta-analysis of trials on aldose reductase inhibitors in diabetic peripheral neuropathy. The Italian Study Group. The St. Vincent Declaration.

    PubMed

    Nicolucci, A; Carinci, F; Cavaliere, D; Scorpiglione, N; Belfiglio, M; Labbrozzi, D; Mari, E; Benedetti, M M; Tognoni, G; Liberati, A

    1996-12-01

    Peripheral neuropathy is one of the most common and disabling long-term sequelae of diabetes mellitus. Aldose reductase inhibitors (ARIs) have been proposed and are increasingly used in many countries for the prevention and treatment of diabetic neuropathy. The aim of this study was to review existing evidence on the effectiveness of ARIs in the treatment of peripheral diabetic neuropathy, with particular reference to the type and clinical relevance of the end point used and to the consistency of results across studies. Thirteen randomized clinical trials (RTCs) comparing ARIs with placebo, published between 1981 and 1993 were included in the meta-analysis. Nerve conduction velocity (NCV) was the only end point reported in all trials. Treatment effect was thus evaluated in terms of NCV mean difference in four different nerves: median motor, median sensory, peroneal motor, and sural sensory. A statistically significant reduction in decline of median motor NCV was present in the treated group as compared to the control group (mean 0.91 ms-1; 95% CI 0.41-1.42 ms-1). For peroneal motor, median sensory, and sural sensory nerves results did not show any clear benefit for patients treated with ARIs. When the analysis was limited to trials with at least 1-year treatment duration, a significant effect was present for peroneal motor NCV (mean 1.24 ms-1; 95% CI 0.32-2.15 ms-1) and a benefit of borderline statistical significance was also present for median motor NCV (mean 0.69 ms-1; 95% CI-0.07-1.45 ms-1). A heterogeneous picture emerged when looking at the results of different studies and serious inconsistencies were also present in the direction of treatment effects among nerves in the same studies. Although the results of 1-year treatment on motor NCV seem encouraging, the uncertainty about the reliability of the end-point employed and the short treatment duration do not allow any clear conclusion about the efficacy of ARIs in the treatment of peripheral diabetic

  17. 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

  18. Effect of a new aldose reductase inhibitor, 8'-chloro-2',3'-dihydrospiro [pyrrolidine-3,6'(5'H)-pyrrolo[1,2,3-de] [1,4]benzoxazine]-2,5,5'- trione (ADN-138), on delayed motor nerve conduction velocity in streptozotocin-diabetic rats.

    PubMed

    Hirata, Y; Fujimori, S; Okada, K

    1988-02-01

    The effects of a chemically new type of aldose reductase inhibitor, ADN-138, on delayed motor nerve conduction velocity (MNCV) and sciatic nerve sorbitol, fructose and myo-inositol levels were studied in streptozotocin-diabetic rats. MNCV in rats was significantly delayed after 3 weeks of diabetes and ADN-138 treatment was started at this point. Treatment of diabetics with ADN-138 at 5 and 20 but not 1 mg/kg/d for 3 weeks resulted in a significant increase in MNCV and reduced sorbitol levels to or below those of nondiabetic controls. However, fructose, though decreased in a dose-dependent manner, was not normalized. The reference drug, Sorbinil, showed similar effects on them. After the 3 weeks of ADN-138(20 mg/kg/d) treatment, diabetics were left on ADN-138 or continued further to be treated with it for 3 weeks. The withdrawal of ADN-138 prevented a further increase in MNCV and restored sorbitol and fructose to nontreated diabetic levels, and myo-inositol levels declined. In contrast, the ADN-138-continued group kept improving its MNCV and normalized sorbitol and myo-inositol. These results suggest that polyol accumulation is responsible for delayed MNCV and that the action of ADN-138 on MNCV reflected reversibility of metabolic function in diabetics.

  19. The aldo-keto reductases (AKRs): Overview.

    PubMed

    Penning, Trevor M

    2015-06-01

    The aldo-keto reductase (AKR) protein superfamily contains >190 members that fall into 16 families and are found in all phyla. These enzymes reduce carbonyl substrates such as: sugar aldehydes; keto-steroids, keto-prostaglandins, retinals, quinones, and lipid peroxidation by-products. Exceptions include the reduction of steroid double bonds catalyzed by AKR1D enzymes (5β-reductases); and the oxidation of proximate carcinogen trans-dihydrodiol polycyclic aromatic hydrocarbons; while the β-subunits of potassium gated ion channels (AKR6 family) control Kv channel opening. AKRs are usually 37kDa monomers, have an (α/β)8-barrel motif, display large loops at the back of the barrel which govern substrate specificity, and have a conserved cofactor binding domain. AKRs catalyze an ordered bi bi kinetic mechanism in which NAD(P)H cofactor binds first and leaves last. In enzymes that favor NADPH, the rate of release of NADP(+) is governed by a slow isomerization step which places an upper limit on kcat. AKRs retain a conserved catalytic tetrad consisting of Tyr55, Asp50, Lys84, and His117 (AKR1C9 numbering). There is conservation of the catalytic mechanism with short-chain dehydrogenases/reductases (SDRs) even though they show different protein folds. There are 15 human AKRs of these AKR1B1, AKR1C1-1C3, AKR1D1, and AKR1B10 have been implicated in diabetic complications, steroid hormone dependent malignancies, bile acid deficiency and defects in retinoic acid signaling, respectively. Inhibitor programs exist world-wide to target each of these enzymes to treat the aforementioned disorders. Inherited mutations in AKR1C and AKR1D1 enzymes are implicated in defects in the development of male genitalia and bile acid deficiency, respectively, and occur in evolutionarily conserved amino acids. The human AKRs have a large number of nsSNPs and splice variants, but in many instances functional genomics is lacking. AKRs and their variants are now poised to be interrogated using

  20. 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.

  1. Aldo-keto reductases in retinoid metabolism: search for substrate specificity and inhibitor selectivity.

    PubMed

    Porté, Sergio; Xavier Ruiz, F; Giménez, Joan; Molist, Iago; Alvarez, Susana; Domínguez, Marta; Alvarez, Rosana; de Lera, Angel R; Parés, Xavier; Farrés, Jaume

    2013-02-25

    Biological activity of natural retinoids requires the oxidation of retinol to retinoic acid (RA) and its binding to specific nuclear receptors in target tissues. The first step of this pathway, the reversible oxidoreduction of retinol to retinaldehyde, is essential to control RA levels. The enzymes of retinol oxidation are NAD-dependent dehydrogenases of the cytosolic medium-chain (MDR) and the membrane-bound short-chain (SDR) dehydrogenases/reductases. Retinaldehyde reduction can be performed by SDR and aldo-keto reductases (AKR), while its oxidation to RA is carried out by aldehyde dehydrogenases (ALDH). In contrast to SDR, AKR and ALDH are cytosolic. A common property of these enzymes is that they only use free retinoid, but not retinoid bound to cellular retinol binding protein (CRBP). The relative contribution of each enzyme type in retinoid metabolism is discussed in terms of the different subcellular localization, topology of membrane-bound enzymes, kinetic constants, binding affinity of CRBP for retinol and retinaldehyde, and partition of retinoid pools between membranes and cytoplasm. The development of selective inhibitors for AKR enzymes 1B1 and 1B10, of clinical relevance in diabetes and cancer, granted the investigation of some structure-activity relationships. Kinetics with the 4-methyl derivatives of retinaldehyde isomers was performed to identify structural features for substrate specificity. Hydrophilic derivatives were better substrates than the more hydrophobic compounds. We also explored the inhibitory properties of some synthetic retinoids, known for binding to retinoic acid receptors (RAR) and retinoid X receptors (RXR). Consistent with its substrate specificity towards retinaldehyde, AKR1B10 was more effectively inhibited by synthetic retinoids than AKR1B1. A RARβ/γ agonist (UVI2008) inhibited AKR1B10 with the highest potency and selectivity, and docking simulations predicted that its carboxyl group binds to the anion-binding pocket. PMID

  2. Functional studies of aldo-keto reductases in Saccharomyces cerevisiae.

    PubMed

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

    2007-03-01

    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

  3. 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

  4. 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.

  5. 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

  6. The aldo-keto reductase superfamily homepage.

    PubMed

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

    2003-02-01

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

  7. Aldo-keto Reductase 1B15 (AKR1B15): a mitochondrial human aldo-keto reductase with activity toward steroids and 3-keto-acyl-CoA conjugates.

    PubMed

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

    2015-03-01

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

  8. Aldo Keto Reductase 1B7 and Prostaglandin F2α Are Regulators of Adrenal Endocrine Functions

    PubMed Central

    Lambert-Langlais, Sarah; Volat, Fanny; Manin, Michèle; Coudoré, François; Val, Pierre; Sahut-Barnola, Isabelle; Ragazzon, Bruno; Louiset, Estelle; Delarue, Catherine; Lefebvre, Hervé; Urade, Yoshihiro; Martinez, Antoine

    2009-01-01

    Prostaglandin F2α (PGF2α), represses ovarian steroidogenesis and initiates parturition in mammals but its impact on adrenal gland is unknown. Prostaglandins biosynthesis depends on the sequential action of upstream cyclooxygenases (COX) and terminal synthases but no PGF2α synthases (PGFS) were functionally identified in mammalian cells. In vitro, the most efficient mammalian PGFS belong to aldo-keto reductase 1B (AKR1B) family. The adrenal gland is a major site of AKR1B expression in both human (AKR1B1) and mouse (AKR1B3, AKR1B7). Thus, we examined the PGF2α biosynthetic pathway and its functional impact on both cortical and medullary zones. Both compartments produced PGF2α but expressed different biosynthetic isozymes. In chromaffin cells, PGF2α secretion appeared constitutive and correlated to continuous expression of COX1 and AKR1B3. In steroidogenic cells, PGF2α secretion was stimulated by adrenocorticotropic hormone (ACTH) and correlated to ACTH-responsiveness of both COX2 and AKR1B7/B1. The pivotal role of AKR1B7 in ACTH-induced PGF2α release and functional coupling with COX2 was demonstrated using over- and down-expression in cell lines. PGF2α receptor was only detected in chromaffin cells, making medulla the primary target of PGF2α action. By comparing PGF2α-responsiveness of isolated cells and whole adrenal cultures, we demonstrated that PGF2α repressed glucocorticoid secretion by an indirect mechanism involving a decrease in catecholamine release which in turn decreased adrenal steroidogenesis. PGF2α may be regarded as a negative autocrine/paracrine regulator within a novel intra-adrenal feedback loop. The coordinated cell-specific regulation of COX2 and AKR1B7 ensures the generation of this stress-induced corticostatic signal. PMID:19809495

  9. 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.

  10. Catalytic Isomerization of Biomass‐Derived Aldoses: A Review

    PubMed Central

    Delidovich, Irina

    2016-01-01

    Abstract 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

  11. 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.

  12. 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

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

    PubMed

    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.

  14. 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

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

    PubMed

    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

  16. 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.

  17. 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

  18. Some physical and immunological properties of ox kidney biliverdin reductase.

    PubMed Central

    Rigney, E M; Phillips, O; Mantle, T J

    1988-01-01

    The liver, kidney and spleen of the mouse and rat and the kidney and spleen of the ox express a monomeric form of biliverdin reductase (Mr 34,000), which in the case of the ox kidney enzyme exists in two forms (pI 5.4 and 5.2) that are probably charge isomers. The livers of the mouse and rats express, in addition, a protein (Mr 46,000) that cross-reacts with antibodies raised against the ox kidney enzyme and may be related to form 2 described by Frydman, Tomaro, Awruch & Frydman [(1983) Biochim. Biophys. Acta 759, 257-263]. Higher-Mr forms appear to exist in the guinea pig and hamster. The ox kidney enzyme has three thiol groups, of which two are accessible to 5,5'-dithiobis-(2-nitrobenzoate) in the native enzyme. Immunocytochemical analysis reveals that biliverdin reductase is localized in proximal tubules of the inner cortex of the rat kidney. Biliverdin reductase antiserum also stains proximal tubules in human and ox kidney. The staining of podocytes in glomeruli of ox kidney with antiserum to aldose reductase is particularly prominent. The localization of biliverdin reductase in the inner cortical zone of rat kidney is similar to that described for glutathione S-transferase YfYf, and it is suggested that one function of this 'intracellular binding protein' may be to maintain a low free concentration of biliverdin to allow biliverdin reductase to operate efficiently. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:3060109

  19. Functional genomic studies of aldo-keto reductases.

    PubMed

    Petrash, J M; Murthy, B S; Young, M; Morris, K; Rikimaru, L; Griest, T A; Harter, T

    2001-01-30

    Aldose reductase (AR) is considered a potential mediator of diabetic complications and is a drug target for inhibitors of diabetic retinopathy and neuropathy in clinical trials. However, the physiological role of this enzyme still has not been established. Since effective inhibition of diabetic complications will require early intervention, it is important to delineate whether AR fulfills a physiological role that cannot be compensated by an alternate aldo-keto reductase. Functional genomics provides a variety of powerful new tools to probe the physiological roles of individual genes, especially those comprising gene families. Several eucaryotic genomes have been sequenced and annotated, including yeast, nematode and fly. To probe the function of AR, we have chosen to utilize the budding yeast Saccharomyces cerevisiae as a potential model system. Unlike Caenorhabditis elegans and D. melanogaster, yeast provides a more desirable system for our studies because its genome is manipulated more readily and is able to sustain multiple gene deletions in the presence of either drug or auxotrophic selectable markers. Using BLAST searches against the human AR gene sequence, we identified six genes in the complete S. cerevisiae genome with strong homology to AR. In all cases, amino acids thought to play important catalytic roles in human AR are conserved in the yeast AR-like genes. All six yeast AR-like open reading frames (ORFs) have been cloned into plasmid expression vectors. Substrate and AR inhibitor specificities have been surveyed on four of the enzyme forms to identify, which are the most functionally similar to human AR. Our data reveal that two of the enzymes (YDR368Wp and YHR104Wp) are notable for their similarity to human AR in terms of activity with aldoses and substituted aromatic aldehydes. Ongoing studies are aimed at characterizing the phenotypes of yeast strains containing single and multiple knockouts of the AR-like genes. PMID:11306085

  20. 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

  1. 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.

  2. Base catalysed isomerisation of aldoses of the arabino and lyxo series in the presence of aluminate.

    PubMed

    Ekeberg, Dag; Morgenlie, Svein; Stenstrøm, Yngve

    2002-04-30

    Base-catalysed isomerisation of aldoses of the arabino and lyxo series in aluminate solution has been investigated. L-Arabinose and D-galactose give L-erythro-2-pentulose (L-ribulose) and D-lyxo-2-hexulose (D-tagatose), respectively, in good yields, whereas lower reactivity is observed for 6-deoxy-D-galactose (D-fucose). From D-lyxose, D-mannose and 6-deoxy-L-mannose (L-rhamnose) are obtained mixtures of ketoses and C-2 epimeric aldoses. Small amounts of the 3-epimers of the ketoses were also formed. 6-Deoxy-L-arabino-2-hexulose (6-deoxy-L-fructose) and 6-deoxy-L-glucose (L-quinovose) were formed in low yields from 6-deoxy-L-mannose and isolated as their O-isopropylidene derivatives. Explanations of the differences in reactivity and course of the reaction have been suggested on the basis of steric effects.

  3. Expression of AKR1C3 and CNN3 as markers for detection of lymph node metastases in colorectal cancer.

    PubMed

    Nakarai, Chiaki; Osawa, Kayo; Akiyama, Minami; Matsubara, Nagahide; Ikeuchi, Hiroki; Yamano, Tomoki; Hirota, Seiichi; Tomita, Naohiro; Usami, Makoto; Kido, Yoshiaki

    2015-08-01

    The aim of the study was to identify a set of discriminating genes that could be used for the prediction of Lymph node (LN) metastasis in human colorectal cancer (CRC), and for this, we compared the whole genome profiles of two CRC cell lines (the primary cell line SW480 and its LN metastatic variant, SW620) and identified eight genes [S100 calcium-binding protein P; aldo-keto reductase family 1(AKR1), member B1 (aldose reductase; AKR1B1); AKR1, member C3 (AKR1C3); calponin 3, acidic; metastasis associated in colon cancer 1; hemoglobin, epsilon 1; trefoil factor 3; and FGGY carbohydrate kinase domain containing]. These genes were examined by quantitative RT-PCR in tissues and LNs in 14 CRC patients and 11 control patients. The level of AKR1C3 mRNA expression was significantly different between the Dukes' stage A, B, and C groups and the control group (p < 0.05, p < 0.001, and p < 0.001) and was also significantly different between Dukes' stage C and A or B groups (p < 0.05 and p < 0.001, respectively). The expression of CNN3 was significantly different between the Dukes' stage C and B or control groups (p < 0.001 and p < 0.01, respectively). There were significant correlations between the expression levels of AKR1C3 and CNN3. AKR1C3 and CNN3 expressions are more accurate and suitable markers for the diagnosis of LN metastasis than the other six genes examined in this study.

  4. 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

  5. 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.

  6. Sequence analysis of frog rho-crystallin by cDNA cloning and sequencing: a member of the aldo-keto reductase family.

    PubMed

    Lu, S F; Pan, F M; Chiou, S H

    1995-09-25

    rho-Crystallin is a major enzyme crystallin present in the lenses of amphibian species with a blocked amino terminus. In order to facilitate the determination of the primary sequence of this taxon-specific crystallin, cDNA mixture was synthesized from the poly(A)+mRNA of bullfrog eye lenses. cDNAs encoding rho-crystallin were then amplified by polymerase chain reaction (PCR) using a new protocol of Rapid Amplification of cDNA Ends (RACE). PCR-amplified product corresponding to rho-crystallin was obtained, which was then subcloned into pUC18 vector and then transformed into E. coli strain JM109. Plasmids purified from the positive clones were prepared for nucleotide sequencing by the automatic fluorescence-based dideoxynucleotide chain-termination method. Sequencing more than 15 clones containing DNA inserts coding for rho-crystallin constructed only one unique and complete full-length reading frame of 975 base pairs covering a deduced protein sequence of 324 amino acids including the universal initiating methionine. It shows 96, 59, 46 and 37 percent sequence similarity to another rho-crystallin from European common frog, bovine prostaglandin-F synthase, human aldose reductase and human aldehyde reductase, respectively, revealing the close relationship between rho-crystallins from related amphibian species and its possible evolutionary relatedness with various aldo-keto reductases. In this study a phylogenetic tree for rho-crystallin and related enzymes is constructed based on multiple-sequence alignment program using a combination of distance matrix and approximate parsimony methods. We have thus established the remote phylogenetic relationship between rho-crystallin and some aldehyde/aldose reductases, which may provide a possible link for the recruitment of this crystallin from detoxification-related enzymes and its physiological role in maintaining a transparent and clear lens.

  7. 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

  8. 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

  9. Effect of thermal stability on protein adsorption to silica using homologous aldo-keto reductases

    PubMed Central

    Felsovalyi, Flora; Patel, Tushar; Mangiagalli, Paolo; Kumar, Sanat K; Banta, Scott

    2012-01-01

    Gaining more insight into the mechanisms governing the behavior of proteins at solid/liquid interfaces is particularly relevant in the interaction of high-value biologics with storage and delivery device surfaces, where adsorption-induced conformational changes may dramatically affect biocompatibility. The impact of structural stability on interfacial behavior has been previously investigated by engineering nonwild-type stability mutants. Potential shortcomings of such approaches include only modest changes in thermostability, and the introduction of changes in the topology of the proteins when disulfide bonds are incorporated. Here we employ two members of the aldo-keto reductase superfamily (alcohol dehydrogenase, AdhD and human aldose reductase, hAR) to gain a new perspective on the role of naturally occurring thermostability on adsorbed protein arrangement and its subsequent impact on desorption. Unexpectedly, we find that during initial adsorption events, both proteins have similar affinity to the substrate and undergo nearly identical levels of structural perturbation. Interesting differences between AdhD and hAR occur during desorption and both proteins exhibit some level of activity loss and irreversible conformational change upon desorption. Although such surface-induced denaturation is expected for the less stable hAR, it is remarkable that the extremely thermostable AdhD is similarly affected by adsorption-induced events. These results question the role of thermal stability as a predictor of protein adsorption/desorption behavior. PMID:22619179

  10. 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

  11. Investigation of the functional role of aldose 1-epimerase in engineered cellobiose utilization.

    PubMed

    Li, Sijin; Ha, Suk-Jin; Kim, Hee Jin; Galazka, Jonathan M; Cate, Jamie H D; Jin, Yong-Su; Zhao, Huimin

    2013-10-10

    Functional expression of a cellodextrin transporter and an intracellular β-glucosidase from Neurospora crassa in Saccharomyces cerevisiae enables simultaneous co-fermentation of cellobiose and non-glucose sugars such as xylose. Here we investigate the functional role of aldose 1-epimerase (AEP) in engineered cellobiose utilization. One AEP (Gal10) and two putative AEPs (Yhr210c and Ynr071c sharing 50.6% and 51.0% amino acid identity with Gal10, respectively) were selected. Deletion of GAL10 led to complete loss of both AEP activity and cell growth on cellobiose, while GAL10 complementation restored the AEP activity and cell growth. In addition, deletion of YHR210C or YNR071C resulted in improved cellobiose utilization. These results suggest that the intracellular mutarotation of β-glucose to α-glucose might be a rate controlling step and Gal10 play a crucial role in cellobiose fermentation by engineered S. cerevisiae. PMID:23954547

  12. 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.

  13. 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.

  14. 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

  15. 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.

  16. 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 ...

  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.

  18. Ribonucleotide Reductase-- a Radical Enzyme

    NASA Astrophysics Data System (ADS)

    Reichard, Peter; Ehrenberg, Anders

    1983-08-01

    Ribonucleotide reductases catalyze the enzymatic formation of deoxyribonucleotides, an obligatory step in DNA synthesis. The native form of the enzyme from Escherichia coli or from mammalian sources contains as part of its polypeptide structure a free tyrosyl radical, stabilized by an iron center. The radical participates in all probability in the catalytic process during the substitution of the hydroxyl group at C-2 of ribose by a hydrogen atom. A second, inactive form of the E. coli reductase lacks the tyrosyl radical. Extracts from E. coli contain activities that interconvert the two forms. The tyrosyl radical is introduced in the presence of oxygen, while anaerobiosis favors its removal, suggesting a regulatory role in DNA synthesis for oxygen.

  19. Nitrate reductase from Rhodopseudomonas sphaeroides.

    PubMed Central

    Kerber, N L; Cardenas, J

    1982-01-01

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

  20. 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.

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

    PubMed Central

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

    1996-01-01

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

  2. 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.

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

    PubMed Central

    Galván, Aurora; Cárdenas, Jacobo; Fernández, Emilio

    1992-01-01

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

  4. Biliverdin reductase isozymes in metabolism.

    PubMed

    O'Brien, Luke; Hosick, Peter A; John, Kezia; Stec, David E; Hinds, Terry D

    2015-04-01

    The biliverdin reductase (BVR) isozymes BVRA and BVRB are cell surface membrane receptors with pleiotropic functions. This review compares, for the first time, the structural and functional differences between the isozymes. They reduce biliverdin, a byproduct of heme catabolism, to bilirubin, display kinase activity, and BVRA, but not BVRB, can act as a transcription factor. The binding motifs present in the BVR isozymes allow a wide range of interactions with components of metabolically important signaling pathways such as the insulin receptor kinase cascades, protein kinases (PKs), and inflammatory mediators. In addition, serum bilirubin levels have been negatively associated with abdominal obesity and hypertriglyceridemia. We discuss the roles of the BVR isozymes in metabolism and their potential as therapeutic targets. PMID:25726384

  5. 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

  6. 5 alpha-reductase deficiency without hypospadias.

    PubMed Central

    Ng, W K; Taylor, N F; Hughes, I A; Taylor, J; Ransley, P G; Grant, D B

    1990-01-01

    A boy aged 4 with penoscrotal hypospadias and his brother aged 12 with micropenis had typical changes of homozygous 5 alpha-reductase deficiency. After three injections of chorionic gonadotrophin there was a trivial rise in plasma dihydrotestosterone with a normal increase in plasma testosterone. Urine steroid chromatography showed abnormally high 5 beta: 5 alpha ratios and 5 alpha-reductase activity was appreciably reduced in genital skin fibroblasts. The results indicate that 5 alpha-reductase deficiency is not invariably associated with genital ambiguity. PMID:2248513

  7. 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 ...

  8. 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.

  9. 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

  10. Differential expression of the enzymatic system controlling synthesis, metabolism, and transport of PGF2 alpha in human fetal membranes.

    PubMed

    Breuiller-Fouché, Michelle; Leroy, Marie-Josèphe; Dubois, Olivier; Reinaud, Pierrette; Chissey, Audrey; Qi, Hong; Germain, Guy; Fortier, Michel A; Charpigny, Gilles

    2010-07-01

    The present study investigated the expression of genes and proteins associated with PGF2alpha biosynthesis, catabolism, and transport in matched amnion and choriodecidua of human term placenta. The concentration of PGF2alpha within fetal membranes depends on the balance between complex enzymatic systems responsible for, respectively, its synthesis-by prostaglandin-endoperoxide synthase 2 (PTGS2) and members of the aldo-keto reductase (AKR) family, AKR1C3 and AKR1B1-and its catabolic inactivation-through hydroxy-prostaglandin-dehydrogenase (HPGD). We observed that AKR1C3 shows equal basal expression (mRNA and protein) in choriodecidua and amnion but that AKR1B1 exhibits preferential expression in the choriodecidua. Expression of HPGD and solute carrier organic anion transporter family member 2A1 (SLCO2A1) was found primarily in the choriodecidua. We also evaluated whether an inflammatory environment induced by the gram-negative bacterial endotoxin lipopolysaccharide (LPS) affects expression of each candidate enzymes. The amnion responded to LPS with a small but significant decrease of AKR1B1 mRNA expression. In contrast, we found a significant increase in PTGS2 and AKR1C3 mRNA expression in choriodecidua after LPS challenge, but such regulation was confirmed only at protein levels for PTGS2 and not for AKR1C3. Our results suggest that the choriodecidua appears to be the main tissue, which expresses maximally all the components (synthesis, degradation, and transport) controlling PGF2alpha levels. PMID:20357271

  11. 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

  12. 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.

  13. Respiratory arsenate reductase as a bidirectional enzyme

    SciTech Connect

    Richey, Christine; Chovanec, Peter; 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.

  14. Evolution of plant defense mechanisms. Relationships of phenylcoumaran benzylic ether reductases to pinoresinol-lariciresinol and isoflavone reductases.

    PubMed

    Gang, D R; Kasahara, H; Xia, Z Q; Vander Mijnsbrugge, K; Bauw, G; Boerjan, W; Van Montagu, M; Davin, L B; Lewis, N G

    1999-03-12

    Pinoresinol-lariciresinol and isoflavone reductase classes are phylogenetically related, as is a third, the so-called "isoflavone reductase homologs." This study establishes the first known catalytic function for the latter, as being able to engender the NADPH-dependent reduction of phenylcoumaran benzylic ethers. Accordingly, all three reductase classes are involved in the biosynthesis of important and related phenylpropanoid-derived plant defense compounds. In this investigation, the phenylcoumaran benzylic ether reductase from the gymnosperm, Pinus taeda, was cloned, with the recombinant protein heterologously expressed in Escherichia coli. The purified enzyme reduces the benzylic ether functionalities of both dehydrodiconiferyl alcohol and dihydrodehydrodiconiferyl alcohol, with a higher affinity for the former, as measured by apparent Km and Vmax values and observed kinetic 3H-isotope effects. It abstracts the 4R-hydride of the required NADPH cofactor in a manner analogous to that of the pinoresinol-lariciresinol reductases and isoflavone reductases. A similar catalytic function was observed for the corresponding recombinant reductase whose gene was cloned from the angiosperm, Populus trichocarpa. Interestingly, both pinoresinol-lariciresinol reductases and isoflavone reductases catalyze enantiospecific conversions, whereas the phenylcoumaran benzylic ether reductase only shows regiospecific discrimination. A possible evolutionary relationship among the three reductase classes is proposed, based on the supposition that phenylcoumaran benzylic ether reductases represent the progenitors of pinoresinol-lariciresinol and isoflavone reductases.

  15. 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

  16. 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

  17. Induction of IFNT-Stimulated Genes by Conceptus-Derived Exosomes during the Attachment Period.

    PubMed

    Nakamura, Keigo; Kusama, Kazuya; Bai, Rulan; Sakurai, Toshihiro; Isuzugawa, Kazuto; Godkin, James D; Suda, Yoshihito; Imakawa, Kazuhiko

    2016-01-01

    Biochemical and/or physical communication between the conceptus and the uterine endometrium is required for conceptus implantation to the maternal endometrium, leading to placentation and the establishment of pregnancy. We previously reported that in vitro co-culture system with bovine trophoblast CT-1 cells, primary uterine endometrial epithelial cells (EECs), and uterine flushings (UFs) mimics in vivo conceptus attachment process. To identify molecules in UFs responsible for this change, we first characterized protein contents of UFs from day 17 cyclic (C17) and pregnant (P17) ewes through the use of two dimensional-Polyacrylamide Gel Electrophoresis (2D-PAGE), followed by Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS) analysis. These analyses identified 266 proteins specific for P17 UFs, from which 172 proteins were identified as exosomal proteins. Among 172 exosomal proteins, 8 proteins that had been identified as exosomal proteins were chosen for further analysis, including macrophage-capping protein (CAPG), aldo-keto reductase family 1, member B1 protein (AKR1B1), bcl-2-like protein 15 (BCL2L15), carbonic anhydrase 2 (CA2), isocitrate dehydrogenase 2 (IDH2), eukaryotic translation elongation factor 2 (EEF2), moesin (MSN), and ezrin (EZR). CAPG and AKR1B1 were again confirmed in P15 and P17 UFs, and more importantly CAPG and AKR1B1, mRNA and protein, were found only in P15 and P17 conceptuses. Moreover, exosomes were isolated from C15, C17, P15, or P17 UFs. Only P15 and P17 exosomes, originated from the conceptus, contained interferon tau (IFNT) as well as CAPG and AKR1B1, and up-regulated STAT1, STAT2, MX1, MX2, BST2, and ISG15 transcripts in EECs. These observations indicate that in addition to endometrial derived exosomes previously described, conceptus-derived exosomes are present in UFs and could function to modify endometrial response. These results suggest that exosomes secreted from conceptuses as well as endometria are involved in cell to

  18. Induction of IFNT-Stimulated Genes by Conceptus-Derived Exosomes during the Attachment Period

    PubMed Central

    Nakamura, Keigo; Kusama, Kazuya; Bai, Rulan; Sakurai, Toshihiro; Isuzugawa, Kazuto; Godkin, James D.; Suda, Yoshihito; Imakawa, Kazuhiko

    2016-01-01

    Biochemical and/or physical communication between the conceptus and the uterine endometrium is required for conceptus implantation to the maternal endometrium, leading to placentation and the establishment of pregnancy. We previously reported that in vitro co-culture system with bovine trophoblast CT-1 cells, primary uterine endometrial epithelial cells (EECs), and uterine flushings (UFs) mimics in vivo conceptus attachment process. To identify molecules in UFs responsible for this change, we first characterized protein contents of UFs from day 17 cyclic (C17) and pregnant (P17) ewes through the use of two dimensional-Polyacrylamide Gel Electrophoresis (2D-PAGE), followed by Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS) analysis. These analyses identified 266 proteins specific for P17 UFs, from which 172 proteins were identified as exosomal proteins. Among 172 exosomal proteins, 8 proteins that had been identified as exosomal proteins were chosen for further analysis, including macrophage-capping protein (CAPG), aldo-keto reductase family 1, member B1 protein (AKR1B1), bcl-2-like protein 15 (BCL2L15), carbonic anhydrase 2 (CA2), isocitrate dehydrogenase 2 (IDH2), eukaryotic translation elongation factor 2 (EEF2), moesin (MSN), and ezrin (EZR). CAPG and AKR1B1 were again confirmed in P15 and P17 UFs, and more importantly CAPG and AKR1B1, mRNA and protein, were found only in P15 and P17 conceptuses. Moreover, exosomes were isolated from C15, C17, P15, or P17 UFs. Only P15 and P17 exosomes, originated from the conceptus, contained interferon tau (IFNT) as well as CAPG and AKR1B1, and up-regulated STAT1, STAT2, MX1, MX2, BST2, and ISG15 transcripts in EECs. These observations indicate that in addition to endometrial derived exosomes previously described, conceptus-derived exosomes are present in UFs and could function to modify endometrial response. These results suggest that exosomes secreted from conceptuses as well as endometria are involved in cell to

  19. Structural prototypes for an extended family of flavoprotein reductases: comparison of phthalate dioxygenase reductase with ferredoxin reductase and ferredoxin.

    PubMed Central

    Correll, C. C.; Ludwig, M. L.; Bruns, C. M.; Karplus, P. A.

    1993-01-01

    The structure of phthalate dioxygenase reductase (PDR), a monomeric iron-sulfur flavoprotein that delivers electrons from NADH to phthalate dioxygenase, is compared to ferredoxin-NADP+ reductase (FNR) and ferredoxin, the proteins that reduce NADP+ in the final reaction of photosystem I. The folding patterns of the domains that bind flavin, NAD(P), and [2Fe-2S] are very similar in the two systems. Alignment of the X-ray structures of PDR and FNR substantiates the assignment of features that characterize a family of flavoprotein reductases whose members include cytochrome P-450 reductase, sulfite and nitrate reductases, and nitric oxide synthase. Hallmarks of this subfamily of flavoproteins, here termed the FNR family, are an antiparallel beta-barrel that binds the flavin prosthetic group, and a characteristic variant of the classic pyridine nucleotide-binding fold. Despite the similarities between FNR and PDR, attempts to model the structure of a dissociable FNR:ferredoxin complex by analogy with PDR reveal features that are at odds with chemical crosslinking studies (Zanetti, G., Morelli, D., Ronchi, S., Negri, A., Aliverti, A., & Curti, B., 1988, Biochemistry 27, 3753-3759). Differences in the binding sites for flavin and pyridine nucleotides determine the nucleotide specificities of FNR and PDR. The specificity of FNR for NADP+ arises primarily from substitutions in FNR that favor interactions with the 2' phosphate of NADP+. Variations in the conformation and sequences of the loop adjoining the flavin phosphate affect the selectivity for FAD versus FMN. The midpoint potentials for reduction of the flavin and [2Fe-2S] groups in PDR are higher than their counterparts in FNR and spinach ferredoxin, by about 120 mV and 260 mV, respectively. Comparisons of the structure of PDR with spinach FNR and with ferredoxin from Anabaena 7120, along with calculations of electrostatic potentials, suggest that local interactions, including hydrogen bonds, are the dominant

  20. 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

  1. 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'.

  2. 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...

  3. Synthesis of Nitrate Reductase in Chlorella

    PubMed Central

    Funkhouser, Edward A.; Shen, Teh-Chien; Ackermann, Renate

    1980-01-01

    Synthesis of nitrate reductase (EC 1.6.6.1) in Chlorella vulgaris was studied under inducing conditions, i.e. with cells grown on ammonia and then transferred to nitrate medium. Cycloheximide (but not chloramphenicol) completely inhibited synthesis of the enzyme, but only if it was added at the start (i.e. at the time of nitrate addition) of the induction period. Cycloheximide inhibition became less effective as induction by nitrate proceeded. Enzyme from small quantities of culture (1 to 3 milliliters of packed cells) was purified to homogeneity with the aid of blue dextran-Sepharose chromatography. Incorporation of radioactivity from labeled arginine into nitrate reductase was measured in the presence and absence of cycloheximide. Conditions were found under which the inhibitor completely blocked the incorporation of labeled amino acid, but only slightly decreased the increase in nitrate reductase activity. The results indicate that synthesis of nitrate reductase from amino acids proceeds by way of a protein precursor which is inactive enzymically. PMID:16661310

  4. Control of dihydrofolate reductase messenger ribonucleic acid production

    SciTech Connect

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

    1981-11-01

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

  5. Augmentation of CFTR maturation by S-nitrosoglutathione reductase.

    PubMed

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

    2016-02-01

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

  6. Steroid 5α-reductase 2 deficiency.

    PubMed

    Mendonca, Berenice B; Batista, Rafael Loch; Domenice, Sorahia; Costa, Elaine M F; Arnhold, Ivo J P; Russell, David W; Wilson, Jean D

    2016-10-01

    Dihydrotestosterone is a potent androgen metabolite formed from testosterone by action of 5α-reductase isoenzymes. Mutations in the type 2 isoenzyme cause a disorder of 46,XY sex development, termed 5α-reductase type 2 deficiency and that was described forty years ago. Many mutations in the encoding gene have been reported in different ethnic groups. In affected 46,XY individuals, female external genitalia are common, but Mullerian ducts regress, and the internal urogenital tract is male. Most affected males are raised as females, but virilization occurs at puberty, and male social sex develops thereafter with high frequency. Fertility can be achieved in some affected males with assisted reproduction techniques, and adults with male social sex report a more satisfactory sex life and quality of life as compared to affected individuals with female social sex. PMID:27224879

  7. Discovery of pinoresinol reductase genes in sphingomonads.

    PubMed

    Fukuhara, Y; Kamimura, N; Nakajima, M; Hishiyama, S; Hara, H; Kasai, D; Tsuji, Y; Narita-Yamada, S; Nakamura, S; Katano, Y; Fujita, N; Katayama, Y; Fukuda, M; Kajita, S; Masai, E

    2013-01-10

    Bacterial genes for the degradation of major dilignols produced in lignifying xylem are expected to be useful tools for the structural modification of lignin in plants. For this purpose, we isolated pinZ involved in the conversion of pinoresinol from Sphingobium sp. strain SYK-6. pinZ showed 43-77% identity at amino acid level with bacterial NmrA-like proteins of unknown function, a subgroup of atypical short chain dehydrogenases/reductases, but revealed only 15-21% identity with plant pinoresinol/lariciresinol reductases. PinZ completely converted racemic pinoresinol to lariciresinol, showing a specific activity of 46±3 U/mg in the presence of NADPH at 30°C. In contrast, the activity for lariciresinol was negligible. This substrate preference is similar to a pinoresinol reductase, AtPrR1, of Arabidopsis thaliana; however, the specific activity of PinZ toward (±)-pinoresinol was significantly higher than that of AtPrR1. The role of pinZ and a pinZ ortholog of Novosphingobium aromaticivorans DSM 12444 were also characterized.

  8. Carbohydrate utilization in Streptococcus thermophilus: characterization of the genes for aldose 1-epimerase (mutarotase) and UDPglucose 4-epimerase.

    PubMed Central

    Poolman, B; Royer, T J; Mainzer, S E; Schmidt, B F

    1990-01-01

    The complete nucleotide sequences of the genes encoding aldose 1-epimerase (mutarotase) (galM) and UDPglucose 4-epimerase (galE) and flanking regions of Streptococcus thermophilus have been determined. Both genes are located immediately upstream of the S. thermophilus lac operon. To facilitate the isolation of galE, a special polymerase chain reaction-based technique was used to amplify the region upstream of galM prior to cloning. The galM protein was homologous to the mutarotase of Acinetobacter calcoaceticus, whereas the galE protein was homologous to UDPglucose 4-epimerase of Escherichia coli and Streptomyces lividans. The amino acid sequences of galM and galE proteins also showed significant similarity with the carboxy-terminal and amino-terminal domains, respectively, of UDPglucose 4-epimerase from Kluyveromyces lactis and Saccharomyces cerevisiae, suggesting that the yeast enzymes contain an additional, yet unidentified (mutarotase) activity. In accordance with the open reading frames of the structural genes, galM and galE were expressed as polypeptides with apparent molecular masses of 39 and 37 kilodaltons, respectively. Significant activities of mutarotase and UDPglucose 4-epimerase were detected in lysates of E. coli cells containing plasmids encoding galM and galE. Expression of galE in E. coli was increased 300-fold when the gene was placed downstream of the tac promoter. The gene order for the gal-lac gene cluster of S. thermophilus is galE-galM-lacS-lacZ. The flanking regions of these genes were searched for consensus promoter sequences and further characterized by primer extension analysis. Analysis of mRNA levels for the gal and lac genes in S. thermophilus showed a strong reduction upon growth in medium containing glucose instead of lactose. The activities of the lac (lactose transport and beta-galactosidase) and gal (UDPglucose 4-epimerase) proteins of lactose- and glucose-grown S. thermophilus cells matched the mRNA levels. Images PMID:1694527

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

    PubMed

    Wei, Yifeng; Li, Bin; Prakash, Divya; Ferry, James G; Elliott, Sean J; Stubbe, JoAnne

    2015-12-01

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

  10. 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

  11. 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.

  12. Radical scavengers as ribonucleotide reductase inhibitors.

    PubMed

    Basu, Arijit; Sinha, Barij Nayan

    2012-01-01

    This paper compiled all the previous reports on radical scavengers, an interesting class of ribonucleotide reductase inhibitors. We have highlighted three key research areas: chemical classification of radical scavengers, structural and functional aspects of the radical site, and progress in drug designing for radical scavengers. Under the chemical classification section, we have recorded the discovery of hydroxyurea followed by discussions on hydroxamic acids, amidoximes, hydroxyguanidines, and phenolic compounds. In the next section, we have compiled the structural information for the radical site obtained from different crystallographic and theoretical studies. Finally, we have included the reported ligand based and structure based drug-designing studies.

  13. A mediated glucose/oxygen enzymatic fuel cell based on printed carbon inks containing aldose dehydrogenase and laccase as anode and cathode.

    PubMed

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

    2012-03-10

    Enzyme electrodes show great potential for many applications, as biosensors and more recently as anodes and cathodes in biocatalytic fuel cells for power generation. Enzymes have advantages over metal catalysts, as they provide high specificity and reaction rates, while operating under mild conditions. Here we report on studies related to development of mass-producible, completely enzymatic printed glucose/oxygen biofuel cells. The cells are based on filter paper coated with conducting carbon inks containing mediators and laccase, for reduction of oxygen, or aldose dehydrogenase, for oxidation of glucose. Mediator performance in these printed formats is compared to relative rate constants for the enzyme-mediator reaction in solution, for a range of anode and cathode mediators. The power output and stability of fuels cells using an acidophilic laccase isolated from Trametes hirsuta is greater, at pH 5, than that for cells based on Melanocarpus albomyces laccase, that shows optimal activity closer to neutral pH, at pH 6. Highest power output, although of limited stability, was observed for ThL/ABTS cathodes, providing a maximum power density of 3.5 μWcm(-2) at 0.34 V, when coupled to an ALDH glucose anode mediated by an osmium complex. The stability of cell voltage above a threshold of 200 mV under a moderate 75 kΩ load is used to benchmark printed fuel cell performance. Highest stability was obtained for a printed fuel cell using osmium complexes as mediators of glucose oxidation by aldose dehydrogenase, and oxygen reduction by T. hirsuta laccase, maintaining cell voltage above 200 mV for 137 h at pH 5. These results provide promising directions for further development of mass-producible, completely enzymatic, printed biofuel cells. PMID:22305173

  14. Structure of an integral membrane sterol reductase from Methylomicrobium alcaliphilum

    PubMed Central

    Li, Xiaochun; Roberti, Rita; Blobel, Günter

    2014-01-01

    Sterols are essential biological molecules in the majority of life forms. Sterol reductases1 including Delta-14 sterol reductase (C14SR), 7-dehydrocholesterol reductase (DHCR7) and 24-dehydrocholesterol reductase (DHCR24) reduce specific carbon-carbon double bonds of the sterol moiety using a reducing cofactor during sterol biosynthesis. Lamin B Receptor2 (LBR), an integral inner nuclear membrane protein, also contains a functional C14SR domain. Here we report the crystal structure of a Delta-14 sterol reductase (maSR1) from the methanotrophic bacterium Methylomicrobium alcaliphilum 20Z, a homolog of human C14SR, LBR, and DHCR7, with the cofactor NADPH. The enzyme contains 10 transmembrane segments (TM). Its catalytic domain comprises the C-terminal half (containing TM6-10) and envelops two interconnected pockets, one of which faces the cytoplasm and houses NADPH, while the other one is accessible from the lipid bilayer. Comparison with a soluble steroid 5β-reductase structure3 suggests that the reducing end of NADPH meets the sterol substrate at the juncture of the two pockets. A sterol reductase activity assay proves maSR1 can reduce the double bond of a cholesterol biosynthetic intermediate demonstrating functional conservation to human C14SR. Therefore, our structure as a prototype of integral membrane sterol reductases provides molecular insight into mutations in DHCR7 and LBR for inborn human diseases. PMID:25307054

  15. 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

  16. Ageing of glutathione reductase in the lens.

    PubMed

    Zhang, W Z; Augusteyn, R C

    1994-07-01

    The distribution of glutathione reductase activity in concentric layers from the lens has been determined as a function of age for 16 species. Primate lenses have almost ten times the level of glutathione reductase found in other species. Comparison with the activity of hexokinase revealed that this is not due to a higher overall rate of metabolism in these lenses. By contrast, the higher activity found in bird and fish lenses reflects a higher metabolic activity in these tissues. In all species, a gradient of activity was observed with the highest specific activity in the outermost cortical fibres, decreasing to virtually no activity in the inner parts of the tissue. No alterations were found in this gradient with increasing age, other than an increase in the amount of nuclear tissue essentially devoid of activity. The maximum activity in the outer cortical fibres was the same, regardless of the age of the lens. The time taken, in different species, for the specific activity to decrease by half, was estimated from the rate of protein accumulation. This time was found to vary from a few days to several years, indicating that the decrease in activity is not due to ageing but rather, it is related to the maturation of fibre cells. These observations are discussed in terms of current concepts of lens ageing and cataract formation. PMID:7835401

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

    PubMed

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

    2002-05-01

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

  18. 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.

  19. 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

  20. 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

  1. Ribonucleotide reductases: essential enzymes for bacterial life

    PubMed Central

    Torrents, Eduard

    2014-01-01

    Ribonucleotide reductase (RNR) is a key enzyme that mediates the synthesis of deoxyribonucleotides, the DNA precursors, for DNA synthesis in every living cell. This enzyme converts ribonucleotides to deoxyribonucleotides, the building blocks for DNA replication, and repair. Clearly, RNR enzymes have contributed to the appearance of genetic material that exists today, being essential for the evolution of all organisms on Earth. The strict control of RNR activity and dNTP pool sizes is important, as pool imbalances increase mutation rates, replication anomalies, and genome instability. Thus, RNR activity should be finely regulated allosterically and at the transcriptional level. In this review we examine the distribution, the evolution, and the genetic regulation of bacterial RNRs. Moreover, this enzyme can be considered an ideal target for anti-proliferative compounds designed to inhibit cell replication in eukaryotic cells (cancer cells), parasites, viruses, and bacteria. PMID:24809024

  2. Ribonucleotide reductases: essential enzymes for bacterial life.

    PubMed

    Torrents, Eduard

    2014-01-01

    Ribonucleotide reductase (RNR) is a key enzyme that mediates the synthesis of deoxyribonucleotides, the DNA precursors, for DNA synthesis in every living cell. This enzyme converts ribonucleotides to deoxyribonucleotides, the building blocks for DNA replication, and repair. Clearly, RNR enzymes have contributed to the appearance of genetic material that exists today, being essential for the evolution of all organisms on Earth. The strict control of RNR activity and dNTP pool sizes is important, as pool imbalances increase mutation rates, replication anomalies, and genome instability. Thus, RNR activity should be finely regulated allosterically and at the transcriptional level. In this review we examine the distribution, the evolution, and the genetic regulation of bacterial RNRs. Moreover, this enzyme can be considered an ideal target for anti-proliferative compounds designed to inhibit cell replication in eukaryotic cells (cancer cells), parasites, viruses, and bacteria. PMID:24809024

  3. Ribonucleotide reductases: essential enzymes for bacterial life.

    PubMed

    Torrents, Eduard

    2014-01-01

    Ribonucleotide reductase (RNR) is a key enzyme that mediates the synthesis of deoxyribonucleotides, the DNA precursors, for DNA synthesis in every living cell. This enzyme converts ribonucleotides to deoxyribonucleotides, the building blocks for DNA replication, and repair. Clearly, RNR enzymes have contributed to the appearance of genetic material that exists today, being essential for the evolution of all organisms on Earth. The strict control of RNR activity and dNTP pool sizes is important, as pool imbalances increase mutation rates, replication anomalies, and genome instability. Thus, RNR activity should be finely regulated allosterically and at the transcriptional level. In this review we examine the distribution, the evolution, and the genetic regulation of bacterial RNRs. Moreover, this enzyme can be considered an ideal target for anti-proliferative compounds designed to inhibit cell replication in eukaryotic cells (cancer cells), parasites, viruses, and bacteria.

  4. Monodehydroascorbate reductase mediates TNT toxicity in plants.

    PubMed

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

    2015-09-01

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

  5. 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

  6. The cytochrome bd respiratory oxygen reductases.

    PubMed

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

    2011-11-01

    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.

  7. Nitrate Reductase-Deficient Mutants in Barley 1

    PubMed Central

    Somers, David A.; Kuo, Tsung-Min; Kleinhofs, Andris; Warner, Robert L.

    1983-01-01

    Nitrate reductase-deficient barley (Hordeum vulgare L.) mutants were assayed for the presence of a functional molybdenum cofactor determined from the activity of the molybdoenzyme, xanthine dehydrogenase, and for nitrate reductase-associated activities. Rocket immunoelectrophoresis was used to detect nitrate reductase cross-reacting material in the mutants. The cross-reacting material levels of the mutants ranged from 8 to 136% of the wild type and were correlated with their nitrate reductase-associated activities, except for nar 1c, which lacked all associated nitrate reductase activities but had 38% of the wild-type cross-reacting material. The cross-reacting material of two nar 1 mutants, as well as nar 2a, Xno 18, Xno 19, and Xno 29, exhibited rocket immunoprecipitates that were similar to the wild-type enzyme indicating structural homology between the mutant and wild-type nitrate reductase proteins. The cross-reacting materials of the seven remaining nar 1 alleles formed rockets only in the presence of purified wild-type nitrate reductase, suggesting structural modifications of the mutant cross-reacting materials. All nar 1 alleles and Xno 29 had xanthine dehydrogenase activity indicating the presence of functional molybdenum cofactors. These results suggest that nar 1 is the structural gene for nitrate reductase. Mutants nar 2a, Xno 18, and Xno 19 lacked xanthine dehydrogenase activity and are considered to be molybdenum cofactor deficient mutants. Cross-reacting material was not detected in uninduced wild-type or mutant extracts, suggesting that nitrate reductase is synthesized de novo in response to nitrate. Images Fig. 1 Fig. 3 PMID:16662774

  8. 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.

  9. Substrate induction of nitrate reductase in barley aleurone layers.

    PubMed

    Ferrari, T E; Varner, J E

    1969-01-01

    Nitrate induces the formation of nitrate reductase activity in barley (Hordeum vulgare L. cv. Himalaya) aleurone layers. Previous work has demonstrated de novo synthesis of alpha-amylase by gibberellic acid in the same tissue. The increase in nitrate reductase activity is inhibited by cycloheximide and 6-methylpurine, but not by actinomycin D. Nitrate does not induce alpha-amylase synthesis, and it has no effect on the gibberellic acid-induced synthesis of alpha-amylase. Also, there is little or no direct effect of gibberellic acid (during the first 6 hr of induction) or of abscisic acid on the nitrate-induced formation of nitrate reductase. Gibberellic acid does interfere with nitrate reductase activity during long-term experiments (greater than 6 hr). However, the time course of this inhibition suggests that the inhibition may be a secondary one. Barley aleurone layers therefore provide a convenient tissue for the study of both substrate- and hormone-induced enzyme formation.

  10. 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

  11. 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

  12. 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

  13. 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.

  14. 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

  15. Role of 5 alpha-reductase in health and disease.

    PubMed

    Randall, V A

    1994-04-01

    The mechanism of androgen action varies in different tissues, but in the majority of androgen target tissues either testosterone or 5 alpha-dihydrotestosterone (DHT) binds to a specific androgen receptor to form a complex that can regulate gene expression. Testosterone is metabolized to DHT by the enzyme 5 alpha-reductase. The autosomal recessive genetic disorder of 5 alpha-reductase deficiency has clearly shown that the requirement for DHT formation varies with different tissues. In this syndrome genetic males contain normal male internal structures including testes, but exhibit ambiguous or female external genitalia at birth; at puberty they undergo partial virilization which includes development of a male gender identity even if brought up as females. Their development suggests that testosterone itself is able to stimulate psychosexual behaviour, development of the embryonic wolffian duct, muscle development, voice deepening, spermatogenesis, and axillary and pubic hair growth; DHT seems to be essential for prostate development and growth, the development of the external genitalia and male patterns of facial and body hair growth or male-pattern baldness. How different hormones operate to regulate genes via the same receptor is currently unknown, but appears to involve cell-specific factors. The 5-alpha-reductase enzyme has proved difficult to isolate biochemically, but recently at least two human isoenzymes have been identified using molecular biological methods. All the various 5 alpha-reductase-deficient kindreds have been shown to have mutations in 5 alpha-reductase 2, the predominant form in the prostate. The biological role of 5 alpha-reductase 1 has not yet been ascertained, but at present it cannot be ruled out that some of the actions ascribed to testosterone are indeed in cells producing DHT via this enzyme. The activity of 5 alpha-reductase is also implicated in benign prostatic hypertrophy, hirsutism and possibly male-pattern baldness; recent evidence

  16. Design and synthesis of polyhydroxy steroids as selective inhibitors against AKR1B10 and molecular docking.

    PubMed

    Chen, Wenli; Chen, Xinying; Zhou, Shujia; Zhang, Hong; Wang, Ling; Xu, Jun; Hu, Xiaopeng; Yin, Wei; Yan, Guangmei; Zhang, Jingxia

    2016-06-01

    AKR1B10 is a member of the human aldo-keto reductase superfamily which is highly expressed in several types of cancers, and has been regarded as a promising cancer therapeutic target. In this paper, a series of polyhydroxy steroids were designed and synthesized to selectively inhibit AKR1B10 activity. The most selective compound, novel compound 6, has an IC50 of 0.83±0.07μM and a selectivity of more than 120-fold for AKR1B10/AKR1B1. Structure-activity relation analyses indicate that hydroxyl at C-19 can significantly improve the selective inhibition of AKR1B10. The binding mode of AKR1B10 and its inhibitors were studied.

  17. Wolinella succinogenes quinol:fumarate reductase and its comparison to E. coli succinate:quinone reductase.

    PubMed

    Lancaster, C Roy D

    2003-11-27

    The three-dimensional structure of Wolinella succinogenes quinol:fumarate reductase (QFR), a dihaem-containing member of the superfamily of succinate:quinone oxidoreductases (SQOR), has been determined at 2.2 A resolution by X-ray crystallography [Lancaster et al., Nature 402 (1999) 377-385]. The structure and mechanism of W. succinogenes QFR and their relevance to the SQOR superfamily have recently been reviewed [Lancaster, Adv. Protein Chem. 63 (2003) 131-149]. Here, a comparison is presented of W. succinogenes QFR to the recently determined structure of the mono-haem containing succinate:quinone reductase from Escherichia coli [Yankovskaya et al., Science 299 (2003) 700-704]. In spite of differences in polypeptide and haem composition, the overall topology of the membrane anchors and their relative orientation to the conserved hydrophilic subunits is strikingly similar. A major difference is the lack of any evidence for a 'proximal' quinone site, close to the hydrophilic subunits, in W. succinogenes QFR.

  18. 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

  19. 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

  20. 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.

  1. DNA damage induction of ribonucleotide reductase.

    PubMed

    Elledge, S J; Davis, R W

    1989-11-01

    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. PMID:2513480

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

    PubMed

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

    2008-07-01

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

  3. Crystal structure of red chlorophyll catabolite reductase: enlargement of the ferredoxin-dependent bilin reductase family.

    PubMed

    Sugishima, Masakazu; Kitamori, Yuka; Noguchi, Masato; Kohchi, Takayuki; Fukuyama, Keiichi

    2009-06-01

    The key steps in the degradation pathway of chlorophylls are the ring-opening reaction catalyzed by pheophorbide a oxygenase and sequential reduction by red chlorophyll catabolite reductase (RCCR). During these steps, chlorophyll catabolites lose their color and phototoxicity. RCCR catalyzes the ferredoxin-dependent reduction of the C20/C1 double bond of red chlorophyll catabolite. RCCR appears to be evolutionarily related to the ferredoxin-dependent bilin reductase (FDBR) family, which synthesizes a variety of phytobilin pigments, on the basis of sequence similarity, ferredoxin dependency, and the common tetrapyrrole skeleton of their substrates. The evidence, however, is not robust; the identity between RCCR and FDBR HY2 from Arabidopsis thaliana is only 15%, and the oligomeric states of these enzymes are different. Here, we report the crystal structure of A. thaliana RCCR at 2.4 A resolution. RCCR forms a homodimer, in which each subunit folds in an alpha/beta/alpha sandwich. The tertiary structure of RCCR is similar to those of FDBRs, strongly supporting that these enzymes evolved from a common ancestor. The two subunits are related by noncrystallographic 2-fold symmetry in which the alpha-helices near the edge of the beta-sheet unique in RCCR participate in intersubunit interaction. The putative RCC-binding site, which was derived by superimposing RCCR onto biliverdin-bound forms of FDBRs, forms an open pocket surrounded by conserved residues among RCCRs. Glu154 and Asp291 of A. thaliana RCCR, which stand opposite each other in the pocket, likely are involved in substrate binding and/or catalysis.

  4. 4-Dimethylaminoazobenzenes: carcinogenicities and reductive cleavage by microsomal azo reductase.

    PubMed

    Lambooy, J P; Koffman, B M

    1985-01-01

    Twenty-four 4-dimethylaminoazobenzenes (DABs) in which systematic structural modifications have been made in the prime ring have been studied for substrate specificity for microsomal azo reductase. The DABs were also evaluated for carcinogenicity and it was found that there was no correlation between carcinogenicity and extent of azo bond cleavage by azo reductase. While any substituent in the prime ring reduces the rate of cleavage of the azo bond relative to the unsubstituted dye, there is a correlation between substituent size and susceptibility to the enzyme. Substituent size was also found to be a significant factor in the induction of hepatomas by the dyes. Preliminary studies have shown that there appears to be a positive correlation between microsomal riboflavin content and the activity of the azo reductase.

  5. 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

  6. 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

  7. Mechanistic imperatives for aldose-ketose isomerization in water: specific, general base- and metal ion-catalyzed isomerization of glyceraldehyde with proton and hydride transfer.

    PubMed

    Nagorski, R W; Richard, J P

    2001-02-01

    The deuterium enrichment of dihydroxyacetone obtained from the aldose-ketose isomerization of D,L-glyceraldehyde in D(2)O at 25 degrees C was determined by (1)H NMR spectroscopy from the integrated areas of the signals for the alpha-CH(2) and alpha-CHD groups of the product. One mole equivalent of deuterium is incorporated into the product when the isomerization is carried out in 150 mM pyrophosphate buffer at pD 8.4, but only 0.6 mol equiv of deuterium is incorporated into the product of isomerization in the presence of 0.01 M deuterioxide ion, so that 40% of the latter isomerization reaction proceeds by the intramolecular transfer of hydride ion. Several pathways were identified for catalysis of the isomerization of glyceraldehyde to give dihydroxyacetone. The isomerization with hydride transfer is strongly catalyzed by added Zn(2+). Deprotonation of glyceraldehyde is rate-determining for isomerization with proton transfer, and this proton-transfer reaction is catalyzed by Brønsted bases. Proton transfer also occurs by a termolecular pathway with catalysis by the combined action of Brønsted bases and Zn(2+). These results show that there is no large advantage to the spontaneous isomerization of glyceraldehyde in alkaline solution with either proton or hydride transfer, and that effective catalytic pathways exist to stabilize the transition states for both of these reactions in water. The existence of separate enzymes that catalyze the isomerization of sugars with hydride transfer and the isomerization of sugar phosphates with proton transfer is proposed to be a consequence of the lack of any large advantage to reaction by either of these pathways for the corresponding nonenzymatic isomerization in water.

  8. 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...

  9. 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...

  10. 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...

  11. 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...

  12. 21 CFR 864.7375 - Glutathione reductase assay.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

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

  13. Domain evolution and functional diversification of sulfite reductases.

    PubMed

    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.

  14. 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)

  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. 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.

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

    PubMed

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

    2012-09-01

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

  19. 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

  20. 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.

  1. Molecular genetics of steroid 5 alpha-reductase 2 deficiency.

    PubMed Central

    Thigpen, A E; Davis, D L; Milatovich, A; Mendonca, B B; Imperato-McGinley, J; Griffin, J E; Francke, U; Wilson, J D; Russell, D W

    1992-01-01

    Two isozymes of steroid 5 alpha-reductase encoded by separate loci catalyze the conversion of testosterone to dihydrotestosterone. Inherited defects in the type 2 isozyme lead to male pseudohermaphroditism in which affected males have a normal internal urogenital tract but external genitalia resembling those of a female. The 5 alpha-reductase type 2 gene (gene symbol SRD5A2) was cloned and shown to contain five exons and four introns. The gene was localized to chromosome 2 band p23 by somatic cell hybrid mapping and chromosomal in situ hybridization. Molecular analysis of the SRD5A2 gene resulted in the identification of 18 mutations in 11 homozygotes, 6 compound heterozygotes, and 4 inferred compound heterozygotes from 23 families with 5 alpha-reductase deficiency. 6 apparent recurrent mutations were detected in 19 different ethnic backgrounds. In two patients, the catalytic efficiency of the mutant enzymes correlated with the severity of the disease. The high proportion of compound heterozygotes suggests that the carrier frequency of mutations in the 5 alpha-reductase type 2 gene may be higher than previously thought. Images PMID:1522235

  2. 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.

  3. ARSENICALS INHIBIT THIOREDOXIN REDUCTASE ACTIVITY IN CULTURED RAT HEPATOCYTES

    EPA Science Inventory

    ARSENICALS INHIBIT THIOREDOXIN REDUCTASE ACTIVITY IN CULTURED RAT HEPATOCYTES.

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

  4. 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

  5. Measurement of nitrous oxide reductase activity in aquatic sediments

    USGS Publications Warehouse

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

    1986-01-01

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

  6. 3-Oxoacyl-[ACP] reductase from oilseed rape (Brassica napus).

    PubMed

    Sheldon, P S; Kekwick, R G; Smith, C G; Sidebottom, C; Slabas, A R

    1992-04-01

    3-Oxoacyl-[ACP] reductase (E.C. 1.1.1.100, alternatively known as beta-ketoacyl-[ACP] reductase), a component of fatty acid synthetase has been purified from seeds of rape by ammonium sulphate fractionation, Procion Red H-E3B chromatography, FPLC gel filtration and high performance hydroxyapatite chromatography. The purified enzyme appears on SDS-PAGE as a number of 20-30 kDa components and has a strong tendency to exist in a dimeric form, particularly when dithiothreitol is not present to reduce disulphide bonds. Cleveland mapping and cross-reactivity with antiserum raised against avocado 3-oxoacyl-[ACP] reductase both indicate that the multiple components have similar primary structures. On gel filtration the enzyme appears to have a molecular mass of 120 kDa suggesting that the native structure is tetrameric. The enzyme has a strong preference for the acetoacetyl ester of acyl carrier protein (Km = 3 microM) over the corresponding esters of the model substrates N-acetyl cysteamine (Km = 35 mM) and CoA (Km = 261 microM). It is inactivated by dilution but this can be partly prevented by the inclusion of NADPH. Using an antiserum prepared against avocado 3-oxoacyl-[ACP] reductase, the enzyme has been visualised inside the plastids of rape embryo and leaf tissues by immunoelectron microscopy. Amino acid sequencing of two peptides prepared by digestion of the purified enzyme with trypsin showed strong similarities with 3-oxoacyl-[ACP] reductase from avocado pear and the Nod G gene product from Rhizobium meliloti.

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

    PubMed

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

    2013-06-01

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

  8. 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.

  9. 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.

  10. (+)-Pinoresinol/(+)-lariciresinol reductase from Forsythia intermedia. Protein purification, cDNA cloning, heterologous expression and comparison to isoflavone reductase.

    PubMed

    Dinkova-Kostova, A T; Gang, D R; Davin, L B; Bedgar, D L; Chu, A; Lewis, N G

    1996-11-15

    Lignans are a widely distributed class of natural products, whose functions and distribution suggest that they are one of the earliest forms of defense to have evolved in vascular plants; some, such as podophyllotoxin and enterodiol, have important roles in cancer chemotherapy and prevention, respectively. Entry into lignan enzymology has been gained by the approximately 3000-fold purification of two isoforms of (+)-pinoresinol/(+)-lariciresinol reductase, a pivotal branchpoint enzyme in lignan biosynthesis. Both have comparable ( approximately 34.9 kDa) molecular mass and kinetic (Vmax/Km) properties and catalyze sequential, NADPH-dependent, stereospecific, hydride transfers where the incoming hydride takes up the pro-R position. The gene encoding (+)-pinoresinol/(+)-lariciresinol reductase has been cloned and the recombinant protein heterologously expressed as a functional beta-galactosidase fusion protein. Its amino acid sequence reveals a strong homology to isoflavone reductase, a key branchpoint enzyme in isoflavonoid metabolism and primarily found in the Fabaceae (angiosperms). This is of great evolutionary significance since both lignans and isoflavonoids have comparable plant defense properties, as well as similar roles as phytoestrogens. Given that lignans are widespread from primitive plants onwards, whereas the isoflavone reductase-derived isoflavonoids are mainly restricted to the Fabaceae, it is tempting to speculate that this branch of the isoflavonoid pathway arose via evolutionary divergence from that giving the lignans.

  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. 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.

  13. 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.

  14. Kinetic measurements of phosphoglucose isomerase and phosphomannose isomerase by direct analysis of phosphorylated aldose-ketose isomers using tandem mass spectrometry

    NASA Astrophysics Data System (ADS)

    Gao, Hong; Chen, Ye; Leary, Julie A.

    2005-02-01

    A mass spectrometry based method for the direct determination of kinetic constants for phosphoglucose isomerase (PGI) and phosphomannose isomerase (PMI) is described. PGI catalyzes the interconversion between glucose-6-phosphate (Glc6P) and fructose-6-phosphate (Fru6P) and PMI performs the same function between mannose-6-phosphate (Man6P) and Fru6P. These two enzymes are essential in the pathways of glycolytic or oxidative metabolism of carbohydrates and have been considered as potential therapeutic targets. Traditionally, they are assayed either by spectrophotometric detection of Glc6P with one or more coupling enzymes or by a colorimetric detection of Fru6P. However, no suitable assay for Man6P has been developed yet to study the reaction of PMI in the direction from Fru6P to Man6P. In the work presented herein, a general assay for the isomeric substrate-product pair between Glc6P and Fru6P or between Man6P and Fru6P was developed, with the aim of directly studying the kinetics of PGI and PMI in both directions. The 6-phosphorylated aldose and ketose isomers were distinguished based on their corresponding tandem mass spectra (MS2) obtained on a quadrupole ion trap mass spectrometer, and a multicomponent quantification method was utilized to determine the composition of binary mixtures. Using this method, the conversion between Fru6P and Glc6P and that between Fru6P and Man6P are directly monitored. The equilibrium constants for the reversible reactions catalyzed by PGI and PMI are measured to be 0.3 and 1.1, respectively, and the kinetic parameters for both substrates of PGI and PMI are also determined. The values of KM and Vmax for Fru6P as substrate of PMI are reported to be 0.15 mM and 7.78 [mu]mol/(min mg), respectively. All other kinetic parameters measured correlate well with those obtained using traditional methods, demonstrating the accuracy and reliability of this assay.

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

    PubMed

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

    2013-11-15

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

  16. The respiratory arsenate reductase from Bacillus selenitireducens strain MLS10

    USGS Publications Warehouse

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

    2003-01-01

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

  17. 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

  18. 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

  19. 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

  20. Cloning and sequence of the human adrenodoxin reductase gene.

    PubMed Central

    Lin, D; Shi, Y F; Miller, W L

    1990-01-01

    Adrenodoxin reductase (ferrodoxin:NADP+ oxidoreductase, EC 1.18.1.2) is a flavoprotein mediating electron transport to all mitochondrial forms of cytochrome P450. We cloned the human adrenodoxin reductase gene and characterized it by restriction endonuclease mapping and DNA sequencing. The entire gene is approximately 12 kilobases long and consists of 12 exons. The first exon encodes the first 26 of the 32 amino acids of the signal peptide, and the second exon encodes the remainder of signal peptide and the apparent FAD binding site. The remaining 10 exons are clustered in a region of only 4.3 kilobases, separated from the first two exons by a large intron of about 5.6 kilobases. Two forms of human adrenodoxin reductase mRNA, differing by the presence or absence of 18 bases in the middle of the sequence, arise from alternate splicing at the 5' end of exon 7. This alternately spliced region is directly adjacent to the NADPH binding site, which is entirely contained in exon 6. The immediate 5' flanking region lacks TATA and CAAT boxes; however, this region is rich in G + C and contains six copies of the sequence GGGCGGG, resembling promoter sequences of "housekeeping" genes. RNase protection experiments show that transcription is initiated from multiple sites in the 5' flanking region, located about 21-91 base pairs upstream from the AUG translational initiation codon. Images PMID:2236061

  1. Structural and functional diversity of ferredoxin-NADP(+) reductases.

    PubMed

    Aliverti, Alessandro; Pandini, Vittorio; Pennati, Andrea; de Rosa, Matteo; Zanetti, Giuliana

    2008-06-15

    Although all ferredoxin-NADP(+) reductases (FNRs) catalyze the same reaction, i.e. the transfer of reducing equivalents between NADP(H) and ferredoxin, they belong to two unrelated families of proteins: the plant-type and the glutathione reductase-type of FNRs. Aim of this review is to provide a general classification scheme for these enzymes, to be used as a framework for the comparison of their properties. Furthermore, we report on some recent findings, which significantly increased the understanding of the structure-function relationships of FNRs, i.e. the ability of adrenodoxin reductase and its homologs to catalyze the oxidation of NADP(+) to its 4-oxo derivative, and the properties of plant-type FNRs from non-photosynthetic organisms. Plant-type FNRs from bacteria and Apicomplexan parasites provide examples of novel ways of FAD- and NADP(H)-binding. The recent characterization of an FNR from Plasmodium falciparum brings these enzymes into the field of drug design.

  2. Modulating hemoglobin nitrite reductase activity through allostery: a mathematical model.

    PubMed

    Rong, Zimei; Alayash, Abdu I; Wilson, Michael T; Cooper, Chris E

    2013-11-30

    The production of nitric oxide by hemoglobin (Hb) has been proposed to play a major role in the control of blood flow. Because of the allosteric nature of hemoglobin, the nitrite reductase activity is a complex function of oxygen partial pressure PO2. We have previous developed a model to obtain the micro rate constants for nitrite reduction by R state (kR) and T state (kT) hemoglobin in terms of the experimental maximal macro rate constant kNmax and the corresponding oxygen concentration PO2max. However, because of the intrinsic difficulty in obtaining accurate macro rate constant kN, from available experiments, we have developed an alternative method to determine the micro reaction rate constants (kR and kT) by fitting the simulated macro reaction rate curve (kN versus PO2) to the experimental data. We then use our model to analyze the effect of pH (Bohr Effect) and blood ageing on the nitrite reductase activity, showing that the fall of bisphosphoglycerate (BPG) during red cell storage leads to increase NO production. Our model can have useful predictive and explanatory power. For example, the previously described enhanced nitrite reductase activity of ovine fetal Hb, in comparison to the adult protein, may be understood in terms of a weaker interaction with BPG and an increase in the value of kT from 0.0087M(-1)s(-1) to 0.083M(-1)s(-1).

  3. 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.

  4. 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

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

    PubMed Central

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

    1999-01-01

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

  6. 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

  7. In Vitro Formation of Nitrate Reductase Using Extracts of the Nitrate Reductase Mutant of Neurospora crassa, nit-1, and Rhodospirillum rubrum

    PubMed Central

    Ketchum, Paul A.; Sevilla, Cynthia L.

    1973-01-01

    In vitro formation of reduced nicotinamide adenine dinucleotide phosphate (NADPH)–nitrate reductase (NADPH: nitrate oxido-reductase, EC 1.6.6.2) has been attained by using extracts of the nitrate reductase mutant of Neurospora crassa, nit-1, and extracts of either photosynthetically or heterotrophically grown Rhodospirillum rubrum, which contribute the constitutive component. The in vitro formation of NADPH-nitrate reductase is characterized by the conversion of the flavin adenine dinucleotide (FAD) stimulated NADPH-cytochrome c reductase, contributed by the N. crassa nit-1 extract from a slower sedimenting form (4.5S) to a faster sedimenting form (7.8S). The 7.8S NADPH-cytochrome c reductase peak coincides in sucrose density gradient profiles with the NADPH–nitrate reductase, FADH2–nitrate reductase and reduced methyl viologen (MVH)–nitrate reductase activities which are also formed in vitro. The constitutive component from R. rubrum is soluble (both in heterotrophically and photosynthetically grown cells), is stimulated by the addition of 10−4 M Na2MoO4 and 10−2 M NaNO3 to cell-free preparations, and has variable activity over the pH range from 3.0 to 9.5. The activity of the constitutive component in some extracts showed a threefold stimulation when the pH was lowered from 6.5 to 4.0. The constitutive activity appears to be associated with a large molecular weight component which sediments as a single peak in sucrose density gradients. However, the constitutive component from R. rubrum is dialyzable and is insensitive to trypsin and protease. These results demonstrate that R. rubrum contains the constitutive component and suggests that it is a low molecular weight, trypsin- and protease-insensitive factor which participates in the in vitro formation of NADPH nitrate reductase. PMID:4270447

  8. 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.

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

    PubMed Central

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

    1990-01-01

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

  10. Selenate reductase activity in Escherichia coli requires Isc iron-sulfur cluster biosynthesis genes.

    PubMed

    Yee, Nathan; Choi, Jessica; Porter, Abigail W; Carey, Sean; Rauschenbach, Ines; Harel, Arye

    2014-12-01

    The selenate reductase in Escherichia coli is a multi-subunit enzyme predicted to bind Fe-S clusters. In this study, we examined the iron-sulfur cluster biosynthesis genes that are required for selenate reductase activity. Mutants devoid of either the iscU or hscB gene in the Isc iron-sulfur cluster biosynthesis pathway lost the ability to reduce selenate. Genetic complementation by the wild-type sequences restored selenate reductase activity. The results indicate the Isc biosynthetic system plays a key role in selenate reductase Fe-S cofactor assembly and is essential for enzyme activity.

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

    PubMed Central

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

    2011-01-01

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

  12. 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

  13. 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

  14. 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

  15. 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.

  16. 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

  17. 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 ...

  18. Regulation of a ribonucleoside reductase during the early generative phase in Acetabularia.

    PubMed

    de Groot, E J; Schweiger, H G

    1985-02-01

    The activity of a ribonucleoside reductase was estimated during the life cycle of Acetabularia. During the early generative phase the enzyme activity was dramatically increased. Regulation of the ribonucleoside reductase was observed even in the absence of the nucleus. The increase in activity was inhibited by chloramphenicol but not by cycloheximide. These results indicate that the enzyme is translated on 70 S ribosomes.

  19. 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.

  20. 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

  1. 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.

  2. 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

  3. 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.

  4. Life with too much polyprenol: polyprenol reductase deficiency.

    PubMed

    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

    2012-04-01

    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.

  5. 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

  6. 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.

  7. 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

  8. 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

  9. Stereospecificity of (+)-pinoresinol and (+)-lariciresinol reductases from Forsythia intermedia.

    PubMed

    Chu, A; Dinkova, A; Davin, L B; Bedgar, D L; Lewis, N G

    1993-12-25

    Pinoresinol/lariciresinol reductase catalyzes the first known example of a highly unusual benzylic ether reduction in plants; its mechanism of hydride transfer is described. The enzyme was found in Forsythia intermedia and catalyzes the presumed regulatory branch-points in the pathway leading to benzylaryltetrahydrofuran, dibenzylbutane, dibenzylbutyrolactone, and aryltetrahydronaphthalene lignans. Using [7,7'-2H2]-pinoresinol and [7,7'-2H3]lariciresinol as substrates, the hydride transfers of the highly unusual reductase were demonstrated to be completely stereospecific (> 99%). The incoming hydrides were found to take up the pro-R position at C-7' (and/or C-7) in lariciresinol and secoisolariciresinol, thereby eliminating the possibility of random hydride delivery to a planar quinone methide intermediate. As might be expected, the mode of hydride abstraction from NADPH was also stereospecific: using [4R-3H] and [4S-3H]NADPH, it was found that only the 4 pro-R hydrogen was abstracted for enzymatic hydride transfer.

  10. 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.

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

    SciTech Connect

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

    1986-11-01

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

  12. 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

  13. Prostates, pates, and pimples. The potential medical uses of steroid 5 alpha-reductase inhibitors.

    PubMed

    Tenover, J S

    1991-12-01

    The steroid 5 alpha-reductase enzyme is responsible for the formation of DHT from testosterone. DHT has been the major androgen implicated in the pathogenesis of benign prostatic hyperplasia, male pattern baldness, acne, and idiopathic female hirsutism. Although specific inhibitors of 5 alpha-reductase are not yet generally available for human use, it is expected that they will become available within the next several years. Based on biochemical, histologic, and anatomic information from animals given 5 alpha-reductase inhibitors, preliminary data on their use in humans, and knowledge gained from men with the inherited 5 alpha-reductase deficiency, it is expected that these 5 alpha-reductase inhibitors may have a major role in the medical management of benign prostatic hyperplasia. In addition, it is possible that these compounds will hold promise for the prevention of male pattern baldness and for the treatment of resistant acne and idiopathic hirsutism. PMID:1723383

  14. 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

  15. The flavin inhibitor diphenyleneiodonium renders Trichomonas vaginalis resistant to metronidazole, inhibits thioredoxin reductase and flavin reductase, and shuts off hydrogenosomal enzymatic pathways.

    PubMed

    Leitsch, David; Kolarich, Daniel; Duchêne, Michael

    2010-05-01

    Infections with the microaerophilic protozoan parasite Trichomonas vaginalis are commonly treated with metronidazole, a 5-nitroimidazole drug. Metronidazole is selectively toxic to microaerophiles and anaerobes because reduction at the drug's nitro group, which is a precondition for toxicity, occurs only quantitatively in these organisms. In our previous work we identified the flavin enzyme thioredoxin reductase as an electron donor to 5-nitroimidazole drugs in T. vaginalis and observed that highly metronidazole-resistant cell lines lack thioredoxin reductase and flavin reductase activities. In this study we added the flavin inhibitor diphenyleneiodonium (DPI) to T. vaginalis cultures in order to test our hypothesis that metronidazole reduction is catalyzed by flavin enzymes, e.g. thioredoxin reductase, and intracellular free flavins. Indeed, within hours, DPI rendered T. vaginalis insensitive to metronidazole concentrations as high as 1mM and prevented the formation of metronidazole adducts with proteins. Thioredoxin reductase activity was absent from DPI-treated cells and flavin reductase activity was sharply decreased. In addition, DPI-treated cells also upregulated the expression of antioxidant enzymes, i.e. thioredoxin peroxidases and superoxide dismutases, and displayed a fundamentally altered metabolism caused by inactivation of pyruvate:ferredoxin oxidoreductase (PFOR) and concomitant upregulation of lactate dehydrogenase (LDH) activity. Thus, the disruption of the cellular flavin metabolism by DPI mediated metabolic steps which are similar to that of cells with metronidazole resistance induced in vitro. Finally, we present direct evidence that the increased expression of antioxidant enzymes is dispensable for acquiring resistance to metronidazole. PMID:20093143

  16. 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

  17. 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

  18. 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

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

    PubMed

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

    2006-11-01

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

  20. 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.

  1. 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.

  2. Biofilm modifies expression of ribonucleotide reductase genes in Escherichia coli.

    PubMed

    Cendra, Maria del Mar; Juárez, Antonio; Torrents, Eduard

    2012-01-01

    Ribonucleotide reductase (RNR) is an essential enzyme for all living organisms since is the responsible for the last step in the synthesis of the four deoxyribonucleotides (dNTPs) necessary for DNA replication and repair. In this work, we have investigated the expression of the three-RNR classes (Ia, Ib and III) during Escherichia coli biofilm formation. We show the temporal and spatial importance of class Ib and III RNRs during this process in two different E. coli wild-type strains, the commensal MG1655 and the enteropathogenic and virulent E2348/69, the prototype for the enteropathogenic E. coli (EPEC). We have established that class Ib RNR, so far considered cryptic, play and important role during biofilm formation. The implication of this RNR class under the specific growth conditions of biofilm formation is discussed. PMID:23050019

  3. A calibration curve for immobilized dihydrofolate reductase activity assay.

    PubMed

    Singh, Priyanka; Morris, Holly; Tivanski, Alexei V; Kohen, Amnon

    2015-09-01

    An assay was developed for measuring the active-site concentration, activity, and thereby the catalytic turnover rate (k cat) of an immobilized dihydrofolate reductase model system (Singh et al., (2015), Anal. Biochem). This data article contains a calibration plot for the developed assay. In the calibration plot rate is plotted as a function of DHFR concentration and shows linear relationship. The concentration of immobilized enzyme was varied by using 5 different size mica chips. The dsDNA concentration was the same for all chips, assuming that the surface area of the mica chip dictates the resulting amount of bound enzyme (i.e. larger sized chip would have more bound DHFR). The activity and concentration of each chip was measured.

  4. 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.

  5. 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

  6. 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

  7. Increased neurotoxicity of arsenic in methylenetetrahydrofolate reductase deficiency.

    PubMed

    Brouwer, O F; Onkenhout, W; Edelbroek, P M; de Kom, J F; de Wolff, F A; Peters, A C

    1992-01-01

    A 16-year-old girl from Surinam presented with mental deterioration and severe paraparesis with areflexia and bilateral Babinski signs. Laboratory examination showed a hyperhomocysteinemia that was caused by 5,10-methylene-tetrahydrofolate reductase (MTHFR) deficiency. In addition, urine samples contained large amounts of arsenic. An open bag with the pesticide copper acetate arsenite was found to be the source of exposure. In remethylation defects such as MTHFR deficiency, the concentration of methyldonors is severely reduced. As arsenic is detoxified by methylation, we suggest that the MTHFR deficiency in this girl might explain the fact that of all family members exposed to arsenic, only she developed severe clinical signs and symptoms of arsenic poisoning.

  8. 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.

  9. Lymphocyte development requires S-nitrosoglutathione reductase1

    PubMed Central

    Yang, Zhiyong; Wang, Zhi-En; Doulias, Paschalis-Thomas; Wei, Wei; Ischiropoulos, Harry; Locksley, Richard M.; Liu, Limin

    2011-01-01

    Nitric oxide is critical to immunity, but its role in the development of the immune system is unknown. Here, we show that S-nitrosoglutathione reductase (GSNOR), a protein key to the control of protein S-nitrosylation, is important for the development of lymphocytes. Genetic deletion of GSNOR in mice results in significant decrease in both T and B lymphocytes in the periphery. In thymus, GSNOR deficiency causes excessive protein S-nitrosylation, increases apoptosis, and reduces the number of CD4 single-positive thymocytes. Lymphopenia andincrease in S-nitrosylation and apoptosis in GSNOR-deficient mice are largely abolished by genetic deletion of inducible nitric oxide synthase (iNOS). Furthermore, the protection of lymphocyte development by GSNOR is apparently intrinsic to hematopoietic cells. Thus GSNOR, likely through regulation of S-nitrosylation and apoptosis, physiologically plays aprotective role in the development of the immune system. PMID:20980633

  10. Purification and characterization of rat lens pyrroline-5-carboxylate reductase.

    PubMed

    Shiono, T; Kador, P F; Kinoshita, J J

    1986-03-19

    delta 1-Pyrroline-5-carboxylate reductase (L-proline:NAD(P)+ 5-oxidoreductase, EC 1.5.1.2) has been purified from rat lens and biochemically characterized. Purification steps included ammonium sulfate fractionation, affinity chromatography on Amicon Matrex Orange A, and gel filtration with Sephadex G-200. These steps were carried out at ambient temperature (22 degrees C) in 20 mM sodium phosphate/potassium phosphate buffer (pH 7.5) containing 10% glycerol, 7 mM mercaptoethanol and 0.5 mM EDTA. The enzyme, purified to apparent homogeneity, displayed a molecular weight of 240 000 by gel chromatography and 30 000 by SDS-polyacrylamide gel electrophoresis. This suggests that the enzyme is composed of eight subunits. The purified enzyme displays a pH optimum between 6.5 and 7.1 and is inhibited by heavy metal ions and p-chloromercuribenzoate. Kinetic studies indicated Km values of 0.62 mM and 0.051 mM for DL-pyrroline-5-carboxylate as substrate when NADH and NADPH respectively were employed as cofactors. The Km values for the cofactors NADH and NADPH with DL-pyrroline-5-carboxylate as substrate were 0.37 mM and 0.006 mM, respectively. With L-pyrroline-5-carboxylate as substrate, Km values of 0.21 mM and 0.022 mM were obtained for NADH and NADPH, respectively. Enzyme activity is potentially inhibited by NADP+ and ATP, suggesting that delta 1-pyrroline-5-carboxylate reductase may be regulated by the energy level and redox state of the lens.

  11. 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.

  12. Expression, purification and enzymatic characterization of Brugia malayi dihydrofolate reductase.

    PubMed

    Perez-Abraham, Romy; Sanchez, Karla Garabiles; Alfonso, Melany; Gubler, Ueli; Siekierka, John J; Goodey, Nina M

    2016-12-01

    Brugia malayi (B. malayi) is one of the three causative agents of lymphatic filariasis, a neglected parasitic disease. Current literature suggests that dihydrofolate reductase is a potential drug target for the elimination of B. malayi. Here we report the recombinant expression and purification of a ∼20 kDa B. malayi dihydrofolate reductase (BmDHFR). A His6-tagged construct was expressed in E. coli and purified by affinity chromatography to yield active and homogeneous enzyme for steady-state kinetic characterization and inhibition studies. The catalytic activity kcat was found to be 1.4 ± 0.1 s(-1), the Michaelis Menten constant KM for dihydrofolate 14.7 ± 3.6 μM, and the equilibrium dissociation constant KD for NADPH 25 ± 24 nM. For BmDHFR, IC50 values for a six DHFR inhibitors were determined to be 3.1 ± 0.2 nM for methotrexate, 32 ± 22 μM for trimethoprim, 109 ± 34 μM for pyrimethamine, 154 ± 46 μM for 2,4-diaminoquinazoline, 771 ± 44 μM for cycloguanil, and >20,000 μM for 2,4-diaminopyrimidine. Our findings suggest that antifolate compounds can serve as inhibitors of BmDHFR. PMID:27544923

  13. Finasteride: the first 5 alpha-reductase inhibitor.

    PubMed

    Sudduth, S L; Koronkowski, M J

    1993-01-01

    Finasteride is a synthetic 4-azasteroid that is a specific competitive inhibitor of 5 alpha-reductase, an intracellular enzyme that converts testosterone to dihydrotestosterone (DHT). It has no binding affinity for androgen receptor sites and itself possesses no androgenic, antiandrogenic, or other steroid hormone-related properties. It is well absorbed after oral administration, with absolute bioavailability in humans of 63% (range 34-108%). The mean time to maximum concentration is 1-2 hours, and it is approximately 90% plasma protein bound. The elimination half-life averages 6-8 hours. The agent is metabolized to a series of five metabolites, of which two are active and possess less than 20% of the 5 alpha-reductase activity of finasteride. Little is known about potential drug interactions, although they appear to be minimal and not clinically relevant. The drug is indicated for the treatment of symptomatic benign prostatic hyperplasia. Its efficacy in regression of prostate gland enlargement is rapid and predictable, although correlation with subsequent improvement in urinary flow and symptoms is highly variable. Dosages of 0.5-100 mg/day regress prostate enlargement; the recommended dosage is 5 mg once/day. Finasteride may hold promise for other DHT-mediated disorders such as acne, facial hirsutism, frontal lobe alopecia, and prostate cancer, but its use in these conditions remains investigational. The frequency of adverse drug events is low, with the most common side effects being impotence, decreased libido, and decreased volume of ejaculate. No reports of intentional overdose have been reported, and dosages of up to 80 mg/day for 3 months have been taken without adverse effect. PMID:7689728

  14. [Decolorization of azo dyes using quinone reductase and quinoid compounds].

    PubMed

    Zhou, Mi; Liu, Guang-Fei; Zhou, Ji-Ti; Jin, Ruo-Fei; Chen, Ming-Xiang; Wang, Yan-Qing

    2009-06-15

    Using quinoid redox mediator and bacterial cellular quinone reductase, we investigated the decolorization ability of gene-engineered strain Escherichia coli YB and the effects of methylhydroquinone (MHQ) pretreatement on decolorization performance of E. coli JM109 and anaerobic sludge. The results indicate that lawsone is an effective accelerator for azo dye decolorization by E. coli YB overexpressing cellular quinone reductase AZR. In the presence of 0.2 mmol x L(-1) lawsone, 75% Amaranth (1 mmol x L(-1)) can be decolorized in 2 h. E. coli YB can also decolorize high concentration of azo dye in the presence of lawsone. Around 50% Amaranth (5 mmol x L(-1)) is decolorized in 8 h. Compared to lawsone, menadione is a less effective mediator. E. coli YB takes 12 h to reach 70% decolorization in the presence of 2.5 mmol x L(-1) menadione. Repeated decolorization studies showed that E. coli YB had stable decolorizing ability in the presence of lawsone. Four rounds of repeated decolorization can be completed in 12 h. Lawsone can also accelerate the decolorization of azo dyes with complex structures such as Acid Scarlet GR and Reactive Brilliant Red K-2BP. With the optimal LQ concentrations, 70% Acid Scarlet GR and Reactive Brilliant Red K-2BP are decolorized in 9 h and 30 h,respectively. Decolorization performances of E. coli JM109 and anaerobic sludge pretreated with MHQ are improved. After MHQ pretreatment,in the presence of lawsone, 80% Amaranth (1 mmol x L(-1)) can be decolorized in 5 h by E. coli JM109, while more than 75% Amaranth can be removed in 11 h by sludge.

  15. 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

  16. Characterization of the nitric oxide reductase from Thermus thermophilus.

    PubMed

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

    2013-07-30

    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 Ca(2+), 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

  17. 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

  18. 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

  19. Glyphosate inhibition of ferric reductase activity in iron deficient sunflower roots.

    PubMed

    Ozturk, Levent; Yazici, Atilla; Eker, Selim; Gokmen, Ozgur; Römheld, Volker; Cakmak, Ismail

    2008-01-01

    Iron (Fe) deficiency is increasingly being observed in cropping systems with frequent glyphosate applications. A likely reason for this is that glyphosate interferes with root uptake of Fe by inhibiting ferric reductase in roots required for Fe acquisition by dicot and nongrass species. This study investigated the role of drift rates of glyphosate (0.32, 0.95 or 1.89 mm glyphosate corresponding to 1, 3 and 6% of the recommended herbicidal dose, respectively) on ferric reductase activity of sunflower (Helianthus annuus) roots grown under Fe deficiency conditions. Application of 1.89 mm glyphosate resulted in almost 50% inhibition of ferric reductase within 6 h and complete inhibition 24 h after the treatment. Even at lower rates of glyphosate (e.g. 0.32 mm and 0.95 mm), ferric reductase was inhibited. Soluble sugar concentration and the NAD(P)H oxidizing capacity of apical roots were not decreased by the glyphosate applications. To our knowledge, this is the first study reporting the effects of glyphosate on ferric reductase activity. The nature of the inhibitory effect of glyphosate on ferric reductase could not be identified. Impaired ferric reductase could be a major reason for the increasingly observed Fe deficiency in cropping systems associated with widespread glyphosate usage.

  20. 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

  1. 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

  2. The 5 alpha-reductase inhibitory components from heartwood of Artocarpus incisus: structure-activity investigations.

    PubMed

    Shimizu, K; Fukuda, M; Kondo, R; Sakai, K

    2000-02-01

    The methanol extract of heartwood of Artocarpus incisus showed potent 5 alpha-reductase inhibitory activity. We investigated the 5 alpha-reductase inhibitory effects of nine compounds isolated from A. incisus. Chlorophorin (IC50 = 37 microM) and artocarpin (IC50 = 85 microM) showed more potent inhibitory effects than did alpha-linolenic acid, which is known as a naturally occurring potent inhibitor. Structure-activity investigations suggested that the presence of an isoprene substituent (prenyl and geranyl) would enhance 5 alpha-reductase inhibitory effects.

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

    PubMed

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

    1993-11-01

    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. PMID:8234296

  4. Age-Specific Peculiarities of Modulation of Blood Aldo-Keto Reductase Isoenzyme Spectrum.

    PubMed

    Davydov, V V

    2015-12-01

    The aldo-keto reductase spectrum of the blood was studied at different stages of ontogeny to elucidate the role of reduction pathway in utilization of the carbonyl products of free radical oxidation in modulation of organism sensitivity to the damaging effect of stress during ontogeny. The studies revealed the age-specific changes in aldo-keto reductase spectrum in the blood. An analogy of the aldo-keto reductase spectrum structure in animals of early maturity and in old rats was found. The appearance of age specificity of the aldo-keto reductase spectrum in the blood creates metabolic prerequisites for changes in the efficiency of utilization of carbonyl products of free radical oxidation via their reductive transformation.

  5. 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.

  6. Participation of NADPH-cytochrome C reductase in thyroid hormone biosynthesis.

    PubMed

    Yamamoto, K; DeGroot, L J

    1975-04-01

    Purified rat liver NADPH-cytochrome c reductase supports iodination of tyrosine in a system including NADPH, cytochrome c and thyroid perioxidase. Catalase inhibits the iodination of tyrosine, while superoxide dismutase has no effect. Antibody developed in the rabbit against purified rat liver NADPH-cytochrome c reductase inhibits both reduction of cytochrome c and tyrosine iodination supported by the enzyme. The antibody forms a single precipitation line with thyroid extract, and inhibits NADPH cytochrome c reductase activity of the thyroid. The antibody partially inhibits iodination in a thyroid mitochondrial-microsomal fraction, but does not inhibit NADH-dependent iodination. The immunochemical studies indicate the participation of NADPH-cytochrome c reductase in thyroidal H2O generation, and the independent existence of NADPH-dependent and NADH-dependent H2O2 generation mechanisms in the thyroid. PMID:235416

  7. Localization and Regulation of Synthesis of Nitrate Reductase in Escherichia coli

    PubMed Central

    Showe, Michael K.; DeMoss, J. A.

    1968-01-01

    The nitrate reductase of Escherichia coli K-12 was localized in a particulate fraction of the cell and it sedimented as if it were bound to a large substructure that is subject to fragmentation during cell disruption procedures. Soluble enzyme, exhibiting a homogenous profile in sucrose gradients, was released from this fraction by an alkaline-heat treatment. Less than 1.5% of total active nitrate reductase apparently occurred in this soluble form during the course of formation of the particulate enzyme. Enzyme synthesis was repressed by aeration in the presence or absence of nitrate. Under anaerobic conditions, nitrate reductase was synthesized at a rate that could be increased 20-fold by the addition of nitrate. When enzyme synthesis was initiated by induction with nitrate or anaerobiosis, biphasic kinetics were obtained. We interpreted the results as evidence for the existence of a redox-sensitive repressor which mediates nitrate reductase regulation. PMID:4869216

  8. Phytoalexin synthesis in soybean cells: elicitor induction of reductase involved in biosynthesis of 6'-deoxychalcone.

    PubMed

    Welle, R; Grisebach, H

    1989-07-01

    Chromatofocusing on Mono P proved to be an efficient purification procedure for the NADPH-dependent reductase from soybean (Glycine max L.) cell cultures which acts together with chalcone synthase in the biosynthesis of 2',4',4-trihydroxychalcone (6'-deoxychalcone). By isoelectric focusing the pI of reductase was determined to be 6.3. Addition of pure soybean reductase to cell-free extracts from stimulated cell cultures of parsley and bean (Phaseolus vulgaris) and from young flowers of Dahlia variabilis caused in each case synthesis of 6'-deoxychalcone. When 4-coumaroyl-CoA was replaced by caffeoyl-CoA in the reductase assay, formation of 2',4',3,4-tetrahydrochalcone (butein) was observed. A polyclonal antireductase antiserum was raised in rabbits and proved to be specific in Ouchterlony diffusion experiments, Western blots and immunotitration. The reductase antiserum showed no cross-reactivity with soybean chalcone synthase (CHS). A biotin/[125I]streptavidin system provided a quantitative Western blot for the reductase. Changes in the activities, amounts of protein, and mRNA activities of reductase and CHS were determined after challenge of soybean cell cultures by elicitor (from Phytophthora megasperma f.sp. glycinea or yeast). For both enzymes a pronounced and parallel increase in activity and amounts of protein was observed after elicitor addition with a maximum at about 16 h after challenge. Parallel increases in mRNA activities occurred earlier. The results indicate a parallel induction of de novo synthesis of reductase and CHS which coact in synthesis of 6'-deoxychalcone. PMID:2500065

  9. 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

  10. Characterization of Streptococcus pneumoniae enoyl-(acyl-carrier protein) reductase (FabK).

    PubMed

    Marrakchi, Hedia; Dewolf, Walter E; Quinn, Chad; West, Joshua; Polizzi, Brian J; So, Chi Y; Holmes, David J; Reed, Shannon L; Heath, Richard J; Payne, David J; Rock, Charles O; Wallis, Nicola G

    2003-03-15

    The enoyl-(acyl-carrier protein) (ACP) reductase catalyses the last step in each cycle of fatty acid elongation in the type II fatty acid synthase systems. An extensively characterized NADH-dependent reductase, FabI, is widely distributed in bacteria and plants, whereas the enoyl-ACP reductase, FabK, is a distinctly different member of this enzyme group discovered in Streptococcus pneumoniae. We were unable to delete the fabK gene from Strep. pneumoniae, suggesting that this is the only enoyl-ACP reductase in this organism. The FabK enzyme was purified and the biochemical properties of the reductase were examined. The visible absorption spectrum of the purified protein indicated the presence of a flavin cofactor that was identified as FMN by MS, and was present in a 1:1 molar ratio with protein. FabK specifically required NADH and the protein activity was stimulated by ammonium ions. FabK also exhibited NADH oxidase activity in the absence of substrate. Strep. pneumoniae belongs to the Bacillus / Lactobacillus / Streptococcus group that includes Staphylococcus aureus and Bacillus subtilis. These two organisms also contain FabK-related genes, suggesting that they may also express a FabK-like enoyl-ACP reductase. However, the genes did not complement a fabI (Ts) mutant and the purified flavoproteins were unable to reduce enoyl-ACP in vitro and did not exhibit NAD(P)H oxidase activity, indicating they were not enoyl-ACP reductases. The restricted occurrence of the FabK enoyl-ACP reductase may be related to the role of substrate-independent NADH oxidation in oxygen-dependent anaerobic energy metabolism.

  11. Measurement of nitrite reductase in leaf tissue of Vigna mungo : A new method.

    PubMed

    Srivastava, R C; Bose, B; Mukerji, D; Mathur, S N; Srivastava, H S

    1979-12-01

    The enzyme nitrite reductase (EC 1.6.6.4) is generally assayed in terms of disappearance of nitrite from the assay medium. We describe a technique which allowed estimation of the enzyme level in leaf tissues of Vigna mungo (L). Hepper in terms of the release of the product (NH3) of the enzyme reaction. The technique is offered as an alternative, possibly more convenient method for assay of nitrite reductase in plant tissue in vivo.

  12. Separation of NADH-fumarate reductase and succinate dehydrogenase activities in Trypanosoma cruzi.

    PubMed

    Christmas, P B; Turrens, J F

    2000-02-15

    A recent review suggested that the activity of NADH-fumarate reductase from trypanosomatids could be catalyzed by succinate dehydrogenase working in reverse (Tielens and van Hellemond, Parasitol. Today 14, 265-271, 1999). The results reported in this study demonstrate that the two activities can easily be separated without any loss in either activity, suggesting that fumarate reductase and succinate dehydrogenase are separate enzymes.

  13. Two isofunctional nitric oxide reductases in Alcaligenes eutrophus H16.

    PubMed Central

    Cramm, R; Siddiqui, R A; Friedrich, B

    1997-01-01

    Two genes, norB and norZ, encoding two independent nitric oxide reductases have been identified in Alcaligenes eutrophus H16. norB and norZ predict polypeptides of 84.5 kDa with amino acid sequence identity of 90%. While norB resides on the megaplasmid pHG1, the norZ gene is located on a chromosomal DNA fragment. Amino acid sequence analysis suggests that norB and norZ encode integral membrane proteins composed of 14 membrane-spanning helices. The region encompassing helices 3 to 14 shows similarity to the NorB subunit of common bacterial nitric oxide reductases, including the positions of six strictly conserved histidine residues. Unlike the Nor enzymes characterized so far from denitrifying bacteria, NorB and NorZ of A. eutrophus contain an amino-terminal extension which may form two additional helices connected by a hydrophilic loop of 203 amino acids. The presence of a NorB/NorZ-like protein was predicted from the genome sequence of the cyanobacterium Synechocystis sp. strain PCC6803. While the common NorB of denitrifying bacteria is associated with a second cytochrome c subunit, encoded by the neighboring gene norC, the nor loci of A. eutrophus and Synechocystis lack adjacent norC homologs. The physiological roles of norB and norZ in A. eutrophus were investigated with mutants disrupted in the two genes. Mutants bearing single-site deletions in norB or norZ were affected neither in aerobic nor in anaerobic growth with nitrate or nitrite as the terminal electron acceptor. Inactivation of both norB and norZ was lethal to the cells under anaerobic growth conditions. Anaerobic growth was restored in the double mutant by introducing either norB or norZ on a broad-host-range plasmid. These results show that the norB and norZ gene products are isofunctional and instrumental in denitrification. PMID:9352929

  14. Isobutyraldehyde production from Escherichia coli by removing aldehyde reductase activity

    PubMed Central

    2012-01-01

    Background Increasing global demand and reliance on petroleum-derived chemicals will necessitate alternative sources for chemical feedstocks. Currently, 99% of chemical feedstocks are derived from petroleum and natural gas. Renewable methods for producing important chemical feedstocks largely remain unaddressed. Synthetic biology enables the renewable production of various chemicals from microorganisms by constructing unique metabolic pathways. Here, we engineer Escherichia coli for the production of isobutyraldehyde, which can be readily converted to various hydrocarbons currently derived from petroleum such as isobutyric acid, acetal, oxime and imine using existing chemical catalysis. Isobutyraldehyde can be readily stripped from cultures during production, which reduces toxic effects of isobutyraldehyde. Results We adopted the isobutanol pathway previously constructed in E. coli, neglecting the last step in the pathway where isobutyraldehyde is converted to isobutanol. However, this strain still overwhelmingly produced isobutanol (1.5 g/L/OD600 (isobutanol) vs 0.14 g/L/OD600 (isobutyraldehyde)). Next, we deleted yqhD which encodes a broad-substrate range aldehyde reductase known to be active toward isobutyraldehyde. This strain produced isobutanol and isobutyraldehyde at a near 1:1 ratio, indicating further native isobutyraldehyde reductase (IBR) activity in E. coli. To further eliminate isobutanol formation, we set out to identify and remove the remaining IBRs from the E. coli genome. We identified 7 annotated genes coding for IBRs that could be active toward isobutyraldehyde: adhP, eutG, yiaY, yjgB, betA, fucO, eutE. Individual deletions of the genes yielded only marginal improvements. Therefore, we sequentially deleted all seven of the genes and assessed production. The combined deletions greatly increased isobutyraldehyde production (1.5 g/L/OD600) and decreased isobutanol production (0.4 g/L/OD600). By assessing production by overexpression of each

  15. 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

  16. 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.

  17. Smith-Lemli-Opitz syndrome is caused by mutations in the 7-dehydrocholesterol reductase gene.

    PubMed Central

    Waterham, H R; Wijburg, F A; Hennekam, R C; Vreken, P; Poll-The, B T; Dorland, L; Duran, M; Jira, P E; Smeitink, J A; Wevers, R A; Wanders, R J

    1998-01-01

    Smith-Lemli-Opitz syndrome is a frequently occurring autosomal recessive developmental disorder characterized by facial dysmorphisms, mental retardation, and multiple congenital anomalies. Biochemically, the disorder is caused by deficient activity of 7-dehydrocholesterol reductase, which catalyzes the final step in the cholesterol-biosynthesis pathway-that is, the reduction of the Delta7 double bond of 7-dehydrocholesterol to produce cholesterol. We identified a partial transcript coding for human 7-dehydrocholesterol reductase by searching the database of expressed sequence tags with the amino acid sequence for the Arabidopsis thaliana sterol Delta7-reductase and isolated the remaining 5' sequence by the "rapid amplification of cDNA ends" method, or 5'-RACE. The cDNA has an open reading frame of 1,425 bp coding for a polypeptide of 475 amino acids with a calculated molecular weight of 54.5 kD. Heterologous expression of the cDNA in the yeast Saccharomyces cerevisiae confirmed that it codes for 7-dehydrocholesterol reductase. Chromosomal mapping experiments localized the gene to chromosome 11q13. Sequence analysis of fibroblast 7-dehydrocholesterol reductase cDNA from three patients with Smith-Lemli-Opitz syndrome revealed distinct mutations, including a 134-bp insertion and three different point mutations, each of which was heterozygous in cDNA from the respective parents. Our data demonstrate that Smith-Lemli-Opitz syndrome is caused by mutations in the gene coding for 7-dehydrocholesterol reductase. PMID:9683613

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

    PubMed Central

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

    2014-01-01

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

  19. Contribution of reductase activity to quinone toxicity in three kinds of hepatic cells.

    PubMed

    Ishihara, Yasuhiro; Tsuji, Kaori; Ishii, Satomi; Kashiwagi, Kyoko; Shimamoto, Norio

    2012-01-01

    Two mechanisms have been proposed to explain quinone cytotoxicity: oxidative stress via the redox cycle, and the arylation of intracellular nucleophiles. The redox cycle is catalyzed by intracellular reductases, and therefore the toxicity of redox cycling quinone is considered to be closely associated with the reductase activity. This study examined the relationship between quinone toxicity and the intracellular reductase activity using 3 kinds of hepatic cells; rat primary hepatocytes, HepG2 and H4IIE. The intracellular reductase activity was; primary hepatocyte >HepG2>H4IIE. The three kinds of cells showed almost the same vulnerability to an arylating quinone, 1,4-naphthoquinone (NQ). However, the susceptibility to a redox cycling quinone, 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) was; primary hepatocyte>HepG2>H4IIE. In addition, the cytotoxicity elicited by DMNQ was significantly attenuated in HepG2 cells and almost completely suppressed in primary hepatocytes by diphenyleneiodonium chloride, a reductase inhibitor. These data suggest that cells with a high reductase activity are susceptible to redox cycling quinones. This study provides essential evidence to assess the toxicity of quinone-based drugs during their developmental processes.

  20. 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.

  1. Purification and characterization of (+)dihydroflavonol (3-hydroxyflavanone) 4-reductase from flowers of Dahlia variabilis.

    PubMed

    Fischer, D; Stich, K; Britsch, L; Grisebach, H

    1988-07-01

    Individual flowers from inflorescences of Dahlia variabilis (cv Scarlet Star) in young developmental stages contained relatively high activity of (+)-dihydroflavonol (DHF) 4-reductase. The DHF reductase was purified from such flowers to apparent homogeneity by a five-step procedure. This included affinity adsorption on Blue Sepharose and elution of the enzyme with NADP+. By gel filtration and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis it was shown that DHF reductase contains only one polypeptide chain with a Mr of about 41,000. The reductase required NADPH as cofactor and catalyzed transfer of the pro-S hydrogen of NADPH to the substrate. Flavanones and dihydroflavonols (3-hydroxyflavanones) were substrates for DHF reductase with pH optima of about 6.0 for flavanones and of about 6.8 for dihydroflavonols. Flavanones were reduced to the corresponding flavan-4-ols and (+)-dihydroflavonols to flavan-3,4-cis-diols. Apparent Michaelis constants determined for (2S)-naringenin, (2S)-eriodicytol, (+)-dihydrokaempferol, (+)-dihydroquercetin, and NADPH were, respectively, 2.3, 2, 10, 15, and 42 microM. V/Km values were higher for dihydroflavonols than for flavanones. Conversion of dihydromyricetin to leucodelphinidin was also catalyzed by the enzyme at a low rate, whereas flavones and flavonols were not accepted as substrates. DHF reductase was not inhibited by metal chelators. PMID:3293532

  2. "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

  3. 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

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

    PubMed

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

    2013-04-01

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

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

    PubMed

    Hartsock, Angela; Shapleigh, James P

    2011-12-01

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

  6. 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

  7. 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.

  8. 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.

  9. Flavodiiron protein from Trichomonas vaginalis hydrogenosomes: the terminal oxygen reductase.

    PubMed

    Smutná, Tamara; Gonçalves, Vera L; Saraiva, Lígia M; Tachezy, Jan; Teixeira, Miguel; Hrdy, Ivan

    2009-01-01

    Trichomonas vaginalis is one of a few eukaryotes that have been found to encode several homologues of flavodiiron proteins (FDPs). Widespread among anaerobic prokaryotes, these proteins are believed to function as oxygen and/or nitric oxide reductases to provide protection against oxidative/nitrosative stresses and host immune responses. One of the T. vaginalis FDP homologues is equipped with a hydrogenosomal targeting sequence and is expressed in the hydrogenosomes, oxygen-sensitive organelles that participate in carbohydrate metabolism and assemble iron-sulfur clusters. The bacterial homologues characterized thus far have been dimers or tetramers; the trichomonad protein is a dimer of identical 45-kDa subunits, each noncovalently binding one flavin mononucleotide. The protein reduces dioxygen to water but is unable to utilize nitric oxide as a substrate, similarly to its closest homologue from another human parasite Giardia intestinalis and related archaebacterial proteins. T. vaginalis FDP is able to accept electrons derived from pyruvate or NADH via ferredoxin and is proposed to play a role in the protection of hydrogenosomes against oxygen. PMID:19011120

  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. Structural Basis for Activation of Class Ib Ribonucleotide Reductase

    SciTech Connect

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

    2010-12-03

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

  12. 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

  13. 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

  14. Intermediate hyperhomocysteinemia resulting from compound heterozygosity of methylenetetrahydrofolate reductase mutations.

    PubMed Central

    Kang, S S; Wong, P W; Bock, H G; Horwitz, A; Grix, A

    1991-01-01

    Four subjects with thermolabile methylenetetrahydrofolate reductase (MTHFR) were discovered among 16 "obligate" heterozygotes for severe MTHFR deficiency and their family members. All four subjects had less than 25% of normal mean MTHFR specific activity in lymphocyte extracts. Three of them with normal serum folate and cyanocobalamin had intermediate hyperhomocysteinemia, and one with high serum folate and cyanocobalamin had no excessive accumulation of serum homocysteine. The biochemical features in these four subjects are distinguishable from subjects homozygous for the thermolabile MTHFR, whose specific activity is approximately 50% of the normal mean, and from heterozygotes for severe MTHFR deficiency, in whom the enzyme is thermostable and has a specific activity of about 50% of the normal mean. We propose that these four subjects are genetic compounds of the allele for the severe mutation and the allele for thermolabile mutation of the MTHFR gene. It is postulated that subjects with this genetic compound are more susceptible to the development of intermediate hyperhomocysteinemia despite normal folate and B12 levels. Nonetheless, hyperhomocysteinemia due to this compound heterozygosity is correctable by oral folic acid therapy. PMID:1998340

  15. 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.

  16. Ribonucleotide reductases: divergent evolution of an ancient enzyme.

    PubMed

    Torrents, Eduard; Aloy, Patrick; Gibert, Isidre; Rodríguez-Trelles, Francisco

    2002-08-01

    Ribonucleotide reductases (RNRs) are uniquely responsible for converting nucleotides to deoxynucleotides in all dividing cells. The three known classes of RNRs operate through a free radical mechanism but differ in the way in which the protein radical is generated. Class I enzymes depend on oxygen for radical generation, class II uses adenosylcobalamin, and the anaerobic class III requires S-adenosylmethionine and an iron-sulfur cluster. Despite their metabolic prominence, the evolutionary origin and relationships between these enzymes remain elusive. This gap in RNR knowledge can, to a major extent, be attributed to the fact that different RNR classes exhibit greatly diverged polypeptide chains, rendering homology assessments inconclusive. Evolutionary studies of RNRs conducted until now have focused on comparison of the amino acid sequence of the proteins, without considering how they fold into space. The present study is an attempt to understand the evolutionary history of RNRs taking into account their three-dimensional structure. We first infer the structural alignment by superposing the equivalent stretches of the three-dimensional structures of representatives of each family. We then use the structural alignment to guide the alignment of all publicly available RNR sequences. Our results support the hypothesis that the three RNR classes diverged from a common ancestor currently represented by the anaerobic class III. Also, lateral transfer appears to have played a significant role in the evolution of this protein family. PMID:12107591

  17. 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

  18. Stimulation of dihydrofolate reductase promoter activity by antimetabolic drugs.

    PubMed Central

    Eastman, H B; Swick, A G; Schmitt, M C; Azizkhan, J C

    1991-01-01

    Dihydrofolate reductase (DHFR; EC 1.5.1.3) is required in folate metabolism for the synthesis of purines, thymidine, and glycine. Although there have been several reports of induction of DHFR enzyme by methotrexate (MTX), a drug that competitively inhibits DHFR, there are no studies reported that examine the effect of MTX on DHFR gene transcription. We have examined the effect of MTX and other inhibitors of DNA synthesis on DHFR transcription using a transient expression assay. MTX stimulates transient expression in a concentration-dependent manner from a hamster DHFR promoter construct containing 150 base pairs 5' to the start of transcription. Addition of either tetrahydrofolate or hypoxanthine plus thymidine prevents the promoter induction in response to MTX, suggesting that stimulation by MTX results from inhibition of these metabolites. Furthermore, two other antimetabolic drugs--fluorodeoxyuridine and hydroxyurea--also stimulate the DHFR promoter in a concentration-dependent manner. In contrast, aphidicolin, which blocks cell growth through inhibition of DNA polymerase alpha, has no effect on the DHFR promoter. The potential relevance of these results to cross-resistance to chemotherapeutic agents and to the process of gene amplification is discussed. Images PMID:1833762

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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

  4. Cheminformatics Models for Inhibitors of Schistosoma mansoni Thioredoxin Glutathione Reductase

    PubMed Central

    Gaba, Sonam; Jamal, Salma; Open Source Drug Discovery Consortium

    2014-01-01

    Schistosomiasis is a neglected tropical disease caused by a parasite Schistosoma mansoni and affects over 200 million annually. There is an urgent need to discover novel therapeutic options to control the disease with the recent emergence of drug resistance. The multifunctional protein, thioredoxin glutathione reductase (TGR), an essential enzyme for the survival of the pathogen in the redox environment has been actively explored as a potential drug target. The recent availability of small-molecule screening datasets against this target provides a unique opportunity to learn molecular properties and apply computational models for discovery of activities in large molecular libraries. Such a prioritisation approach could have the potential to reduce the cost of failures in lead discovery. A supervised learning approach was employed to develop a cost sensitive classification model to evaluate the biological activity of the molecules. Random forest was identified to be the best classifier among all the classifiers with an accuracy of around 80 percent. Independent analysis using a maximally occurring substructure analysis revealed 10 highly enriched scaffolds in the actives dataset and their docking against was also performed. We show that a combined approach of machine learning and other cheminformatics approaches such as substructure comparison and molecular docking is efficient to prioritise molecules from large molecular datasets. PMID:25629082

  5. 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

  6. Biodegradation of explosives by transgenic plants expressing pentaerythritol tetranitrate reductase.

    PubMed

    French, C E; Rosser, S J; Davies, G J; Nicklin, S; Bruce, N C

    1999-05-01

    Plants offer many advantages over bacteria as agents for bioremediation; however, they typically lack the degradative capabilities of specially selected bacterial strains. Transgenic plants expressing microbial degradative enzymes could combine the advantages of both systems. To investigate this possibility in the context of bioremediation of explosive residues, we generated transgenic tobacco plants expressing pentaerythritol tetranitrate reductase, an enzyme derived from an explosive-degrading bacterium that enables degradation of nitrate ester and nitroaromatic explosives. Seeds from transgenic plants were able to germinate and grow in the presence of 1 mM glycerol trinitrate (GTN) or 0.05 mM trinitrotoluene, at concentrations that inhibited germination and growth of wild-type seeds. Transgenic seedlings grown in liquid medium with 1 mM GTN showed more rapid and complete denitration of GTN than wild-type seedlings. This example suggests that transgenic plants expressing microbial degradative genes may provide a generally applicable strategy for bioremediation of organic pollutants in soil. PMID:10331811

  7. 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

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

    PubMed

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

    2011-10-15

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

  9. 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.

  10. 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.

  11. Atypical features of Thermus thermophilus succinate:quinone reductase.

    PubMed

    Kolaj-Robin, Olga; Noor, Mohamed R; O'Kane, Sarah R; Baymann, Frauke; Soulimane, Tewfik

    2013-01-01

    The Thermus thermophilus succinate:quinone reductase (SQR), serving as the respiratory complex II, has been homologously produced under the control of a constitutive promoter and subsequently purified. The detailed biochemical characterization of the resulting wild type (wt-rcII) and His-tagged (rcII-His(8)-SdhB and rcII-SdhB-His(6)) complex II variants showed the same properties as the native enzyme with respect to the subunit composition, redox cofactor content and sensitivity to the inhibitors malonate, oxaloacetate, 3-nitropropionic acid and nonyl-4-hydroxyquinoline-N-oxide (NQNO). The position of the His-tag determined whether the enzyme retained its native trimeric conformation or whether it was present in a monomeric form. Only the trimer exhibited positive cooperativity at high temperatures. The EPR signal of the [2Fe-2S] cluster was sensitive to the presence of substrate and showed an increased rhombicity in the presence of succinate in the native and in all recombinant forms of the enzyme. The detailed analysis of the shape of this signal as a function of pH, substrate concentration and in the presence of various inhibitors and quinones is presented, leading to a model for the molecular mechanism that underlies the influence of succinate on the rhombicity of the EPR signal of the proximal iron-sulfur cluster.

  12. 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.

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

    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.

  14. 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.

  15. 5α-Reductase Type 3 Expression in Human Benign and Malignant Tissues: A Comparative Analysis During Prostate Cancer Progression

    PubMed Central

    Godoy, Alejandro; Kawinski, Elzbieta; Li, Yun; Oka, Daizo; Alexiev, Borislav; Azzouni, Faris; Titus, Mark A.; Mohler, James L.

    2015-01-01

    BACKGROUND A third isozyme of human 5α-steroid reductase, 5α-reductase-3, was identified in prostate tissue at the mRNA level. However, the levels of 5α-reductase-3 protein expression and its cellular localization in human tissues remain unknown. METHODS A specific monoclonal antibody was developed, validated, and used to characterize for the first time the expression of 5α-reductase-3 protein in 18 benign and 26 malignant human tissue types using immunostaining analyses. RESULTS AND CONCLUSIONS In benign tissues, 5α-reductase-3 immunostaining was high in conventional androgen-regulated human tissues, such as skeletal muscle and prostate. However, high levels of expression also were observed in non-conventional androgen-regulated tissues, which suggest either multiples target tissues for androgens or different functions of 5α-reductase-3 among human tissues. In malignant tissues, 5α-reductase-3 immunostaining was ubiquitous but particularly over-expressed in some cancers compared to their benign counterparts, which suggests a potential role for 5α-reductase-3 as a biomarker of malignancy. In benign prostate, 5α-reductase-3 immunostaining was localized to basal epithelial cells, with no immunostaining observed in secretory/luminal epithelial cells. In high-grade prostatic intraepithelial neoplasia (HGPIN), 5α-reductase-3 immunostaining was localized in both basal epithelial cells and neoplastic epithelial cells characteristic of HGPIN. In androgen-stimulated and castration-recurrent prostate cancer (CaP), 5α-reductase-3 immunostaining was present in most epithelial cells and at similar levels, and at levels higher than observed in benign prostate. Analyses of expression and functionality of 5α-reductase-3 in human tissues may prove useful for development of treatment for benign prostatic enlargement and prevention and treatment of CaP. PMID:21557268

  16. ADP-ribosylation of dinitrogenase reductase from Clostridium pasteurianum prevents its inhibition of nitrogenase from Azotobacter vinelandii.

    PubMed

    Murrell, S A; Lowery, R G; Ludden, P W

    1988-04-15

    The effect of ADP-ribosylation of dinitrogenase reductase on its binding to dinitrogenase was investigated. Dinitrogenase reductase from Clostridium pasteurianum (Cp2) was a substrate for the ADP-ribosyltransferase and the dinitrogenase-reductase-activating glycohydrolase from Rhodospirillum rubrum. ADP-ribosylation inactivated Cp2 and prevented its formation of a tight complex with dinitrogenase from Azotobacter vinelandii (Av1). The complex between Cp2 and Av1 could not be ADP-ribosylated once it formed.

  17. Reduction of amphetamine hydroxylamine and other aliphatic hydroxylamines by benzamidoxime reductase and human liver microsomes.

    PubMed

    Clement, B; Behrens, D; Möller, W; Cashman, J R

    2000-10-01

    For the reduction of N-hydroxylated derivatives of strongly basic functional groups, such as amidines, guanidines, and aminohydrazones, an oxygen-insensitive liver microsomal system, the benzamidoxime reductase, has been described. To reconstitute the complete activity of the benzamidoxime reductase, the system required cytochrome b(5), NADH-cytochrome b(5)-reductase, and the benzamidoxime reductase, a cytochrome P450 enzyme, which has been purified to homogeneity from pig liver. It was not known if this enzyme system was also capable of reducing aliphatic hydroxylamines. The N-hydroxylation of aliphatic amines is a well-known metabolic process. It was of interest to study the possibility of benzamidoxime reductase reducing N-hydroxylated metabolites of aliphatic amines back to the parent compound. Overall, N-hydroxylation and reduction would constitute a futile metabolic cycle. As examples of medicinally relevant compounds, the hydroxylamines of methamphetamine, amphetamine, and N-methylamine as model compounds were investigated. Formation of methamphetamine and amphetamine was analyzed by newly developed HPLC methods. All three hydroxylamines were easily reduced by benzamidoxime reductase to their parent amines with reduction rates of 220.6 nmol min(-1) (mg of protein)(-1) for methamphetamine, 5.25 nmol min(-1) (mg of protein)(-1) for amphetamine, and 153 nmol min(-1) (mg of protein)(-1) for N-methylhydroxylamine. Administration of synthetic hydroxylamines of amphetamine and methamphetamine to primary rat neuronal cultures produced frank cell toxicity. Compared with amphetamine or the oxime of amphetamine, the hydroxylamines were significantly more toxic to primary neuronal cells. The benzamidoxime reductase is therefore involved in the detoxication of these reactive hydroxylamines.

  18. The two-domain structure of 5'-adenylylsulfate (APS) reductase from Enteromorpha intestinalis is a requirement for efficient APS reductase activity.

    PubMed

    Kim, Sung-Kun; Gomes, Varinnia; Gao, Yu; Chandramouli, Kala; Johnson, Michael K; Knaff, David B; Leustek, Thomas

    2007-01-16

    5'-Adenylylsulfate (APS) reductase from Enteromorpha intestinalis (EiAPR) is composed of two domains that function together to reduce APS to sulfite. The carboxyl-terminal domain functions as a glutaredoxin that mediates the transfer of electrons from glutathione to the APS reduction site on the amino-terminal domain. To study the basis for the interdomain interaction, a heterologous system was constructed in which the C domain of EiAPR was fused to the carboxyl terminus of the APS reductase from Pseudomonas aeruginosa (PaAPR), an enzyme that normally uses thioredoxin as an electron donor and is incapable of using glutathione for this function. The hybrid enzyme, which retains the [4Fe-4S] cluster from PaAPR, was found to use both thioredoxin and glutathione as an electron donor for APS reduction. The ability to use glutathione was enhanced by the addition of Na2SO4 to the reaction buffer, a property that the hybrid enzyme shares with EiAPR. When the C domain was added as a separate component, it was much less efficient in conferring PaAPR with the ability to use glutathione as an electron donor, despite the fact that the separately expressed C domain functioned in two activities that are typical for glutaredoxins, hydroxyethyl disulfide reduction and electron donation to ribonucleotide reductase. These results suggest that the physical connection of the reductase and C domain on a single polypeptide is critical for the electron-transfer reaction. Moreover, the effect of Na2SO4 suggests that a water-ordering component of the reaction milieu is critical for the catalytic function of plant-type APS reductases by promoting the interdomain interaction.

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

    PubMed

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

    2014-01-01

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

  20. Mechanistic studies of ribonucleoside triphosphate reductase from Lactobacillus leichmannii

    SciTech Connect

    Harris, G.M.

    1984-01-01

    The mechanism of action of the adenosylcobalamin (AdoCbl)-dependent ribonucleoside triphosphate reductase (RTPR) was investigated using isotope effect and substrate specificity studies. These experiments were conducted on RTPR purified by a new method from Lactobacillus leichmannii. Isotope effect studies using (3{prime}-{sup 3}H)UTP and (3{prime}-{sup 3}H)ATP demonstrated that the 3{prime} C-H bond of the nucleotide is cleaved in order to cleave the 2{prime} C-OH bond. AdoCbl does not act as a direct H abstractor from the 3{prime} position of the substrate, but instead is thought to act as a radical chain initiator to generate an amino acid radical on the enzyme. Further support for this enzyme mediated cleavage of the 3{prime} C-H bond of the nucleotide and the novel role of AdoCbl came from studies using (3{prime}{sup 3}H)2{prime}-chloro-2{prime}-deoxyuridine 5{prime}-triphosphate ((3{prime}-{sup 3}H)CIUTP). Evidence is presented that during the course of this reaction, the {sup 3}H abstracted from the 3{prime} position of (3{prime}-{sup 3}H)CIUTP was either exchanged with the solvent or returned to the {beta} face of the 2{prime} position to produce (2{prime}{sup 3}H)-2{prime}-deoxy-3{prime}-ketoUTP. This result demonstrates that RTPR is capable of catalyzing a rearrangement reaction. The significance of the RTPR-catalyzed rearrangement with respect to the AdoCbl-dependent enzymes which catalyze rearrangements is discussed.

  1. 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

  2. Pyranopterin Coordination Controls Molybdenum Electrochemistry in Escherichia coli Nitrate Reductase*

    PubMed Central

    Wu, Sheng-Yi; Rothery, Richard A.; Weiner, Joel H.

    2015-01-01

    We test the hypothesis that pyranopterin (PPT) coordination plays a critical role in defining molybdenum active site redox chemistry and reactivity in the mononuclear molybdoenzymes. The molybdenum atom of Escherichia coli nitrate reductase A (NarGHI) is coordinated by two PPT-dithiolene chelates that are defined as proximal and distal based on their proximity to a [4Fe-4S] cluster known as FS0. We examined variants of two sets of residues involved in PPT coordination: (i) those interacting directly or indirectly with the pyran oxygen of the bicyclic distal PPT (NarG-Ser719, NarG-His1163, and NarG-His1184); and (ii) those involved in bridging the two PPTs and stabilizing the oxidation state of the proximal PPT (NarG-His1092 and NarG-His1098). A S719A variant has essentially no effect on the overall Mo(VI/IV) reduction potential, whereas the H1163A and H1184A variants elicit large effects (ΔEm values of −88 and −36 mV, respectively). Ala variants of His1092 and His1098 also elicit large ΔEm values of −143 and −101 mV, respectively. An Arg variant of His1092 elicits a small ΔEm of +18 mV on the Mo(VI/IV) reduction potential. There is a linear correlation between the molybdenum Em value and both enzyme activity and the ability to support anaerobic respiratory growth on nitrate. These data support a non-innocent role for the PPT moieties in controlling active site metal redox chemistry and catalysis. PMID:26297003

  3. 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

  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. The Effect of Protein Mass Modulation on Human Dihydrofolate Reductase.

    PubMed

    Francis, Kevin; Sapienza, Paul J; Lee, Andrew L; Kohen, Amnon

    2016-02-23

    Dihydrofolate reductase (DHFR) from Escherichia coli has long served as a model enzyme with which to elucidate possible links between protein dynamics and the catalyzed reaction. Such physical properties of its human counterpart have not been rigorously studied so far, but recent computer-based simulations suggest that these two DHFRs differ significantly in how closely coupled the protein dynamics and the catalyzed C-H → C hydride transfer step are. To test this prediction, two contemporary probes for studying the effect of protein dynamics on catalysis were combined here: temperature dependence of intrinsic kinetic isotope effects (KIEs), which are sensitive to the physical nature of the chemical step, and protein mass modulation, which slows down fast dynamics (femto- to picosecond time scale) throughout the protein. The intrinsic H/T KIEs of human DHFR, like those of E. coli DHFR, are shown to be temperature-independent in the range from 5 to 45 °C, indicating fast sampling of donor and acceptor distances (DADs) at the reaction's transition state (or tunneling ready state, TRS). Mass modulation of these enzymes through isotopic labeling with (13)C, (15)N, and (2)H at nonexchangeable hydrogens yields an 11% heavier enzyme. The additional mass has no effect on the intrinsic KIEs of the human enzyme. This finding indicates that the mass modulation of the human DHFR affects neither DAD distribution nor the DAD's conformational sampling dynamics. Furthermore, reduction in the enzymatic turnover number and the dissociation rate constant for the product indicate that the isotopic substitution affects kinetic steps that are not the catalyzed C-H → C hydride transfer. The findings are discussed in terms of fast dynamics and their role in catalysis, the comparison of calculations and experiments, and the interpretation of isotopically modulated heavy enzymes in general.

  6. 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

  7. Metabolism of bupropion by carbonyl reductases in liver and intestine.

    PubMed

    Connarn, Jamie N; Zhang, Xinyuan; Babiskin, Andrew; Sun, Duxin

    2015-07-01

    Bupropion's metabolism and the formation of hydroxybupropion in the liver by cytochrome P450 2B6 (CYP2B6) has been extensively studied; however, the metabolism and formation of erythro/threohydrobupropion in the liver and intestine by carbonyl reductases (CR) has not been well characterized. The purpose of this investigation was to compare the relative contribution of the two metabolism pathways of bupropion (by CYP2B6 and CR) in the subcellular fractions of liver and intestine and to identify the CRs responsible for erythro/threohydrobupropion formation in the liver and the intestine. The results showed that the liver microsome generated the highest amount of hydroxybupropion (Vmax = 131 pmol/min per milligram, Km = 87 μM). In addition, liver microsome and S9 fractions formed similar levels of threohydrobupropion by CR (Vmax = 98-99 pmol/min per milligram and Km = 186-265 μM). Interestingly, the liver has similar capability to form hydroxybupropion (by CYP2B6) and threohydrobupropion (by CR). In contrast, none of the intestinal fractions generate hydroxybupropion, suggesting that the intestine does not have CYP2B6 available for metabolism of bupropion. However, intestinal S9 fraction formed threohydrobupropion to the extent of 25% of the amount of threohydrobupropion formed by liver S9 fraction. Enzyme inhibition and Western blots identified that 11β-dehydrogenase isozyme 1 in the liver microsome fraction is mainly responsible for the formation of threohydrobupropion, and in the intestine AKR7 may be responsible for the same metabolite formation. These quantitative comparisons of bupropion metabolism by CR in the liver and intestine may provide new insight into its efficacy and side effects with respect to these metabolites.

  8. The potato R locus codes for dihydroflavonol 4-reductase.

    PubMed

    Zhang, Yongfei; Cheng, Shuping; De Jong, Darlene; Griffiths, Helen; Halitschke, Rayko; De Jong, Walter

    2009-09-01

    The potato R locus is required for the production of red pelargonidin-based anthocyanin pigments in potato (Solanum tuberosum L.). Red color also requires tissue-specific regulatory genes, such as D (for expression in tuber skin) and F (expression in flowers). A related locus, P, is required for production of blue/purple anthocyanins; P is epistatic to R. We have previously reported that the dihydroflavonol 4-reductase gene (dfr) co-segregates with R. To test directly whether R corresponds to dfr, we placed the allele of dfr associated with red color under the control of the CaMV 35S promoter and introduced it into the potato cultivar Prince Hairy (genotype dddd rrrr P-), which has white tubers and pale blue flowers. Transgenic Prince Hairy tubers remained white, but flower color changed to purple. Three independent transgenic lines, as well as a vector-transformed line, were then crossed with the red-skinned variety Chieftain (genotype D-R-pppp), to establish populations that segregated for D, R, P, and the dfr transgene or empty vector. Markers were used to genotype progeny at D and R. Progeny carrying the empty vector in the genetic background D-rrrr produced white or purple tubers, while progeny with the same genotype and the dfr transgene produced red or purple tubers. HPLC and LC-MS/MS analyses of anthocyanins present in Chieftain and in a red-skinned progeny clone with the dfr transgene in a D-rrrr background revealed no qualitative differences. Thus, dfr can fully complement R, both in terms of tuber color and anthocyanin composition.

  9. Tales of Dihydrofolate Binding to R67 Dihydrofolate Reductase.

    PubMed

    Duff, Michael R; Chopra, Shaileja; Strader, Michael Brad; Agarwal, Pratul K; Howell, Elizabeth E

    2016-01-12

    Homotetrameric R67 dihydrofolate reductase possesses 222 symmetry and a single active site pore. This situation results in a promiscuous binding site that accommodates either the substrate, dihydrofolate (DHF), or the cofactor, NADPH. NADPH interacts more directly with the protein as it is larger than the substrate. In contrast, the p-aminobenzoyl-glutamate tail of DHF, as monitored by nuclear magnetic resonance and crystallography, is disordered when bound. To explore whether smaller active site volumes (which should decrease the level of tail disorder by confinement effects) alter steady state rates, asymmetric mutations that decreased the half-pore volume by ∼35% were constructed. Only minor effects on k(cat) were observed. To continue exploring the role of tail disorder in catalysis, 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide-mediated cross-linking between R67 DHFR and folate was performed. A two-folate, one-tetramer complex results in the loss of enzyme activity where two symmetry-related K32 residues in the protein are cross-linked to the carboxylates of two bound folates. The tethered folate could be reduced, although with a ≤30-fold decreased rate, suggesting decreased dynamics and/or suboptimal positioning of the cross-linked folate for catalysis. Computer simulations that restrain the dihydrofolate tail near K32 indicate that cross-linking still allows movement of the p-aminobenzoyl ring, which allows the reaction to occur. Finally, a bis-ethylene-diamine-α,γ-amide folate adduct was synthesized; both negatively charged carboxylates in the glutamate tail were replaced with positively charged amines. The K(i) for this adduct was ∼9-fold higher than for folate. These various results indicate a balance between folate tail disorder, which helps the enzyme bind substrate while dynamics facilitates catalysis. PMID:26637016

  10. 5 alpha-reductase inhibitors and prostatic disease.

    PubMed

    Schröder, F H

    1994-08-01

    5 alpha-Reductase inhibitors are a new class of substances with very specific effects on type I and type II 5 alpha R which may be of use in the treatment of skin disease, such as male pattern baldness, male acne and hirsutism, as well as prostatic hyperplasia and prostate cancer. At least two types of 5 alpha R inhibitors with a different pH optimum have been described. cDNA encoding for both the type I and the type II enzyme has been cloned. Most of the orally effective 5 alpha R inhibitors belong to the class of 4-azasteroids. The radical substituted in the 17 position of the steroid ring seems to be related to species specific variations and to the types of 5 alpha R enzymes in different species and organ systems. 5 alpha R inhibitors lead to a decrease of plasma DHT by about 65% while there is a slight rise in plasma testosterone. The decrease of tissue DHT in the ventral prostate of the intact rat, the dog and in humans is more pronounced and amounts to about 85%. There is a reciprocal rise of tissue T in these systems. The application of an inhibitor of 5 alpha R type II leads to a shrinkage of BPH in men by about 30%. In the rat a similar shrinkage accompanied by a significant decrease of total organ DNA occurs. This decrease, however, is not as pronounced as can be achieved with castration.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7522999

  11. Rational Design of a Structural and Functional Nitric Oxide Reductase

    SciTech Connect

    Yeung, N.; Lin, Y; Gao, Y; Zhao, X; Russell, B; Lei, L; Miner, L; Robinson, H; Lu, Y

    2009-01-01

    Protein design provides a rigorous test of our knowledge about proteins and allows the creation of novel enzymes for biotechnological applications. Whereas progress has been made in designing proteins that mimic native proteins structurally, it is more difficult to design functional proteins. In comparison to recent successes in designing non-metalloproteins, it is even more challenging to rationally design metalloproteins that reproduce both the structure and function of native metalloenzymes. This is because protein metal-binding sites are much more varied than non-metal-containing sites, in terms of different metal ion oxidation states, preferred geometry and metal ion ligand donor sets. Because of their variability, it has been difficult to predict metal-binding site properties in silico, as many of the parameters, such as force fields, are ill-defined. Therefore, the successful design of a structural and functional metalloprotein would greatly advance the field of protein design and our understanding of enzymes. Here we report a successful, rational design of a structural and functional model of a metalloprotein, nitric oxide reductase (NOR), by introducing three histidines and one glutamate, predicted as ligands in the active site of NOR, into the distal pocket of myoglobin. A crystal structure of the designed protein confirms that the minimized computer model contains a haem/non-haem FeB centre that is remarkably similar to that in the crystal structure. This designed protein also exhibits NO reduction activity, and so models both the structure and function of NOR, offering insight that the active site glutamate is required for both iron binding and activity. These results show that structural and functional metalloproteins can be rationally designed in silico.

  12. The Effect of Protein Mass Modulation on Human Dihydrofolate Reductase.

    PubMed

    Francis, Kevin; Sapienza, Paul J; Lee, Andrew L; Kohen, Amnon

    2016-02-23

    Dihydrofolate reductase (DHFR) from Escherichia coli has long served as a model enzyme with which to elucidate possible links between protein dynamics and the catalyzed reaction. Such physical properties of its human counterpart have not been rigorously studied so far, but recent computer-based simulations suggest that these two DHFRs differ significantly in how closely coupled the protein dynamics and the catalyzed C-H → C hydride transfer step are. To test this prediction, two contemporary probes for studying the effect of protein dynamics on catalysis were combined here: temperature dependence of intrinsic kinetic isotope effects (KIEs), which are sensitive to the physical nature of the chemical step, and protein mass modulation, which slows down fast dynamics (femto- to picosecond time scale) throughout the protein. The intrinsic H/T KIEs of human DHFR, like those of E. coli DHFR, are shown to be temperature-independent in the range from 5 to 45 °C, indicating fast sampling of donor and acceptor distances (DADs) at the reaction's transition state (or tunneling ready state, TRS). Mass modulation of these enzymes through isotopic labeling with (13)C, (15)N, and (2)H at nonexchangeable hydrogens yields an 11% heavier enzyme. The additional mass has no effect on the intrinsic KIEs of the human enzyme. This finding indicates that the mass modulation of the human DHFR affects neither DAD distribution nor the DAD's conformational sampling dynamics. Furthermore, reduction in the enzymatic turnover number and the dissociation rate constant for the product indicate that the isotopic substitution affects kinetic steps that are not the catalyzed C-H → C hydride transfer. The findings are discussed in terms of fast dynamics and their role in catalysis, the comparison of calculations and experiments, and the interpretation of isotopically modulated heavy enzymes in general. PMID:26813442

  13. Pyranopterin Coordination Controls Molybdenum Electrochemistry in Escherichia coli Nitrate Reductase.

    PubMed

    Wu, Sheng-Yi; Rothery, Richard A; Weiner, Joel H

    2015-10-01

    We test the hypothesis that pyranopterin (PPT) coordination plays a critical role in defining molybdenum active site redox chemistry and reactivity in the mononuclear molybdoenzymes. The molybdenum atom of Escherichia coli nitrate reductase A (NarGHI) is coordinated by two PPT-dithiolene chelates that are defined as proximal and distal based on their proximity to a [4Fe-4S] cluster known as FS0. We examined variants of two sets of residues involved in PPT coordination: (i) those interacting directly or indirectly with the pyran oxygen of the bicyclic distal PPT (NarG-Ser(719), NarG-His(1163), and NarG-His(1184)); and (ii) those involved in bridging the two PPTs and stabilizing the oxidation state of the proximal PPT (NarG-His(1092) and NarG-His(1098)). A S719A variant has essentially no effect on the overall Mo(VI/IV) reduction potential, whereas the H1163A and H1184A variants elicit large effects (ΔEm values of -88 and -36 mV, respectively). Ala variants of His(1092) and His(1098) also elicit large ΔEm values of -143 and -101 mV, respectively. An Arg variant of His(1092) elicits a small ΔEm of +18 mV on the Mo(VI/IV) reduction potential. There is a linear correlation between the molybdenum Em value and both enzyme activity and the ability to support anaerobic respiratory growth on nitrate. These data support a non-innocent role for the PPT moieties in controlling active site metal redox chemistry and catalysis.

  14. 5 alpha-reductase deficiency in patients with micropenis.

    PubMed

    Gad, Y Z; Nasr, H; Mazen, I; Salah, N; el-Ridi, R

    1997-03-01

    The enzyme 5 alpha-reductase (5 alpha R), by virtue of its peripheral 5 alpha-reduction of testosterone (T) to dihydrotestosterone (DHT), is believed to play a major role in the differentiation and the subsequent growth of the penis. However, recent studies have reported 5 alpha R deficiency (5 alpha RD) in patients with isolated micropenis and hypothesized that 5 alpha RD is not invariably associated with genital ambiguity. In Egypt, 5 alpha RD has been reported frequently among intersex patients. The aim of this study was to assess the role of 5 alpha RD in the development of micropenis among Egyptian patients with abnormal sexual development. The study included 29 patients who were categorized into three groups (isolated micropenis, 9 patients; microphallus with genital ambiguity, 11 patients; genital ambiguity with normal-sized phallus, 9 patients). Activity of 5 alpha R was assessed by estimating T/DHT ratios in the basal state in pubertal subjects and following human chorionic gonadotropin (HCG) stimulation test in prepubertals. The results showed that the incidence of 5 alpha RD was much higher in cases of ambiguous genitalia with micropenis (5 families out of 10, 50%) than in those with isolated microphallus (1/9, 11.1%) or those with ambiguous genitalia and normal-sized phallus (1/8, 12.5%). In conclusion, the study showed that isolated micropenis is a heterogeneous disorder and that 5 alpha RD, despite its relative prevalence in Egypt, has a minimal role in the aetiology. On the other hand, 5 alpha RD seems to correlate with penile length in intersex cases.

  15. Nitrate reductase activity in heme-deficient mutants of Staphylococcus aureus.

    PubMed Central

    Burke, K A; Lascelles, J

    1976-01-01

    Mutants H-14 and H-18 of Staphylococcus aureus require hemin for growth on glycerol and other nonfermentable substrates. H-14 also responds to delta-aminolevulinate. Heme-deficient cells grown in the presence of nitrate do not have lactate-nitrate reductase activity but gain this activity when incubated with hemin in buffer and glucose. Lactate-nitrate reductase activity is also restored to the membrane fraction from such cells by incubation with hemin and dithiothreitol; addition of adenosine 5'-triphosphate has no effect upon the restoration. Cells grown with nitrate in the absence of hemin have two to five times more reduced benzyl viologen-nitrate reductase activity than do those grown with hemin. The activity increases throughout the growth period in the absence of hemin, but with hemin present enzyme formation ceases before the end of growth. There was no evidence of enzyme destruction. The distribution of nitrate reductase activity between membrane and cytoplasm was similar in cells grown with and without hemin; 70 to 90% was in the cytoplasm. It is concluded that heme-deficient staphylococci form apo-cytochrome b, which readily combines in vitro with its prosthetic group to restore normal function. The avaliability of the heme prosthetic group influences the formation of nitrate reductase. PMID:1262303

  16. 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

  17. Regulation of 5alpha-reductase isoforms by oxytocin in the rat ventral prostate.

    PubMed

    Assinder, S J; Johnson, C; King, K; Nicholson, H D

    2004-12-01

    Oxytocin (OT) is present in the male reproductive tract, where it is known to modulate contractility, cell growth, and steroidogenesis. Little is known about how OT regulates these processes. This study describes the localization of OT receptor in the rat ventral prostate and investigates if OT regulates gene expression and/or activity of 5alpha-reductase isoforms I and II. The ventral prostates of adult male Wistar rats were collected following daily sc administration of saline (control), OT, a specific OT antagonist or both OT plus antagonist for 3 d. Expression of the OT receptor was identified in the ventral prostate by RT-PCR and Western blot, and confirmed to be a single active binding site by radioreceptor assay. Immunohistochemistry localized the receptor to the epithelium of prostatic acini and to the stromal tissue. Real-time RT-PCR determined that OT treatment significantly reduced expression of 5alpha-reductase I but significantly increased 5alpha-reductase II expression in the ventral prostate. Activity of both isoforms of 5alpha-reductase was significantly increased by OT, resulting in increased concentration of prostatic dihydrotestosterone. In conclusion, OT is involved in regulating conversion of testosterone to the biologically active dihydrotestosterone in the rat ventral prostate. It does so by differential regulation of 5alpha-reductase isoforms I and II.

  18. Recombinant pinoresinol-lariciresinol reductases from western red cedar (Thuja plicata) catalyze opposite enantiospecific conversions.

    PubMed

    Fujita, M; Gang, D R; Davin, L B; Lewis, N G

    1999-01-01

    Although the heartwood of woody plants represents the main source of fiber and solid wood products, essentially nothing is known about how the biological processes leading to its formation are initiated and regulated. Accordingly, a reverse transcription-polymerase chain reaction-guided cloning strategy was employed to obtain genes encoding pinoresinol-lariciresinol reductases from western red cedar (Thuja plicata) as a means to initiate the study of its heartwood formation. (+)-Pinoresinol-(+)-lariciresinol reductase from Forsythia intermedia was used as a template for primer construction for reverse transcription-polymerase chain reaction amplifications, which, when followed by homologous hybridization cloning, resulted in the isolation of two distinct classes of putative pinoresinol-lariciresinol reductase cDNA clones from western red cedar. A representative of each class was expressed as a fusion protein with beta-galactosidase and assayed for enzymatic activity. Using both deuterated and radiolabeled (+/-)-pinoresinols as substrates, it was established that each class of cDNA encoded a pinoresinol-lariciresinol reductase of different (opposite) enantiospecificity. Significantly, the protein from one class converted (+)-pinoresinol into (-)-secoisolariciresinol, whereas the other utilized the opposite (-)-enantiomer to give the corresponding (+)-form. This differential substrate specificity raises important questions about the role of each of these individual reductases in heartwood formation, such as whether they are expressed in different cells/tissues or at different stages during heartwood development.

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

    PubMed

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

    2012-05-01

    Recent research on respiratory chain of the parasitic helminth, Ascaris suum has shown that the mitochondrial NADH-fumarate reductase system (fumarate respiration), which is composed of complex I (NADH-rhodoquinone reductase), rhodoquinone and complex II (rhodoquinol-fumarate reductase) plays an important role in the anaerobic energy metabolism of adult parasites inhabiting hosts. The enzymes in these parasite-specific pathways are potential target for chemotherapy. We isolated a novel compound, nafuredin, from Aspergillus niger, which inhibits NADH-fumarate reductase in helminth mitochondria at nM order. It competes for the quinone-binding site in complex I and shows high selective toxicity to the helminth enzyme. Moreover, nafuredin exerts anthelmintic activity against Haemonchus contortus in in vivo trials with sheep indicating that mitochondrial complex I is a promising target for chemotherapy. In addition to complex I, complex II is a good target because its catalytic direction is reverse of succinate-ubiquionone reductase in the host complex II. Furthermore, we found atpenin and flutolanil strongly and specifically inhibit mitochondrial complex II. Interestingly, fumarate respiration was found not only in the parasites but also in some types of human cancer cells. Analysis of the mitochondria from the cancer cells identified an anthelminthic as a specific inhibitor of the fumarate respiration. Role of isoforms of human complex II in the hypoxic condition of cancer cells and fetal tissues is a challenge. This article is part of a Special Issue entitled Biochemistry of Mitochondria, Life and Intervention 2010. PMID:22226661

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

    PubMed Central

    Beierlein, Jennifer M.; Frey, Kathleen M.; Bolstad, David B.; Pelphrey, Phillip M.; Joska, Tammy M.; Smith, Adrienne E.; Priestley, Nigel D.; Wright, Dennis L.; Anderson, Amy C.

    2008-01-01

    Bacillus anthracis, the causative agent of anthrax, poses a significant biodefense danger. Serious limitations in approved therapeutics and the generation of resistance have produced a compelling need for new therapeutic agents against this organism. Bacillus anthracis is known to be insensitive to the clinically used antifolate, trimethoprim, because of a lack of potency against the dihydrofolate reductase enzyme. Herein, we describe a novel lead series of B. anthracis dihydrofolate reductase inhibitors characterized by an extended trimethoprim-like scaffold. The best lead compound adds only 22 Da to the molecular weight and is 82-fold more potent than trimethoprim. An X-ray crystal structure of this lead compound bound to B. anthracis dihydrofolate reductase in the presence of NADPH was determined to 2.25 Å resolution. The structure reveals several features that can be exploited for further development of this lead series. PMID:19007108

  1. Crystallization and preliminary X-ray crystallographic analysis of Sulfolobus solfataricus thioredoxin reductase

    SciTech Connect

    Ruggiero, Alessia; Ruocco, Maria Rosaria; Grimaldi, Pasquale; Arcari, Paolo; Masullo, Mariorosario; Zagari, Adriana; Vitagliano, Luigi

    2005-10-01

    The thioredoxin reductase isolated from S. solfataricus has been crystallized. Diffraction data have been collected from the wild type and from the NADP-bound form of the enzyme to 1.80 and 1.95 Å, respectively. The structure of the thioredoxin reductase has been solved using MAD diffraction data. A thermostable thioredoxin reductase isolated from Sulfolobus solfataricus (SsTrxR) has been successfully crystallized in the absence and in the presence of NADP. Two different crystal forms have been obtained. Crystals of the form that yields higher resolution data (1.8 Å) belong to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 76.77, b = 120.68, c = 126.85 Å. The structure of the enzyme has been solved by MAD methods using the anomalous signal from the Se atoms of selenomethionine-labelled SsTrxR.

  2. Denitrification by plant roots? New aspects of plant plasma membrane-bound nitrate reductase.

    PubMed

    Eick, Manuela; Stöhr, Christine

    2012-10-01

    A specific form of plasma membrane-bound nitrate reductase in plants is restricted to roots. Two peptides originated from plasma membrane integral proteins isolated from Hordeum vulgare have been assigned as homologues to the subunit NarH of respiratory nitrate reductase of Escherichia coli. Corresponding sequences have been detected for predicted proteins of Populus trichocarpa with high degree of identities for the subunits NarH (75%) and NarG (65%), however, with less accordance for the subunit NarI. These findings coincide with biochemical properties, particularly in regard to the electron donors menadione and succinate. Together with the root-specific and plasma membrane-bound nitrite/NO reductase, nitric oxide is produced under hypoxic conditions in the presence of nitrate. In this context, a possible function in nitrate respiration of plant roots and an involvement of plants in denitrification processes are discussed.

  3. Structure of Physarum polycephalum cytochrome b 5 reductase at 1.56 Å resolution

    PubMed Central

    Kim, Sangwoo; Suga, Michihiro; Ogasahara, Kyoko; Ikegami, Terumi; Minami, Yoshiko; Yubisui, Toshitsugu; Tsukihara, Tomitake

    2007-01-01

    Physarum polycephalum cytochrome b 5 reductase catalyzes the reduction of cytochrome b 5 by NADH. The structure of P. polycephalum cytochrome b 5 reductase was determined at a resolution of 1.56 Å. The molecular structure was compared with that of human cytochrome b 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. PMID:17401193

  4. 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

  5. Crystallization and preliminary X-ray crystallographic studies of pig heart carbonyl reductase

    SciTech Connect

    Aoki, Ken-ichi; Tanaka, Nobutada; Ishikura, Shuhei; Araki, Naoko; Imamura, Yorishige; Hara, Akira; Nakamura, Kazuo T.

    2006-10-01

    Pig heart carbonyl reductase has been crystallized in the presence of NADPH. Diffraction data have been collected using synchrotron radiation. Pig heart carbonyl reductase (PHCR), which belongs to the short-chain dehydrogenase/reductase (SDR) family, has been crystallized by the hanging-drop vapour-diffusion method. Two crystal forms (I and II) have been obtained in the presence of NADPH. Form I crystals belong to the tetragonal space group P4{sub 2}, with unit-cell parameters a = b = 109.61, c = 94.31 Å, and diffract to 1.5 Å resolution. Form II crystals belong to the tetragonal space group P4{sub 1}2{sub 1}2, with unit-cell parameters a = b = 120.10, c = 147.00 Å, and diffract to 2.2 Å resolution. Both crystal forms are suitable for X-ray structure analysis at high resolution.

  6. 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.

  7. Interplay between the cis-prenyltransferases and polyprenol reductase in the yeast Saccharomyces cerevisiae.

    PubMed

    Szkopinska, Anna; Swiezewska, Ewa; Rytka, Joanna

    2006-01-01

    Dolichol formation is examined in three Saccharomyces cerevisiae strains with mutations in the ERG20 gene encoding farnesyl diphosphate synthase (mevalonic acid pathway) and/or the ERG9 gene encoding squalene synthase (sterol synthesis pathway) differing in the amount and chain length of the polyisoprenoids synthesized. Our results suggest that the activities of two yeast cis-prenyltransferases Rer2p and Srt1p and polyprenol reductase are not co-regulated and that reductase may be the rate-limiting enzyme in dolichol synthesis if the amount of polyisoprenoids synthesized exceeds a certain level. We demonstrate that reductase preferentially acts on typical polyprenols with 13-18 isoprene residues but can reduce much longer polyprenols with even 32 isoprene residues.

  8. 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

  9. 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.

  10. 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.

  11. 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

  12. Side chain conformational averaging in human dihydrofolate reductase.

    PubMed

    Tuttle, Lisa M; Dyson, H Jane; Wright, Peter E

    2014-02-25

    The three-dimensional structures of the dihydrofolate reductase enzymes from Escherichia coli (ecDHFR or ecE) and Homo sapiens (hDHFR or hE) are very similar, despite a rather low level of sequence identity. Whereas the active site loops of ecDHFR undergo major conformational rearrangements during progression through the reaction cycle, hDHFR remains fixed in a closed loop conformation in all of its catalytic intermediates. To elucidate the structural and dynamic differences between the human and E. coli enzymes, we conducted a comprehensive analysis of side chain flexibility and dynamics in complexes of hDHFR that represent intermediates in the major catalytic cycle. Nuclear magnetic resonance relaxation dispersion experiments show that, in marked contrast to the functionally important motions that feature prominently in the catalytic intermediates of ecDHFR, millisecond time scale fluctuations cannot be detected for hDHFR side chains. Ligand flux in hDHFR is thought to be mediated by conformational changes between a hinge-open state when the substrate/product-binding pocket is vacant and a hinge-closed state when this pocket is occupied. Comparison of X-ray structures of hinge-open and hinge-closed states shows that helix αF changes position by sliding between the two states. Analysis of χ1 rotamer populations derived from measurements of (3)JCγCO and (3)JCγN couplings indicates that many of the side chains that contact helix αF exhibit rotamer averaging that may facilitate the conformational change. The χ1 rotamer adopted by the Phe31 side chain depends upon whether the active site contains the substrate or product. In the holoenzyme (the binary complex of hDHFR with reduced nicotinamide adenine dinucleotide phosphate), a combination of hinge opening and a change in the Phe31 χ1 rotamer opens the active site to facilitate entry of the substrate. Overall, the data suggest that, unlike ecDHFR, hDHFR requires minimal backbone conformational rearrangement as

  13. 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

  14. Adverse Effects and Safety of 5-alpha Reductase Inhibitors (Finasteride, Dutasteride): A Systematic Review

    PubMed Central

    Hirshburg, Jason M.; Kelsey, Petra A.; Therrien, Chelsea A.; Gavino, A. Carlo; Reichenberg, Jason S.

    2016-01-01

    Finasteride and dutasteride, both 5-alpha reductase inhibitors, are considered first-line treatment for androgenetic hair loss in men and used increasingly in women. In each case, patients are expected to take the medications indefinitely despite the lack of research regarding long-term adverse effects. Concerns regarding the adverse effects of these medications has led the United States National Institutes of Health to add a link for post-finasteride syndrome to its Genetic and Rare Disease Information Center. Herein, the authors report the results of a literature search reviewing adverse events of 5-alpha reductase inhibitors as they relate to prostate cancer, psychological effects, sexual health, and use in women. Several large studies found no increase in incidence of prostate cancer, a possible increase of high-grade cancer when detected, and no change in survival rate with 5-alpha reductase inhibitor use. Currently, there is no direct link between 5-alpha reductase inhibitor use and depression; however, several small studies have led to depression being listed as a side effect on the medication packaging. Sexual effects including erectile dysfunction and decreased libido and ejaculate were reported in as many as 3.4 to 15.8 percent of men. To date, there are very few studies evaluating 5-alpha reductase inhibitor use in women. Risks include birth defects in male fetuses if used in pregnancy, decreased libido, headache, gastrointestinal discomfort, and isolated reports of changes in menstruation, acne, and dizziness. Overall, 5-alpha reductase inhibitors were well-tolerated in both men and women, but not without risk, highlighting the importance of patient education prior to treatment. PMID:27672412

  15. The biochemical and phenotypic characterization of females homozygous for 5 alpha-reductase-2 deficiency.

    PubMed

    Katz, M D; Cai, L Q; Zhu, Y S; Herrera, C; DeFillo-Ricart, M; Shackleton, C H; Imperato-McGinley, J

    1995-11-01

    The biochemical and physiologic manifestations of decreased 5 alpha-dihydrotestosterone (DHT) in females are characterized. Three females from the large Dominican kindred with 5 alpha-reductase-2 deficiency were identified as homozygous for a point mutation (R246W, C-->T) on exon 5 of the 5 alpha-reductase-2 gene by single strand DNA conformational polymorphism analysis and DNA sequence analysis. Body hair was decreased; there was no history of acne. Despite delayed menarche, all were fertile, and two had twins. Urinary 5 beta/5 alpha C19 and C21 steroid metabolite ratios were elevated. Plasma testosterone was normal to elevated, with low DHT, resulting in an increased testosterone/DHT ratio. 3 alpha,5 alpha-Androstanediol glucuronide was low. Menstrual cycle profiling performed in two subjects showed ovulatory gonadotropin peaks. Sebum production was normal. 5 alpha-Reductase-2-deficient homozygotic females demonstrate the importance of DHT in the physiology and pathophysiology of body hair growth. Normal sebum implies regulation by the 5 alpha-reductase-1 isoenzyme. Delayed puberty suggests involvement of 5 alpha-reductase-2 in menarche at the hypothalamic/pituitary and/or ovarian level. As two had nonidentical twins, DHT and/or the DHT/estradiol ratio may regulate follicular development, with lower levels permitting more than one dominant follicle per cycle and higher levels impairing follicular development and ovulation. Thus, females with 5 alpha-reductase-2 deficiency highlight a role for DHT in hirsutism and/or menstrual disorders. PMID:7593420

  16. Adverse Effects and Safety of 5-alpha Reductase Inhibitors (Finasteride, Dutasteride): A Systematic Review

    PubMed Central

    Hirshburg, Jason M.; Kelsey, Petra A.; Therrien, Chelsea A.; Gavino, A. Carlo; Reichenberg, Jason S.

    2016-01-01

    Finasteride and dutasteride, both 5-alpha reductase inhibitors, are considered first-line treatment for androgenetic hair loss in men and used increasingly in women. In each case, patients are expected to take the medications indefinitely despite the lack of research regarding long-term adverse effects. Concerns regarding the adverse effects of these medications has led the United States National Institutes of Health to add a link for post-finasteride syndrome to its Genetic and Rare Disease Information Center. Herein, the authors report the results of a literature search reviewing adverse events of 5-alpha reductase inhibitors as they relate to prostate cancer, psychological effects, sexual health, and use in women. Several large studies found no increase in incidence of prostate cancer, a possible increase of high-grade cancer when detected, and no change in survival rate with 5-alpha reductase inhibitor use. Currently, there is no direct link between 5-alpha reductase inhibitor use and depression; however, several small studies have led to depression being listed as a side effect on the medication packaging. Sexual effects including erectile dysfunction and decreased libido and ejaculate were reported in as many as 3.4 to 15.8 percent of men. To date, there are very few studies evaluating 5-alpha reductase inhibitor use in women. Risks include birth defects in male fetuses if used in pregnancy, decreased libido, headache, gastrointestinal discomfort, and isolated reports of changes in menstruation, acne, and dizziness. Overall, 5-alpha reductase inhibitors were well-tolerated in both men and women, but not without risk, highlighting the importance of patient education prior to treatment.

  17. Adverse Effects and Safety of 5-alpha Reductase Inhibitors (Finasteride, Dutasteride): A Systematic Review.

    PubMed

    Hirshburg, Jason M; Kelsey, Petra A; Therrien, Chelsea A; Gavino, A Carlo; Reichenberg, Jason S

    2016-07-01

    Finasteride and dutasteride, both 5-alpha reductase inhibitors, are considered first-line treatment for androgenetic hair loss in men and used increasingly in women. In each case, patients are expected to take the medications indefinitely despite the lack of research regarding long-term adverse effects. Concerns regarding the adverse effects of these medications has led the United States National Institutes of Health to add a link for post-finasteride syndrome to its Genetic and Rare Disease Information Center. Herein, the authors report the results of a literature search reviewing adverse events of 5-alpha reductase inhibitors as they relate to prostate cancer, psychological effects, sexual health, and use in women. Several large studies found no increase in incidence of prostate cancer, a possible increase of high-grade cancer when detected, and no change in survival rate with 5-alpha reductase inhibitor use. Currently, there is no direct link between 5-alpha reductase inhibitor use and depression; however, several small studies have led to depression being listed as a side effect on the medication packaging. Sexual effects including erectile dysfunction and decreased libido and ejaculate were reported in as many as 3.4 to 15.8 percent of men. To date, there are very few studies evaluating 5-alpha reductase inhibitor use in women. Risks include birth defects in male fetuses if used in pregnancy, decreased libido, headache, gastrointestinal discomfort, and isolated reports of changes in menstruation, acne, and dizziness. Overall, 5-alpha reductase inhibitors were well-tolerated in both men and women, but not without risk, highlighting the importance of patient education prior to treatment. PMID:27672412

  18. 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

  19. 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 ...

  20. 5 alpha-reductase and aromatase inhibitory constituents from Brassica rapa L. pollen.

    PubMed

    Li, Yong-Hui; Yang, Yi-Fang; Li, Kun; Jin, Li-Li; Yang, Nian-Yun; Kong, De-Yun

    2009-04-01

    In the screening of biologically active constituents from Brassica rapa pollen, the supercritical CO(2) fluid extract (SFE-CO(2)) showed potent 5 alpha-reductase and aromatase inhibiting activity. The SFE-CO(2) extract was separated by various chromatographic methods to give two new phytosterol derivatives, 24-methylenecholesterol linolenate (1) and cycloeucalenol linolenate (2), as well as eight known compounds, 24-methylenecholesterol palmitate (3), cycloeucalenol (4), pollinastanol (5), 24-methylenecholesterol (6), linolenic acid (7), palmitic acid (8), monolinolein (9) and monopalmitin (10), compounds 7 and 9 showed potent 5 alpha-reductase inhibitory activity; compounds 1-6 and 10 showed potent aromatase inhibitory activity.

  1. 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.

  2. 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

  3. 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.

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

    PubMed

    Plancarte, Agustin; Nava, Gabriela

    2015-02-01

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

  5. Is decreased libido associated with the use of HMG-CoA-reductase inhibitors?

    PubMed Central

    de Graaf, L; Brouwers, A H P M; Diemont, W L

    2004-01-01

    Aims and methods To describe patients with decreased libido during use of a HMG-CoA-reductase-inhibitor, and to discuss causality and pharmacological hypotheses for this association by analysis of the adverse drug reactions (ADR) database of the Netherlands Pharmacovigilance Centre Lareb. Results Eight patients were identified as having decreased libido during use of statins. In two of these cases testosterone levels were determined and appeared to be decreased. Conclusion Decreased libido is a probable adverse drug reaction of HMG-CoA-reductase-inhibitors and is reversible. The ADR may be caused by low serum testosterone levels, mainly due to intracellular cholesterol depletion. PMID:15327593

  6. 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.

  7. 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

  8. The activation state of nitrate reductase is not always correlated with total nitrate reductase activity in leaves

    PubMed

    Man; Abd-El Baki GK; Stegmann; Weiner; Kaiser

    1999-10-01

    The relation between nitrate reductase (NR; EC 1.6.6.1) activity, activation state and NR protein in leaves of barley (Hordeum vulgare L.) seedlings was investigated. Maximum NR activity (NRA(max)) and NR protein content (Western blotting) were modified by growing plants hydroponically at low (0.3 mM) or high (10 mM) nitrate supply. In addition, plants were kept under short-day (8 h light/16 h dark) or long-day (16 h light/8 h dark) conditions in order to manipulate the concentration of nitrate stored in the leaves during the dark phase, and the concentrations of sugars and amino acids accumulated during the light phase, which are potential signalling compounds. Plants were also grown under phosphate deficiency in order to modify their glucose-6-phosphate content. In high-nitrate/long-day conditions, NRA(max) and NR protein were almost constant during the whole light period. Low-nitrate/long-day plants had only about 30% of the NRA(max) and NR protein of high-nitrate plants. In low-nitrate/long-day plants, NRA(max) and NR protein decreased strongly during the second half of the light phase. The decrease was preceded by a strong decrease in the leaf nitrate content. Short daylength generally led to higher nitrate concentrations in leaves. Under short-day/low-nitrate conditions, NRA(max) was slightly higher than under long-day conditions and remained almost constant during the day. This correlated with maintenance of higher nitrate concentrations during the short light period. The NR activation state in the light was very similar in high-nitrate and low-nitrate plants, but dark inactivation was twice as high in the high-nitrate plants. Thus, the low NRA(max) in low-nitrate/long-day plants was slightly compensated by a higher activation state of NR. Such a partial compensation of a low NR(max) by a higher dark activation state was not observed with phosphate-depleted plants. Total leaf concentrations of sugars, of glutamine and glutamate and of glucose-6-phosphate did

  9. 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

  10. 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

  11. 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

  12. Proton and electron pathways in the bacterial nitric oxide reductase.

    PubMed

    Hendriks, Janneke H M; Jasaitis, Audrius; Saraste, Matti; Verkhovsky, Michael I

    2002-02-19

    Electron- and proton-transfer reactions in bacterial nitric oxide reductase (NOR) have been investigated by optical spectroscopy and electrometry. In liposomes, NOR does not show any generation of an electric potential during steady-state turnover. This electroneutrality implies that protons are taken up from the same side of the membrane as electrons during catalysis. Intramolecular electron redistribution after photolysis of the partially reduced CO-bound enzyme shows that the electron transfer in NOR has the same pathway as in the heme-copper oxidases. The electron is transferred from the acceptor site, heme c, via a low-spin heme b to the binuclear active site (heme b3/FeB). The electron-transfer rate between hemes c and b is (3 +/- 2) x 10(4) s(-1). The rate of electron transfer between hemes b and b3 is too fast to be resolved (>10(6) s(-1)). Only electron transfer between heme c and heme b is coupled to the generation of an electric potential. This implies that the topology of redox centers in NOR is comparable to that in the heme-copper cytochrome oxidases. The optical and electrometric measurements allow identification of the intermediate states formed during turnover of the fully reduced enzyme, as well as the associated proton and electron movement linked to the NO reduction. The first phase (k = 5 x 10(5) s(-1)) is electrically silent, and characterized by the disappearance of absorbance at 433 nm and the appearance of a broad peak at 410 nm. We assign this phase to the formation of a ferrous NO adduct of heme b3. NO binding is followed by a charge separation phase (k = 2.2 x 10(5) s(-1)). We suggest that the formation of this intermediate that is not linked to significant optical changes involves movement of charged side chains near the active site. The next step creates a negative potential with a rate constant of approximately 3 x 10(4) s(-1) and a weak optical signature. This is followed by an electrically silent phase with a rate constant of 5 x 10

  13. Tissue distribution and ontogeny of steroid 5 alpha-reductase isozyme expression.

    PubMed Central

    Thigpen, A E; Silver, R I; Guileyardo, J M; Casey, M L; McConnell, J D; Russell, D W

    1993-01-01

    The synthesis of dihydrotestosterone is catalyzed by steroid 5 alpha-reductase isozymes, designated types 1 and 2. Mutation of type 2 results in male pseudohermaphroditism, in which the external genitalia are phenotypically female at birth. Two striking and unexplained features of this disorder are that external genitalia of affected males undergo virilization during puberty and that these individuals have less temporal hair regression. The tissue-specific and developmental expression patterns of the 5 alpha-reductase isozymes were investigated by immunoblotting. The type 1 isozyme is not detectable in the fetus, is transiently expressed in newborn skin and scalp, and permanently expressed in skin from the time of puberty. There was no qualitative difference in 5 alpha-reductase type 1 expression between adult balding vs. nonbalding scalp. The type 2 isozyme is transiently expressed in skin and scalp of newborns. Type 2 is the predominant isozyme detectable in fetal genital skin, male accessory sex glands, and in the prostate, including benign prostatic hyperplasia and prostate adenocarcinoma tissues. Both isozymes are expressed in the liver, but only after birth. These results are consistent with 5 alpha-reductase type 1 being responsible for virilization in type 2-deficient subjects during puberty, and suggest that the type 2 isozyme may be an initiating factor in development of male pattern baldness. Images PMID:7688765

  14. 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

  15. 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

  16. Primary ∆4-3-oxosteroid 5β-reductase deficiency: two cases in China.

    PubMed

    Zhao, Jing; Fang, Ling-Juan; Setchell, Kenneth D R; Chen, Rui; Li, Li-Ting; Wang, Jian-She

    2012-12-21

    Aldo-keto reductase 1D1 (AKR1D1) deficiency, a rare but life-threatening form of bile acid deficiency, has not been previously described in China. Here, we describe the first two primary ∆4-3-oxosteroid 5β-reductase deficiency patients in Mainland China diagnosed by fast atom bombardment-mass spectroscopy of urinary bile acids and confirmed by genetic analysis. A high proportion of atypical 3-oxo-∆4-bile acids in the urine indicated a deficiency in ∆4-3-oxosteroid 5β-reductase. All of the coding exons and adjacent intronic sequence of the AKR1D1 gene were sequenced using peripheral lymphocyte genomic DNA of two patients and one of the patient's parents. One patient exhibited compound heterozygous mutations: c.396C>A and c.722A>T, while the other was heterozygous for the mutation c.797G>A. Based on these mutations, a diagnosis of primary ∆4-3-oxosteroid 5β-reductase deficiency could be confirmed. With ursodeoxycholic acid treatment and fat-soluble vitamin supplements, liver function tests normalized rapidly, and the degree of hepatomegaly was markedly reduced in both patients.

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

    PubMed Central

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

    2014-01-01

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

  18. Copper-dependent inhibition and oxidative inactivation with affinity cleavage of yeast glutathione reductase.

    PubMed

    Murakami, Keiko; Tsubouchi, Ryoko; Fukayama, Minoru; Yoshino, Masataka

    2014-06-01

    Effects of copper on the activity and oxidative inactivation of yeast glutathione reductase were analyzed. Glutathione reductase from yeast was inhibited by cupric ion and more potently by cuprous ion. Copper ion inhibited the enzyme noncompetitively with respect to the substrate GSSG and NADPH. The Ki values of the enzyme for Cu(2+) and Cu(+) ion were determined to be 1 and 0.35 μM, respectively. Copper-dependent inactivation of glutathione reductase was also analyzed. Hydrogen peroxide and copper/ascorbate also caused an inactivation with the cleavage of peptide bond of the enzyme. The inactivation/fragmentation of the enzyme was prevented by addition of catalase, suggesting that hydroxyl radical produced through the cuprous ion-dependent reduction of oxygen is responsible for the inactivation/fragmentation of the enzyme. SDS-PAGE and TOF-MS analysis confirmed eight fragments, which were further determined to result from the cleavage of the Met17-Ser18, Asn20-Thr21, Glu251-Gly252, Ser420-Pro421, Pro421-Thr422 bonds of the enzyme by amino-terminal sequencing analysis. Based on the kinetic analysis and no protective effect of the substrates, GSSG and NADPH on the copper-mediated inactivation/fragmentation of the enzyme, copper binds to the sites apart from the substrate-sites, causing the peptide cleavage by hydroxyl radical. Copper-dependent oxidative inactivation/fragmentation of glutathione reductase can explain the prooxidant properties of copper under the in vivo conditions.

  19. 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.

  20. 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

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

    PubMed

    Rižner, Tea Lanišnik; Penning, Trevor M

    2014-01-01

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

  2. 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)

  3. 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

  4. Identification and molecular characterization of mitochondrial ferredoxins and ferredoxin reductase from Arabidopsis.

    PubMed

    Takubo, Keiko; Morikawa, Tomomi; Nonaka, Yasuki; Mizutani, Masaharu; Takenaka, Shigeo; Takabe, Keiji; Takahashi, Masa-aki; Ohta, Daisaku

    2003-07-01

    We have identified and characterized novel types of ferredoxin and ferredoxin reductase from Arabidopsis. Among a number of potential ferredoxin reductase genes in the Arabidopsis genome, AtMFDR was identified to encode a homologue of mitochondrial ferredoxin reductase, and AtMFDX1 and AtMFDX2 were predicted to code for proteins similar to mitochondrial ferredoxin. First, we isolated cDNAs for these proteins and expressed them in heterologous systems of insect cells and Escherichia coli, respectively. The recombinant AtMFDX1 and AtMFDR proteins exhibited spectral properties characteristic of ferredoxin and ferredoxin reductase, respectively, and a pair of recombinant AtMFDX1 and AtMFDR proteins was sufficient to transfer electrons from NAD(P)H to cytochrome c in vitro. Subcellular fractionation analyses suggested membrane association of AtMFDR protein, and protein-gel blot analyses and transient expression studies of green fluorescence protein fusions indicated mitochondrial localization of AtMFDX1 and AtMFDR. RNA-gel blot analyses revealed that the accumulation levels of AtMFDXs and AtMFDR gene transcripts were specifically high in flowers, while protein-gel blot analysis demonstrated substantial accumulation of AtMFDR protein in leaf, stem, and flower. Possible physiological roles of these mitochondrial electron transfer components are discussed in relation to redox dependent metabolic pathways in plants.

  5. 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

  6. Non-heme iron hydroperoxo species in superoxide reductase as a catalyst for oxidation reactions.

    PubMed

    Rat, S; Ménage, S; Thomas, F; Nivière, V

    2014-11-25

    The non-heme high-spin ferric iron hydroperoxo species formed in superoxide reductase catalyzes oxidative aldehyde deformylation through its nucleophilic character. This species also acts as an electrophile to catalyze oxygen atom transfer in sulfoxidation reactions, highlighting the oxidation potential of non-heme iron hydroperoxo species.

  7. 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 ...

  8. Identification and in vitro characterization of a Marek’s disease virus encoded ribonucleotide reductase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Marek’s disease virus (MDV) encodes a ribonucleotide reductase (RR), a key regulatory enzyme in the DNA synthesis pathway. The gene coding for the RR of MDV is located in the unique long (UL) region of the genome. The large subunit is encoded by UL39 (RR1) and is predicted to comprise 860 amino acid...

  9. 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

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

    PubMed Central

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

    2012-01-01

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

  11. Discovering Echinococcus granulosus thioredoxin glutathione reductase inhibitors through site-specific dynamic combinatorial chemistry.

    PubMed

    Saiz, Cecilia; Castillo, Valerie; Fontán, Pablo; Bonilla, Mariana; Salinas, Gustavo; Rodríguez-Haralambides, Alejandra; Mahler, S Graciela

    2014-02-01

    In this study, we report a strategy using dynamic combinatorial chemistry for targeting the thioredoxin (Trx)-reductase catalytic site on Trx glutathione reductase (TGR), a pyridine nucleotide thiol-disulfide oxido-reductase. We chose Echinococcus granulosus TGR since it is a bottleneck enzyme of platyhelminth parasites and a validated pharmacological target. A dynamic combinatorial library (DCL) was constructed based on thiol-disulfide reversible exchange. We demonstrate the use of 5-thio-2-nitrobenzoic acid (TNB) as a non-covalent anchor fragment in a DCL templated by E. granulosus TGR. The heterodimer of TNB and bisthiazolidine (2af) was identified, upon library analysis by HPLC (IC50 = 24 μM). Furthermore, 14 analogs were synthetically prepared and evaluated against TGR. This allowed the study of a structure-activity relationship and the identification of a disulfide TNB-tricyclic bisthiazolidine (2aj) as the best enzyme inhibitor in these series, with an IC50 = 24 μM. Thus, our results validate the use of DCL for targeting thiol-disulfide oxido-reductases.

  12. Molecular cloning and expression of the human delta7-sterol reductase.

    PubMed

    Moebius, F F; Fitzky, B U; Lee, J N; Paik, Y K; Glossmann, H

    1998-02-17

    Inhibitors of the last steps of cholesterol biosynthesis such as AY9944 and BM15766 severely impair brain development. Their molecular target is the Delta7-sterol reductase (EC 1.3.1.21), suspected to be defective in the Smith-Lemli-Opitz syndrome, a frequent inborn disorder of sterol metabolism. Molecular cloning of the cDNA revealed that the human enzyme is a membrane-bound protein with a predicted molecular mass of 55 kDa and six to nine putative transmembrane segments. The protein is structurally related to plant and yeast sterol reductases. In adults the ubiquitously transcribed mRNA is most abundant in adrenal gland, liver, testis, and brain. The Delta7-sterol reductase is the ultimate enzyme of cholesterol biosynthesis in vertebrates and is absent from yeast. Microsomes from Saccharomyces cerevisiae strains heterologously expressing the human cDNA remove the C7-8 double bond in 7-dehydrocholesterol. The conversion to cholesterol depends on NADPH and is potently inhibited by AY9944 (IC50 0.013 microM), BM15766 (IC50 1.2 microM), and triparanol (IC50 14 microM). Our work paves the way to clarify whether a defect in the delta7-sterol reductase gene underlies the Smith-Lemli-Opitz syndrome. PMID:9465114

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

    PubMed Central

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

    1998-01-01

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

  14. 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...

  15. 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

  16. 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...

  17. Genes encoding vitamin-K epoxide reductase are present in Drosophila and trypanosomatid protists.

    PubMed

    Robertson, Hugh M

    2004-10-01

    Vitamin-K epoxide reductase is encoded by the VKORC1 gene in mammals and other vertebrates, which also have a paralog, VKORC1L1. Single homologs are present in basal deuterostome and insect genomes, including Drosophila, and three trypanosomatid protists. VKOR is therefore an ancient gene/protein that can be studied in the Drosophila model system.

  18. Purification and characterization of a soybean flour-inducible ferredoxin reductase of Streptomyces griseus.

    PubMed Central

    Ramachandra, M; Seetharam, R; Emptage, M H; Sariaslani, F S

    1991-01-01

    We have purified an NADH-dependent ferredoxin reductase from crude extracts of Streptomyces griseus cells grown in soybean flour-enriched medium. The purified protein has a molecular weight of 60,000 as determined by sodium dodecyl sulfate gel electrophoresis. The enzyme requires Mg2+ ion for catalytic activity in reconstituted assays, and its spectral properties resemble those of many other flavin adenine dinucleotide-containing flavoproteins. A relatively large number of hydrophobic amino acid residues are found by amino acid analysis, and beginning with residue 7, a consensus flavin adenine dinucleotide binding sequence, GXGXXGXXXA, is revealed in this protein. In the presence of NADH, the ferredoxin reductase reduces various electron acceptors such as cytochrome c, potassium ferricyanide, dichlorophenolindophenol, and nitroblue tetrazolium. However, only cytochrome c reduction by the ferredoxin reductase is enhanced by the addition of ferredoxin. In the presence of NADH, S. griseus ferredoxin and cytochrome P-450soy, the ferredoxin reductase mediates O dealkylation of 7-ethoxycoumarin. Images FIG. 2 PMID:1938912

  19. Copper-dependent inhibition and oxidative inactivation with affinity cleavage of yeast glutathione reductase.

    PubMed

    Murakami, Keiko; Tsubouchi, Ryoko; Fukayama, Minoru; Yoshino, Masataka

    2014-06-01

    Effects of copper on the activity and oxidative inactivation of yeast glutathione reductase were analyzed. Glutathione reductase from yeast was inhibited by cupric ion and more potently by cuprous ion. Copper ion inhibited the enzyme noncompetitively with respect to the substrate GSSG and NADPH. The Ki values of the enzyme for Cu(2+) and Cu(+) ion were determined to be 1 and 0.35 μM, respectively. Copper-dependent inactivation of glutathione reductase was also analyzed. Hydrogen peroxide and copper/ascorbate also caused an inactivation with the cleavage of peptide bond of the enzyme. The inactivation/fragmentation of the enzyme was prevented by addition of catalase, suggesting that hydroxyl radical produced through the cuprous ion-dependent reduction of oxygen is responsible for the inactivation/fragmentation of the enzyme. SDS-PAGE and TOF-MS analysis confirmed eight fragments, which were further determined to result from the cleavage of the Met17-Ser18, Asn20-Thr21, Glu251-Gly252, Ser420-Pro421, Pro421-Thr422 bonds of the enzyme by amino-terminal sequencing analysis. Based on the kinetic analysis and no protective effect of the substrates, GSSG and NADPH on the copper-mediated inactivation/fragmentation of the enzyme, copper binds to the sites apart from the substrate-sites, causing the peptide cleavage by hydroxyl radical. Copper-dependent oxidative inactivation/fragmentation of glutathione reductase can explain the prooxidant properties of copper under the in vivo conditions. PMID:24671306

  20. 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...

  1. Monoterpene metabolism. Cloning, expression, and characterization of menthone reductases from peppermint.

    PubMed

    Davis, Edward M; Ringer, Kerry L; McConkey, Marie E; Croteau, Rodney

    2005-03-01

    (-)-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 K(m) values of 674 mum, > 1 mm, and 10 mum for menthone, isomenthone, and NADPH, respectively, with a k(cat) 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 K(m) values of 3.0 mum, 41 mum, and 0.12 mum for menthone, isomenthone, and NADPH, respectively, with a k(cat) of 0.6 s(-1). The respective activities of

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

    PubMed

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

    2013-11-01

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

  3. 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

  4. Sequence diversity and enzyme activity of ferric-chelate reductase LeFRO1 in tomato.

    PubMed

    Kong, Danyu; Chen, Chunlin; Wu, Huilan; Li, Ye; Li, Junming; Ling, Hong-Qing

    2013-11-20

    Ferric-chelate reductase which functions in the reduction of ferric to ferrous iron on root surface is a critical protein for iron homeostasis in strategy I plants. LeFRO1 is a major ferric-chelate reductase involved in iron uptake in tomato. To identify the natural variations of LeFRO1 and to assess their effect on the ferric-chelate reductase activity, we cloned the coding sequences of LeFRO1 from 16 tomato varieties collected from different regions, and detected three types of LeFRO1 (LeFRO1(MM), LeFRO1(Ailsa) and LeFRO1(Monita)) with five amino acid variations at the positions 21, 24, 112, 195 and 582. Enzyme activity assay revealed that the three types of LeFRO1 possessed different ferric-chelate reductase activity (LeFRO1(Ailsa) > LeFRO1(MM) > LeFRO1(Monita)). The 112th amino acid residue Ala of LeFRO1 is critical for maintaining the high activity of ferric-chelate reductase, because modification of this amino acid resulted in a significant reduction of enzyme activity. Further, we showed that the combination of the amino acid residue Ile at the site 24 with Lys at the site 582 played a positive role in the enzyme activity of LeFRO1. In conclusion, the findings are helpful to understand the natural adaptation mechanisms of plants to iron-limiting stress, and may provide new knowledge to select and manipulate LeFRO1 for improving the iron deficiency tolerance in tomato.

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

    PubMed

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

    2005-03-01

    The enzyme 5alpha-reductase is responsible for the conversion of testosterone (T) to its more potent androgen dihydrotestosterone (DHT). This steroid had been implicated in androgen-dependent diseases such as: benign prostatic hyperplasia, prostate cancer, acne and androgenic alopecia. The inhibition of 5alpha-reductase enzyme offers a potentially useful treatment for these diseases. In this study, we report the synthesis and pharmacological evaluation of several new 3-substituted pregna-4, 16-diene-6, 20-dione derivatives. These compounds were prepared from the commercially available 16-dehydropregnenolone acetate. The biological activity of the new steroidal derivatives was determined in vivo as well as in vitro experiments. In vivo experiments, the anti-androgenic effect of the steroids was demonstrated by the decrease of the weight of the prostate gland of gonadectomized hamster treated with T plus finasteride or the new steroids. The IC50 value of these steroids was determined by measuring the conversion of radio labeled T to DHT. The results of this study carried out with 5alpha-reductase enzyme from hamster and human prostate showed that four of the six steroidal derivatives (5, 7, 9, 10) exhibited much higher 5alpha-reductase inhibitory activity, as indicated by the IC50 values than the presently used Proscar 3 (finasteride). The comparison of the weight of the hamster's prostate gland indicated that compound 5 had a comparable weight decrease as finasteride. The overall data of this study showed very clearly those compounds 5, 7, 9, 10 are good inhibitors for the 5alpha-reductase enzyme. PMID:15763601

  6. Protein method for investigating mercuric reductase gene expression in aquatic environments.

    PubMed

    Ogunseitan, O A

    1998-02-01

    A colorimetric assay for NADPH-dependent, mercuric ion-specific oxidoreductase activity was developed to facilitate the investigation of mercuric reductase gene expression in polluted aquatic ecosystems. Protein molecules extracted directly from unseeded freshwater and samples seeded with Pseudomonas aeruginosa PU21 (Rip64) were quantitatively assayed for mercuric reductase activity in microtiter plates by stoichiometric coupling of mercuric ion reduction to a colorimetric redox chain through NADPH oxidation. Residual NADPH was determined by titration with phenazine methosulfate-catalyzed reduction of methyl thiazolyl tetrazolium to produce visible formazan. Spectrophotometric determination of formazan concentration showed a positive correlation with the amount of NADPH remaining in the reaction mixture (r2 = 0.99). Mercuric reductase activity in the protein extracts was inversely related to the amount of NADPH remaining and to the amount of formazan produced. A qualitative nitrocellulose membrane-based version of the method was also developed, where regions of mercuric reductase activity remained colorless against a stained-membrane background. The assay detected induced mercuric reductase activity from 10(2) CFU, and up to threefold signal intensity was detected in seeded freshwater samples amended with mercury compared to that in mercury-free samples. The efficiency of extraction of bacterial proteins from the freshwater samples was (97 +/- 2)% over the range of population densities investigated (10(2) to 10(8) CFU/ml). The method was validated by detection of enzyme activity in protein extracts of water samples from a polluted site harboring naturally occurring mercury-resistant bacteria. The new method is proposed as a supplement to the repertoire of molecular techniques available for assessing specific gene expression in heterogeneous microbial communities impacted by mercury pollution.

  7. 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

  8. Purification and characterization of an NADPH-cytochrome P450 (cytochrome c) reductase from spearmint (Mentha spicata) glandular trichomes.

    PubMed

    Ponnamperuma, K; Croteau, R

    1996-05-01

    Solubilized NADPH-cytochrome c (P450) reductase was purified to homogeneity from an extract of spearmint (Mentha spicata) glandular trichomes by dye-ligand interaction chromatography on Matrex-Gel Red A and affinity chromatography on 2', 5'-adenosine diphosphate agarose. SDS-PAGE of the purified enzyme preparation revealed the presence of two similar proteins with masses of 82 kDa (major) and 77 kDa (minor) that crossreacted on immunoblot analysis with polyclonal antibodies directed against NADPH-cytochrome P450 reductase from Jerusalem artichoke and from mung bean. Complete immunoinhibition of reductase activity was observed with both types of polyclonal antibodies, while only partial inhibition of activity resulted using a family of monoclonal antibodies directed against the Jerusalem artichoke cytochrome P450 reductase. Inhibition of the spearmint oil gland cytochrome c reductase was also observed with the diphenyliodonium ion. The K(m) values for the cosubstrates NADPH and cytochrome c were 6.2 and 3.7 microM, respectively, and the pH optimum for activity was at 8.5. The NADPH-cytochrome c reductase reconstituted NADPH-dependent (-)-4S-limonene-6-hydroxylase activity in the presence of cytochrome P450, purified from the microsomal fraction of spearmint oil gland cells and dilauroyl phosphatidyl choline. These characteristics establish the identity of the purified enzyme as a NADPH-cytochrome P450 reductase.

  9. 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

  10. 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.

  11. Assignment of the human dihydrofolate reductase gene to the q11. -->. q22 region of chromosome 5

    SciTech Connect

    Funanage, V.L.; Myoda, T.T.; Moses, P.A.; Cowell, H.R.

    1984-10-01

    Cells from a dihydrofolate reductase-deficit Chinese hamster ovary cell line were hybridized to human fetal skin fibroblast cells. Nineteen dihydrofolate reductase-positive hybrid clones were isolated and characterized. Cytogenetic and biochemical analyses of these clones have shown that the human dihydrofolate reductase (DHFR) gene is located on chromosome 5. Three of these hybrid cell lines contained different terminal deletions of chromosome 5. An analysis of the breakpoints of these deletions has demonstrated that the DHFR gene resides in the q11..-->..q22 region.

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

    PubMed

    Ono, S; Hirano, H

    1984-04-01

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

  13. Spinach nitrite reductase. Purification and properties of a siroheme-containing iron-sulfur enzyme.

    PubMed

    Vega, J M; Kamin, H

    1977-02-10

    Ferredoxin-nitrite reductase (EC 1.7.7.1.) from spinach has been purified to homogeneity with a specific activity of 110 units/mg of protein. The enzyme, Mr = 61,000 has 3 iron atoms (of which one is in siroheme) and 2 labile sulfides, i.e. 1 (Fe2-S2) per molecule, with absorption maxima at 276, 386 (Soret), 573 (alpha), and 690 nm, with an E386 of 3.97 X 10(4) M-1-cm-1, and A276/A386 absorptivity ratio of 1.8. Anaerobic addition of dithionite results in the loss of the 690 nm peak and the splitting of the 573 nm absorption band into two broad peaks at 545 and 585 nm. Reduction by dithionite is enhanced by cyanide (Fig. 7) and requires about 3 electron eq per mol of enzyme. With nitrite or hydroxylamine (substrates of the enzyme), cyanide (a competitive inhibitor with respect to nitrite), or sulfite, the 690 nm absorption band of substrate-free enzyme disappears and the absorbance in the Soret and alpha region are altered. The high spin EPR signals disappear (J. M. Vega, H. Kamin, N. R. Orme-Johnson, and W. H. Orme-Johnson, unpublished observations). Titration permits calculation of 1 mol of nitrite bound/mol of enzyme with a Kdiss of 3.2 X 10(-6) M. Dithionite-reduced enzyme also forms complexes with added nitrite, hydroxylamine, or cyanide, characterized by marked alterations in the 573 (alpha) absorption band. THus, substrates or competitive inhibitors can be bound to the oxidized or reduced enzyme forms. CO inhibits nitrite reductase and forms a complex with reduced enzyme (epsilonmax at 395, 543, and 585 nm). Formation or dissociation of the spectrophotometrically detectable CO complex correlates with inhibition or inhibition-reversal of nitrite reduction catalysis. During steady state turnover with dithionite and nitrite, the enzyme forms a complex with added nitrite with absorption difference maxima at 445, 538, and 580 nm with respect to reduced enzyme. When nearly all substrate is depleted the spectrum of a new species appears, indicating that nitrite

  14. Purification and Characterization of the Nitrate Reductase from the Diatom Thalassiosira pseudonana1

    PubMed Central

    Amy, Nancy K.; Garrett, Reginald H.

    1974-01-01

    The assimilatory nitrate reductase (NADH: nitrate oxidoreductase, E.C. 1.6.6.2.) from the marine diatom Thalassiosira pseudonana, Hasle and Heimdal, has been purified 200-fold and characterized. The regulation of nitrate reductase in response to various conditions of nitrogen nutrition has been investigated. Nitrate reductase activity is repressed by the presence of ammonium in vivo, and its synthesis is derepressed when ammonium is absent. The derepression process is sensitive to cycloheximide and apparently requires protein synthesis. Repression of enzyme activity by ammonium is neither inhibited nor delayed by the presence of cycloheximide. In vitro, ammonium does not inhibit enzyme activity. NADH is the physiological electron donor for the enzyme in a flavin-dependent reaction. Spectral studies have indicated the presence of a b-type cytochrome associated with the enzyme. It is possible to observe enzymatic oxidation-reduction reactions which represent partial functions of the over-all electron transport capacity of this enzyme. Nitrate reductase will accept electrons from artificial electron donors such as reduced methyl viologen in a flavin-independent reaction. Further, dithionitereduced flavin adenine dinucleotide can donate electrons to the enzyme to reduce nitrate to nitrite. Finally, the nitrate reductase will exhibit a diaphorase activity and reduce the artificial electron acceptor mammalian cytochrome c in flavin-adeninedinucleotide-dependent reaction. Inhibition studies with potassium cyanide, sodium azide, and o-phenanthroline have yielded indirect evidence for metal component (s) of the enzyme. The inhibition of the NADH-requiring enzyme activities by p-hydroxymercuribenzoate has shown that an essential sulfhydryl group is involved in the initial portion of the electron transport. Heat treatment exerts an effect similar to the p-hydroxymercuribenzoate inhibition; namely, the NADH-requiring activities are rapidly inactivated, whereas the terminal

  15. Coordinated response of renal medullary enzymes regulating net sorbitol production in diuresis and antidiuresis.

    PubMed

    Sands, J M; Schrader, D C

    1990-07-01

    The renal response to changes in hydration includes variation in intracellular sorbitol, a major inner medullary osmolyte. To examine the mechanism for changes in net sorbitol production, we measured activities of enzymes regulating sorbitol production (aldose reductase) and degradation (sorbitol dehydrogenase) in untreated, water diuretic, and antidiuretic (water restriction and/or vasopressin administration) rats. Collecting duct segments dissected from collagenase-treated kidneys of Sprague-Dawley rats were divided into outer medullary and three distinct inner medullary regions. Aldose reductase activity increased during antidiuresis and decreased during diuresis. In contrast, sorbitol dehydrogenase activity was very low during antidiuresis and increased during diuresis. These changes in enzyme activity were found after 3 days, but not after 1 day, of water diuresis/antidiuresis. Enzyme activity changed only in the deepest 50% of the inner medullary collecting duct. Thus, there is coordinated regulation of aldose reductase and sorbitol dehydrogenase activities so that (a) during water diuresis, aldose reductase activity decreases while sorbitol dehydrogenase activity increases; and (b) during antidiuresis (water restriction and/or vasopressin administration), aldose reductase activity increases while sorbitol dehydrogenase activity remains low. We conclude that long-term osmoregulation in response to physiologic stimuli involves both aldose reductase and sorbitol dehydrogenase activities in rat terminal inner medullary collecting duct segments.

  16. A conservative region of the mercuric reductase gene (mera) as a molecular marker of bacterial mercury resistance

    PubMed Central

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

    2008-01-01

    The most common bacterial mercury resistance mechanism is based on the reduction of Hg(II) to Hg0, which is dependent of the mercuric reductase enzyme (MerA) activity. The use of a 431 bp fragment of a conservative region of the mercuric reductase (merA) gene was applied as a molecular marker of this mechanism, allowing the identification of mercury resistant bacterial strains. PMID:24031221

  17. Male pseudohermaphroditism due to 5 alpha-reductase-2 deficiency in an Arab kindred.

    PubMed Central

    al-Attia, H. M.

    1997-01-01

    Six Arabs subjects (three postpubertal, two prepubertal and one pubertal) from three interrelated Omani families with male pseudohermaphroditism due to 5 alpha-reductase-2 deficiency were evaluated. These subjects had been raised as girls since birth as they were born with a clitoral-like phallus and ambiguous external genitalia of pseudovaginal perineoscrotal hypospadias with separate urethral and vaginal orifices. They underwent variable degrees of increased muscular habitus and phallic enlargement during puberty and beyond. Gynaecomastia was absent and the body and facial hair was insignificant. After diagnosis, a transition to male social sex occurred in two cases, one of which was interventional. Two retained the female social sex, one of which was also interventional, while the other two maintained an equivocal gender status. This report provides new data on the characterisation of 5 alpha-reductase-2 deficiency in various clusters. Images Figure 2 Figure 3 PMID:9497950

  18. 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.

  19. 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

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

    PubMed

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

    2014-08-01

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

  1. 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

  2. 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

  3. 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.

  4. 5-Alpha-Reductase 2 Deficiency in a Woman with Primary Amenorrhea

    PubMed Central

    Kalantar Hormozi, Mohammadreza; Iranparvar Alamdari, Manouchehr; Tavosi, Zahra

    2013-01-01

    Steroid 5-alpha-reductase 2 deficiency is a rare disorder leading to male pseudohermaphroditism, a condition characterized by incomplete differentiation of male genitalia in 46,XY patients. Here, we report a case of a 21-year-old woman from Ardabil who presented with primary amenorrhea, ambiguous genitalia, and lack of breast development. All of the serum hormone profiles were normal except for raised serum total testosterone. Testosterone to DHT ratio (T/DHT) was elevated before (15.72) and further increased after hCG stimulation (32.46). A chromosomal study revealed a 46,XY karyotype. A bilateral gonadectomy, recessive cliteroplasty, urethroplasty, and vaginoplasty were performed and hormonal replacement therapy using estrogen was started. In conclusion, the diagnosis of 5-alpha-reductase 2 deficiency may be suspected in infants with ambiguous genitalia or in adolescents or young adults with the characteristic phenotype and serum hormone profiles. PMID:24383016

  5. 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

  6. Changes in cerebrospinal fluid levels of malondialdehyde and glutathione reductase activity in multiple sclerosis.

    PubMed

    Calabrese, V; Raffaele, R; Cosentino, E; Rizza, V

    1994-01-01

    The chemical composition of human cerebrospinal fluid (CSF) is considered to reflect brain metabolism. In this study we measured malondialdehyde (MDA) levels and the activity of enzymes involved in antioxidative processes, glutathione reductase and glutathione peroxidase, in human cerebrospinal fluid of multiple-sclerosis (MS) patients and normal healthy volunteers. Our results indicated that the cerebrospinal fluid in MS showed significantly higher endogenous levels of MDA than the control, as well as a much greater resistance to in-vitro stimulation test. In addition, we found the activity of GSH reductase significantly increased, about twice the control values, whereas the activity of glutathione peroxidase was markedly decreased as compared to control values. Our findings suggest that in MS the activity of antioxidant enzymes is modified, and indicates the conceivable possibility of a pathogenic role of oxidative stress in the determinism of the disease. PMID:7607784

  7. 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

  8. Comparative Modeling and Molecular Dynamics Simulation of Substrate Binding in Human Fatty Acid Synthase: Enoyl Reductase and β-Ketoacyl Reductase Catalytic Domains

    PubMed Central

    John, Arun; Krishnakumar, Subramanian

    2015-01-01

    Fatty acid synthase (FASN, EC 2.3.1.85), is a multi-enzyme dimer complex that plays a critical role in lipogenesis. This lipogenic enzyme has gained importance beyond its physiological role due to its implications in several clinical conditions-cancers, obesity, and diabetes. This has made FASN an attractive pharmacological target. Here, we have attempted to predict the theoretical models for the human enoyl reductase (ER) and β-ketoacyl reductase (KR) domains based on the porcine FASN crystal structure, which was the structurally closest template available at the time of this study. Comparative modeling methods were used for studying the structure-function relationships. Different validation studies revealed the predicted structures to be highly plausible. The respective substrates of ER and KR domains-namely, trans-butenoyl and β-ketobutyryl-were computationally docked into active sites using Glide in order to understand the probable binding mode. The molecular dynamics simulations of the apo and holo states of ER and KR showed stable backbone root mean square deviation trajectories with minimal deviation. Ramachandran plot analysis showed 96.0% of residues in the most favorable region for ER and 90.3% for the KR domain, respectively. Thus, the predicted models yielded significant insights into the substrate binding modes of the ER and KR catalytic domains and will aid in identifying novel chemical inhibitors of human FASN that target these domains. PMID:25873848

  9. Co-expression of monodehydroascorbate reductase and dehydroascorbate reductase from Brassica rapa effectively confers tolerance to freezing-induced oxidative stress.

    PubMed

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

    2013-10-01

    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.

  10. 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

    2016-01-01

    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.

  11. 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.

  12. 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

  13. Ajoene is an inhibitor and subversive substrate of human glutathione reductase and Trypanosoma cruzi trypanothione reductase: crystallographic, kinetic, and spectroscopic studies.

    PubMed

    Gallwitz, H; Bonse, S; Martinez-Cruz, A; Schlichting, I; Schumacher, K; Krauth-Siegel, R L

    1999-02-11

    Ajoene ((E,Z)-4,5,9-trithiadodeca-1,6,11-triene 9-oxide), a garlic-derived natural compound, is a covalent inhibitor as well as a substrate of human glutathione reductase (GR) and Trypanosoma cruzi trypanothione reductase (TR). The 2.1-A resolution crystal structure of GR inhibited by (E)-ajoene revealed a mixed disulfide between the active site Cys58 and the CH2=CH-CH2-SO-CH2-CH=CH-S moiety of ajoene. The modified enzyme has a markedly increased oxidase activity when compared to free GR. GR reduces (Z)-ajoene with a kcat/Km of 6.8 x 10(3) M-1 s-1 yielding 4,5,9-trithiadodeca-1, 6,11-triene (deoxyajoene) and 4,8,9,13-tetrathiahexadeca-1,6,10, 15-tetraene as stable reaction products. The reaction leads also to the formation of single-electron reduced products and concomitantly superoxide anion radicals as shown by coupling the reaction to the reduction of cytochrome c. The interactions between the flavoenzymes and ajoene are expected to increase the oxidative stress of the respective cell. The antiparasitic and cytostatic actions of ajoene may at least in part be due to the multiple effects on key enzymes of the antioxidant thiol metabolism.

  14. Up regulation of glyoxylate reductase/hydroxypyruvate reductase (GRHPR) is associated with intestinal epithelial cells apoptosis in TNBS-induced experimental colitis.

    PubMed

    Zong, Chunyan; Nie, Xiaoke; Zhang, Dongmei; Ji, Qianqian; Qin, Yongwei; Wang, Liang; Jiang, Dawei; Gong, Chen; Liu, Yifei; Zhou, Guoxiong

    2016-05-01

    Glyoxylate reductase/hydroxypyruvate reductase (GRHPR), which exists mainly in the liver, is a D-2-hydroxy-acid dehydrogenase that plays a critical role in the formation of primary hyperoxaluria type 2 (PH2). Here, we investigated GRHPR expression and its potential role in both human Crohn's disease (CD) and experimental colitis. Murine experimental colitis models were established by administration of trinitrobenzenesulphonic acid (TNBS). As shown by Western blot, significant up-regulation of GRHPR was found in TNBS-treated mice as compared with normal controls. Immunohistochemistry (IHC) also showed increased GRHPR expression, and the molecule was located in intestinal epithelial cells (IECs). This phenomenon also occurred in patients with Crohn's disease. Besides, in an in vitro study, human IEC line HT-29 cells cultured with tumor necrosis factor α (TNF-α) were used to evaluate the changes in expression of GRHPR. Moreover, overexpression of GRHPR was accompanied by active caspase-3 and cleaved poly ADP-ribose polymerase (PARP) accumulation. Furthermore, knock-down GRHPR could inhibit the accumulation of active caspase-3 and cleaved PARP as shown by Western blot in TNF-α treated HT-29 cells. Flow cytometry assay indicated that interference of GRHPR led to increasing apoptosis of IECs. These data suggested that GRHPR might exert its pro-apoptosis function in IECs. Thus, GRHPR might play an important role in regulating IECs apoptosis, and might be a potential therapeutic target for CD. PMID:26997491

  15. 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