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Sample records for 1a dihydroorotate dehydrogenase

  1. Mechanism of flavin reduction in the class 1A dihydroorotate dehydrogenase from Lactococcus lactis

    Fagan, Rebecca L; Jensen, Kaj Frank; Björnberg, Olof; Palfey, Bruce A

    2007-01-01

    is concerted or stepwise was addressed for the class 1A enzyme from Lactococcus lactis by determining kinetic isotope effects (KIEs) on flavin reduction in anaerobic stopped-flow experiments. Isotope effects were determined at two pH values. At pH 7.0, KIEs were approximately 2-fold for DHO labeled....... These isotope effects are consistent with a concerted oxidation of DHO. The pH dependence of reduction was also determined, and a pKa of 8.3 was found. This pKa can be attributed to the ionization of the active site cysteine which deprotonates C5 of DHO during the reaction. To further investigate the...... importance of the active site base, two site-directed mutants were also studied: Cys130Ala (removal of the active site base) and Cys130Ser (replacement with the active site base used by class 2 DHODs). Both mutant enzymes exhibited binding affinities for DHO similar to that of the wild-type enzyme. Reduction...

  2. Two different dihydroorotate dehydrogenases from yeast Saccharomyees kluyveri

    Zameitat, E.; Knecht, Wolfgang; Piskur, Jure; Loffler, M.

    2004-01-01

    Genes for two structurally and functionally different dihydroorotate dehydrogenases (DHODHs, EC 1.3.99.11), catalyzing the fourth step of pyrimidine biosynthesis, have been previously found in yeast Saccharomyces klujveri. One is closely related to the Schizosaccharomyces pombe mitochondrial family...

  3. Biochemical and molecular characterization of the pyrimidine biosynthetic enzyme dihydroorotate dehydrogenase from Toxoplasma gondii

    Triana, Miryam Andrea Hortua; Huynh, My-Hang; Manuel F Garavito; Fox, Barbara A.; Bzik, David J.; Vern B Carruthers; Löffler, Monika; Zimmermann, Barbara H.

    2012-01-01

    The pyrimidine biosynthesis pathway in the protozoan pathogen Toxoplasma gondii is essential for parasite growth during infection. To investigate the properties of dihydroorotate dehydrogenase (TgDHOD), the fourth enzyme in the T. gondii pyrimidine pathway, we expressed and purified recombinant TgDHOD. TgDHOD exhibited a specific activity of 84 U/mg, a kcat of 89 sec−1, a Km = 60 μM for L-dihydroorotate, and a Km = 29 μM for decylubiquinone (QD). Quinones lacking or having short isoprenoid si...

  4. Biochemical characterization of recombinant dihydroorotate dehydrogenase from the opportunistic pathogenic yeast Candida albicans

    Zameitat, E.; Gojkovic, Zoran; Knecht, Wolfgang;

    2006-01-01

    Candida albicans is the most prevalent yeast pathogen in humans, and recently it has become increasingly resistant to the current antifungal agents. In this study we investigated C. albicans dihydroorotate dehydrogenase (DHODH, EC 1.3.99.11), which catalyzes the fourth step of de novo pyrimidine ...

  5. Structure of Dihydroorotate Dehydrogenase B: Electron Transfer between Two Flavin Groups Bridged by an Iron-Sulphur Cluster

    Rowland, Poul; Nørager, Sofie; Jensen, Kaj Frank; Larsen, Sine

    2000-01-01

    BACKGROUND: The fourth step and only redox reaction in pyrimidine de novo biosynthesis is catalyzed by the flavoprotein dihydroorotate dehydrogenase (DHOD). Based on their sequences, DHODs are grouped into two major families. Lactococcus lactis is one of the few organisms with two DHODs, A and B...... to the NADPH ferredoxin reductase superfamily. The binding site for NAD+ has been deduced from the similarity to these proteins. The orotate binding in DHODB is similar to that in the family 1A enzymes. The close proximity of the three redox centers makes it possible to propose a possible electron...

  6. Novel Inhibitors of Plasmodium falciparum Dihydroorotate Dehydrogenase with Anti-malarial Activity in the Mouse Model*

    Booker, Michael L.; Bastos, Cecilia M.; Kramer, Martin L.; Barker, Robert H.; Skerlj, Renato; Sidhu, Amar Bir; Deng, Xiaoyi; Celatka, Cassandra; Cortese, Joseph F.; Guerrero Bravo, Jose E.; Crespo Llado, Keila N.; Serrano, Adelfa E.; Angulo-Barturen, Iñigo; Jiménez-Díaz, María Belén; Viera, Sara

    2010-01-01

    Plasmodium falciparum, the causative agent of the most deadly form of human malaria, is unable to salvage pyrimidines and must rely on de novo biosynthesis for survival. Dihydroorotate dehydrogenase (DHODH) catalyzes the rate-limiting step in the pyrimidine biosynthetic pathway and represents a potential target for anti-malarial therapy. A high throughput screen and subsequent medicinal chemistry program identified a series of N-alkyl-5-(1H-benzimidazol-1-yl)thiophene-2-carboxamides with low ...

  7. Expression, purification and crystallization of Trypanosoma cruzi dihydroorotate dehydrogenase complexed with orotate

    Inaoka, Daniel Ken; Takashima, Eizo; Osanai, Arihiro; Shimizu, Hironari [Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); Nara, Takeshi; Aoki, Takashi [Department of Parasitology, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan); Harada, Shigeharu [Department of Applied Biology, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585 (Japan); Kita, Kiyoshi, E-mail: kitak@m.u-tokyo.ac.jp [Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan)

    2005-10-01

    The Trypanosoma cruzi dihydroorotate dehydrogenase, a key enzyme in pyrimidine de novo biosynthesis and redox homeostasis, was crystallized in complex with its first reaction product, orotate. Dihydroorotate dehydrogenase (DHOD) catalyzes the oxidation of dihydroorotate to orotate, the fourth step and the only redox reaction in the de novo biosynthesis of pyrimidine. DHOD from Trypanosoma cruzi (TcDHOD) has been expressed as a recombinant protein in Escherichia coli and purified to homogeneity. Crystals of the TcDHOD–orotate complex were grown at 277 K by the sitting-drop vapour-diffusion technique using polyethylene glycol 3350 as a precipitant. The crystals diffract to better than 1.8 Å resolution using synchrotron radiation (λ = 0.900 Å). X-ray diffraction data were collected at 100 K and processed to 1.9 Å resolution with 98.2% completeness and an overall R{sub merge} of 7.8%. The TcDHOD crystals belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 67.87, b = 71.89, c = 123.27 Å. The presence of two molecules in the asymmetric unit (2 × 34 kDa) gives a crystal volume per protein weight (V{sub M}) of 2.2 Å{sup 3} Da{sup −1} and a solvent content of 44%.

  8. Expression, purification and crystallization of Trypanosoma cruzi dihydroorotate dehydrogenase complexed with orotate

    The Trypanosoma cruzi dihydroorotate dehydrogenase, a key enzyme in pyrimidine de novo biosynthesis and redox homeostasis, was crystallized in complex with its first reaction product, orotate. Dihydroorotate dehydrogenase (DHOD) catalyzes the oxidation of dihydroorotate to orotate, the fourth step and the only redox reaction in the de novo biosynthesis of pyrimidine. DHOD from Trypanosoma cruzi (TcDHOD) has been expressed as a recombinant protein in Escherichia coli and purified to homogeneity. Crystals of the TcDHOD–orotate complex were grown at 277 K by the sitting-drop vapour-diffusion technique using polyethylene glycol 3350 as a precipitant. The crystals diffract to better than 1.8 Å resolution using synchrotron radiation (λ = 0.900 Å). X-ray diffraction data were collected at 100 K and processed to 1.9 Å resolution with 98.2% completeness and an overall Rmerge of 7.8%. The TcDHOD crystals belong to the orthorhombic space group P212121, with unit-cell parameters a = 67.87, b = 71.89, c = 123.27 Å. The presence of two molecules in the asymmetric unit (2 × 34 kDa) gives a crystal volume per protein weight (VM) of 2.2 Å3 Da−1 and a solvent content of 44%

  9. Biochemical and molecular characterization of the pyrimidine biosynthetic enzyme dihydroorotate dehydrogenase from Toxoplasma gondii.

    Hortua Triana, Miryam Andrea; Huynh, My-Hang; Garavito, Manuel F; Fox, Barbara A; Bzik, David J; Carruthers, Vern B; Löffler, Monika; Zimmermann, Barbara H

    2012-08-01

    The pyrimidine biosynthesis pathway in the protozoan pathogen Toxoplasma gondii is essential for parasite growth during infection. To investigate the properties of dihydroorotate dehydrogenase (TgDHOD), the fourth enzyme in the T. gondii pyrimidine pathway, we expressed and purified recombinant TgDHOD. TgDHOD exhibited a specific activity of 84U/mg, a k(cat) of 89s(-1), a K(m)=60μM for l-dihydroorotate, and a K(m)=29μM for decylubiquinone (Q(D)). Quinones lacking or having short isoprenoid side chains yielded lower k(cat)s than Q(D). As expected, fumarate was a poor electron acceptor for this family 2 DHOD. The IC(50)s determined for A77-1726, the active derivative of the human DHOD inhibitor leflunomide, and related compounds MD249 and MD209 were, 91μM, 96μM, and 60μM, respectively. The enzyme was not significantly affected by brequinar or TTFA, known inhibitors of human DHOD, or by atovaquone. DSM190, a known inhibitor of Plasmodium falciparum DHOD, was a poor inhibitor of TgDHOD. TgDHOD exhibits a lengthy 157-residue N-terminal extension, consistent with a potential organellar targeting signal. We constructed C-terminally c-myc tagged TgDHODs to examine subcellular localization of TgDHOD in transgenic parasites expressing the tagged protein. Using both exogenous and endogenous expression strategies, anti-myc fluorescence signal colocalized with antibodies against the mitochondrial marker ATPase. These findings demonstrate that TgDHOD is associated with the parasite's mitochondrion, revealing this organelle as the site of orotate production in T. gondii. The TgDHOD gene appears to be essential because while gene tagging was successful at the TgDHOD gene locus, attempts to delete the TgDHOD gene were not successful in the KU80 background. Collectively, our study suggests that TgDHOD is an excellent target for the development of anti-Toxoplasma drugs. PMID:22580100

  10. The crystal structure of Lactococcus lactis dihydroorotate dehydrogenase A complexed with the enzyme reaction product throws light on its enzymatic function

    Rowland, Paul; Bjørnberg, Olof; Nielsen, Finn S.; Jensen, Kaj Frank; Larsen, Sine

    1998-01-01

    Dihydroorotate dehydrogenases (DHODs) catalyze the oxidation of (S)-dihydroorotate to orotate, the fourth step and only redox reaction in the de novo biosynthesis of pyrimidine nucleotides. A description is given of the crystal structure of Lactococcus lactis dihydroorotate dehydrogenase A (DHODA......) complexed with the product of the enzyme reaction orotate. The structure of the complex to 2.0 A resolution has been compared with the structure of the native enzyme. The active site of DHODA is known to contain a water filled cavity buried beneath a highly conserved and flexible loop. In the complex the...

  11. Biochemical characterization of recombinant dihydroorotate dehydrogenase from the opportunistic pathogenic yeast Candida albicans

    Zameitat, E.; Gojkovic, Zoran; Knecht, Wolfgang; Piskur, Jure; Loffler, M.

    2006-01-01

    Candida albicans is the most prevalent yeast pathogen in humans, and recently it has become increasingly resistant to the current antifungal agents. In this study we investigated C. albicans dihydroorotate dehydrogenase (DHODH, EC 1.3.99.11), which catalyzes the fourth step of de novo pyrimidine...... lacks the targeting sequence and the transmembrane domain, were subcloned from C. albicans, recombinantly expressed in Escherichia coli, purified, and characterized for their kinetics and substrate specificity. An inhibitor screening with 28 selected compounds was performed. Only the dianisidine...... derivative, redoxal, and the biphenyl quinoline-carboxylic acid derivative, brequinar sodium, which are known to be potent inhibitors of mammalian DHODH, markedly reduced C. albicans DHODH activity. This study provides a background for the development of antipyrimidines with high efficacy for decreasing in...

  12. Site directed spin labeling studies of Escherichia coli dihydroorotate dehydrogenase N-terminal extension

    Couto, Sheila G. [Instituto de Fisica de Sao Carlos, Universidade de Sao Paulo, Av. Trabalhador Sao-carlense 400, C.P. 369, 13560-970, Sao Carlos, SP (Brazil); Grupo de Biofisica e Fisica Aplicada a Medicina, Instituto de Fisica, Universidade Federal de Goias, Campus Samambaia, C.P. 131, 74001-970, Goiania, GO (Brazil); Cristina Nonato, M. [Laboratorio de Cristalografia de Proteinas, Faculdade de Ciencias Farmaceuticas de Ribeirao Preto, Universidade de Sao Paulo, Av. do Cafe S/N, 14040-903, Ribeirao Preto, SP (Brazil); Costa-Filho, Antonio J., E-mail: ajcosta@ffclrp.usp.br [Instituto de Fisica de Sao Carlos, Universidade de Sao Paulo, Av. Trabalhador Sao-carlense 400, C.P. 369, 13560-970, Sao Carlos, SP (Brazil); Departamento de Fisica, Faculdade de Filosofia, Ciencias e Letras de Ribeirao Preto, Av. Bandeirantes 3900, 14040-901, Ribeirao Preto, SP (Brazil)

    2011-10-28

    Highlights: Black-Right-Pointing-Pointer EcDHODH is a membrane-associated enzyme and a promising target for drug design. Black-Right-Pointing-Pointer Enzyme's N-terminal extension is responsible for membrane association. Black-Right-Pointing-Pointer N-terminal works as a molecular lid regulating access to the protein interior. -- Abstract: Dihydroorotate dehydrogenases (DHODHs) are enzymes that catalyze the fourth step of the de novo synthesis of pyrimidine nucleotides. In this reaction, DHODH converts dihydroorotate to orotate, using a flavine mononucleotide as a cofactor. Since the synthesis of nucleotides has different pathways in mammals as compared to parasites, DHODH has gained much attention as a promising target for drug design. Escherichia coli DHODH (EcDHODH) is a family 2 DHODH that interacts with cell membranes in order to promote catalysis. The membrane association is supposedly made via an extension found in the enzyme's N-terminal. In the present work, we used site directed spin labeling (SDSL) to specifically place a magnetic probe at positions 2, 5, 19, and 21 within the N-terminal and thus monitor, by using Electron Spin Resonance (ESR), dynamics and structural changes in this region in the presence of a membrane model system. Overall, our ESR spectra show that the N-terminal indeed binds to membranes and that it experiences a somewhat high flexibility that could be related to the role of this region as a molecular lid controlling the entrance of the enzyme's active site and thus allowing the enzyme to give access to quinones that are dispersed in the membrane and that are necessary for the catalysis.

  13. Novel Inhibitors of Plasmodium falciparum Dihydroorotate Dehydrogenase with Anti-malarial Activity in the Mouse Model

    Booker, Michael L.; Bastos, Cecilia M.; Kramer, Martin L.; Barker, Jr., Robert H.; Skerlj, Renato; Sidhu, Amar Bir; Deng, Xiaoyi; Celatka, Cassandra; Cortese, Joseph F.; Guerrero Bravo, Jose E.; Crespo Llado, Keila N.; Serrano, Adelfa E.; Angulo-Barturen, Iñigo; Jiménez-Díaz, María Belén; Viera, Sara; Garuti, Helen; Wittlin, Sergio; Papastogiannidis, Petros; Lin, Jing-wen; Janse, Chris J.; Khan, Shahid M.; Duraisingh, Manoj; Coleman, Bradley; Goldsmith, Elizabeth J.; Phillips, Margaret A.; Munoz, Benito; Wirth, Dyann F.; Klinger, Jeffrey D.; Wiegand, Roger; Sybertz, Edmund (Leiden-MC); (Puerto Rico); (STPHI); (Harvard); (GSK); (Genzyme); (UTSMC)

    2010-11-22

    Plasmodium falciparum, the causative agent of the most deadly form of human malaria, is unable to salvage pyrimidines and must rely on de novo biosynthesis for survival. Dihydroorotate dehydrogenase (DHODH) catalyzes the rate-limiting step in the pyrimidine biosynthetic pathway and represents a potential target for anti-malarial therapy. A high throughput screen and subsequent medicinal chemistry program identified a series of N-alkyl-5-(1H-benzimidazol-1-yl)thiophene-2-carboxamides with low nanomolar in vitro potency against DHODH from P. falciparum, P. vivax, and P. berghei. The compounds were selective for the parasite enzymes over human DHODH, and x-ray structural data on the analog Genz-667348, demonstrated that species selectivity could be attributed to amino acid differences in the inhibitor-binding site. Compounds from this series demonstrated in vitro potency against the 3D7 and Dd2 strains of P. falciparum, good tolerability and oral exposure in the mouse, and ED{sub 50} values in the 4-day murine P. berghei efficacy model of 13-21 mg/kg/day with oral twice-daily dosing. In particular, treatment with Genz-667348 at 100 mg/kg/day resulted in sterile cure. Two recent analogs of Genz-667348 are currently undergoing pilot toxicity testing to determine suitability as clinical development candidates.

  14. A long-duration dihydroorotate dehydrogenase inhibitor (DSM265) for prevention and treatment of malaria.

    Phillips, Margaret A; Lotharius, Julie; Marsh, Kennan; White, John; Dayan, Anthony; White, Karen L; Njoroge, Jacqueline W; El Mazouni, Farah; Lao, Yanbin; Kokkonda, Sreekanth; Tomchick, Diana R; Deng, Xiaoyi; Laird, Trevor; Bhatia, Sangeeta N; March, Sandra; Ng, Caroline L; Fidock, David A; Wittlin, Sergio; Lafuente-Monasterio, Maria; Benito, Francisco Javier Gamo; Alonso, Laura Maria Sanz; Martinez, Maria Santos; Jimenez-Diaz, Maria Belen; Bazaga, Santiago Ferrer; Angulo-Barturen, Iñigo; Haselden, John N; Louttit, James; Cui, Yi; Sridhar, Arun; Zeeman, Anna-Marie; Kocken, Clemens; Sauerwein, Robert; Dechering, Koen; Avery, Vicky M; Duffy, Sandra; Delves, Michael; Sinden, Robert; Ruecker, Andrea; Wickham, Kristina S; Rochford, Rosemary; Gahagen, Janet; Iyer, Lalitha; Riccio, Ed; Mirsalis, Jon; Bathhurst, Ian; Rueckle, Thomas; Ding, Xavier; Campo, Brice; Leroy, Didier; Rogers, M John; Rathod, Pradipsinh K; Burrows, Jeremy N; Charman, Susan A

    2015-07-15

    Malaria is one of the most significant causes of childhood mortality, but disease control efforts are threatened by resistance of the Plasmodium parasite to current therapies. Continued progress in combating malaria requires development of new, easy to administer drug combinations with broad-ranging activity against all manifestations of the disease. DSM265, a triazolopyrimidine-based inhibitor of the pyrimidine biosynthetic enzyme dihydroorotate dehydrogenase (DHODH), is the first DHODH inhibitor to reach clinical development for treatment of malaria. We describe studies profiling the biological activity, pharmacological and pharmacokinetic properties, and safety of DSM265, which supported its advancement to human trials. DSM265 is highly selective toward DHODH of the malaria parasite Plasmodium, efficacious against both blood and liver stages of P. falciparum, and active against drug-resistant parasite isolates. Favorable pharmacokinetic properties of DSM265 are predicted to provide therapeutic concentrations for more than 8 days after a single oral dose in the range of 200 to 400 mg. DSM265 was well tolerated in repeat-dose and cardiovascular safety studies in mice and dogs, was not mutagenic, and was inactive against panels of human enzymes/receptors. The excellent safety profile, blood- and liver-stage activity, and predicted long half-life in humans position DSM265 as a new potential drug combination partner for either single-dose treatment or once-weekly chemoprevention. DSM265 has advantages over current treatment options that are dosed daily or are inactive against the parasite liver stage. PMID:26180101

  15. Structural Plasticity of Malaria Dihydroorotate Dehydrogenase Allows Selective Binding of Diverse Chemical Scaffolds

    Malaria remains a major global health burden and current drug therapies are compromised by resistance. Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) was validated as a new drug target through the identification of potent and selective triazolopyrimidine-based DHODH inhibitors with anti-malarial activity in vivo. Here we report x-ray structure determination of PfDHODH bound to three inhibitors from this series, representing the first of the enzyme bound to malaria specific inhibitors. We demonstrate that conformational flexibility results in an unexpected binding mode identifying a new hydrophobic pocket on the enzyme. Importantly this plasticity allows PfDHODH to bind inhibitors from different chemical classes and to accommodate inhibitor modifications during lead optimization, increasing the value of PfDHODH as a drug target. A second discovery, based on small molecule crystallography, is that the triazolopyrimidines populate a resonance form that promotes charge separation. These intrinsic dipoles allow formation of energetically favorable H-bond interactions with the enzyme. The importance of delocalization to binding affinity was supported by site-directed mutagenesis and the demonstration that triazolopyrimidine analogs that lack this intrinsic dipole are inactive. Finally, the PfDHODH-triazolopyrimidine bound structures provide considerable new insight into species-selective inhibitor binding in this enzyme family. Together, these studies will directly impact efforts to exploit PfDHODH for the development of anti-malarial chemotherapy.

  16. The Role of Dihydroorotate Dehydrogenase in Apoptosis Induction in Response to Inhibition of the Mitochondrial Respiratory Chain Complex III

    Khutornenko, A.; Dalina, A.; Chernyak, B.; Chumakov, P; Evstafieva, A.

    2014-01-01

    A mechanism for the induction of programmed cell death (apoptosis) upon dysfunction of the mitochondrial respiratory chain has been studied. Previously, we had found that inhibition of mitochondrial cytochrome bc1, a component of the electron transport chain complex III, leads to activation of tumor suppressor p53, followed by apoptosis induction. The mitochondrial respiratory chain is coupled to the de novo pyrimidine biosynthesis pathway via the mitochondrial enzyme dihydroorotate dehydroge...

  17. In Vitro Resistance Selections for Plasmodium falciparum Dihydroorotate Dehydrogenase Inhibitors Give Mutants with Multiple Point Mutations in the Drug-binding Site and Altered Growth*

    Ross, Leila S.; Gamo, Francisco Javier; Lafuente-Monasterio, Maria José; Singh, Onkar M. P.; Rowland, Paul; Wiegand, Roger C.; Wirth, Dyann F

    2014-01-01

    Malaria is a preventable and treatable disease; yet half of the world's population lives at risk of infection, and an estimated 660,000 people die of malaria-related causes every year. Rising drug resistance threatens to make malaria untreatable, necessitating both the discovery of new antimalarial agents and the development of strategies to identify and suppress the emergence and spread of drug resistance. We focused on in-development dihydroorotate dehydrogenase (DHODH) inhibitors. Characte...

  18. Lactococcus lactis Dihydroorotate Dehydrogenase A Mutants Reveal Important Facets of the Enzymatic Function

    Nørager, Sofie Charlotte; Arent, S; Björnberg, Olof;

    2003-01-01

    and 1B, and class 2. This division corresponds to differences in cellular location and the nature of the electron acceptor. Herein we report a study of Lactococcus lactis DHODA, a representative of the class 1A enzymes. Based on the DHODA structure we selected seven residues that are highly conserved...

  19. Development of a human dihydroorotate dehydrogenase (hDHODH pharma-similarity index approach with scaffold-hopping strategy for the design of novel potential inhibitors.

    Kuei-Chung Shih

    Full Text Available Human dihydroorotate dehydrogenase (hDHODH is a class-2 dihydroorotate dehydrogenase. Because it is extensively used by proliferating cells, its inhibition in autoimmune and inflammatory diseases, cancers, and multiple sclerosis is of substantial clinical importance. In this study, we had two aims. The first was to develop an hDHODH pharma-similarity index approach (PhSIA using integrated molecular dynamics calculations, pharmacophore hypothesis, and comparative molecular similarity index analysis (CoMSIA contour information techniques. The approach, for the discovery and design of novel inhibitors, was based on 25 diverse known hDHODH inhibitors. Three statistical methods were used to verify the performance of hDHODH PhSIA. Fischer's cross-validation test provided a 98% confidence level and the goodness of hit (GH test score was 0.61. The q(2, r(2, and predictive r(2 values were 0.55, 0.97, and 0.92, respectively, for a partial least squares validation method. In our approach, each diverse inhibitor structure could easily be aligned with contour information, and common substructures were unnecessary. For our second aim, we used the proposed approach to design 13 novel hDHODH inhibitors using a scaffold-hopping strategy. Chemical features of the approach were divided into two groups, and the Vitas-M Laboratory fragment was used to create de novo inhibitors. This approach provides a useful tool for the discovery and design of potential inhibitors of hDHODH, and does not require docking analysis; thus, our method can assist medicinal chemists in their efforts to identify novel inhibitors.

  20. Structure-Guided Lead Optimization of Triazolopyrimidine-Ring Substituents Identifies Potent Plasmodium falciparum Dihydroorotate Dehydrogenase Inhibitors with Clinical Candidate Potential

    Coteron, Jose M.; Marco, Maria; Esquivias, Jorge; Deng, Xiaoyi; White, Karen L.; White, John; Koltun, Maria; El Mazouni, Farah; Kokkonda, Sreekanth; Katneni, Kasiram; Bhamidipati, Ravi; Shackleford, David M.; Angulo-Barturen, Inigo; Ferrer, Santiago B.; Jimenez-Diaz, Maria Belen; Gamo, Francisco-Javier; Goldsmith, Elizabeth J.; Charman, William N.; Bathurst, Ian; Floyd, David; Matthews, David; Burrows, Jeremy N.; Rathod, Pradipsinh K.; Charman, Susan A.; Phillips, Margaret A. (UWASH); (MMV, Switzerland); (GSK); (Monash); (UW); (UTSMC)

    2012-02-27

    Drug therapy is the mainstay of antimalarial therapy, yet current drugs are threatened by the development of resistance. In an effort to identify new potential antimalarials, we have undertaken a lead optimization program around our previously identified triazolopyrimidine-based series of Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) inhibitors. The X-ray structure of PfDHODH was used to inform the medicinal chemistry program allowing the identification of a potent and selective inhibitor (DSM265) that acts through DHODH inhibition to kill both sensitive and drug resistant strains of the parasite. This compound has similar potency to chloroquine in the humanized SCID mouse P. falciparum model, can be synthesized by a simple route, and rodent pharmacokinetic studies demonstrated it has excellent oral bioavailability, a long half-life and low clearance. These studies have identified the first candidate in the triazolopyrimidine series to meet previously established progression criteria for efficacy and ADME properties, justifying further development of this compound toward clinical candidate status.

  1. Androgen regulation of aldehyde dehydrogenase 1A3 (ALDH1A3) in androgen responsive human prostate cancer cell LNCaP.

    Previous gene array data from our laboratory identified the retinoic acid (RA) biosynthesis enzyme aldehyde dehydrogenase 1A3 (ALDH1A3) as a putative androgen-responsive gene in prostate cancer epithelial cells (LNCaP). In the present study we attempted to identify if any of the three ALDH1A/RA synt...

  2. A new type of didhydroorotate dehydrogenase, type 1S, from the thermoacidophilic archaeon Sulfolobus solfataricus

    Sørensen, P. G.; Dandanell, Gert

    2002-01-01

    Dihydroorotate dehydrogenase (DHOD) (EC 1.3.3.1) from the thermoacidophilic archaeon Sulfolobus solfataricus P2 (DSM 1617) was partially purified 3,158-fold, characterized, and the encoding genes identified. Based on enzymological as well as phylogenetic methods, dihydroorotate dehydrogenase from S....... solfataricus (DHODS) represents a new type of DHOD, type 1S. Furthermore, it is unable to use any of the (type-specific) natural electron acceptors employed by all other presently known DHODs. DHODS shows optimal activity at 70°C in the pH range 7-8.5. It is capable of using ferricyanide, 2...

  3. OsHSD1, a hydroxysteroid dehydrogenase, is involved in cuticle formation and lipid homeostasis in rice.

    Zhang, Zhe; Cheng, Zhi-Jun; Gan, Lu; Zhang, Huan; Wu, Fu-Qing; Lin, Qi-Bing; Wang, Jiu-Lin; Wang, Jie; Guo, Xiu-Ping; Zhang, Xin; Zhao, Zhi-Chao; Lei, Cai-Lin; Zhu, Shan-Shan; Wang, Chun-Ming; Wan, Jian-Min

    2016-08-01

    Cuticular wax, a hydrophobic layer on the surface of all aerial plant organs, has essential roles in plant growth and survival under various environments. Here we report a wax-deficient rice mutant oshsd1 with reduced epicuticular wax crystals and thicker cuticle membrane. Quantification of the wax components and fatty acids showed elevated levels of very-long-chain fatty acids (VLCFAs) and accumulation of soluble fatty acids in the leaves of the oshsd1 mutant. We determined the causative gene OsHSD1, a member of the short-chain dehydrogenase reductase family, through map-based cloning. It was ubiquitously expressed and responded to cold stress and exogenous treatments with NaCl or brassinosteroid analogs. Transient expression of OsHSD1-tagged green fluorescent protein revealed that OsHSD1 localized to both oil bodies and endoplasmic reticulum (ER). Dehydrogenase activity assays demonstrated that OsHSD1 was an NAD(+)/NADP(+)-dependent sterol dehydrogenase. Furthermore, OsHSD1 mutation resulted in faster protein degradation, but had no effect on the dehydrogenase activity. Together, our data indicated that OsHSD1 plays a specialized role in cuticle formation and lipid homeostasis, probably by mediating sterol signaling. This work provides new insights into oil-body associated proteins involved in wax and lipid metabolism. PMID:27297988

  4. Deficient Expression of Aldehyde Dehydrogenase 1A1 Is Consistent with Increased Sensitivity of Gorlin Syndrome Patients to Radiation Carcinogenesis

    Wright, Aaron T.; Magnaldo, Thierry; Sontag, Ryan L.; Anderson, Lindsey N.; Sadler, Natalie C.; Piehowski, Paul D.; Gache, Yannick; Weber, Thomas J.

    2015-06-01

    Human phenotypes that are highly susceptible to radiation carcinogenesis have been identified. Sensitive phenotypes often display robust regulation of molecular features that modify biological response, which can facilitate identification of relevant pathways/networks. Here we interrogate primary dermal fibroblasts isolated from Gorlin syndrome patients (GDFs), who display a pronounced tumorigenic response to radiation, in comparison to normal human dermal fibroblasts (NHDFs). Our approach exploits newly developed thiol-reactive probes with a flexible click chemistry functional group to define changes in protein thiol profiles in live cell studies, which minimizes artifacts associated with cell lysis. We observe qualitative differences in protein thiol profiles by SDS-PAGE analysis when detection by iodoacetamide vs maleimide probe chemistries are compared, and pretreatment of cells with hydrogen peroxide eliminates detection of the majority of SDS-PAGE bands. Redox probes revealed deficient expression of an apparent 55 kDa protein thiol in GDFs from independent donors, compared with NHDFs. Proteomics tentatively identified this protein as aldehyde dehydrogenase 1A1 (ALDH1A1), a key enzyme regulating retinoic acid synthesis, and this deficiency was confirmed by Western blot. Redox probes revealed additional protein thiol differences between GDFs and NHDFs, including radiation responsive annexin family members. Our results indicate a multifactorial basis for the unusual sensitivity of Gorlin syndrome to radiation carcinogenesis, and the pathways identified have plausible implications for radiation health effects.

  5. The role of aldehyde dehydrogenase-1 (ALDH1A1 polymorphisms in harmful alcohol consumption in a Finnish population

    Lind Penelope A

    2008-09-01

    Full Text Available Abstract Liver cystolic aldehyde dehydrogenase 1 (ALDH1A1 has been previously associated with both alcohol dependence and alcohol consumption behaviour, and has been implicated in alcohol-induced flushing and alcohol sensitivity in Caucasians. The present study tested for association between ALDH1A1 and alcohol consumption behaviour and susceptibility to problem drinking or alcohol dependence in Finnish cohorts of unrelated male subjects recruited from alcoholism clinical treatment facilities (n = 104 and from the general population (n = 201. All participants completed the Alcohol Use Disorder Identification Test (AUDIT and were genotyped for eight single nucleotide polymorphisms (SNPs within or flanking ALDH1A1. To test for association between alcohol consumption behaviour and these polymorphisms, we used generalised linear models and haplotypic analysis. Three SNPs were nominally associated (rs348449, p = 0.043; rs610529, p = 0.013; rs348479, p = 0.025 with the quantitative AUDIT score, which evaluates alcohol consumption behaviour. Two-locus (rs6I0529-rs2288087 haplotype analysis increased the strength of association with AUDIT score (p = 0.00I5. Additionally, rs348449 is highly associated with problem drinking (allelic odds ratio [OR] 7.87, 95 per cent confidence interval [CI] 1.67-37.01 but due to the low minor allele frequency (0.01 and 0.07 in controls and problem drinkers, respectively, more samples are required to validate this observation. Conversely, rs348479 (p = 0.019 and rs6I0529 (allelic OR 0.65, 95 per cent CI 0.43-0.98; genotypic OR 0.32, 95 per cent CI 0.12-0.84 are implicated in alcohol dependence status. This study provides further evidence for a role for ALDH1A1 in alcohol consumption behaviour, including problem drinking and possibly alcohol dependence, in our Finnish population.

  6. Multiple states of the Tyr318Leu mutant of dihydroorotate dehydrogenase revealed by single molecule kinetics

    Shi, J.; Palfey, B.A.; Dertouzos, J.;

    2004-01-01

    single enzyme molecules through the characteristic on-off fluorescence signal, which corresponds to flavin mononucleotide (FMN) interconverting between the oxidized and reduced states during turnover. Our single-molecule data provide evidence of a distinct static heterogeneity in the enzymatic activity......, with some molecules going through the on-off cycles 5-fold faster than others, however, there is no detectable dynamic disorder in DHOD turnover. When 0.1% reduced Triton X-100, a detergent that more closely simulates the natural membrane environment, is added, our data suggest the degree of static...... molecular heterogeneity is reduced. The observation of static heterogeneity suggests that the enzyme, which associates with the membrane in vivo, is present in distinct conformations that result in different catalytic efficiencies. The alternate conformations are most likely the result of the loss of van...

  7. Metabolism of trans, trans-muconaldehyde, a cytotoxic metabolite of benzene, in mouse liver by alcohol dehydrogenase Adh1 and aldehyde reductase AKR1A4

    The reductive metabolism of trans, trans-muconaldehyde, a cytotoxic metabolite of benzene, was studied in mouse liver. Using an HPLC-based stopped assay, the primary reduced metabolite was identified as 6-hydroxy-trans, trans-2,4-hexadienal (OH/CHO) and the secondary metabolite as 1,6-dihydroxy-trans, trans-2,4-hexadiene (OH/OH). The main enzymes responsible for the highest levels of reductase activity towards trans, trans-muconaldehyde were purified from mouse liver soluble fraction first by Q-sepharose chromatography followed by either blue or red dye affinity chromatography. In mouse liver, trans, trans-muconaldehyde is predominantly reduced by an NADH-dependent enzyme, which was identified as alcohol dehydrogenase (Adh1). Kinetic constants obtained for trans, trans-muconaldehyde with the native Adh1 enzyme showed a V max of 2141 ± 500 nmol/min/mg and a K m of 11 ± 4 μM. This enzyme was inhibited by pyrazole with a K I of 3.1 ± 0.57 μM. Other fractions were found to contain muconaldehyde reductase activity independent of Adh1, and one enzyme was identified as the NADPH-dependent aldehyde reductase AKR1A4. This showed a V max of 115 nmol/min/mg and a K m of 15 ± 2 μM and was not inhibited by pyrazole

  8. MFE1, a Member of the Peroxisomal Hydroxyacyl Coenzyme A Dehydrogenase Family, Affects Fatty Acid Metabolism Necessary for Morphogenesis in Dictyostelium spp.

    Matsuoka, Satomi; Saito, Tamao; Kuwayama, Hidekazu; Morita, Naoki; Ochiai, Hiroshi; Maeda, Mineko

    2003-01-01

    β-Oxidation of long-chain fatty acids and branched-chain fatty acids is carried out in mammalian peroxisomes by a multifunctional enzyme (MFE) or d-bifunctional protein, with separate domains for hydroxyacyl coenzyme A (CoA) dehydrogenase, enoyl-CoA hydratase, and steroid carrier protein SCP2. We have found that Dictyostelium has a gene, mfeA, encoding MFE1 with homology to the hydroxyacyl-CoA dehydrogenase and SCP2 domains. A separate gene, mfeB, encodes MFE2 with homology to the enoyl-CoA h...

  9. CvADH1, a member of short-chain alcohol dehydrogenase family, is inducible by gibberellin and sucrose in developing watermelon seeds.

    Kim, Joonyul; Kang, Hong-Gyu; Jun, Sung-Hoon; Lee, Jinwon; Yim, Jieun; An, Gynheung

    2003-01-01

    To understand the molecular mechanisms that control seed formation, we selected a seed-preferential gene (CvADH1) from the ESTs of developing watermelon seeds. RNA blot analysis and in situ localization showed that CvADH1 was preferentially expressed in the nucellar tissue. The CvADH1 protein shared about 50% homology with short-chain alcohol dehydrogenase including ABA2 in Arabidopsis thaliana, stem secoisolariciresinol dehydrogenase in Forsythia intermedia, and 3beta-hydroxysterol dehydrogenase in Digitalis lanata. We investigated gene-expression levels in seeds from both normally pollinated fruits and those made parthenocarpic via N-(2-chloro-4-pyridyl)-N'-phenylurea treatment, the latter of which lack zygotic tissues. Whereas the transcripts of CvADH1 rapidly started to accumulate from about the pre-heart stage in normal seeds, they were not detectable in the parthenocarpic seeds. Treating the parthenogenic fruit with GA(3) strongly induced gene expression, up to the level accumulated in pollinated seeds. These results suggest that the CvADH1 gene is induced in maternal tissues by signals made in the zygotic tissues, and that gibberellin might be one of those signals. We also observed that CvADH1 expression was induced by sucrose in the parthenocarpic seeds. Therefore, we propose that the CvADH1 gene is inducible by gibberellin, and that sucrose plays an important role in the maternal tissues of watermelon during early seed development. PMID:12552151

  10. Glucose-6-phosphate dehydrogenase

    ... this page: //medlineplus.gov/ency/article/003671.htm Glucose-6-phosphate dehydrogenase test To use the sharing features on this page, please enable JavaScript. Glucose-6-phosphate dehydrogenase (G6PD) is a type of ...

  11. Plant Formate Dehydrogenase

    John Markwell

    2005-01-10

    The research in this study identified formate dehydrogenase, an enzyme that plays a metabolic role on the periphery of one-carbon metabolism, has an unusual localization in Arabidopsis thaliana and that the enzyme has an unusual kinetic plasticity. These properties make it possible that this enzyme could be engineered to attempt to engineer plants with an improved photosynthetic efficiency. We have produced transgenic Arabidopsis and tobacco plants with increased expression of the formate dehydrogenase enzyme to initiate further studies.

  12. Glucose-6-phosphate dehydrogenase deficiency

    ... this page: //medlineplus.gov/ency/article/000528.htm Glucose-6-phosphate dehydrogenase deficiency To use the sharing features on this page, please enable JavaScript. Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a condition ...

  13. Molybdenum center of xanthine dehydrogenase

    Cyanolysis of native, oxidized xanthine dehydrogenase is known to inactivate the enzyme by removing a unique sulfur as thiocyanate. Chemical, genetic, and spectroscopic evidence indicates that this sulfur is a terminal ligand of Mo and is present in native xanthine dehydrogenase, but not in cyanolyzed xanthine dehydrogenase or native sulfite oxidase. A procedure for rapid, reproducible, and quantitative reconstitution of desulfo Mo hydroxylases with sulfide was developed. The cyanolyzable sulfur of xanthine dehydrogenase was specifically radiolabeled with 35sulfide using this procedure. Various chemical properties of the cyanolyzable sulfur could be determined with the radiolabelled enzyme. The data support the conclusion that the cyanolyzable sulfur is a terminal sulfur ligand of the Mo atoms, and is not part of an organic moiety. Application of the resulfuration procedure to crude extracts of Drosophila melanogaster ma-1 flies, which are pleiotropically deficient in xanthine dehydrogenase and aldehyde oxidase, led to the emergence of these enzyme activities. Evidence for the identity of in vitro reconstituted xanthine dehydrogenase from ma-1 mutants with wild type enzyme is presented. A system for efficient reconstitution of the apo-subunits of the molybdoenzyme nitrate reductase from the Neurospora crassa mutant nit-1 with molybdenum cofactor from denatured purified molybdoenzymes in the absence of exogenous molybdate was developed

  14. Glucose-6-Phosphate Dehydrogenase Deficiency Overview

    ... Drugs GARD Information Navigator FAQs About Rare Diseases Glucose-6-phosphate dehydrogenase deficiency Title Other Names: G6PD ... G6PD deficiency Categories: Newborn Screening Summary Summary Listen Glucose 6 phosphate dehydrogenase (G6PD) deficiency is a hereditary ...

  15. Glucose-6-Phosphate Dehydrogenase Deficiency Overview

    ... Information Center (GARD) Print friendly version Glucose-6-phosphate dehydrogenase deficiency Table of Contents Overview Symptoms Cause ... National Institutes of Health. Overview Listen Glucose 6 phosphate dehydrogenase (G6PD) deficiency is a hereditary condition in ...

  16. Sorbitol dehydrogenase is a zinc enzyme.

    Jeffery, J; Chesters, J; C. Mills; P.J. Sadler; Jörnvall, H

    1984-01-01

    Evidence is given that tetrameric sorbitol dehydrogenase from sheep liver contains one zinc atom per subunit, most probably located at the active site, and no other specifically bound zinc or iron atom. In alcohol dehydrogenases that are structurally related to sorbitol dehydrogenase, more than one zinc atom per subunit can complicate investigations of zinc atom function. Therefore, sorbitol dehydrogenase will be particularly valuable for defining the precise roles of zinc in alcohol and poly...

  17. Specific biotinylation of IMP dehydrogenase

    Hoefler, B. Christopher; Gollapalli, Deviprasad R.; Hedstrom, Lizbeth

    2011-01-01

    IMP dehydrogenase (IMPDH) catalyzes a critical step in guanine nucleotide biosynthesis. IMPDH also has biological roles that are distinct from its enzymatic function. We report a biotin-linked reagent that selectively labels IMPDH and is released by dithiothreitol. This reagent will be invaluable in elucidating the moonlighting functions of IMPDH.

  18. Studies on 2-oxoacid dehydrogenase multienzyme complexes of Azotobacter vinelandii

    Bosma, H.J.

    1984-01-01

    In this thesis, some studies on the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase multienzyme complexes of Azotobacter vinelandii are described; the emphasis strongly lies on the pyruvate dehydrogenase complex.A survey of the literature on 2-oxoacid dehydrogenase complexes is given in chap

  19. The three-dimensional structure of the ternary complex of Corynebacterium glutamicum diaminopimelate dehydrogenase-NADPH-L-2-amino-6-methylene-pimelate.

    Cirilli, M.; Scapin, G.; Sutherland, A; Vederas, J. C.; Blanchard, J. S.

    2000-01-01

    The three-dimensional (3D) structure of Corynebacterium glutamicum diaminopimelate D-dehydrogenase in a ternary complex with NADPH and L-2-amino-6-methylene-pimelate has been solved and refined to a resolution of 2.1 A. L-2-Amino-6-methylene-pimelate was recently synthesized and shown to be a potent competitive inhibitor (5 microM) vs. meso-diaminopimelate of the Bacillus sphaericus dehydrogenase (Sutherland et al., 1999). Diaminopimelate dehydrogenase catalyzes the reversible NADP+ -dependen...

  20. Microbial alcohol dehydrogenases: identification, characterization and engineering

    Machielsen, M.P.

    2007-01-01

    Keywords: alcohol dehydrogenase, laboratory evolution, rational protein engineering, Pyrococcus furiosus, biocatalysis, characterization, computational design, thermostability.   Alcohol dehydrogeases (ADHs) catalyze the interconversion of alcohols, aldehydes and ketones. They display a wide variety

  1. Screening of aspartate dehydrogenase of bacteria

    Fukuda, Shoko; Okamura, Tokumitsu; Yasumasa, Izumi; Takeno, Tomomi; Ohsugi, Masahiro

    2001-01-01

    Fifty-two strains of bacteria cultured under aerobic conditions and 12 strains of bacteria cultured under anaerobic conditions demonstrated high activity staining of aspartate dehydrogenase with NAD^+. Four strains of bacteria cultured under aerobic conditions and 7 strains of bacteria cultured under anaerobic conditions demonstrated high activity staining of aspartate dehydrogenase with NADP^+. Seven strains of bacteria cultured under aerobic conditions and 4 strains of bacteria cultured und...

  2. Phosphorylation site on yeast pyruvate dehydrogenase complex

    The pyruvate dehydrogenase complex was purified to homogeneity from baker's yeast (Saccharomyces cerevisiae). Yeast cells were disrupted in a Manton-Gaulin laboratory homogenizer. The pyruvate dehydrogenase complex was purified by fractionation with polyethylene glycol, isoelectric precipitation, ultracentrifugation and chromatography on hydroxylapatite. Final purification of the yeast pyruvate dehydrogenase complex was achieved by cation-exchange high pressure liquid chromatography (HPLC). No endogenous pyruvate dehydrogenase kinase activity was detected during the purification. However, the yeast pyruvate dehydrogenase complex was phosphorylated and inactivated with purified pyruvate dehydrogenase kinase from bovine kidney. Tryptic digestion of the 32P-labeled complex yielded a single phosphopeptide which was purified to homogeniety. The tryptic digest was subjected to chromatography on a C-18 reverse phase HPLC column with a linear gradient of acetonitrile. Radioactive fractions were pooled, concentrated, and subjected to anion-exchange HPLC. The column was developed with a linear gradient of ammonium acetate. Final purification of the phosphopeptide was achieved by chromatography on a C-18 reverse phase HPLC column developed with a linear gradient of acetonitrile. The amino acid sequence of the homogeneous peptide was determined by manual modified Edman degradation

  3. An autosomal glucose-6-phosphate dehydrogenase (hexose-6-phosphate dehydrogenase) polymorphism in human saliva.

    Tan, S G; Ashton, G C

    1976-01-01

    Glucose-6-phosphate dehydrogenase (hexose-6-phosphate dehydrogenase) from human saliva has been demonstrated by the zymogram technique. Three phenotypes were found. Family and population studies suggested that these phenotypes are the products of an autosomal locus with two alleles Sgd-1 and Sgd-2. PMID:950237

  4. Inducible xylitol dehydrogenases in enteric bacteria.

    Doten, R C; Mortlock, R P

    1985-01-01

    Morganella morganii ATCC 25829, Providencia stuartii ATCC 25827, Serratia marcescens ATCC 13880, and Erwinia sp. strain 4D2P were found to induce a xylitol dehydrogenase when grown on a xylitol-containing medium. The xylitol dehydrogenases were partially purified from the four strains, and those from M. morganii ATCC 25829, P. stuartii ATCC 25827, and S. marcescens ATCC 13880 were all found to oxidize xylitol to D-xylulose. These three enzymes had KmS for xylitol of 7.1 to 16.4 mM and molecul...

  5. Yeast surface display of dehydrogenases in microbial fuel-cells.

    Gal, Idan; Schlesinger, Orr; Amir, Liron; Alfonta, Lital

    2016-12-01

    Two dehydrogenases, cellobiose dehydrogenase from Corynascus thermophilus and pyranose dehydrogenase from Agaricus meleagris, were displayed for the first time on the surface of Saccharomyces cerevisiae using the yeast surface display system. Surface displayed dehydrogenases were used in a microbial fuel cell and generated high power outputs. Surface displayed cellobiose dehydrogenase has demonstrated a midpoint potential of -28mV (vs. Ag/AgCl) at pH=6.5 and was used in a mediator-less anode compartment of a microbial fuel cell producing a power output of 3.3μWcm(-2) using lactose as fuel. Surface-displayed pyranose dehydrogenase was used in a microbial fuel cell and generated high power outputs using different substrates, the highest power output that was achieved was 3.9μWcm(-2) using d-xylose. These results demonstrate that surface displayed cellobiose dehydrogenase and pyranose dehydrogenase may successfully be used in microbial bioelectrochemical systems. PMID:27459246

  6. Three-dimensional structures of the three human class I alcohol dehydrogenases

    Niederhut, Monica S.; Gibbons, Brian J.; Perez-Miller, Samantha; Hurley, Thomas D.

    2001-01-01

    In contrast with other animal species, humans possess three distinct genes for class I alcohol dehydrogenase and show polymorphic variation in the ADH1B and ADH1C genes. The three class I alcohol dehydrogenase isoenzymes share ∼93% sequence identity but differ in their substrate specificity and their developmental expression. We report here the first three-dimensional structures for the ADH1A and ADH1C*2 gene products at 2.5 and 2.0 Å, respectively, and the structure of the ADH1B*1 gene produ...

  7. Microbial alcohol dehydrogenases: identification, characterization and engineering

    Machielsen, M.P.

    2007-01-01

    Keywords: alcohol dehydrogenase, laboratory evolution, rational protein engineering, Pyrococcus furiosus, biocatalysis, characterization, computational design, thermostability.   Alcohol dehydrogeases (ADHs) catalyze the interconversion of alcohols, aldehydes and ketones. They display a wide variety of substrate specificities and are involved in an astonishingly wide range of metabolic processes, in all living organisms. Besides the scientific interest in ADHs, they are also attractive biocat...

  8. Genetics Home Reference: pyruvate dehydrogenase deficiency

    ... the most common cause of pyruvate dehydrogenase deficiency , accounting for approximately 80 percent of cases. These mutations ... deficiency ClinicalTrials.gov (1 link) ClinicalTrials.gov Scientific articles on PubMed (1 link) PubMed OMIM (5 links) ...

  9. Genetics Home Reference: lactate dehydrogenase deficiency

    ... throughout the body and is important for creating energy for cells. There are five different forms of this enzyme, each made up of four ... and lactate dehydrogenase-B subunits make up the different forms of the ... large amounts of energy during high-intensity physical activity when the body's ...

  10. The Genetics of Alcohol Metabolism: Role of Alcohol Dehydrogenase and Aldehyde Dehydrogenase Variants

    Edenberg, Howard J

    2007-01-01

    The primary enzymes involved in alcohol metabolism are alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH). Both enzymes occur in several forms that are encoded by different genes; moreover, there are variants (i.e., alleles) of some of these genes that encode enzymes with different characteristics and which have different ethnic distributions. Which ADH or ALDH alleles a person carries influence his or her level of alcohol consumption and risk of alcoholism. Researchers to date pri...

  11. Characterization of xylitol dehydrogenase from Debaryomyces hansenii

    Girio, F.M.; Amaral-Collaco, M.T. [INETI, Lisboa (Portugal); Pelica, F. [ITQB, Oeiras (Portugal)

    1996-01-01

    The xylitol dehydrogenase (EC 1.1.1.9) from xylose-grown cells of Debaryomyces hansenii was partially purified in two chromatographic steps, and characterization studies were carried out in order to investigate the role of the xylitol dehydrogenase-catalyzed step in the regulation of D-xylose metabolism. The enzyme was most active at pH 9.0-9.5, and exhibited a broad polyol specificity. The Michaelis constants for xylitol and NAD{sup +} were 16.5 and 0.55 mM, respectively. Ca{sup 2+}, Mg{sup 2+}, and Mn{sup 2+} did not affect the enzyme activity. Conversely, Zn{sup 2+}, Cd{sup 2+}, and Co{sup 2+} strongly inhibited the enzyme activity. It was concluded that NAD{sup +}-xylitol dehydrogenase from D. hansenii has similarities with other xylose-fermenting yeasts in respect to optimal pH, substrate specificity, and K{sub m} value for xylitol, and therefore should be named L-iditol:NAD{sup +}-5-oxidoreductase (EC 1.1.1.14). The reason D. hansenii is a good xylitol producer is not because of its value of K for xylitol, which is low enough to assure its fast oxidation by NAD{sup +}-xylitol dehydrogenase. However, a higher K{sub m} value of xylitol dehydrogenase for NAD{sup +} compared to the K{sub m} values of other xylose-fermenting yeasts may be responsible for the higher xylitol yields. 22 refs., 4 figs., 2 tabs.

  12. Phosphorylation-dephosphorylation of yeast pyruvate dehydrogenase

    Pyruvate dehydrogenase complex (PDC) was purified to homogeneity from baker's yeast (Saccharomyces cerevisiae). No pyruvate dehydrogenase (PDH) kinase activity was detected at any stage of the purification. However, the purified PDC was phosphorylated and inactivated by purified PDH kinase from bovine kidney mitochondria, Mg2+, and [γ-32P]ATP. The protein-bound radioactivity was localized in the PDH α subunit. The phosphorylated, inactivated PDC was dephosphorylated and reactivated with purified bovine PDH phosphatase, Mg2+, and Ca2+. From a tryptic digest of phosphorylated yeast PDC a radioactive peptide was isolated by anion and reverse phase HPLC. The sequence of this tetradecapeptide is Tyr-Gly-Gly-His-Ser(P)-Met-Ser-Asp-Pro-Gly-Thr-Thr-Tyr-Arg. This sequence is very similar to the sequence of a tryptic phosphopeptide derived from the α subunit of bovine kidney and heart PDH: Tyr-His-Gly-His-Ser(P)-Met-Ser-Asp-Pro-Gly-Val-Ser-Tyr-Arg

  13. Alcohol dehydrogenase activity in immobilized yeast cells

    A method for the immobilization of Saccharomyces cerevisiae was developed and the activity of alcohol dehydrogenase of the immobilized cells was determined. The treatment of the yeast cells with 1 % toluene followed by irradiation with acrylamide and bisacrylamide resulted in a high activity of alcohol dehydrogenase in the immobilized cells. The enzyme of the immobilized cells was stable in the pH range of 7.5 - 8.0 and the optimum pH opposed to be 8.5. Although the immobilized cells showed a rather low level of thermostability, it is suggested that they could be used for a long period of time at a temperature of 27 deg C. The immobilized cells did not exhibit any loss in the enzyme activity when stored at 4 deg C or -20 deg C. (author)

  14. Glucose 6 phosphate dehydrogenase deficiency Review

    Şaşmaz, İlgen

    2009-01-01

    Glucose 6 phosphate dehydrogenase G6PD is the first enzyme of the pentose phosphate pathway providing reducing power to all cells in the form of reduced form of nicotinamide adenine dinucleotide phosphate G6PD deficiency is the most common human enzyme defect being present in more than 400 million people worldwide G6PD deficiency is an X linked hereditary genetic defect caused by mutations in the G6PD gene Clinical presentations include acute hemolytic anemia chronic hemolytic anemia neonatal...

  15. Dihydrodiol dehydrogenase and polycyclic aromatic hydrocarbon metabolism

    Smithgall, T.E.

    1986-01-01

    Carcinogenic activation of polycyclic aromatic hydrocarbons by microsomal monoxygenases proceeds through trans-dihydrodiol metabolites to diol-epoxide ultimate carcinogens. This thesis directly investigated the role of dihydrodiol dehydrogenase, a cytosolic NAD(P)-linked oxidoreductase, in the detoxification of polycyclic aromatic trans-dihydrodiols. A wide variety of non-K-region trans-dihydrodiols were synthesized and shown to be substrates for the homogeneous rat liver dehydrogenase, including several potent proximate carcinogens derived from 7,12-dimethylbenz(a)anthracene, 5-methylchrysene, and benzo(a)pyrene. Since microsomal activation of polycyclic aromatic hydrocarbons is highly stereospecific, the stereochemical course of enzymatic trans-dihydrodiol oxidation was monitored using circular dichroism spectropolarimetry. The major product formed from the dehydrogenase-catalyzed oxidation of the trans-1,2-dihydrodiol of naphthalene was characterized using UV, IR, NMR, and mass spectroscopy, and appears to be 4-hydroxy-1,2-naphthoquinone. Mass spectral analysis suggests that an analogous hydroxylated o-quinone is formed as the major product of benzo(a)pyrene-7,8-dihydrodiol oxidation. Enzymatic oxidation of trans-dihydrodiols was shown to be potently inhibited by all of the major classes of the nonsteroidal antiinflammatory drugs. Enhancement of trans-dihydrodiol proximate carcinogen oxidation may protect against possible adverse effects of the aspirin-like drugs, and help maintain the balance between activation and detoxification of polycyclic aromatic hydrocarbons.

  16. Idiopathic intracranial hypertension, hormones, and 11β-hydroxysteroid dehydrogenases

    Markey, Keira A; Uldall, Maria; Botfield, Hannah; Cato, Liam D; Miah, Mohammed A L; Hassan-Smith, Ghaniah; Jensen, Rigmor H; Gonzalez, Ana M; Sinclair, Alexandra J

    2016-01-01

    Idiopathic intracranial hypertension (IIH) results in raised intracranial pressure (ICP) leading to papilledema, visual dysfunction, and headaches. Obese females of reproductive age are predominantly affected, but the underlying pathological mechanisms behind IIH remain unknown. This review provides an overview of pathogenic factors that could result in IIH with particular focus on hormones and the impact of obesity, including its role in neuroendocrine signaling and driving inflammation. Despite occurring almost exclusively in obese women, there have been a few studies evaluating the mechanisms by which hormones and adipokines exert their effects on ICP regulation in IIH. Research involving 11β-hydroxysteroid dehydrogenase type 1, a modulator of glucocorticoids, suggests a potential role in IIH. Improved understanding of the complex interplay between adipose signaling factors such as adipokines, steroid hormones, and ICP regulation may be key to the understanding and future management of IIH.

  17. Lactate dehydrogenase X, malate dehydrogenase and total protein in rat spermatozoa during epididymal transit.

    Vermouth, N T; Carriazo, C S; Ponce, R H; Blanco, A

    1986-01-01

    Lactate dehydrogenase isozyme X (LDH X), malate dehydrogenase (MDH) and total soluble protein have been determined in lysates of spermatozoa isolated from caput, corpus and cauda of rat epididymis. Transit of spermatozoa through epididymis is accompanied by a reduction of LDH X, MDH and total protein per cell in sexually rested animals. The profiles of reduction along epididymal segments are different for the three variables studied. Mating with receptive females during the 5 days prior to determinations increases significantly the levels of MDH in spermatozoa from all sections of epididymis and produces increase of total soluble protein in the cells contained in cauda. PMID:3956158

  18. Molecular cloning of gluconobacter oxydans DSM 2003 xylitol dehydrogenase gene

    Sadeghi, H. Mir Mohammad; Ahmadi, R; Aghaabdollahian, S.; Mofid, M.R.; Ghaemi, Y.; Abedi, D

    2011-01-01

    Due to the widespread applications of xylitol dehydrogenase, an enzyme used for the production of xylitol, the present study was designed for the cloning of xylitol dehydrogenase gene from Glcunobacter oxydans DSM 2003. After extraction of genomic DNA from this bacterium, xylitol dehydrogenase gene was replicated using polymerase chain reaction (PCR). The amplified product was entered into pTZ57R cloning vector by T/A cloning method and transformation was performed by heat shocking of the E. ...

  19. Molecular genetic analysis of human alcohol dehydrogenase

    Duester, G; Wesley Hatfield, G.; Smith, M.

    1985-01-01

    Human alcohol dehydrogenase (ADH) consists of a complex group of isozymes encoded by at least five non-identical genes, two of which have previously been shown through enzymatic analysis to possess polymorphic variants. Using a cDNA probe the ADH2gene encoding the β subunit of human ADH was mapped to human chromosome 4. The cDNA probe for ADH2 was also used to detect a restriction fragment length polymorphism present in human populations. This polymorphism may help establish whether certain A...

  20. Lactate dehydrogenase in sickle cell disease.

    Stankovic Stojanovic, Katia; Lionnet, François

    2016-07-01

    Lactate dehydrogenase (LDH) activity is elevated in many pathological states. Interest in LDH activity in sickle cell disease (SCD) has developed out of an increased comprehension of the pathophysiological process and the clinical course of the disease. Elevated LDH activity in SCD comes from various mechanisms, especially intravascular hemolysis, as well as ischemia-reperfusion damage and tissular necrosis. Intravascular hemolysis is associated with vasoconstriction, platelet activation, endothelial damage, and vascular complications. LDH has been used as a diagnostic and prognostic factor of acute and chronic complications. In this review we have evaluated the literature where LDH activity was examined during steady-state or acute conditions in SCD. PMID:27138446

  1. NAD(H recycling activity of an engineered bifunctional enzyme galactose dehydrogenase/lactate dehydrogenase

    2006-03-01

    Full Text Available A chimeric bifunctional enzyme composing of galactose dehydrogenase (galDH; from Pseudomonas fluorescens and lactate dehydrogenase (LDH; from Bacillus stearothermophilus was successfully constructed. The chimeric galDH/LDH possessed dual characteristics of both galactose dehydrogenase and lactate dehydrogenase activities while exhibiting hexameric rearrangement with a molecular weight of approximately 400 kDa. In vitro observations showed that the chimeric enzyme was able to recycle NAD with a continuous production of lactate without any externally added NADH. Two fold higher recycling rate (0.3 mM/h than that of the native enzyme was observed at pH values above 8.5. Proximity effects became especially pronounced during the recycling assay when diffusion hindrance was induced by polyethylene glycol. All these findings open up a high feasibility to apply the NAD(H recycling system for metabolic engineering purposes e.g. as a model to gain a better understanding on the molecular proximity process and as the routes for synthesizing of numerous high-value-added compounds.

  2. Recommended nomenclature for the vertebrate alcohol dehydrogenase gene family.

    Duester, G; Farrés, J; Felder, M R; Holmes, R S; Höög, J O; Parés, X; Plapp, B V; Yin, S J; Jörnvall, H

    1999-08-01

    The alcohol dehydrogenase (ADH) gene family encodes enzymes that metabolize a wide variety of substrates, including ethanol, retinol, other aliphatic alcohols, hydroxysteroids, and lipid peroxidation products. Studies on 19 vertebrate animals have identified ADH orthologs across several species, and this has now led to questions of how best to name ADH proteins and genes. Seven distinct classes of vertebrate ADH encoded by non-orthologous genes have been defined based upon sequence homology as well as unique catalytic properties or gene expression patterns. Each class of vertebrate ADH shares 80% sequence identity such as the case for class I ADH where humans have three class I ADH genes, horses have two, and mice have only one. Presented here is a nomenclature that uses the widely accepted vertebrate ADH class system as its basis. It follows the guidelines of human and mouse gene nomenclature committees, which recommend coordinating names across species boundaries and eliminating Roman numerals and Greek symbols. We recommend that enzyme subunits be referred to by the symbol "ADH" (alcohol dehydrogenase) followed by an Arabic number denoting the class; i.e. ADH1 for class I ADH. For genes we recommend the italicized root symbol "ADH" for human and "Adh" for mouse, followed by the appropriate Arabic number for the class; i.e. ADH1 or Adh1 for class I ADH genes. For organisms where multiple species-specific isoenzymes exist within a class, we recommend adding a capital letter after the Arabic number; i.e. ADH1A, ADH1B, and ADH1C for human alpha, beta, and gamma class I ADHs, respectively. This nomenclature will accommodate newly discovered members of the vertebrate ADH family, and will facilitate functional and evolutionary studies. PMID:10424757

  3. Aldehyde dehydrogenase protein superfamily in maize.

    Zhou, Mei-Liang; Zhang, Qian; Zhou, Ming; Qi, Lei-Peng; Yang, Xiong-Bang; Zhang, Kai-Xuan; Pang, Jun-Feng; Zhu, Xue-Mei; Shao, Ji-Rong; Tang, Yi-Xiong; Wu, Yan-Min

    2012-11-01

    Maize (Zea mays ssp. mays L.) is an important model organism for fundamental research in the agro-biotechnology field. Aldehydes were generated in response to a suite of environmental stresses that perturb metabolism including salinity, dehydration, desiccation, and cold and heat shock. Many biologically important aldehydes are metabolized by the superfamily of NAD(P)(+)-dependent aldehyde dehydrogenases. Here, starting from the database of Z. mays, we identified 28 aldehyde dehydrogenase (ALDH) genes and 48 transcripts by the in silico cloning method using the ALDH-conserved domain amino acid sequence of Arabidopsis and rice as a probe. Phylogenetic analysis shows that all 28 members of the ALDH gene families were classified to ten distinct subfamilies. Microarray data and quantitative real-time PCR analysis reveal that ZmALDH9, ZmALDH13, and ZmALDH17 genes involve the function of drought stress, acid tolerance, and pathogens infection. These results suggested that these three ZmALDH genes might be potentially useful in maize genetic improvement. PMID:22983498

  4. HISTIDINE MUTAGENESIS OF ARABIDOPSIS THALIANA PYRUVATE DEHYDROGENASE KINASE

    Pyruvate dehydrogenase kinase (PDK) is the primary regulator of flux through the mitochondrial pyruvate dehydrogenase complex (PDC). Analysis of the primary amino acid sequences of PDK from various sources reveals that these enzymes include the five domains characteristic of prokaryotic two-compone...

  5. Studies on the structure and function of pyruvate dehydrogenase complexes

    Abreu, de R.A.

    1978-01-01

    The aim of the present investigation was to obtain more information of the structure and function of the pyruvate dehydrogenase complexes from Azotobacter vinelandii and Escherichia coli.In chapter 2 a survey is given of the recent literature on pyruvate dehydrogenase complexes.In chapter 3 results

  6. INFLUENCE OF SELECTED PHARMACEUTICALS ON ACTIVATED SLUDGE DEHYDROGENASE ACTIVITY

    Agnieszka Tomska

    2016-06-01

    The aim of this work was to evaluate the effect of selected antibiotics - sulfanilamide and erythromycin on activated sludge dehydrogenase activity with use of trifenyltetrazolinum chloride (TTC test. Dehydrogenases activity is an indicator of biochemical activity of microorganisms present in activated sludge or the ability to degrade organic compounds in waste water. TTC test is particularly useful for the regularity of the course of treatment, in which the presence of inhibitors of biochemical reactions and toxic compounds are present. It was observed that the dehydrogenase activity decreases with the increase of a antibiotics concentration. The lowest value of the dehydrogenase activity equal to 32.4 μmol TF / gMLSS obtained at sulfanilamide concentration 150mg / l. For this sample, an inhibition of dehydrogenase activity was 31%.

  7. Nuclear Magnetic Resonance Approaches in the Study of 2-Oxo Acid Dehydrogenase Multienzyme Complexes—A Literature Review

    Mulchand S. Patel

    2013-09-01

    Full Text Available The 2-oxoacid dehydrogenase complexes (ODHc consist of multiple copies of three enzyme components: E1, a 2-oxoacid decarboxylase; E2, dihydrolipoyl acyl-transferase; and E3, dihydrolipoyl dehydrogenase, that together catalyze the oxidative decarboxylation of 2-oxoacids, in the presence of thiamin diphosphate (ThDP, coenzyme A (CoA, Mg2+ and NAD+, to generate CO2, NADH and the corresponding acyl-CoA. The structural scaffold of the complex is provided by E2, with E1 and E3 bound around the periphery. The three principal members of the family are pyruvate dehydrogenase (PDHc, 2-oxoglutarate dehydrogenase (OGDHc and branched-chain 2-oxo acid dehydrogenase (BCKDHc. In this review, we report application of NMR-based approaches to both mechanistic and structural issues concerning these complexes. These studies revealed the nature and reactivity of transient intermediates on the enzymatic pathway and provided site-specific information on the architecture and binding specificity of the domain interfaces using solubilized truncated domain constructs of the multi-domain E2 component in its interactions with the E1 and E3 components. Where studied, NMR has also provided information about mobile loops and the possible relationship of mobility and catalysis.

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

    Margit Winkler

    2013-08-01

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

  9. Fast internal dynamics in alcohol dehydrogenase

    Large-scale domain motions in alcohol dehydrogenase (ADH) have been observed previously by neutron spin-echo spectroscopy (NSE). We have extended the investigation on the dynamics of ADH in solution by using high-resolution neutron time-of-flight (TOF) and neutron backscattering (BS) spectroscopy in the incoherent scattering range. The observed hydrogen dynamics were interpreted in terms of three mobility classes, which allowed a simultaneous description of the measured TOF and BS spectra. In addition to the slow global protein diffusion and domain motions observed by NSE, a fast internal process could be identified. Around one third of the protons in ADH participate in the fast localized diffusive motion. The diffusion coefficient of the fast internal motions is around two third of the value of the surrounding D2O solvent. It is tempting to associate the fast internal process with solvent exposed amino acid residues with dangling side chains

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

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

    2013-01-01

    Enzymes of the non-conventional yeast Yarrowia lipolytica seem to be tailor-made for the conversion of lipophilic substrates. Herein, we cloned and overexpressed the Zn-dependent alcohol dehydrogenase ADH2 from Yarrowia lipolytica in Escherichia coli. The purified enzyme was characterized in vitro. The substrate scope for YlADH2 mediated oxidation and reduction was investigated spectrophotometrically and the enzyme showed a broader substrate range than its homolog from Saccharomyces cerevisia...

  11. Malate dehydrogenases from actinomycetes: structural comparison of Thermoactinomyces enzyme with other actinomycete and Bacillus enzymes.

    Smith, K.; Sundaram, T K; Kernick, M

    1984-01-01

    Malate dehydrogenases from bacteria belonging to the genus Thermoactinomyces are tetrameric, like those from Bacillus spp., and exhibit a high degree of structural homology to Bacillus malate dehydrogenase as judged by immunological cross-reactivity. Malate dehydrogenases from other actinomycetes are dimers and do not cross-react with antibodies to Bacillus malate dehydrogenase.

  12. Immunochemical properties of NAD+-linked glycerol dehydrogenases from Escherichia coli and Klebsiella pneumoniae.

    Tang, J. C.; Forage, R G; Lin, E C

    1982-01-01

    An NAD+-linked glycerol dehydrogenase hyperproduced by a mutant of Escherichia coli K-12 was found to be immunochemically homologous to a minor glycerol dehydrogenase of unknown physiological function in Klebsiella pneumoniae 1033, but not to the glycerol dehydrogenase of the dha system responsible for anaerobic dissimilation of glycerol or to the 2,3-butanediol dehydrogenase of K. pneumoniae.

  13. Crystal structure of the NADP+-dependent aldehyde dehydrogenase from Vibrio harveyi: structural implications for cofactor specificity and affinity.

    Ahvazi, B; Coulombe, R; Delarge, M; Vedadi, M; Zhang, L; Meighen, E; Vrielink, A

    2000-01-01

    Aldehyde dehydrogenase from the bioluminescent bacterium, Vibrio harveyi, catalyses the oxidation of long-chain aliphatic aldehydes to acids. The enzyme is unique compared with other forms of aldehyde dehydrogenase in that it exhibits a very high specificity and affinity for the cofactor NADP(+). Structural studies of this enzyme and comparisons with other forms of aldehyde dehydrogenase provide the basis for understanding the molecular features that dictate these unique properties and will enhance our understanding of the mechanism of catalysis for this class of enzyme. The X-ray structure of aldehyde dehydrogenase from V. harveyi has been solved to 2.5-A resolution as a partial complex with the cofactor NADP(+) and to 2. 1-A resolution as a fully bound 'holo' complex. The cofactor preference exhibited by different forms of the enzyme is predominantly determined by the electrostatic environment surrounding the 2'-hydroxy or the 2'-phosphate groups of the adenosine ribose moiety of NAD(+) or NADP(+), respectively. In the NADP(+)-dependent structures the presence of a threonine and a lysine contribute to the cofactor specificity. In the V. harveyi enzyme an arginine residue (Arg-210) contributes to the high cofactor affinity through a pi stacking interaction with the adenine ring system of the cofactor. Further differences between the V. harveyi enzyme and other aldehyde dehydrogenases are seen in the active site, in particular a histidine residue which is structurally conserved with phosphorylating glyceraldehyde-3-phosphate dehydrogenase. This may suggest an alternative mechanism for activation of the reactive cysteine residue for nucleophilic attack. PMID:10903148

  14. Cloning and Polymorphisms of Yak Lactate Dehydrogenase b Gene

    Yaou Xu

    2013-06-01

    Full Text Available The main objective of this work was to study the unique polymorphisms of the lactate dehydrogenase-1 (LDH1 gene in yak (Bos grunniens. Native polyacrylamide gel electrophoresis revealed three phenotypes of LDH1 (a tetramer of H subunit in yak heart and longissimus muscle extracts. The corresponding gene, ldhb, encoding H subunits of three LDH1 phenotypes was obtained by RT-PCR. A total of six nucleotide differences were detected in yak ldhb compared with that of cattle, of which five mutations cause amino acid substitutions. Sequence analysis shows that the G896A and C689A, mutations of ldhb gene, result in alterations of differently charged amino acids, and create the three phenotypes (F, M, and S of yak LDH1. Molecular modeling of the H subunit of LDH indicates that the substituted amino acids are not located within NAD+ or substrate binding sites. PCR-RFLP examination of G896A mutation demonstrated that most LDH1-F samples are actually heterozygote at this site. These results help to elucidate the molecular basis and genetic characteristic of the three unique LDH1 phenotypes in yak.

  15. Genetics Home Reference: 3-hydroxyacyl-CoA dehydrogenase deficiency

    ... as seizures, life-threatening heart and breathing problems, coma, and sudden death. This condition may explain some ... hydroxyacyl-CoA dehydrogenase United Mitochondrial Disease Foundation: Treatments & Therapies These resources from MedlinePlus offer information about the ...

  16. Crystallization behaviour of glyceraldehyde dehydrogenase from Thermoplasma acidophilum

    Lermark, L.; Degtjarik, Oksana; Steffler, F.; Sieber, V.; Kutá-Smatanová, Ivana

    2015-01-01

    Roč. 71, č. 12 (2015), s. 1475-1480. ISSN 2053-230X Institutional support: RVO:67179843 Keywords : TaAlDH * Thermoplasma acidophilum * bioproduction * cell-free enzyme cascade * glyceraldehyde dehydrogenase Subject RIV: CE - Biochemistry

  17. Glucose 6 phosphate dehydrogenase deficiency in adults

    Objective: To determine the frequency of glucose-6-phosphate dehydrogenase (G6PD) deficiency in adults presented with anemia. Subjects and Methods: Eighteen months admission data was reviewed for G6PD deficiency as a cause of anemia. Anemia was defined by world health organization (WHO) criteria as haemoglobin less than 11.3 gm%. G6PD activity was measured by Sigma dye decolorisation method. All patients were screened for complications of hemolysis and its possible cause. Patients with more than 13 years of age were included in the study. Results: Out of 3600 patients admitted, 1440 were found anaemic and 49 as G6PD deficient. So the frequency of G6PD deficiency in anaemic patients was 3.4% and the overall frequency is 1.36%. G6PD deficiency among males and females was three and six percent respectively. Antimalarials and antibiotics containing sulphonamide group were the most common precipitating factors for hemolysis. Anemia and jaundice were the most common presentations while malaria was the most common associated disease. Acute renal failure was the most severe complication occurring in five patients with two deaths. Conclusion: G6PD deficiency is a fairly common cause of anemia with medicine as common precipitating factor for hemolysis. Such complications can be avoided with early recognition of the disease and avoiding indiscriminate use of medicine. (author)

  18. Inhibitors of 17beta-hydroxysteroid dehydrogenase type 1.

    Brozic, P; Lanisnik Risner, T; Gobec, S

    2008-01-01

    Carcinogenesis of hormone-related cancers involves hormone-stimulated cell proliferation, which increases the number of cell divisions and the opportunity for random genetic errors. In target tissues, steroid hormones are interconverted between their potent, high affinity forms for their respective receptors and their inactive, low affinity forms. One group of enzymes responsible for these interconversions are the hydroxysteroid dehydrogenases, which regulate ligand access to steroid receptors and thus act at a pre-receptor level. As part of this group, the 17beta-hydroxysteroid dehydrogenases catalyze either oxidation of hydroxyl groups or reduction of keto groups at steroid position C17. The thoroughly characterized 17beta-hydroxysteroid dehydrogenase type 1 activates the less active estrone to estradiol, a potent ligand for estrogen receptors. This isoform is expressed in gonads, where it affects circulating levels of estradiol, and in peripheral tissue, where it regulates ligand occupancy of estrogen receptors. Inhibitors of 17beta-hydroxysteroid dehydrogenase type 1 are thus highly interesting potential therapeutic agents for the control of estrogen-dependent diseases such as endometriosis, as well as breast and ovarian cancers. Here, we present the review on the recent development of inhibitors of 17beta-hydroxysteroid dehydrogenase type 1 published and patented since the previous review of 17beta-hydroxysteroid dehydrogenase inhibitors of Poirier (Curr. Med. Chem., 2003, 10, 453). These inhibitors are divided into two separate groups according to their chemical structures: steroidal and non-steroidal 17beta-hydroxysteroid dehydrogenase type 1 inhibitors. Their estrogenic/ proliferative activities and selectivities over other 17beta-hydroxysteroid dehydrogenases that are involved in local regulation of estrogen action (types 2, 7 and 12) are also presented. PMID:18220769

  19. Aromatic amine dehydrogenase, a second tryptophan tryptophylquinone enzyme.

    Govindaraj, S; Eisenstein, E.; Jones, L. H.; Sanders-Loehr, J; Chistoserdov, A Y; Davidson, V L; Edwards, S. L.

    1994-01-01

    Aromatic amine dehydrogenase (AADH) catalyzes the oxidative deamination of aromatic amines including tyramine and dopamine. AADH is structurally similar to methylamine dehydrogenase (MADH) and possesses the same tryptophan tryptophylquinone (TTQ) prosthetic group. AADH exhibits an alpha 2 beta 2 structure with subunit molecular weights of 39,000 and 18,000 and with a quinone covalently attached to each beta subunit. Neither subunit cross-reacted immunologically with antibodies to the correspo...

  20. Soluble aldehyde dehydrogenase and metabolism of aldehydes by soybean bacteroids.

    Peterson, J. B.; LaRue, T A

    1982-01-01

    A soluble aldehyde dehydrogenase (EC 1.2.1.3) was partially purified from Rhizobium japonicum bacteroids and from free-living R. japonicum 61A76. The enzyme was activated by NAD+, NADH, and dithiothreitol, and it reduced NAD(P)+. Acetaldehyde, propionaldehyde, butyraldehyde, benzaldehyde, and succinic semialdehyde were substrates. The Km for straight-chain aldehydes decreased with increasing carbon chain length. The aldehyde dehydrogenase was inhibited by 6-cyanopurine, but not by metronidazo...

  1. Malate dehydrogenase activity in human seminal plasma and spermatozoa homogenates

    Hulya Leventerler

    2013-08-01

    Full Text Available Purpose: Malate Dehydrogenase is an important enzyme of the Krebs cycle, most cells require this enzyme for their metabolic activity. We evaluated the Malate Dehydrogenase (NAD/NADP activity in human seminal plasma and sperm homogenates in normozoospermic, fertile and infertile males. Also glucose and fructose concentrations were determined in the seminal plasma samples. Material and Methods: Malate Dehydrogenase (NAD/NADP activity in human seminal plasma and sperm homogenates of normozoospermic and infertile males was determined by spectrophotometric method. Semen analysis was considered according to the WHO Criteria. Results: Malat Dehydrogenase-NAD value in seminal plasma (the mean ± SD, mU/ml of asthenoteratospermic (40.0±25.7 and azospermic (38.0±43.6 groups were significantly lower than normozoospermic, (93.9±52.1 males. Malat Dehydrogenase-NAD value in sperm homogenates (the mean ± SD, mU/ 20x106 sperm of teratospermic group (136.8±61.8 was significantly higher compared to the normozoospermic (87.3±26.5 males. Glucose concentration (mg/dl in asthenoteratospermic (4.0±1.4 and azospermic (15.4±6.4 groups were significantly higher than fertile (2.0±2.1 males. Also fructose concentration (mg/dl in asthenoteratospermic (706.6±143.3 and azospermic (338.1±228.2 groups were significantly high compared to the normozoospermic (184.7±124.8 group. Conclusion: Sperm may be some part of the source of Malat Dehydrogenase activity in semen. Malat Dehydrogenase activity in seminal plasma has an important role on energy metabolism of sperm. Intermediate substrates of Krebs cycle might have been produced under the control of Malat Dehydrogenase and these substrates may be important for sperm motility and male infertility. [Cukurova Med J 2013; 38(4.000: 648-658

  2. In vitro inhibition of 10-formyltetrahydrofolate dehydrogenase activity by acetaldehyde

    Mun, Ju-Ae; Doh, Eunjin; Min, Hyesun

    2008-01-01

    Alcoholism has been associated with folate deficiency in humans and laboratory animals. Previous study showed that ethanol feeding reduces the dehydrogenase and hydrolase activity of 10-formyltetrahydrofolate dehydrogenase (FDH) in rat liver. Hepatic ethanol metabolism generates acetaldehyde and acetate. The mechanisms by which ethanol and its metabolites produce toxicity within the liver cells are unknown. We purified FDH from rat liver and investigated the effect of ethanol, acetaldehyde an...

  3. Structural basis for cellobiose dehydrogenase action during oxidative cellulose degradation

    Tan, Tien-Chye; Kracher, Daniel; Gandini, Rosaria; Sygmund, Christoph; Kittl, Roman; Haltrich, Dietmar; Hallberg, B Martin; Ludwig, Roland; Divne, Christina

    2015-01-01

    A new paradigm for cellulose depolymerization by fungi focuses on an oxidative mechanism involving cellobiose dehydrogenases (CDH) and copper-dependent lytic polysaccharide monooxygenases (LPMO); however, mechanistic studies have been hampered by the lack of structural information regarding CDH. CDH contains a haem-binding cytochrome (CYT) connected via a flexible linker to a flavin-dependent dehydrogenase (DH). Electrons are generated from cellobiose oxidation catalysed by DH and shuttled vi...

  4. Molecular determinants of the cofactor specificity of ribitol dehydrogenase, a short-chain dehydrogenase/reductase

    Moon, Hee-Jung; Tiwari, Manish Kumar; Singh, Ranjitha;

    2012-01-01

    Ribitol dehydrogenase from Zymomonas mobilis (ZmRDH) catalyzes the conversion of ribitol to d-ribulose and concomitantly reduces NAD(P)(+) to NAD(P)H. A systematic approach involving an initial sequence alignment-based residue screening, followed by a homology model-based screening and site-direc...... insights into the function of the Ser156 residue were obtained by substituting it with other hydrophobic nonpolar or polar amino acids. Substituting Ser156 with the negatively charged amino acids (Asp and Glu) altered the cofactor specificity of ZmRDH toward NAD(+) (S156D, [k...

  5. Structural studies on dihydrolipoyl transacetylase : the core component of the pyruvate dehydrogenase complex of Azotobacter vinelandii.

    Hanemaaijer, R.

    1988-01-01

    The studies described in this thesis deal with the structure of the Azotobactervinelandii dihydrolipoyl transacetylase, the core component (E 2 ) of the pyruvate dehydrogenase complex. in all organisms the pyruvate dehydrogenase complex is closely related to the 2-oxoglutarate dehydrogenase complex and, if present, the branched-chain 2-oxoacid dehydrogenase complex. These enzyme complexes are large multimeric structures. The smallest known is the pyruvate dehydrogenase complex from A.vineland...

  6. Functional analysis of a cinnamyl alcohol dehydrogenase involved in lignin biosynthesis in wheat

    Ma, Qing-Hu

    2010-01-01

    Cinnamyl alcohol dehydrogenase (CAD) catalyses the final step in the biosynthesis of monolignols. In the present study, a cDNA encoding a CAD was isolated from wheat, designated as TaCAD1. A genome-wide data mining in the wheat EST database revealed another 10 CAD-like homologues, namely TaCAD2 to TaCAD11. A phylogenetic analysis showed that TaCAD1 belonged to the bona fide CAD group involved in lignin synthesis. Two other putative CADs from the wheat genome (TaCAD2 and TaCAD4) also belonged ...

  7. Assessment of toxicity using dehydrogenases activity and mathematical modeling.

    Matyja, Konrad; Małachowska-Jutsz, Anna; Mazur, Anna K; Grabas, Kazimierz

    2016-07-01

    Dehydrogenase activity is frequently used to assess the general condition of microorganisms in soil and activated sludge. Many studies have investigated the inhibition of dehydrogenase activity by various compounds, including heavy metal ions. However, the time after which the measurements are carried out is often chosen arbitrarily. Thus, it can be difficult to estimate how the toxic effects of compounds vary during the reaction and when the maximum of the effect would be reached. Hence, the aim of this study was to create simple and useful mathematical model describing changes in dehydrogenase activity during exposure to substances that inactivate enzymes. Our model is based on the Lagergrens pseudo-first-order equation, the rate of chemical reactions, enzyme activity, and inactivation and was created to describe short-term changes in dehydrogenase activity. The main assumption of our model is that toxic substances cause irreversible inactivation of enzyme units. The model is able to predict the maximum direct toxic effect (MDTE) and the time to reach this maximum (TMDTE). In order to validate our model, we present two examples: inactivation of dehydrogenase in microorganisms in soil and activated sludge. The model was applied successfully for cadmium and copper ions. Our results indicate that the predicted MDTE and TMDTE are more appropriate than EC50 and IC50 for toxicity assessments, except for long exposure times. PMID:27021434

  8. Characterization and purification of carbon monoxide dehydrogenase from Methanosarcina barkeri

    Carbon monoxide-dependent production of H2, CO2, and CH4 was detected in crude cell extracts of acetate-grown Methanosarcina barkeri. This metabolic transformation was associated with an active methyl viologen-linked CO dehydrogenase activity (5 to 10 U/mg of protein). Carbon monoxide dehydrogenase activity was inhibited 85% by 10 μM KCN and was rapidly inactivated by O2. The enzyme was nearly homogenous after 20-fold purification, indicating that a significant proportion of soluble cell protein was CO dehydrogenase (ca. 5%). The native purified enzyme displayed a molecular weight of 232,000 and a two-subunit composition of 92,000 and 18,000 daltons. The enzyme was shown to contain nickel by isolation of radioactive CO dehydrogenase from cells grown in 63Ni. Analysis of enzyme kinetic properties revealed an apparent K/sub m/ of 5 mM for CO and a V/sub max/ of 1300 U/mg of protein. The spectral properties of the enzyme were similar to those published for CO dehydrogenase from acetogenic anaerobes. The physiological functions of the enzyme are discussed

  9. Targeting aldehyde dehydrogenase: a potential approach for cell labeling

    Vaidyanathan, Ganesan [Department of Radiology, Duke University Medical Center, Box 3808, Durham, NC 27710 (United States)], E-mail: ganesan.v@duke.edu; Song, Haijing; Affleck, Donna; McDougald, Darryl L. [Department of Radiology, Duke University Medical Center, Box 3808, Durham, NC 27710 (United States); Storms, Robert W. [Division of Cellular Therapy, Department of Medicine, Duke University Medical Center, Durham, NC 27710 (United States); Zalutsky, Michael R.; Chin, Bennett B. [Department of Radiology, Duke University Medical Center, Box 3808, Durham, NC 27710 (United States)

    2009-11-15

    Introduction: To advance the science and clinical application of stem cell therapy, the availability of a highly sensitive, quantitative and translational method for tracking stem cells would be invaluable. Because hematopoetic stem cells express high levels of the cytosolic enzyme aldehyde dehydrogenase-1A1 (ALDH1), we sought to develop an agent that is specific to ALDH1 and thus to cells expressing the enzyme. Such an agent might be also helpful in identifying tumors that are resistant to cyclophosphomide chemotherapy because ALDH1 is known to be responsible for this resistance. Methods: We developed schemes for the synthesis of two radioiodinated aldehdyes - N-formylmethyl-5-[*I]iodopyridine-3-carboxamide ([*I]FMIC) and 4-diethylamino-3-[*I]iodobenzaldehyde ([*I]DEIBA)-at no-carrier-added levels from their respective tin precursors. These agents were evaluated using pure ALDH1 and tumor cells that expressed the enzyme. Results: The average radiochemical yields for the synthesis of [{sup 125}I]FMIC and [{sup 125}I]DEIBA were 70{+-}5% and 47{+-}14%, respectively. ALDH1 converted both compounds to respective acids suggesting their suitability as ALDH1 imaging agents. Although ability of ALDH1 within the cells to oxidize one of these substrates was shown, specific uptake in ALDH-expressing tumor cells could not be demonstrated. Conclusion: To pursue this approach for ALDH1 imaging, radiolabeled aldehydes need to be designed such that, in addition to being good substrates for ALDH1, the cognate products should be sufficiently polar so as to be retained within the cells.

  10. Targeting aldehyde dehydrogenase: a potential approach for cell labeling

    Introduction: To advance the science and clinical application of stem cell therapy, the availability of a highly sensitive, quantitative and translational method for tracking stem cells would be invaluable. Because hematopoetic stem cells express high levels of the cytosolic enzyme aldehyde dehydrogenase-1A1 (ALDH1), we sought to develop an agent that is specific to ALDH1 and thus to cells expressing the enzyme. Such an agent might be also helpful in identifying tumors that are resistant to cyclophosphomide chemotherapy because ALDH1 is known to be responsible for this resistance. Methods: We developed schemes for the synthesis of two radioiodinated aldehdyes - N-formylmethyl-5-[*I]iodopyridine-3-carboxamide ([*I]FMIC) and 4-diethylamino-3-[*I]iodobenzaldehyde ([*I]DEIBA)-at no-carrier-added levels from their respective tin precursors. These agents were evaluated using pure ALDH1 and tumor cells that expressed the enzyme. Results: The average radiochemical yields for the synthesis of [125I]FMIC and [125I]DEIBA were 70±5% and 47±14%, respectively. ALDH1 converted both compounds to respective acids suggesting their suitability as ALDH1 imaging agents. Although ability of ALDH1 within the cells to oxidize one of these substrates was shown, specific uptake in ALDH-expressing tumor cells could not be demonstrated. Conclusion: To pursue this approach for ALDH1 imaging, radiolabeled aldehydes need to be designed such that, in addition to being good substrates for ALDH1, the cognate products should be sufficiently polar so as to be retained within the cells.

  11. Testis-specific glyceraldehyde-3-phosphate dehydrogenase: origin and evolution

    Frishman Dmitrij

    2011-06-01

    Full Text Available Abstract Background Glyceraldehyde-3-phosphate dehydrogenase (GAPD catalyses one of the glycolytic reactions and is also involved in a number of non-glycolytic processes, such as endocytosis, DNA excision repair, and induction of apoptosis. Mammals are known to possess two homologous GAPD isoenzymes: GAPD-1, a well-studied protein found in all somatic cells, and GAPD-2, which is expressed solely in testis. GAPD-2 supplies energy required for the movement of spermatozoa and is tightly bound to the sperm tail cytoskeleton by the additional N-terminal proline-rich domain absent in GAPD-1. In this study we investigate the evolutionary history of GAPD and gain some insights into specialization of GAPD-2 as a testis-specific protein. Results A dataset of GAPD sequences was assembled from public databases and used for phylogeny reconstruction by means of the Bayesian method. Since resolution in some clades of the obtained tree was too low, syntenic analysis was carried out to define the evolutionary history of GAPD more precisely. The performed selection tests showed that selective pressure varies across lineages and isoenzymes, as well as across different regions of the same sequences. Conclusions The obtained results suggest that GAPD-1 and GAPD-2 emerged after duplication during the early evolution of chordates. GAPD-2 was subsequently lost by most lineages except lizards, mammals, as well as cartilaginous and bony fishes. In reptilians and mammals, GAPD-2 specialized to a testis-specific protein and acquired the novel N-terminal proline-rich domain anchoring the protein in the sperm tail cytoskeleton. This domain is likely to have originated by exonization of a microsatellite genomic region. Recognition of the proline-rich domain by cytoskeletal proteins seems to be unspecific. Besides testis, GAPD-2 of lizards was also found in some regenerating tissues, but it lacks the proline-rich domain due to tissue-specific alternative splicing.

  12. Aminotransferase and glutamate dehydrogenase activities in lactobacilli and streptococci.

    Peralta, Guillermo Hugo; Bergamini, Carina Viviana; Hynes, Erica Rut

    2016-01-01

    Aminotransferases and glutamate dehydrogenase are two main types of enzymes involved in the initial steps of amino acid catabolism, which plays a key role in the cheese flavor development. In the present work, glutamate dehydrogenase and aminotransferase activities were screened in twenty one strains of lactic acid bacteria of dairy interest, either cheese-isolated or commercial starters, including fifteen mesophilic lactobacilli, four thermophilic lactobacilli, and two streptococci. The strains of Streptococcus thermophilus showed the highest glutamate dehydrogenase activity, which was significantly elevated compared with the lactobacilli. Aspartate aminotransferase prevailed in most strains tested, while the levels and specificity of other aminotransferases were highly strain- and species-dependent. The knowledge of enzymatic profiles of these starter and cheese-isolated cultures is helpful in proposing appropriate combinations of strains for improved or increased cheese flavor. PMID:27266631

  13. Crystal structure of homoisocitrate dehydrogenase from Schizosaccharomyces pombe

    Bulfer, Stacie L.; Hendershot, Jenna M.; Trievel, Raymond C. (Michigan); (UCSF)

    2013-09-18

    Lysine biosynthesis in fungi, euglena, and certain archaebacteria occurs through the {alpha}-aminoadipate pathway. Enzymes in the first steps of this pathway have been proposed as potential targets for the development of antifungal therapies, as they are absent in animals but are conserved in several pathogenic fungi species, including Candida, Cryptococcus, and Aspergillus. One potential antifungal target in the {alpha}-aminoadipate pathway is the third enzyme in the pathway, homoisocitrate dehydrogenase (HICDH), which catalyzes the divalent metal-dependent conversion of homoisocitrate to 2-oxoadipate (2-OA) using nicotinamide adenine dinucleotide (NAD{sup +}) as a cofactor. HICDH belogns to a family of {beta}-hydroxyacid oxidative decarboxylases that includes malate dehydrogenase, tartrate dehydrogenase, 6-phosphogluconate dehydrogenase, isocitrate dehydrogenase (ICDH), and 3-isopropylmalte dehydrogenase (IPMDH). ICDH and IPMDH are well-characterized enzymes that catalyze the decarboxylation of isocitrate to yield 2-oxoglutarate (2-OG) in the citric acid cycle and the conversion of 3-isopropylmalate to 2-oxoisovalerate in the leucine biosynthetic pathway, respectively. Recent structural and biochemical studies of HICDH reveal that this enzyme shares sequence, structural, and mechanistic homology with ICDH and IPMDH. To date, the only published structures of HICDH are from the archaebacteria Thermus thermophilus (TtHICDH). Fungal HICDHs diverge from TtHICDH in several aspects, including their thermal stability, oligomerization state, and substrate specificity, thus warranting further characterization. To gain insights into these differences, they determined crystal structures of a fungal Schizosaccharomyces pombe HICDH (SpHICDH) as an apoenzyme and as a binary complex with additive tripeptide glycyl-glycyl-glycine (GGG) to 1.55 {angstrom} and 1.85 {angstrom} resolution, respectively. Finally, a comparison of the SpHICDH and TtHICDH structures reveal differences in

  14. Properties of Lactate Dehydrogenase in a Psychrophilic Marine Bacterium

    Mitchell, P; Yen, H. C.; Mathemeier, P. F.

    1985-01-01

    Lactate dehydrogenase (EC 1.1.1.27) from Vibrio marinus MP-1 was purified 15-fold and ammonium activated. The optimum pH for pyruvate reduction was 7.4. Maximum lactate dehydrogenase activity occurred at 10 to 15 degrees C, and none occurred at 40 degrees C. The crude-extract enzyme was stable between 15 and 20 degrees C and lost 50% of its activity after 60 min at 45 degrees C. The partially purified enzyme was stable between 8 and 15 degrees C and lost 50% of its activity after 60 min at 30...

  15. Reversible inactivation of CO dehydrogenase with thiol compounds

    Highlights: • Rather large thiols (e.g. coenzyme A) can reach the active site of CO dehydrogenase. • CO- and H2-oxidizing activity of CO dehydrogenase is inhibited by thiols. • Inhibition by thiols was reversed by CO or upon lowering the thiol concentration. • Thiols coordinate the Cu ion in the [CuSMo(=O)OH] active site as a third ligand. - Abstract: Carbon monoxide dehydrogenase (CO dehydrogenase) from Oligotropha carboxidovorans is a structurally characterized member of the molybdenum hydroxylase enzyme family. It catalyzes the oxidation of CO (CO + H2O → CO2 + 2e− + 2H+) which proceeds at a unique [CuSMo(=O)OH] metal cluster. Because of changing activities of CO dehydrogenase, particularly in subcellular fractions, we speculated whether the enzyme would be subject to regulation by thiols (RSH). Here we establish inhibition of CO dehydrogenase by thiols and report the corresponding Ki-values (mM): L-cysteine (5.2), D-cysteine (9.7), N-acetyl-L-cysteine (8.2), D,L-homocysteine (25.8), L-cysteine–glycine (2.0), dithiothreitol (4.1), coenzyme A (8.3), and 2-mercaptoethanol (9.3). Inhibition of the enzyme was reversed by CO or upon lowering the thiol concentration. Electron paramagnetic resonance spectroscopy (EPR) and X-ray absorption spectroscopy (XAS) of thiol-inhibited CO dehydrogenase revealed a bimetallic site in which the RSH coordinates to the Cu-ion as a third ligand ([MoVI(=O)OH(2)SCuI(SR)S-Cys]) leaving the redox state of the Cu(I) and the Mo(VI) unchanged. Collectively, our findings establish a regulation of CO dehydrogenase activity by thiols in vitro. They also corroborate the hypothesis that CO interacts with the Cu-ion first. The result that thiol compounds much larger than CO can freely travel through the substrate channel leading to the bimetallic cluster challenges previous concepts involving chaperone function and is of importance for an understanding how the sulfuration step in the assembly of the bimetallic cluster might proceed

  16. Reversible inactivation of CO dehydrogenase with thiol compounds

    Kreß, Oliver [Department of Microbiology, University of Bayreuth, 95440 Bayreuth (Germany); Gnida, Manuel [Department of Chemistry, University of Paderborn, 33098 Paderborn (Germany); Pelzmann, Astrid M. [Department of Microbiology, University of Bayreuth, 95440 Bayreuth (Germany); Marx, Christian [Institute of Biochemistry and Biophysics, Friedrich-Schiller-University of Jena, 07745 Jena (Germany); Meyer-Klaucke, Wolfram [Department of Chemistry, University of Paderborn, 33098 Paderborn (Germany); Meyer, Ortwin, E-mail: Ortwin.Meyer@uni-bayreuth.de [Department of Microbiology, University of Bayreuth, 95440 Bayreuth (Germany)

    2014-05-09

    Highlights: • Rather large thiols (e.g. coenzyme A) can reach the active site of CO dehydrogenase. • CO- and H{sub 2}-oxidizing activity of CO dehydrogenase is inhibited by thiols. • Inhibition by thiols was reversed by CO or upon lowering the thiol concentration. • Thiols coordinate the Cu ion in the [CuSMo(=O)OH] active site as a third ligand. - Abstract: Carbon monoxide dehydrogenase (CO dehydrogenase) from Oligotropha carboxidovorans is a structurally characterized member of the molybdenum hydroxylase enzyme family. It catalyzes the oxidation of CO (CO + H{sub 2}O → CO{sub 2} + 2e{sup −} + 2H{sup +}) which proceeds at a unique [CuSMo(=O)OH] metal cluster. Because of changing activities of CO dehydrogenase, particularly in subcellular fractions, we speculated whether the enzyme would be subject to regulation by thiols (RSH). Here we establish inhibition of CO dehydrogenase by thiols and report the corresponding K{sub i}-values (mM): L-cysteine (5.2), D-cysteine (9.7), N-acetyl-L-cysteine (8.2), D,L-homocysteine (25.8), L-cysteine–glycine (2.0), dithiothreitol (4.1), coenzyme A (8.3), and 2-mercaptoethanol (9.3). Inhibition of the enzyme was reversed by CO or upon lowering the thiol concentration. Electron paramagnetic resonance spectroscopy (EPR) and X-ray absorption spectroscopy (XAS) of thiol-inhibited CO dehydrogenase revealed a bimetallic site in which the RSH coordinates to the Cu-ion as a third ligand ([Mo{sup VI}(=O)OH{sub (2)}SCu{sup I}(SR)S-Cys]) leaving the redox state of the Cu(I) and the Mo(VI) unchanged. Collectively, our findings establish a regulation of CO dehydrogenase activity by thiols in vitro. They also corroborate the hypothesis that CO interacts with the Cu-ion first. The result that thiol compounds much larger than CO can freely travel through the substrate channel leading to the bimetallic cluster challenges previous concepts involving chaperone function and is of importance for an understanding how the sulfuration step in

  17. Prostaglandin dehydrogenase and the initiation of labor.

    Challis, J R; Patel, F A; Pomini, F

    1999-01-01

    In summary, these studies have suggested that prostaglandin dehydrogenase may have a central role to play in the mechanisms which determine biologically active prostaglandin concentrations within human fetal membranes and placenta at the time of labor, at term or preterm. Moreover, our studies indicate that the regulation of PGDH may by multifactorial (figure 3). In certain regions of the membranes, we suggest that PGDH expression may be influenced by levels of anti-inflammatory and pro-inflammatory cytokines. In other regions of the membranes, we suggest that PGDH may be regulated at a transcriptional level by competing activities of progesterone and cortisol. The action of progesterone could be effected through systemically-derived steroid, or by locally synthesized steroid, acting in a paracrine and/or autocrine fashion. The effects of cortisol in placenta must be due to glucocorticoid derived from the maternal or fetal compartment, since the placenta lacks the hydroxylases required for endogenous cortisol production. However, metabolism of cortisol by 11 beta-HSD-2 reduces the potency of this glucocorticoid in placental tissue. In chorion however, cortisol may be formed locally, from cortisone, in addition to its being derived from the maternal circulation and/or from the amniotic fluid. Our current studies do not allow us to delineate whether the effects of progesterone and cortisol on PGDH are exerted through the glucocorticoid receptor (GR) or progesterone receptor (PR) or both. It is possible that through pregnancy, PGDH activity is maintained by progesterone acting either through low levels of PR in membranes, or, more likely, acting through GR. At term, elevated levels of cortisol compete with and displace progesterone from GR, resulting in inhibition of PGDH transcription and activity. In this way, local withdrawal of progesterone action would be effected within human intrauterine tissues, without requiring changes in systemic, circulating progesterone

  18. Preparation of 15N-labeled L-alanine by coupling the alanine dehydrogenase and alcohol dehydrogenase reactions

    A simple enzymatic procedure for the preparation of L-[15N]alanine, one of the metabolically most active amino acids in all types of cells, is reported. The procedure is based on the coupling of two reactions, one catalyzed by bacterial alanine dehydrogenase, the second catalyzed by yeast alcohol dehydrogenase. An impediment in the use of this procedure could be the high cost of commercial AlaDH. However, the enzyme is widespread in the Bacillus species and partially purified samples, adequate preparative purposes, could be obtained relatively easily by chromatography on blue-Sepharose. (Auth.)

  19. Enhanced clearance of lactic dehydrogenase-5 in severe combined immunodeficiency (SCID) mice: effect of lactic dehydrogenase virus on enzyme clearance.

    Hayashi, T.; Ozaki, M.; Mori, I; Saito, M; Itoh, T.; Yamamoto, H.

    1992-01-01

    The lactic dehydrogenase (LDH) level in plasma and the clearance of LDH in C.B-17 scid (severe combined immunodeficiency; SCID) mice were compared with those in C.B-17 or BALB/cCrSlc mice with or without lactic dehydrogenase virus (LDV) infection. The resting enzyme level in SCID mice showed little difference from that in C.B-17 or BALB/cCrSlc mice. The degree of increased plasma LDH level in SCID mice was lower than that in C.B-17 and BALB/cCrSlc mice after LDV infection. To assess the mecha...

  20. Polymorphisms of alcohol dehydrogenase 2 and aldehyde dehydrogenase 2 and colorectal cancer risk in Chinese males

    Chang-Ming Gao; Keitaro Matsuo; Nobuyuki Hamajima; Kazuo Tajima; Toshiro Takezaki; Jian-Zhong Wu; Xiao-Mei Zhang; Hai-Xia Cao; Jian-Hua Ding; Yan-Ting Liu; Su-Ping Li; Jia Cao

    2008-01-01

    AIM: To evaluate the relationship between drinking and polymorphisms of alcohol dehydrogenase 2 (ADH2) and/or aldehyde dehydrogenase 2 (ALDH2) for risk of colorectal cancer (CRC) in Chinese males.METHODS: A case-control study was conducted in 190 cases and 223 population-based controls.ADH2 Arg47His (G-A) and ALDH2 Glu487Lys (G-A) genotypes were identified by PCR and denaturing high-performance liquid chromatography (DHPLC).Information on smoking and drinking was collected and odds ratio (OR) was estimated.RESULTS: The ADH2 A/A and ALDH2 G/G genotypes showed moderately increased CRC risk. The age- and smoking-adjusted OR for ADH2 A/A relative to G/A and G/G was 1.60 (95% CI=1.08-2.36), and the adjusted OR for ALDH2 G/G relative to G/A and A/A was 1.79 (95% CI=1.19-2.69). Significant interactions between ADH2,ALDH2 and drinking were observed. As compared to the subjects with ADH2 G and ALDH2 A alleles, those with ADH2 A/A and ALDH2 G/G genotypes had a significantly increased OR (3.05, 95% CI= 1.67-5.57). The OR for CRC among drinkers with the ,4DH2 A/A genotype was increased to 3.44 (95% CI= 1.84-6.42) compared with non-drinkers with the ADH2 G allele. The OR for CRC among drinkers with theALDH2 G/G genotype was also increased to 2.70 (95% CI= 1.57-4.66) compared with non-drinkers with the ALDH2 A allele.CONCLUSION: Polymorphisms of the ADH2 and ALDH2 genes are significantly associated with CRC risk. There are also significant gene-gene and geneenvironment interactions between drinking and ADH2 and ALDH2 polymorphisms regarding CRC risk in Chinese males.

  1. The Crystal Structure of Aquifex aeolicus Prephenate Dehydrogenase Reveals the Mode of Tyrosine Inhibition*

    Sun, Warren; Shahinas, Dea; Bonvin, Julie; Hou, Wenjuan; Kimber, Matthew S.; Turnbull, Joanne; Christendat, Dinesh

    2009-01-01

    TyrA proteins belong to a family of dehydrogenases that are dedicated to l-tyrosine biosynthesis. The three TyrA subclasses are distinguished by their substrate specificities, namely the prephenate dehydrogenases, the arogenate dehydrogenases, and the cyclohexadienyl dehydrogenases, which utilize prephenate, l-arogenate, or both substrates, respectively. The molecular mechanism responsible for TyrA substrate selectivity and regulation is unknown. To further our underst...

  2. Alcohol and Aldehyde Dehydrogenases: Retinoid Metabolic Effects in Mouse Knockout Models

    Kumar, Sandeep; Sandell, Lisa L.; Trainor, Paul A; Koentgen, Frank; Duester, Gregg

    2011-01-01

    Retinoic acid (RA) is the active metabolite of vitamin A (retinol) that controls growth and development. The first step of RA synthesis is controlled by enzymes of the alcohol dehydrogenase (ADH) and retinol dehydrogenase (RDH) families that catalyze oxidation of retinol to retinaldehyde. The second step of RA synthesis is controlled by members of the aldehyde dehydrogenase (ALDH) family also known as retinaldehyde dehydrogenase (RALDH) that further oxidize retinaldehyde to produce RA. RA fun...

  3. Purification and characterization of xylitol dehydrogenase from Fusarium oxysporum

    Panagiotou, Gianni; Kekos, D.; Macris, B.J.;

    2002-01-01

    An NAD(+)-dependent xylitol dehydrogenase (XDH) from Fusarium oxysporum, a key enzyme in the conversion of xylose to ethanol, was purified to homogeneity and characterised. It was homodimeric with a subunit of M-r 48 000, and pI 3.6. It was optimally active at 45degreesC and pH 9-10. It was fully...

  4. 21 CFR 862.1420 - Isocitric dehydrogenase test system.

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Isocitric dehydrogenase test system. 862.1420 Section 862.1420 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry...

  5. 21 CFR 862.1440 - Lactate dehydrogenase test system.

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Lactate dehydrogenase test system. 862.1440 Section 862.1440 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry...

  6. 21 CFR 862.1380 - Hydroxybutyric dehydrogenase test system.

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Hydroxybutyric dehydrogenase test system. 862.1380 Section 862.1380 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry...

  7. Medium-chain acyl-CoA dehydrogenase deficiency

    Waddell, Leigh; Wiley, Veronica; Carpenter, Kevin; Bennetts, Bruce; Angel, Lyn; Andresen, Brage S; Wilcken, Bridget

    2006-01-01

    The fatty acid oxidation disorder most commonly identified by tandem mass spectrometry newborn screening is the potentially fatal medium-chain acyl-CoA dehydrogenase deficiency (MCAD). In clinically presenting cases, 80% are homozygous for the common mutation, c.985A > G and 18% heterozygous. We ...

  8. Cloning and expression of chicken 20-hydroxysteroid dehydrogenase

    Bryndová, Jana; Klusoňová, Petra; Kučka, Marek; Vagnerová, Karla; Mikšík, Ivan; Pácha, Jiří

    2006-01-01

    Roč. 37, č. 3 (2006), s. 453-462. ISSN 0952-5041 R&D Projects: GA AV ČR(CZ) IAA6011201 Grant ostatní: GA UK(CZ) 216/2004 Institutional research plan: CEZ:AV0Z50110509 Keywords : 20-hydroxysteroid dehydrogenase * SDR family Subject RIV: CE - Biochemistry Impact factor: 2.988, year: 2006

  9. Cloning and expression of chicken 20beta hydroxysteroid dehydrogenase

    Klusoňová, Petra; Kučka, Marek; Bryndová, Jana; Vagnerová, Karla; Mikšík, Ivan; Pácha, Jiří

    Seefeld, 2006. [International Symposium of the Journal of Steroid Biochemistry and Molecular Biology /17./. 31.05.2006-03.06.2006, Seefeld] R&D Projects: GA AV ČR(CZ) IAA6011201 Keywords : 20beta hydroxysteroid dehydrogenase * chicken Subject RIV: ED - Physiology

  10. Cloning and characterization of a ribitol dehydrogenase from Zymomonas mobilis

    Moon, Hee-Jung; Tiwari, Manish; Jeya, Marimuthu;

    2010-01-01

    Ribitol dehydrogenase (RDH) catalyzes the conversion of ribitol to D-ribulose. A novel RDH gene was cloned from Zymomonas mobilis subsp. mobilis ZM4 and overexpressed in Escherichia coli BL21(DE3). DNA sequence analysis revealed an open reading frame of 795 bp, capable of encoding a polypeptide...

  11. Molecular cloning of gluconobacter oxydans DSM 2003 xylitol dehydrogenase gene

    Sadeghi, H. Mir Mohammad; Ahmadi, R.; Aghaabdollahian, S.; Mofid, M.R.; Ghaemi, Y.; Abedi, D.

    2011-01-01

    Due to the widespread applications of xylitol dehydrogenase, an enzyme used for the production of xylitol, the present study was designed for the cloning of xylitol dehydrogenase gene from Glcunobacter oxydans DSM 2003. After extraction of genomic DNA from this bacterium, xylitol dehydrogenase gene was replicated using polymerase chain reaction (PCR). The amplified product was entered into pTZ57R cloning vector by T/A cloning method and transformation was performed by heat shocking of the E. coli XL1-blue competent cells. Following plasmid preparation, the cloned gene was digested out and ligated into the expression vector pET-22b(+). Electrophoresis of PCR product showed a 789 bp band. Recombinant plasmid (rpTZ57R) was then constructed. This plasmid was double digested with XhoI and EcoRI resulting in 800 bp and 2900 bp bands. The obtained insert was ligated into pET-22b(+) vector and its orientation was confirmed with XhoI and BamHI restriction enzymes. In conclusion, in the present study the recombinant expression vector containing xylitol dehydrogenase gene has been constructed and can be used for the production of this enzyme in high quantities. PMID:22110522

  12. Cofactor engineering of Lactobacillus brevis alcohol dehydrogenase by computational design

    Machielsen, M.P.; Looger, L.L.; Raedts, J.G.J.; Dijkhuizen, S.; Hummel, W.; Henneman, H.G.; Daussmann, T.; Oost, van der J.

    2009-01-01

    The R-specific alcohol dehydrogenase from Lactobacillus brevis (Lb-ADH) catalyzes the enantioselective reduction of prochiral ketones to the corresponding secondary alcohols. It is stable and has broad substrate specificity. These features make this enzyme an attractive candidate for biotechnologica

  13. Crystallization and preliminary X-ray analysis of aldehyde dehydrogenase from Vibrio harveyi.

    Croteau, N.; Vedadi, M.; Delarge, M.; Meighen, E.; Abu-Abed, M.; Howell, P. L.; Vrielink, A.

    1996-01-01

    Aldehyde dehydrogenase from Vibrio harveyi catalyzes the oxidation of long-chain aliphatic aldehydes to acids. The enzyme is unique among the family of aldehyde dehydrogenases in that it exhibits much higher specificity for the cofactor NADP+ than for NAD+. The sequence of this form of the enzyme varies significantly from the NAD+ dependent forms, suggesting differences in the three-dimensional structure that may be correlated to cofactor specificity. Crystals of the enzyme have been grown both in the presence and absence of NADP+ using the hanging drop vapor diffusion technique. In order to improve crystal size and quality, iterative seeding techniques were employed. The crystals belong to space group P2(1), with unit cell dimensions a = 79.4 A, b = 131.1 A, c = 92.2 A, and beta = 92.4 degrees. Freezing the crystal to 100 K has enabled a complete set of data to be collected using a rotating anode source (lambda = 1.5418 A). The crystals diffract to a minimum d-spacing of 2.6 A resolution. Based on density calculations, two homodimers of molecular weight 110 kDa are estimated to be present in the asymmetric unit. Self-rotation functions show the presence of 3 noncrystallographic twofold symmetry axes. PMID:8897616

  14. Succinate dehydrogenase subunit D and succinate dehydrogenase subunit B mutation analysis in canine phaeochromocytoma and paraganglioma.

    Holt, D E; Henthorn, P; Howell, V M; Robinson, B G; Benn, D E

    2014-07-01

    Phaeochromocytomas (PCs) are tumours of the adrenal medulla chromaffin cells. Paragangliomas (PGLs) arise in sympathetic ganglia (previously called extra-adrenal PCs) or in non-chromaffin parasympathetic ganglia cells that are usually non-secretory. Parenchymal cells from these tumours have a common embryological origin from neural crest ectoderm. Several case series of canine PCs and PGLs have been published and a link between the increased incidence of chemoreceptor neoplasia in brachycephalic dog breeds and chronic hypoxia has been postulated. A similar link to hypoxia in man led to the identification of germline heterozygous mutations in the gene encoding succinate dehydrogenase subunit D (SDHD) and subsequently SDHA, SDHB and SDHC in similar tumours. We investigated canine PCs (n = 6) and PGLs (n = 2) for SDHD and SDHB mutations and in one PGL found a somatic SDHD mutation c.365A>G (p.Lys122Arg) in exon 4, which was not present in normal tissue from this brachycephalic dog. Two PCs were heterozygous for both c.365A>G (p.Lys122Arg) mutation and an exon 3 silent variant c.291G>A. We also identified the heterozygous SDHB exon 2 mutation c.113G>A (p.Arg38Gln) in a PC. These results illustrate that genetic mutations may underlie tumourigenesis in canine PCs and PGLs. The spontaneous nature of these canine diseases and possible association of PGLs with hypoxia in brachycephalic breeds may make them an attractive model for studying the corresponding human tumours. PMID:24813157

  15. [Cooperative properties of D-glyceraldehyde-3-phosphate dehydrogenase].

    Nagradova, N K

    1977-03-01

    The structure of the active center of glyceraldehyde-3-phosphate dehydrogenase and the arrangement of subunits in the tetrameric molecule is delineated. The mechanism of cooperative effects in the oligomer is considered, and the involvement of various regions of the active center and of different-subunit contact area in the realization of the cooperative phenomena is discussed. A special attention is paid to the effect of NAD+ bound to one of the subunits of the tetramer on the structure of an adjacent subunit and to the problem of the participation of the coenzyme in the creation of anion-binding sites of the enzyme. The conditions of reversible dissociation of the tetrameric apoenzyme molecule into dimers are depicted, and the role of NAD+ in the organization of the quaternary structure of the dehydrogenase is discussed. The problem of catalytic activity of the dimeric form of the enzyme is argued. PMID:193581

  16. Molecular properties of succinate dehydrogenase isolated from Micrococcus luteus (lysodeikticus).

    Crowe, B A; Owen, P.(Imperial College London, London, United Kingdom)

    1983-01-01

    Succinate dehydrogenase (EC 1.3.99.1) of Micrococcus luteus was selectively precipitated from Triton X-100-solubilized membranes by using specific antiserum. The precipitated enzyme contained equimolar amounts of four polypeptides with apparent molecular weights of 72,000, 30,000, 17,000, and 15,000. The 72,000 polypeptide possessed a covalently bound flavin prosthetic group and appeared to be strongly antigenic as judged by immunoprinting experiments. Low-temperature absorption spectroscopy ...

  17. Visual evoked potentials in succinate semialdehyde dehydrogenase (SSADH) Deficiency

    Di Rosa, G.; Malaspina, P; P. Blasi(INAF Arcetri); Dionisi-Vici, C.; Rizzo, C; Tortorella, G; Crutchfield, S. R.; Gibson, K. M.

    2009-01-01

    In mammals, increased GABA in the central nervous system has been associated with abnormalities of visual evoked potentials (VEPs), predominantly manifested as increased latency of the major positive component P100. Accordingly, we hypothesized that patients with a defect in GABA metabolism, succinate semialdehyde dehydrogenase (SSADH) deficiency (in whom supraphysiological levels of GABA accumulate), would manifest VEP anomalies. We evaluated VEPs on two patients with confirmed SSADH deficie...

  18. Glucose-6-Phosphate Dehydrogenase Deficiency in Nigerian Children

    Olatundun Williams; Daniel Gbadero; Grace Edowhorhu; Ann Brearley; Tina Slusher; Lund, Troy C.

    2013-01-01

    Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human enzymopathy and in Sub-Saharan Africa, is a significant cause of infection- and drug-induced hemolysis and neonatal jaundice. Our goals were to determine the prevalence of G6PD deficiency among Nigerian children of different ethnic backgrounds and to identify predictors of G6PD deficiency by analyzing vital signs and hematocrit and by asking screening questions about symptoms of hemolysis. We studied 1,122 children (...

  19. Aldehyde dehydrogenase inhibition as a pathogenic mechanism in Parkinson disease

    Fitzmaurice, Arthur G.; Rhodes, Shannon L.; Lulla, Aaron; Murphy, Niall P.; Lam, Hoa A.; O’Donnell, Kelley C.; Barnhill, Lisa; Casida, John E.; Cockburn, Myles; Sagasti, Alvaro; Stahl, Mark C.; Maidment, Nigel T; Ritz, Beate; Bronstein, Jeff. M.

    2013-01-01

    Parkinson disease (PD) is a neurodegenerative disorder particularly characterized by the loss of dopaminergic neurons in the substantia nigra. Pesticide exposure has been associated with PD occurrence, and we previously reported that the fungicide benomyl interferes with several cellular processes potentially relevant to PD pathogenesis. Here we propose that benomyl, via its bioactivated thiocarbamate sulfoxide metabolite, inhibits aldehyde dehydrogenase (ALDH), leading to accumulation of the...

  20. Glucose-6-phosphate dehydrogenase deficiency. WHO Working Group.

    1989-01-01

    Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the commonest enzyme disorder of human beings and a globally important cause of neonatal jaundice, which can lead to kernicterus and death or spastic cerebral palsy. It can also lead to life-threatening haemolytic crises in childhood and at later ages, by interacting with specific drugs and with fava beans in the diet. The complications of G6PD deficiency can largely be prevented by education and information, and neonatal jaundice can be ...

  1. Aldehyde Dehydrogenases in Cellular Responses to Oxidative/electrophilic Stress

    Singh, Surendra; Brocker, Chad; Koppaka, Vindhya; Ying, Chen; Jackson, Brian; Matsumoto, Akiko; Thompson, David C.; Vasiliou, Vasilis

    2012-01-01

    Reactive oxygen species (ROS) are continuously generated within living systems and the inability to manage ROS load leads to elevated oxidative stress and cell damage. Oxidative stress is coupled to the oxidative degradation of lipid membranes, also known as lipid peroxidation. This process generates over 200 types of aldehydes, many of which are highly reactive and toxic. Aldehyde dehydrogenases (ALDHs) metabolize endogenous and exogenous aldehydes and thereby mitigate oxidative/electrophili...

  2. Direct Observation of Correlated Interdomain Motion in Alcohol Dehydrogenase

    Biehl, R.; Hoffmann, B.; Monkenbusch, M.; Falus, P.; Préost, S.; Merkel, R.; Richter, D.

    2008-01-01

    Interdomain motions in proteins are essential to enable or promote biochemical function. Neutron spinecho spectroscopy is used to directly observe the domain dynamics of the protein alcohol dehydrogenase. The collective motion of domains as revealed by their coherent form factor relates to the cleft opening dynamics between the binding and the catalytic domains enabling binding and release of the functional important cofactor. The cleft opening mode hardens as a result of an overall stiffenin...

  3. Prognostic values of aldehyde dehydrogenase 1 isoenzymes in ovarian cancer

    Ma YM; Zhao S

    2016-01-01

    Yu-mei Ma,1 Shan Zhao2 1Department of Pathology, 2Department of Cancer Second Division, The Second Hospital of Hebei Medical University, Shijiazhuang City, People’s Republic of China Abstract: Aldehyde dehydrogenase 1 (ALDH1) activity has been used as a functional stem cell marker to isolate cancer stem cells in different cancer types, including ovarian cancer. However, which ALDH1’s isoenzymes are contributing to ALDH1 activity in ovarian cancer remains elusive. In addition, th...

  4. Detailed kinetics and regulation of mammalian 2-oxoglutarate dehydrogenase

    Dash Ranjan K; Pradhan Ranjan K; Qi Feng; Beard Daniel A

    2011-01-01

    Abstract Background Mitochondrial 2-oxoglutarate (α-ketoglutarate) dehydrogenase complex (OGDHC), a key regulatory point of tricarboxylic acid (TCA) cycle, plays vital roles in multiple pathways of energy metabolism and biosynthesis. The catalytic mechanism and allosteric regulation of this large enzyme complex are not fully understood. Here computer simulation is used to test possible catalytic mechanisms and mechanisms of allosteric regulation of the enzyme by nucleotides (ATP, ADP), pH, an...

  5. Neonatal screening for glucose-6-phosphate dehydrogenase deficiency: sex distribution.

    Kaplan, M.; Hammerman, C; Kvit, R; Rudensky, B; Abramov, A.

    1994-01-01

    Eight hundred and six newborn infants at high risk for glucose-6-phosphate dehydrogenase (G-6-PD) deficiency were screened; 30.2% of the boys and 10.4% of the girls had severe G-6-PD deficiency. Surprisingly, 14% of the enzyme deficient girls had a father from a low risk ethnic group. Girls of high risk mothers should be screened for G-6-PD deficiency regardless of paternal origin.

  6. Characterization of the rat Class 3 aldehyde dehydrogenase gene promoter.

    Xie, Y Q; Takimoto, K; Pitot, H. C.; Miskimins, W K; Lindahl, R

    1996-01-01

    The Class 3 aldehyde dehydrogenase gene (ALDH-3) is differentially expressed. Expression is either constitutive or xenobiotic inducible via an aromatic hydrocarbon (Ah) receptor-mediated pathway, depending upon the tissue. A series of studies were performed to examine the regulation of rat ALDH-3 basal expression. DNase I footprint analysis identified four DNA regions within the proximal 1 kb of the 5' flanking region of rat ALDH-3 which interact with regulatory proteins. Reporter gene and ge...

  7. Regulation of human class I alcohol dehydrogenases by bile acids

    Langhi, Cédric; Pedraz-Cuesta, Elena; Haro, Diego; Marrero, Pedro F; Rodríguez, Joan C.

    2013-01-01

    Class I alcohol dehydrogenases (ADH1s) are the rate-limiting enzymes for ethanol and vitamin A (retinol) metabolism in the liver . Because previous studies have shown that human ADH1 enzymes may participate in bile acid metabolism, we investigated whether the bile acid-activated nuclear receptor farnesoid X receptor (FXR) regulates ADH1 genes. In human hepatocytes, both the endogenous FXR ligand chenodeoxycholic acid and synthetic FXR-specific agonist GW4064 increased ADH1 mRNA, protein, and ...

  8. Optic neuropathy in a patient with pyruvate dehydrogenase deficiency

    Small, Juan E. [Massachusetts General Hospital and Harvard Medical School, Department of Radiology, Boston, MA (United States); Gonzalez, Guido E. [Massachusetts Eye and Ear Infirmary and Harvard Medical School, Department of Radiology, Boston, MA (United States); Clinica Alemana de Santiago, Departmento de Imagenes, Santiago (Chile); Nagao, Karina E.; Walton, David S. [Massachusetts Eye and Ear Infirmary and Harvard Medical School, Department of Ophthalmology, Boston, MA (United States); Caruso, Paul A. [Massachusetts Eye and Ear Infirmary and Harvard Medical School, Department of Radiology, Boston, MA (United States)

    2009-10-15

    Pyruvate dehydrogenase (PDH) deficiency is a genetic disorder of mitochondrial metabolism. The clinical manifestations range from severe neonatal lactic acidosis to chronic neurodegeneration. Optic neuropathy is an uncommon clinical sequela and the imaging findings of optic neuropathy in these patients have not previously been described. We present a patient with PDH deficiency with bilateral decreased vision in whom MRI demonstrated bilateral optic neuropathy and chiasmopathy. (orig.)

  9. Hydroxysteroid dehydrogenases (HSDs) in bacteria: a bioinformatic perspective.

    Kisiela, Michael; Skarka, Adam; Ebert, Bettina; Maser, Edmund

    2012-03-01

    Steroidal compounds including cholesterol, bile acids and steroid hormones play a central role in various physiological processes such as cell signaling, growth, reproduction, and energy homeostasis. Hydroxysteroid dehydrogenases (HSDs), which belong to the superfamily of short-chain dehydrogenases/reductases (SDR) or aldo-keto reductases (AKR), are important enzymes involved in the steroid hormone metabolism. HSDs function as an enzymatic switch that controls the access of receptor-active steroids to nuclear hormone receptors and thereby mediate a fine-tuning of the steroid response. The aim of this study was the identification of classified functional HSDs and the bioinformatic annotation of these proteins in all complete sequenced bacterial genomes followed by a phylogenetic analysis. For the bioinformatic annotation we constructed specific hidden Markov models in an iterative approach to provide a reliable identification for the specific catalytic groups of HSDs. Here, we show a detailed phylogenetic analysis of 3α-, 7α-, 12α-HSDs and two further functional related enzymes (3-ketosteroid-Δ(1)-dehydrogenase, 3-ketosteroid-Δ(4)(5α)-dehydrogenase) from the superfamily of SDRs. For some bacteria that have been previously reported to posses a specific HSD activity, we could annotate the corresponding HSD protein. The dominating phyla that were identified to express HSDs were that of Actinobacteria, Proteobacteria, and Firmicutes. Moreover, some evolutionarily more ancient microorganisms (e.g., Cyanobacteria and Euryachaeota) were found as well. A large number of HSD-expressing bacteria constitute the normal human gastro-intestinal flora. Another group of bacteria were originally isolated from natural habitats like seawater, soil, marine and permafrost sediments. These bacteria include polycyclic aromatic hydrocarbons-degrading species such as Pseudomonas, Burkholderia and Rhodococcus. In conclusion, HSDs are found in a wide variety of microorganisms including

  10. Retinol dehydrogenase 10 is indispensible for spermatogenesis in juvenile males

    Tong, Ming-Han; Yang, Qi-En; Davis, Jeffrey C.; Griswold, Michael D.

    2012-01-01

    Retinoic acid (RA), an active vitamin A derivative, is essential for mammalian spermatogenesis. Genetic studies have revealed that oxidation of vitamin A to retinal by retinol dehydrogenase 10 (RDH10) is critical for embryonic RA biosynthesis. However, physiological roles of RDH10 in postnatal RA synthesis remain unclear, given that Rdh10 loss-of-function mutations lead to early embryonic lethality. We conducted in vivo genetic studies of Rdh10 in postnatal mouse testes and found that an RDH1...

  11. Fatty acids and the regulation of pyruvate dehydrogenase interconversion

    Stewart, Melanie Ann.

    1997-01-01

    This thesis presents evidence for a novel mechanism of regulation of pyruvate dehydrogenase (PDH) kinase by fatty acids and also results of a study of muscle triacylglycerol concentration. In animals regulation of PDH complex activity is central to the selection of respiratory fuels and to the conservation of glucose during carbohydrate deprivation. The principal means of regulation of PDH complex is interconversion of phosphorylated (inactive) and dephosphorylated (active) fo...

  12. A guide to 17beta-hydroxysteroid dehydrogenases.

    Adamski, J; Jakob, F J

    2001-01-22

    17beta-Hydroxysteroid dehydrogenases (17beta-HSD) are pivotal in controlling the biological potency of steroid hormones by catalyzing oxidation or reduction at position 17. Several 17beta-HSDs may as well metabolize further substrates including alcohols, bile acids, fatty acids and retinols. This review summarizes recent progress in the field of 17beta-HSD research provides an update of nomenclature. PMID:11165003

  13. Retinal Dehydrogenase 12 (RDH12) Mutations in Leber Congenital Amaurosis

    Perrault, Isabelle; Hanein, Sylvain; Gerber, Sylvie; Barbet, Fabienne; Ducroq, Dominique; Dollfus, Helene; Hamel, Christian,; Dufier, Jean-Louis; Munnich, Arnold; Kaplan, Josseline; Rozet, Jean-Michel

    2004-01-01

    Leber congenital amaurosis (LCA), the most early-onset and severe form of all inherited retinal dystrophies, is responsible for congenital blindness. Ten LCA genes have been mapped, and seven of these have been identified. Because some of these genes are involved in the visual cycle, we regarded the retinal pigment epithelium and photoreceptor-specific retinal dehydrogenase (RDH) genes as candidate genes in LCA. Studying a series of 110 unrelated patients with LCA, we found mutations in the p...

  14. Encapsulation of Alcohol Dehydrogenase in Mannitol by Spray Drying

    Hirokazu Shiga; Hiromi Joreau; Tze Loon Neoh; Takeshi Furuta; Hidefumi Yoshii

    2014-01-01

    The retention of the enzyme activity of alcohol dehydrogenase (ADH) has been studied in various drying processes such as spray drying. The aim of this study is to encapsulate ADH in mannitol, either with or without additive in order to limit the thermal denaturation of the enzyme during the drying process. The retention of ADH activity was investigated at different drying temperatures. When mannitol was used, the encapsulated ADH was found inactive in all the dried powders. This is presumably...

  15. Cloning, purification and crystallization of Thermus thermophilus proline dehydrogenase

    Cloning, purification and crystallization of T. thermophilus proline dehydrogenase is reported. The detergent n-octyl β-d-glucopyranoside was used to reduce polydispersity, which enabled crystallization. Nature recycles l-proline by converting it to l-glutamate. This four-electron oxidation process is catalyzed by the two enzymes: proline dehydrogenase (PRODH) and Δ1-pyrroline-5-carboxylate dehydrogenase. This note reports the cloning, purification and crystallization of Thermus thermophilus PRODH, which is the prototype of a newly discovered superfamily of bacterial monofunctional PRODHs. The results presented here include production of a monodisperse protein solution through use of the detergent n-octyl β-d-glucopyranoside and the growth of native crystals that diffracted to 2.3 Å resolution at Advanced Light Source beamline 4.2.2. The space group is P212121, with unit-cell parameters a = 82.2, b = 89.6, c = 94.3 Å. The asymmetric unit is predicted to contain two protein molecules and 46% solvent. Molecular-replacement trials using a fragment of the PRODH domain of the multifunctional Escherichia coli PutA protein as the search model (24% amino-acid sequence identity) did not produce a satisfactory solution. Therefore, the structure of T. thermophilus PRODH will be determined by multiwavelength anomalous dispersion phasing using a selenomethionyl derivative

  16. A glycolate dehydrogenase in the mitochondria of Arabidopsis thaliana.

    Bari, Rafijul; Kebeish, Rashad; Kalamajka, Rainer; Rademacher, Thomas; Peterhänsel, Christoph

    2004-03-01

    The fixation of molecular O2 by the oxygenase activity of Rubisco leads to the formation of phosphoglycolate in the chloroplast that is further metabolized in the process of photorespiration. The initial step of this pathway is the oxidation of glycolate to glyoxylate. Whereas in higher plants this reaction takes place in peroxisomes and is dependent on oxygen as a co-factor, most algae oxidize glycolate in the mitochondria using organic co-factors. The identification and characterization of a novel glycolate dehydrogenase in Arabidopsis thaliana is reported here. The enzyme is dependent on organic co-factors and resembles algal glycolate dehydrogenases in its enzymatic properties. Mutants of E. coli incapable of glycolate oxidation can be complemented by overexpression of the Arabidopsis open reading frame. The corresponding RNA accumulates preferentially in illuminated leaves, but was also found in other tissues investigated. A fusion of the N-terminal part of the Arabidopsis glycolate dehydrogenase to red fluorescent protein accumulates in mitochondria when overexpressed in the homologous system. Based on these results it is proposed that the basic photorespiratory system of algae is conserved in higher plants. PMID:14966218

  17. Novel yeast cell dehydrogenase activity assay in situ.

    Berłowska, Joanna; Kregiel, Dorota; Klimek, Leszek; Orzeszyna, Bartosz; Ambroziak, Wojciech

    2006-01-01

    The aim of this research was to develop a suitable method of succinate dehydrogenase activity assay in situ for different industrial yeast strains. For this purpose different compounds: EDTA, Triton X-100, sodium deoxycholate, digitonin, nystatin and beta-mercaptoethanol were used. The permeabilization process was controlled microscopically by primuline staining. Enzyme assay was conducted in whole yeast cells with Na-succinate as substrate, phenazine methosulfate (PMS) as electron carrier and in the presence one of two different tetrazolium salts: tetrazolium blue chloride (BT) or cyanoditolyl tetrazolium chloride (CTC) reduced during the assay. In comparabile studies of yeast vitality the amount of intracellular ATP was determined according to luciferin/luciferase method. During the succinate dehydrogenase assay in intact yeast cells without permeabilization, BT formazans were partially visualized in the cells, but CTC formazans appeared to be totally extracellular or associated with the plasma membrane. Under these conditions there was no linear relationship between formazan color intensity signal and yeast cell density. From all chemical compounds tested, only digitonin was effective in membrane permeabilization without negative influence on cell morphology. Furthermore, with digitonin-treated cells a linear relationship between formazan color intensity signal and yeast cell number was noticed. Significant decreasing of succinate dehydrogenase activity and ATP content were observed during aging of the tested yeast strains. PMID:17419290

  18. Pyruvate Dehydrogenase Kinases: Therapeutic Targets for Diabetes and Cancers

    Nam Ho Jeoung

    2015-06-01

    Full Text Available Impaired glucose homeostasis is one of the risk factors for causing metabolic diseases including obesity, type 2 diabetes, and cancers. In glucose metabolism, pyruvate dehydrogenase complex (PDC mediates a major regulatory step, an irreversible reaction of oxidative decarboxylation of pyruvate to acetyl-CoA. Tight control of PDC is critical because it plays a key role in glucose disposal. PDC activity is tightly regulated using phosphorylation by pyruvate dehydrogenase kinases (PDK1 to 4 and pyruvate dehydrogenase phosphatases (PDP1 and 2. PDKs and PDPs exhibit unique tissue expression patterns, kinetic properties, and sensitivities to regulatory molecules. During the last decades, the up-regulation of PDKs has been observed in the tissues of patients and mammals with metabolic diseases, which suggests that the inhibition of these kinases may have beneficial effects for treating metabolic diseases. This review summarizes the recent advances in the role of specific PDK isoenzymes on the induction of metabolic diseases and describes the effects of PDK inhibition on the prevention of metabolic diseases using pharmacological inhibitors. Based on these reports, PDK isoenzymes are strong therapeutic targets for preventing and treating metabolic diseases.

  19. Deficiency of retinaldehyde dehydrogenase 1 induces BMP2 and increases bone mass in vivo.

    Shriram Nallamshetty

    Full Text Available The effects of retinoids, the structural derivatives of vitamin A (retinol, on post-natal peak bone density acquisition and skeletal remodeling are complex and compartment specific. Emerging data indicates that retinoids, such as all trans retinoic acid (ATRA and its precursor all trans retinaldehyde (Rald, exhibit distinct and divergent transcriptional effects in metabolism. Despite these observations, the role of enzymes that control retinoid metabolism in bone remains undefined. In this study, we examined the skeletal phenotype of mice deficient in retinaldehyde dehydrogenase 1 (Aldh1a1, the enzyme responsible for converting Rald to ATRA in adult animals. Bone densitometry and micro-computed tomography (µCT demonstrated that Aldh1a1-deficient (Aldh1a1(-/- female mice had higher trabecular and cortical bone mass compared to age and sex-matched control C57Bl/6 wild type (WT mice at multiple time points. Histomorphometry confirmed increased cortical bone thickness and demonstrated significantly higher bone marrow adiposity in Aldh1a1(-/- mice. In serum assays, Aldh1a1(-/- mice also had higher serum IGF-1 levels. In vitro, primary Aldh1a1(-/- mesenchymal stem cells (MSCs expressed significantly higher levels of bone morphogenetic protein 2 (BMP2 and demonstrated enhanced osteoblastogenesis and adipogenesis versus WT MSCs. BMP2 was also expressed at higher levels in the femurs and tibias of Aldh1a1(-/- mice with accompanying induction of BMP2-regulated responses, including expression of Runx2 and alkaline phosphatase, and Smad phosphorylation. In vitro, Rald, which accumulates in Aldh1a1(-/- mice, potently induced BMP2 in WT MSCs in a retinoic acid receptor (RAR-dependent manner, suggesting that Rald is involved in the BMP2 increases seen in Aldh1a1 deficiency in vivo. Collectively, these data implicate Aldh1a1 as a novel determinant of cortical bone density and marrow adiposity in the skeleton in vivo through modulation of BMP signaling.

  20. Immunological study of lactate dehydrogenase from Streptococcus mutans and evidence of common antigenic domains with lactate dehydrogenases from lactic bacteria.

    Sommer, P.; Klein, J P; Ogier, J. A.; Frank, R M

    1986-01-01

    Rabbit polyclonal antibodies directed against purified Streptococcus mutans L-(+)-lactate dehydrogenase reacted with the purified enzyme, giving a marked deviation of its kinetic parameters. The enzyme affinity for pyruvate or NADH decreased in the presence of antibody, the affinity for fructose 1,6-diphosphate (FDP) appeared to be slightly affected, and the cooperativity of the ligand binding was lowered. A partial protective effect was observed when the enzyme was preincubated with FDP prio...

  1. Pyruvate Dehydrogenase and Pyruvate Dehydrogenase Kinase Expression in Non Small Cell Lung Cancer and Tumor-Associated Stroma

    Michael I. Koukourakis

    2005-01-01

    Full Text Available Pyruvate dehydrogenase (PDH catalyzes the conversion of pyruvate to acetyl-coenzyme A, which enters into the Krebs cycle, providing adenosine triphosphate (ATP to the cell. PDH activity is under the control of pyruvate dehydrogenase kinases (PDKs. Under hypoxic conditions, conversion of pyruvate to lactate occurs, a reaction catalyzed by lactate dehydrogenase 5 (LDH5. In cancer cells, however, pyruvate is transformed to lactate occurs, regardless of the presence of oxygen (aerobic glycolysis/Warburg effect. Although hypoxic intratumoral conditions account for HIFia stabilization and induction of anaerobic metabolism, recent data suggest that high pyruvate concentrations also result in HIFia stabilization independently of hypoxia. In the present immunohistochemical study, we provide evidence that the PDH/PDK pathway is repressed in 73% of non small cell lung carcinomas, which may be a key reason for HIFia stabilization and “aerobic glycolysis.” However, about half of PDHdeficient carcinomas are not able to switch on the HIF pathway, and patients harboring these tumors have an excellent postoperative outcome. A small subgroup of clinically aggressive tumors maintains a coherent PDH and HIF/LDH5 expression. In contrast to cancer cells, fibroblasts in the tumor-supporting stroma exhibit an intense PDH but reduced PDK1 expression favoring maximum PDH activity. This means that stroma may use lactic acid produced by tumor cells, preventing the creation of an intolerable intratumoral acidic environment at the same time.

  2. Evidence for distinct dehydrogenase and isomerase sites within a single 3. beta. -hydroxysteroid dehydrogenase/5-ene-4-ene isomerase protein

    Luu-The, V.; Takahashi, Masakazu; de Launoit, Y.; Dumont, M.; Lachance, Y.; Labrie, F. (Laval Univ., Quebec City, Quebec (Canada))

    1991-09-10

    Complementary DNA encoding human 3{beta}-hydroxysteroid dehydrogenase/5-ene-4-ene isomerase (3-{beta}-HSD) has been expressed in transfected GH{sub 4}C{sub 1} with use of the cytomegalovirus promoter. The activity of the expressed protein clearly shows that both dehydrogenase and isomerase enzymatic activities are present within a single protein. However, such findings do not indicate whether the two activities reside within one or two closely related catalytic sites. With use of ({sup 3}H)-5-androstenedione, the intermediate compound in dehydroepiandrosterone (DHEA) transformation into 4-androstenedione by 3{beta}-HSD, the present study shows that 4MA (N,N-diethyl-4-methyl-3-oxo-4-aza-5{alpha}-androstane-17{beta}-carboxamide) and its analogues of 5-androstenedione to 4-androstenedione with an approximately 1,000-fold higher K{sub i} value. The present results thus strongly suggest that dehydrogenase and isomerase activities are present at separate sites on the 3-{beta}-HSD protein. Such data suggest that the irreversible step in the transformation of DHEA to 4-androstenedione is due to a separate site possessing isomerase activity that converts the 5-ene-3-keto to a much more stable 4-ene-3-keto configuration.

  3. Reduction of 3-mercaptopyruvate in rat liver is catalyzed by lactate dehydrogenase.

    Ohta,Jun; Ubuka,Toshihiko

    1989-01-01

    It has been assumed that the in vivo reduction of 3-mercaptopyruvate, an intermediate of cysteine metabolism, to 3-mercaptolactate is catalyzed by lactate dehydrogenase (EC 1.1.1.27) though no definitive evidence has been presented. In order to examine this assumption, reduction of 3-mercaptopyruvate and its inhibition were studied using rat liver homogenate, lactate dehydrogenase purified from rat liver and anti-lactate dehydrogenase antiserum. Reduction of 3-mercaptopyruvate was actively ca...

  4. 6-Phosphogluconate Dehydrogenase Mechanism: EVIDENCE FOR ALLOSTERIC MODULATION BY SUBSTRATE

    Hanau, Stefania; Montin, Katy; Cervellati, Carlo; Magnani, Morena; Dallocchio, Franco

    2010-01-01

    The reductive carboxylation of ribulose-5-phosphate (Ru5P) by 6-phosphogluconate dehydrogenase (6PGDH) from Candida utilis was investigated using kinetic isotope effects. The intrinsic isotope effect for proton abstraction from Ru5P was found at 4.9 from deuterium isotope effects on V and V/K and from tritium isotope effects on V/K. The presence of 6-phosphogluconate (6PG) in the assay mixture changes the magnitude of the observed isotope effects. In the absence of 6PG D(V/K) and D(V) are 1.6...

  5. In vitro hydrogen production by glucose dehydrogenase and hydrogenase

    Woodward, J. [Oak Ridge National Lab., TN (United States)

    1996-10-01

    A new in vitro enzymatic pathway for the generation of molecular hydrogen from glucose has been demonstrated. The reaction is based upon the oxidation of glucose by Thermoplasma acidophilum glucose dehydrogenase with the concomitant oxidation of NADPH by Pyrococcus furiosus hydrogenase. Stoichiometric yields of hydrogen were produced from glucose with continuous cofactor recycle. This simple system may provide a method for the biological production of hydrogen from renewable sources. In addition, the other product of this reaction, gluconic acid, is a high-value commodity chemical.

  6. Glucose-6-phosphate dehydrogenase (G6PD) Deficiency

    DD Farhud"; L Yazdanpanah

    2008-01-01

    "nGlucose-6-phosphate dehydrogenase (G6PD) Deficiency is the most prevalent enzymopathy in mankind. It has sex-linked in­heritance. This enzyme exists in all cells.  G6PD deficiency increases the sensitivity of red blood cells to oxidative dam­age. G6PD deficiency was discovered in 1950 when some people suffered hemolytic anemia as a result of taking antimalar­ial drugs (primaquin). Most people with G6PD deficiency do not have any symptoms, till they are ...

  7. Evaluation of predictive tests for screening for dihydropyrimidine dehydrogenase deficiency.

    van Staveren, M C; Guchelaar, H Jan; van Kuilenburg, A B P; Gelderblom, H; Maring, J G

    2013-10-01

    5-Fluorouracil (5-FU) is rapidly degraded by dihyropyrimidine dehydrogenase (DPD). Therefore, DPD deficiency can lead to severe toxicity or even death following treatment with 5-FU or capecitabine. Different tests based on assessing DPD enzyme activity, genetic variants in DPYD and mRNA variants have been studied for screening for DPD deficiency, but none of these are implemented broadly into clinical practice. We give an overview of the tests that can be used to detect DPD deficiency and discuss the advantages and disadvantages of these tests. PMID:23856855

  8. Functional characterization of a vanillin dehydrogenase in Corynebacterium glutamicum

    Ding, Wei; Si, Meiru; Zhang, Weipeng; Zhang, Yaoling; Chen, Can; Zhang, Lei; Lu, Zhiqiang; Chen, Shaolin; Shen, Xihui

    2015-01-01

    Vanillin dehydrogenase (VDH) is a crucial enzyme involved in the degradation of lignin-derived aromatic compounds. Herein, the VDH from Corynebacterium glutamicum was characterized. The relative molecular mass (Mr) determined by SDS-PAGE was ~51kDa, whereas the apparent native Mr values revealed by gel filtration chromatography were 49.5, 92.3, 159.0 and 199.2kDa, indicating the presence of dimeric, trimeric and tetrameric forms. Moreover, the enzyme showed its highest level of activity towar...

  9. [Genetic variations in alcohol dehydrogenase, drinking habits and alcoholism

    Tolstrup, J.S.; Rasmussen, S.; Tybjaerg-Hansen, A.;

    2008-01-01

    Alcohol is degraded primarily by alcohol dehydrogenase (ADH), and genetic variation that affects the rate of alcohol degradation is found in ADH1B and ADH1C. By genotyping 9,080 white men and women from the general population, we found that men and women with ADH1B slow versus fast alcohol...... degradation drank approximately 30% more alcohol per week and had a higher risk of everyday and heavy drinking, and of alcoholism. Individuals with ADH1C slow versus fast alcohol degradation had a higher risk of heavy drinking Udgivelsesdato: 2008/8/25...

  10. Genetic Control of Malate Dehydrogenase Isozymes in Maize

    Goodman, M. M.; Stuber, C. W.; Lee, C N; Johnson, F. M.

    1980-01-01

    At least six nuclear loci are responsible for the genetic control of malate dehydrogenase (L-malate: NAD oxidoreductase; EC 1.1.1.37; MDH) in coleoptiles of maize. Three independently segregating loci (Mdh1, Mdh2, Mdh3) govern the production of MDH isozymes resistant to inactivation by ascorbic acid and found largely or solely in the mitochondria. A rare recessive allele found at a fourth nuclear locus (mmm) causes increased electrophoretic mobility of the MDH isozymes governed by the Mdh1, M...

  11. Evolution of D-lactate dehydrogenase activity from glycerol dehydrogenase and its utility for D-lactate production from lignocellulose.

    Wang, Qingzhao; Ingram, Lonnie O; Shanmugam, K T

    2011-11-22

    Lactic acid, an attractive, renewable chemical for production of biobased plastics (polylactic acid, PLA), is currently commercially produced from food-based sources of sugar. Pure optical isomers of lactate needed for PLA are typically produced by microbial fermentation of sugars at temperatures below 40 °C. Bacillus coagulans produces L(+)-lactate as a primary fermentation product and grows optimally at 50 °C and pH 5, conditions that are optimal for activity of commercial fungal cellulases. This strain was engineered to produce D(-)-lactate by deleting the native ldh (L-lactate dehydrogenase) and alsS (acetolactate synthase) genes to impede anaerobic growth, followed by growth-based selection to isolate suppressor mutants that restored growth. One of these, strain QZ19, produced about 90 g L(-1) of optically pure D(-)-lactic acid from glucose in < 48 h. The new source of D-lactate dehydrogenase (D-LDH) activity was identified as a mutated form of glycerol dehydrogenase (GlyDH; D121N and F245S) that was produced at high levels as a result of a third mutation (insertion sequence). Although the native GlyDH had no detectable activity with pyruvate, the mutated GlyDH had a D-LDH specific activity of 0.8 μmoles min(-1) (mg protein)(-1). By using QZ19 for simultaneous saccharification and fermentation of cellulose to D-lactate (50 °C and pH 5.0), the cellulase usage could be reduced to 1/3 that required for equivalent fermentations by mesophilic lactic acid bacteria. Together, the native B. coagulans and the QZ19 derivative can be used to produce either L(+) or D(-) optical isomers of lactic acid (respectively) at high titers and yields from nonfood carbohydrates. PMID:22065761

  12. Engineering of pyranose dehydrogenase for increased oxygen reactivity.

    Iris Krondorfer

    Full Text Available Pyranose dehydrogenase (PDH, a member of the GMC family of flavoproteins, shows a very broad sugar substrate specificity but is limited to a narrow range of electron acceptors and reacts extremely slowly with dioxygen as acceptor. The use of substituted quinones or (organometals as electron acceptors is undesirable for many production processes, especially of food ingredients. To improve the oxygen reactivity, site-saturation mutagenesis libraries of twelve amino acids around the active site of Agaricus meleagris PDH were expressed in Saccharomyces cerevisiae. We established high-throughput screening assays for oxygen reactivity and standard dehydrogenase activity using an indirect Amplex Red/horseradish peroxidase and a DCIP/D-glucose based approach. The low number of active clones confirmed the catalytic role of H512 and H556. Only one position was found to display increased oxygen reactivity. Histidine 103, carrying the covalently linked FAD cofactor in the wild-type, was substituted by tyrosine, phenylalanine, tryptophan and methionine. Variant H103Y was produced in Pichia pastoris and characterized and revealed a five-fold increase of the oxygen reactivity.

  13. Phenylbutyrate Therapy for Pyruvate Dehydrogenase Complex Deficiency and Lactic Acidosis

    Ferriero, Rosa; Manco, Giuseppe; Lamantea, Eleonora; Nusco, Edoardo; Ferrante, Mariella I.; Sordino, Paolo; Stacpoole, Peter W.; Lee, Brendan; Zeviani, Massimo; Brunetti-Pierri, Nicola

    2014-01-01

    Lactic acidosis is a build-up of lactic acid in the blood and tissues, which can be due to several inborn errors of metabolism as well as nongenetic conditions. Deficiency of pyruvate dehydrogenase complex (PDHC) is the most common genetic disorder leading to lactic acidosis. Phosphorylation of specific serine residues of the E1α subunit of PDHC by pyruvate dehydrogenase kinase (PDK) inactivates the enzyme, whereas dephosphorylation restores PDHC activity. We found that phenylbutyrate enhances PDHC enzymatic activity in vitro and in vivo by increasing the proportion of unphosphorylated enzyme through inhibition of PDK. Phenylbutyrate given to C57B6/L wild-type mice results in a significant increase in PDHC enzyme activity and a reduction of phosphorylated E1α in brain, muscle, and liver compared to saline-treated mice. By means of recombinant enzymes, we showed that phenylbutyrate prevents phosphorylation of E1α through binding and inhibition of PDK, providing a molecular explanation for the effect of phenylbutyrate on PDHC activity. Phenylbutyrate increases PDHC activity in fibroblasts from PDHC-deficient patients harboring various molecular defects and corrects the morphological, locomotor, and biochemical abnormalities in the noam631 zebrafish model of PDHC deficiency. In mice, phenylbutyrate prevents systemic lactic acidosis induced by partial hepatectomy. Because phenylbutyrate is already approved for human use in other diseases, the findings of this study have the potential to be rapidly translated for treatment of patients with PDHC deficiency and other forms of primary and secondary lactic acidosis. PMID:23467562

  14. Coenzyme- and His-tag-induced crystallization of octopine dehydrogenase

    The crystal structure of octopine dehydrogenase revealed a specific role of the His5 tag in inducing the crystal contacts required for successful crystallization. Over the last decade, protein purification has become more efficient and standardized through the introduction of affinity tags. The choice and position of the tag, however, can directly influence the process of protein crystallization. Octopine dehydrogenase (OcDH) without a His tag and tagged protein constructs such as OcDH-His5 and OcDH-LEHis6 have been investigated for their crystallizability. Only OcDH-His5 yielded crystals; however, they were multiple. To improve crystal quality, the cofactor NADH was added, resulting in single crystals that were suitable for structure determination. As shown by the structure, the His5 tag protrudes into the cleft between the NADH and l-arginine-binding domains and is mainly fixed in place by water molecules. The protein is thereby stabilized to such an extent that the formation of crystal contacts can proceed. Together with NADH, the His5 tag obviously locks the enzyme into a specific conformation which induces crystal growth

  15. A straightforward radiometric technique for measuring IMP dehydrogenase.

    Cooney, D A; Wilson, Y; McGee, E

    1983-04-15

    [2-3H]Inosinic acid ([2-3H]IMP) has been biosynthesized in good yield from [2-3H]hypoxanthine and PRPP via the action of a partially purified preparation of hypoxanthine/guanine phosphoribosyl transferase from mouse brain. The product was purified in one step by ascending paper chromatography, and used to assess the activity of IMP dehydrogenase. To conduct the assay, tritiated substrate is admixed with enzyme in a final volume of 10 microliters; NAD is present to serve as cofactor for the reaction, and allopurinol to inhibit the oxidation of any hypoxanthine generated as a consequence of side reactions. After an appropriate period of incubation, the 3H2O arising from the oxidation of tritiated IMP via [3H]NAD is isolated by quantitative microdistillation. Performed as described, the assay is facile, sensitive, and accurate, with the capability of detecting the dehydrogenation of as little as 1 pmol of [3H]IMP. Using it, measurements have been made of IMP dehydrogenase in a comprehensive array of mouse organs. Of these, pancreas contained the enzyme at the highest specific activity. PMID:6135372

  16. Orthodontic Force Application in Correlation with Salivary Lactate Dehydrogenase Activity

    Erik Husin

    2013-07-01

    Full Text Available Orthodontic tooth movement generate mechanical forces to periodontal ligament and alveolar bone. The forces correlate with initial responses of periodontal tissues and involving many metabolic changes. One of the metabolic changes detected in saliva is lactate dehydrogenase (LDH activity. Objectives: To evaluate the correlation between orthodontic interrupted force application, lactate dehydrogenase activity and the distance of tooth movement. Methods: upper premolar, pre-retraction of upper canine and 1, 7, 14, 21 and 28 days post-retraction of upper canine with 100g interrupted orthodontic force. Results: duration of force (F=11.926 p 14 and 28 days post-retraction of canine. The region of retraction correlated with the distance of tooth movement (F=7.377 p=0.007. The duration of force correlated with the distance of tooth movement (F=66.554 p=0.000. retraction of canine. Conclusion: This study concluded that orthodontic interrupted force application on canine could increase the distance of tooth movement and LDH activity in saliva.

  17. Crystal structure of a chimaeric bacterial glutamate dehydrogenase.

    Oliveira, Tânia; Sharkey, Michael A; Engel, Paul C; Khan, Amir R

    2016-06-01

    Glutamate dehydrogenases (EC 1.4.1.2-4) catalyse the oxidative deamination of L-glutamate to α-ketoglutarate using NAD(P)(+) as a cofactor. The bacterial enzymes are hexameric, arranged with 32 symmetry, and each polypeptide consists of an N-terminal substrate-binding segment (domain I) followed by a C-terminal cofactor-binding segment (domain II). The catalytic reaction takes place in the cleft formed at the junction of the two domains. Distinct signature sequences in the nucleotide-binding domain have been linked to the binding of NAD(+) versus NADP(+), but they are not unambiguous predictors of cofactor preference. In the absence of substrate, the two domains move apart as rigid bodies, as shown by the apo structure of glutamate dehydrogenase from Clostridium symbiosum. Here, the crystal structure of a chimaeric clostridial/Escherichia coli enzyme has been determined in the apo state. The enzyme is fully functional and reveals possible determinants of interdomain flexibility at a hinge region following the pivot helix. The enzyme retains the preference for NADP(+) cofactor from the parent E. coli domain II, although there are subtle differences in catalytic activity. PMID:27303899

  18. High-pressure-induced water penetration into 3-isopropylmalate dehydrogenase

    Structures of 3-isopropylmalate dehydrogenase were determined at pressures ranging from 0.1 to 650 MPa. Comparison of these structures gives a detailed picture of the swelling of a cavity at the dimer interface and the generation of a new cleft on the molecular surface, which are accompanied by water penetration. Hydrostatic pressure induces structural changes in proteins, including denaturation, the mechanism of which has been attributed to water penetration into the protein interior. In this study, structures of 3-isopropylmalate dehydrogenase (IPMDH) from Shewanella oneidensis MR-1 were determined at about 2 Å resolution under pressures ranging from 0.1 to 650 MPa using a diamond anvil cell (DAC). Although most of the protein cavities are monotonically compressed as the pressure increases, the volume of one particular cavity at the dimer interface increases at pressures over 340 MPa. In parallel with this volume increase, water penetration into the cavity could be observed at pressures over 410 MPa. In addition, the generation of a new cleft on the molecular surface accompanied by water penetration could also be observed at pressures over 580 MPa. These water-penetration phenomena are considered to be initial steps in the pressure-denaturation process of IPMDH

  19. High-pressure-induced water penetration into 3-isopropylmalate dehydrogenase

    Nagae, Takayuki; Kawamura, Takashi [Nagoya University, (Japan); Chavas, Leonard M. G. [High Energy Research Organization (KEK), (Japan); Niwa, Ken; Hasegawa, Masashi [Nagoya University, (Japan); Kato, Chiaki [Japan Agency for Marine-Earth Science and Technology (JAMSTEC), (Japan); Watanabe, Nobuhisa, E-mail: nobuhisa@nagoya-u.jp [Nagoya University, (Japan); Nagoya University, (Japan)

    2012-03-01

    Structures of 3-isopropylmalate dehydrogenase were determined at pressures ranging from 0.1 to 650 MPa. Comparison of these structures gives a detailed picture of the swelling of a cavity at the dimer interface and the generation of a new cleft on the molecular surface, which are accompanied by water penetration. Hydrostatic pressure induces structural changes in proteins, including denaturation, the mechanism of which has been attributed to water penetration into the protein interior. In this study, structures of 3-isopropylmalate dehydrogenase (IPMDH) from Shewanella oneidensis MR-1 were determined at about 2 Å resolution under pressures ranging from 0.1 to 650 MPa using a diamond anvil cell (DAC). Although most of the protein cavities are monotonically compressed as the pressure increases, the volume of one particular cavity at the dimer interface increases at pressures over 340 MPa. In parallel with this volume increase, water penetration into the cavity could be observed at pressures over 410 MPa. In addition, the generation of a new cleft on the molecular surface accompanied by water penetration could also be observed at pressures over 580 MPa. These water-penetration phenomena are considered to be initial steps in the pressure-denaturation process of IPMDH.

  20. Inhibition of Horse Liver Alcohol Dehydrogenase by Methyltin Compounds

    Pavel V. Bychkov

    2004-01-01

    Full Text Available The study of inorganic tin (SnCl2, SnCl4 and methyltin compounds (MeSnCl3, Me2SnCI2, Me3SnCl effects on the enzymatic activity of alcohol dehydrogenase (ADH in the reaction of ethanol oxidation has been carried out. The experimental results of the study show that inorganic tin and methyltin substances induce slight inhibition of the catalytic activity of horse liver alcohol dehydrogenase (HLADH, unable to be improved during pre-incubation with the enzyme. The conditions for carrying out the kinetic investigation of the mentioned phenomenon were optimized and as it turned out the mechanism of methyltin trichloride action, as the most effective methyltin inhibitor, is more complex than the proposed interaction of the metal atom with SH-groups of the enzyme protein. It was demonstrated that the tin compounds act in the same manner as methylmercury compounds and might serve as oxidative agents towards the co-enzyme NADH. Kinetic data on MeSnCl3 were calculated. Data acquired on NAD-dependent ADH from horse liver and those regarding NAD-dependent LDH from sturgeon liver were compared.

  1. Structural basis for cellobiose dehydrogenase action during oxidative cellulose degradation.

    Tan, Tien-Chye; Kracher, Daniel; Gandini, Rosaria; Sygmund, Christoph; Kittl, Roman; Haltrich, Dietmar; Hällberg, B Martin; Ludwig, Roland; Divne, Christina

    2015-01-01

    A new paradigm for cellulose depolymerization by fungi focuses on an oxidative mechanism involving cellobiose dehydrogenases (CDH) and copper-dependent lytic polysaccharide monooxygenases (LPMO); however, mechanistic studies have been hampered by the lack of structural information regarding CDH. CDH contains a haem-binding cytochrome (CYT) connected via a flexible linker to a flavin-dependent dehydrogenase (DH). Electrons are generated from cellobiose oxidation catalysed by DH and shuttled via CYT to LPMO. Here we present structural analyses that provide a comprehensive picture of CDH conformers, which govern the electron transfer between redox centres. Using structure-based site-directed mutagenesis, rapid kinetics analysis and molecular docking, we demonstrate that flavin-to-haem interdomain electron transfer (IET) is enabled by a haem propionate group and that rapid IET requires a closed CDH state in which the propionate is tightly enfolded by DH. Following haem reduction, CYT reduces LPMO to initiate oxygen activation at the copper centre and subsequent cellulose depolymerization. PMID:26151670

  2. Structural basis for cellobiose dehydrogenase action during oxidative cellulose degradation

    Tan, Tien-Chye; Kracher, Daniel; Gandini, Rosaria; Sygmund, Christoph; Kittl, Roman; Haltrich, Dietmar; Hällberg, B. Martin; Ludwig, Roland; Divne, Christina

    2015-01-01

    A new paradigm for cellulose depolymerization by fungi focuses on an oxidative mechanism involving cellobiose dehydrogenases (CDH) and copper-dependent lytic polysaccharide monooxygenases (LPMO); however, mechanistic studies have been hampered by the lack of structural information regarding CDH. CDH contains a haem-binding cytochrome (CYT) connected via a flexible linker to a flavin-dependent dehydrogenase (DH). Electrons are generated from cellobiose oxidation catalysed by DH and shuttled via CYT to LPMO. Here we present structural analyses that provide a comprehensive picture of CDH conformers, which govern the electron transfer between redox centres. Using structure-based site-directed mutagenesis, rapid kinetics analysis and molecular docking, we demonstrate that flavin-to-haem interdomain electron transfer (IET) is enabled by a haem propionate group and that rapid IET requires a closed CDH state in which the propionate is tightly enfolded by DH. Following haem reduction, CYT reduces LPMO to initiate oxygen activation at the copper centre and subsequent cellulose depolymerization. PMID:26151670

  3. Toxicity of Nitrification Inhibitors on Dehydrogenase Activity in Soils

    Ferisman Tindaon

    2011-01-01

    Full Text Available The objective of this research was to determine the effects of nitrification inhibitors (NIs such as 3,4-dimethylpyrazolephosphate=DMPP, 4-Chlor-methylpyrazole phosphate=ClMPP and dicyandiamide,DCD which might be expected to inhibit microbial activity, on dehydrogenase activity (DRA,in three different soils in laboratory conditions. Dehydrogenase activity were assessed via reduction of 2-p-Iodophenyl-3-p-nitrophenyl-5-phenyltetrazoliumchloride (INT. The toxicity and dose response curve of three NIs were quantified under laboratory conditions using a loamy clay, a sandy loam and a sandy soil. The quantitative determination of DHA was carried out spectrophotometrically. In all experiments, the influence of 5-1000 times the base concentration were examined. To evaluate the rate of inhibition with the increasing NI concentrations, dose reponse curves were presented and no observable effect level =NOEL, as well as effective dose ED10 and ED 50(10% and 50% inhibition were calculated. The NOEL for common microbial activity such as DHA was about 30–70 times higher than base concentration in all investigated soils. ClMPP exhibited the strongest influence on the non target microbial processes in the three soils if it compare to DMPP and DCD. The NOEL,ED10 and ED50 values higher in clay than in loamy or sandy soil. The NIs were generally most effective in sandy soils. The three NIs considered at the present state of knowledge as environmentally safe in use.

  4. New recombinant bacterium comprises a heterologous gene encoding glycerol dehydrogenase and/or an up-regulated native gene encoding glycerol dehydrogenase, useful for producing ethanol

    2010-01-01

    TECHNOLOGY FOCUS - BIOTECHNOLOGY - Preparation (claimed): Producing recombinant bacterium having enhanced ethanol production characteristics when cultivated in growth medium comprising glycerol comprises: (a) transforming a parental bacterium by (i) the insertion of a heterologous gene encoding...... glycerol dehydrogenase; and/or (ii) up-regulating a native gene encoding glycerol dehydrogenase; and (b) obtaining the recombinant bacterium. Preferred Bacterium: In the recombinant bacterium above, the inserted heterologous gene and/or the up-regulated native gene is encoding a glycerol dehydrogenase...... dehydrogenase encoding region of the bacterium, or is inserted into a phosphotransacetylase encoding region of the bacterium, or is inserted into an acetate kinase encoding region of the bacterium. It is operably linked to an inducible, a regulated or a constitutive promoter. The up-regulated glycerol...

  5. The oxyanion hole of Pseudomonas fluorescens mannitol 2-dehydrogenase: a novel structural motif for electrostatic stabilisation in alcohol dehydrogenase active sites

    Klimacek, Mario; Nidetzky, B

    2009-01-01

    Abstract The side chains of Asn-191 and Asn-300 constitute a characteristic structural motif of the active site of Pseudomonas fluorescens mannitol 2-dehydrogenase that lacks precedent in known alcohol dehydrogenases and resembles the canonical oxyanion binding pocket of serine proteases. We have used steady-state and transient kinetic studies of the effects of varied pH and deuterium isotopic substitutions in substrates and solvent on the enzymatic rates to delineate catalytic con...

  6. Effect of the allelic variants of aldehyde dehydrogenase ALDH2*2 and alcohol dehydrogenase ADH1B*2 on blood acetaldehyde concentrations

    Peng Giia-Sheun; Yin Shih-Jiun

    2009-01-01

    Abstract Alcoholism is a complex behavioural disorder. Molecular genetics studies have identified numerous candidate genes associated with alcoholism. It is crucial to verify the disease susceptibility genes by correlating the pinpointed allelic variations to the causal phenotypes. Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) are the principal enzymes responsible for ethanol metabolism in humans. Both ADH and ALDH exhibit functional polymorphisms among racial populations; the...

  7. 21 CFR 864.7360 - Erythrocytic glucose-6-phosphate dehydrogenase assay.

    2010-04-01

    ... assay. 864.7360 Section 864.7360 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND... § 864.7360 Erythrocytic glucose-6-phosphate dehydrogenase assay. (a) Identification. An erythrocytic glucose-6-phosphate dehydrogenase assay is a device used to measure the activity of the enzyme...

  8. Cloning and mRNA Expression of NADH Dehydrogenase during Ochlerotatus taeniorhynchus Development and Pesticide Response

    NADH dehydrogenase, the largest of the respiratory complexes, is the first enzyme of the mitochondrial electron transport chain. We have cloned and sequenced cDNA of NADH dehydrogenase gene from Ochlerotatus (Ochlerotatus) taeniorhynchus (Wiedemann) adult (GeneBank Accession number: FJ458415). The ...

  9. Evidence for a nickel-containing carbon monoxide dehydrogenase in Methanobrevibacter arboriphilicus.

    Hammel, K E; Cornwell, K L; Diekert, G B; Thauer, R K

    1984-01-01

    In growing cultures of Methanobrevibacter arboriphilicus (Methanobrevibacter arboriphilus), the synthesis of active carbon monoxide dehydrogenase required nickel. The 21-fold-purified enzyme from 63Ni-labeled cells of M. arboriphilicus comigrated with 63Ni during gel filtration. These results provide evidence that the carbon monoxide dehydrogenase of methanogens is a nickel protein.

  10. Role and structural characterization of plant aldehyde dehydrogenases from family 2 and family 7

    Končitíková, R.; Vigouroux, A.; Kopečná, M.; Andree, T.; Bartoš, Jan; Šebela, M.; Moréra, S.; Kopečný, D.

    2015-01-01

    Roč. 468, Part: 1 (2015), s. 109-123. ISSN 0264-6021 R&D Projects: GA ČR GA15-22322S; GA MŠk(CZ) LO1204 Institutional support: RVO:61389030 Keywords : aldehyde dehydrogenase 2 (ALDH2) * aldehyde dehydrogenase 7 (ALDH7) * benzaldehyde Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.396, year: 2014

  11. Soil dehydrogenase activity in the presence of chromium (III) and (VI)

    Wolińska A.; Stępniewska Z.

    2005-01-01

    The paper presents the influence of chromium forms (III) and (VI) on the soil dehydrogenase activity. Enzyme activities can be considered effective indicators of soil quality changes resulting from environmental stress or management practices. It was found that chromium compounds have detrimental effects on soil dehydrogenase activity. After the addition of chromium, a rapid and significant decrease in enzymatic activities was observed.

  12. A 'random steady-state' model for the pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase enzyme complexes

    The multienzyme complexes, pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase, involved in the central metabolism of Escherichia coli consist of multiple copies of three different enzymes, E1, E2 and E3, that cooperate to channel substrate intermediates between their active sites. The E2 components form the core of the complex, while a mixture of E1 and E3 components binds to the core. We present a random steady-state model to describe catalysis by such multienzyme complexes. At a fast time scale, the model describes the enzyme catalytic mechanisms of substrate channeling at a steady state, by polynomially approximating the analytic solution of a biochemical master equation. At a slower time scale, the structural organization of the different enzymes in the complex and their random binding/unbinding to the core is modeled using methods from equilibrium statistical mechanics. Biologically, the model describes the optimization of catalytic activity by substrate sharing over the entire enzyme complex. The resulting enzymatic models illustrate the random steady state (RSS) for modeling multienzyme complexes in metabolic pathways

  13. Evaluation of alcohol dehydrogenase and aldehyde dehydrogenase enzymes as bi-enzymatic anodes in a membraneless ethanol microfluidic fuel cell

    Galindo-de-la-Rosa, J.; Arjona, N.; Arriaga, L. G.; Ledesma-García, J.; Guerra-Balcázar, M.

    2015-12-01

    Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (AldH) enzymes were immobilized by covalent binding and used as the anode in a bi-enzymatic membraneless ethanol hybrid microfluidic fuel cell. The purpose of using both enzymes was to optimize the ethanol electro-oxidation reaction (EOR) by using ADH toward its direct oxidation and AldH for the oxidation of aldehydes as by-products of the EOR. For this reason, three enzymatic bioanode configurations were evaluated according with the location of enzymes: combined, vertical and horizontally separated. In the combined configuration, a current density of 16.3 mA cm-2, a voltage of 1.14 V and a power density of 7.02 mW cm-2 were obtained. When enzymes were separately placed in a horizontal and vertical position the ocp drops to 0.94 V and to 0.68 V, respectively. The current density also falls to values of 13.63 and 5.05 mA cm-2. The decrease of cell performance of bioanodes with separated enzymes compared with the combined bioanode was of 31.7% and 86.87% for the horizontal and the vertical array.

  14. Identification and Overexpression of a Bifunctional Aldehyde/Alcohol Dehydrogenase Responsible for Ethanol Production in Thermoanaerobacter mathranii

    Yao, Shuo; Just Mikkelsen, Marie

    2010-01-01

    ethanol as a fermentation product, while other adh knockout strains showed no significant difference from the wild type. Further analysis revealed that the ΔadhE strain was defective in aldehyde dehydrogenase activity, but still maintained alcohol dehydrogenase activity. This showed that AdhE is the major......Thermoanaerobacter mathranii contains four genes, adhA, adhB, bdhA and adhE, predicted to code for alcohol dehydrogenases involved in ethanol metabolism. These alcohol dehydrogenases were characterized as NADP(H)-dependent primary alcohol dehydrogenase (AdhA), secondary alcohol dehydrogenase (Adh......B), butanol dehydrogenase (BdhA) and NAD(H)-dependent bifunctional aldehyde/alcohol dehydrogenase (AdhE), respectively. Here we observed that AdhE is an important enzyme responsible for ethanol production in T. mathranii based on the constructed adh knockout strains. An adhE knockout strain fails to produce...

  15. A Case of Hyperammonemia Associated with High Dihydropyrimidine Dehydrogenase Activity

    Nagaharu, Keiki; Ikemura, Kenji; Yamashita, Yoshiki; Oda, Hiroyasu; Ishihara, Mikiya; Sugawara, Yumiko; Tamaru, Satoshi; Mizuno, Toshiro; Katayama, Naoyuki

    2016-01-01

    Over the past decades, 5-Fluorouracil (5-FU) has been widely used to treat several types of carcinoma, including esophageal squamous cell carcinoma. In addition to its common side effects, including diarrhea, mucositis, neutropenia, and anemia, 5-FU treatment has also been reported to cause hyperammonemia. However, the exact mechanism responsible for 5-FU-induced hyperammonemia remains unknown. We encountered an esophageal carcinoma patient who developed hyperammonemia when receiving 5-FU-containing chemotherapy but did not exhibit any of the other common adverse effects of 5-FU treatment. At the onset of hyperammonemia, laboratory tests revealed high dihydropyrimidine dehydrogenase (DPD) activity and rapid 5-FU clearance. Our findings suggested that 5-FU hypermetabolism may be one of the key mechanisms responsible for hyperammonemia during 5-FU treatment. PMID:27195162

  16. Mellemkaedet acyl-CoA dehydrogenase (MCAD)-mangel

    Gregersen, N; Winter, V; Andresen, B S;

    1992-01-01

    today considered more common than previously anticipated, since the incidence of patients with MCAD enzyme deficiency in Denmark is estimated to 1/27,000 newborns, or two new cases annually. The relationship between the enzyme defect (gene defect) and the clinical expression of the disease is a main......Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is a potentially fatal metabolic disease, which is characterized by non-ketotic hypoglycemia and lethargy. The disease manifests itself by periodic attacks in connection with infections and periods of fasting, or suddenly as unexpected child......-card constitute today a certain and specific diagnosis for the disease in 75% of all cases. In the remaining 25% the mutation analysis is supplemented with urine metabolite studies by gas chromatography/mass spectrometry, and with measurements of enzyme activities in cultured skin fibroblasts. The disease is...

  17. Phosphorylation of formate dehydrogenase in potato tuber mitochondria

    Bykova, N.V.; Stensballe, A.; Egsgaard, H.;

    2003-01-01

    Two highly phosphorylated proteins were detected after two-dimensional (blue native/SDS-PAGE) gel electrophoretic separation of the matrix fraction isolated from potato tuber mitochondria. These two phosphoproteins were identified by mass spectrometry as formate dehydrogenase (FDH) and the E1alpha...... extent of phosphorylation of both FDH and PDH was strongly decreased by NAD+, formate, and pyruvate, indicating that reversible phosphorylation of FDH and PDHs was regulated in a similar fashion. At low oxygen concentrations inside the intact potato tubers, FDH activity was strongly increased relative to...... cytochrome c oxidase activity pointing to a possible involvement of FDH in hypoxic metabolism. Computational sequence analysis indicated that a conserved local sequence motif of pyruvate formate-lyase is found in the Arabidopsis thaliana genome, and this enzyme might be the source of formate for FDH in...

  18. IMP Dehydrogenase: Structural Schizophrenia and an Unusual Base

    Hedstrom,L.; Gan, L.

    2006-01-01

    Textbooks describe enzymes as relatively rigid templates for the transition state of a chemical reaction, and indeed an enzyme such as chymotrypsin, which catalyzes a relatively simple hydrolysis reaction, is reasonably well described by this model. Inosine monophosphate dehydrogenase (IMPDH) undergoes a remarkable array of conformational transitions in the course of a complicated catalytic cycle, offering a dramatic counterexample to this view. IMPDH displays several other unusual mechanistic features, including an Arg residue that may act as a general base catalyst and a dynamic monovalent cation site. Further, IMPDH appears to be involved in 'moon-lighting' functions that may require additional conformational states. How the balance between conformational states is maintained and how the various conformational states interconvert is only beginning to be understood.

  19. Glucose-6 phosphate dehydrogenase deficiency and psychotic illness

    Vijender Singh

    2012-01-01

    Full Text Available Mr. T, a 28-year-old unmarried male, a diagnosed case of Glucose-6 Phosphate Dehydrogenase (G6PD deficiency since childhood, presented with 13 years of psychotic illness and disturbed biological functions. He showed poor response to antipsychotics and mood stabilizers and had three prior admissions to Psychiatry. There was a family history of psychotic illness. The General Physical Examination and Systemic Examination were unremarkable. Mental Status Examination revealed increased psychomotor activity, pressure of speech, euphoric affect, prolixity, delusion of persecution, delusion of grandiosity, delusion of control, thought withdrawal and thought insertion, and second and third person auditory hallucinations, with impaired judgment and insight. A diagnosis of schizophrenia paranoid type, with a differential diagnosis of schizoaffective disorder manic subtype, was made. This case is being reported for its rarity and atypicality of clinical presentation, as well as a course of psychotic illness in the G6PD Deficiency state,with its implications on management.

  20. Frostbite: A Novel Presentation of Glucose-6-Phosphate Dehydrogenase Deficiency?

    Bowles, Justin M; Joas, Chris; Head, Steven

    2015-01-01

    Acute hemolytic anemia (AHA) due to glucose 6-phosphate dehydrogenase (G6PD) deficiency has rarely been recognized as a contributor to the development of frostbite. We discuss a case of frostbite in a 32-year-old male Marine with G6PD deficiency during military training on Mount McKinley in Alaska, which eventually led to a permanent disability. In this report, the pathophysiology of G6PD deficiency, the effects of hemolytic anemia, and factors that contribute to frostbite will be discussed, as well as the clinical findings, treatment course, and the outcome of this case. The patient was evacuated and admitted to Alaska Regional Hospital. He was treated for fourth-degree frostbite, ultimately resulting in the complete or partial amputation of all toes. Although it cannot be proved that AHA occurred in this patient, this case potentially adds frostbite to the list of rare but possible clinical presentations of G6PD deficiency. PMID:26360347

  1. Kawasaki disease with Glucose-6-Phosphate Dehydrogenase deficiency, case report.

    Obeidat, Hesham Radi; Al-Dossary, Sahar; Asseri, Abdulsalam

    2015-09-01

    Kawasaki disease (KD) is an acute, self-limited vasculitis of unknown etiology that occurs predominantly in infants and children younger than 5 years of age. Coronary artery abnormalities are the most serious complication. Based on the literatures infusion of Intravenous Immunoglobulin of 2 g/kg and a high dose of oral aspirin up to 100 mg/kg/day are the standard treatment for Kawasaki disease in the acute stage, and should be followed by antiplatelet dose of aspirin for thrombocytosis. Glucose-6-Phosphate Dehydrogenase (G6PD) deficiency is an inherited X-linked hereditary disorder, and aspirin can induce hemolysis in patients with G6PD deficiency. We report a case of a 5 year and 8 month old male with KD and G6PD deficiency. PMID:27134550

  2. Microbial metabolic activity in soil as measured by dehydrogenase determinations

    Casida, L. E., Jr.

    1977-01-01

    The dehydrogenase technique for measuring the metabolic activity of microorganisms in soil was modified to use a 6-h, 37 C incubation with either glucose or yeast extract as the electron-donating substrate. The rate of formazan production remained constant during this time interval, and cellular multiplication apparently did not occur. The technique was used to follow changes in the overall metabolic activities of microorganisms in soil undergoing incubation with a limiting concentration of added nutrient. The sequence of events was similar to that obtained by using the Warburg respirometer to measure O2 consumption. However, the major peaks of activity occurred earlier with the respirometer. This possibly is due to the lack of atmospheric CO2 during the O2 consumption measurements.

  3. Engineered PQQ-Glucose Dehydrogenase as a Universal Biosensor Platform.

    Guo, Zhong; Murphy, Lindy; Stein, Viktor; Johnston, Wayne A; Alcala-Perez, Siro; Alexandrov, Kirill

    2016-08-17

    Biosensors with direct electron output hold promise for nearly seamless integration with portable electronic devices. However, so far, they have been based on naturally occurring enzymes that significantly limit the spectrum of detectable analytes. Here, we present a novel biosensor architecture based on analyte-driven intermolecular recombination and activity reconstitution of a re-engineered component of glucometers: PQQ-glucose dehydrogenase. We demonstrate that this sensor architecture can be rapidly adopted for the detection of immunosuppressant drugs, α-amylase protein, or protease activity of thrombin and Factor Xa. The biosensors could be stored in dried form without appreciable loss of activity. We further show that ligand-induced activity of the developed biosensors could be directly monitored by chronoamperometry, enabling construction of disposable sensory electrodes. We expect that this architecture could be expanded to the detection of other biochemical activities, post-translational modifications, nucleic acids, and inorganic molecules. PMID:27463000

  4. Alcohol dehydrogenase polymorphism in barrel cactus populations of Drosophila mojavensis.

    Cleland, S; Hocutt, G D; Breitmeyer, C M; Markow, T A; Pfeiler, E

    1996-07-01

    Starch gel electrophoresis revealed that the alcohol dehydrogenase (ADH-2) locus was polymorphic in two populations (from Agua Caliente, California and the Grand Canyon, Arizona) of cactophilic Drosophila mojavensis that utilize barrel cactus (Ferocactus acanthodes) as a host plant. Electromorphs representing products of a slow (S) and a fast (F) allele were found in adult flies. The frequency of the slow allele was 0.448 in flies from Agua Caliente and 0.659 in flies from the Grand Canyon. These frequencies were intermediate to those of the low (Baja California peninsula, Mexico) and high (Sonora, Mexico and southern Arizona) frequency Adh-2S populations of D. mojavensis that utilize different species of host cacti. PMID:8765684

  5. Lactate dehydrogenase assay for assessment of polycation cytotoxicity

    Parhamifar, Ladan; Andersen, Helene; Moghimi, Seyed Moien

    2013-01-01

    Cellular toxicity and/or cell death entail complex mechanisms that require detailed evaluation for proper characterization. A detailed mechanistic assessment of cytotoxicity is essential for design and construction of more effective polycations for nucleic acid delivery. A single toxicity assay...... cannot stand alone in determining the type and extent of damage or cell death mechanism. In this chapter we describe a lactate dehydrogenase (LDH) assay for high-throughput screening that can be used as a starting point for further detailed cytotoxicity determination. LDH release is considered an early...... event in necrosis but a late event in apoptosis. An accurate temporal assessment of the toxic responses is crucial as late apoptosis may convert into necrosis as well as in situations where cell death is initiated without any visible cell morphological changes or responses in assays measuring late...

  6. Circadian rhythm of lactate dehydrogenase in rat testis.

    Vermouth, N T; Ponce, R H; Carriazo, C S; Blanco, A

    1984-01-01

    Activity of total lactate dehydrogenase (LDH) and of the isozyme X (LDH X or C4) have been determined at 2 hr intervals during 24 hr cycles in testis of adult rats maintained since birth in a photoperiod of 14 hr light: 10 hr dark. LDH X activity of epididymal sections (caput, corpus and cauda) from the same animals was also determined. Total LDH and LDH X activities in testis exhibited circadian rhythms with different timing. LDH X in the three portions of epididymis showed diurnal variations similar to those in testis. Rats subjected to constant light or constant dark presented marked modifications of LDH X profiles, indicating that the photoperiod plays a synchronizer role. While total soluble proteins did not show variations in testis of rats exposed to the photoperiod, a circadian rhythm was demonstrated in animals maintained in constant light or dark. PMID:6467917

  7. Over-Expression, Purification and Crystallization of Human Dihydrolipoamide Dehydrogenase

    Hong, Y. S.; Ciszak, Ewa; Patel, Mulchand

    2000-01-01

    Dehydrolipoamide dehydrogenase (E3; dihydrolipoan-tide:NAD+ oxidoreductase, EC 1.8.1.4) is a common catalytic component found in pyruvate dehydrogenase complex, alpha-ketoglutarate dehydrogenase complex, and branched-chain cc-keto acid dehydrogenase complex. E3 is also a component (referred to as L protein) of the glycine cleavage system in bacterial metabolism (2). Active E3 forms a homodimer with four distinctive subdomain structures (FAD binding, NAD+ binding, central and interface domains) with non-covalently but tightly bound FAD in the holoenzyme. Deduced amino acids from cloned full-length human E3 gene showed a total of 509 amino acids with a leader sequence (N-terminal 35 amino acids) that is excised (mature form) during transportation of expressed E3 into mitochondria membrane. So far, three-dimensional structure of human E3 has not been reported. Our effort to achieve the elucidation of the X-ray crystal structure of human E3 will be presented. Recombinant pPROEX-1 expression vector (from GIBCO BRL Life Technologies) having the human E3 gene without leader sequence was constructed by Polymerase Chain Reaction (PCR) and subsequent ligation, and cloned in E.coli XL1-Blue by transformation. Since pPROEX-1 vector has an internal His-tag (six histidine peptide) located at the upstream region of a multicloning site, one-step affinity purification of E3 using nickelnitriloacetic acid (Ni-NTA) agarose resin, which has a strong affinity to His-tag, was feasible. Also a seven-amino-acid spacer peptide and a recombinant tobacco etch virus protease recognition site (seven amino acids peptide) found between His-tag and first amino acid of expressed E3 facilitated the cleavage of His-tag from E3 after the affinity purification. By IPTG induction, ca. 15 mg of human E3 (mature form) was obtained from 1L LB culture with overnight incubation at 25C. Over 98% of purity of E3 from one-step Ni-NTA agarose affinity purification was confirmed by SDS-PAGE analysis. For

  8. Cardiac-specific succinate dehydrogenase deficiency in Barth syndrome.

    Dudek, Jan; Cheng, I-Fen; Chowdhury, Arpita; Wozny, Katharina; Balleininger, Martina; Reinhold, Robert; Grunau, Silke; Callegari, Sylvie; Toischer, Karl; Wanders, Ronald Ja; Hasenfuß, Gerd; Brügger, Britta; Guan, Kaomei; Rehling, Peter

    2015-01-01

    Barth syndrome (BTHS) is a cardiomyopathy caused by the loss of tafazzin, a mitochondrial acyltransferase involved in the maturation of the glycerophospholipid cardiolipin. It has remained enigmatic as to why a systemic loss of cardiolipin leads to cardiomyopathy. Using a genetic ablation of tafazzin function in the BTHS mouse model, we identified severe structural changes in respiratory chain supercomplexes at a pre-onset stage of the disease. This reorganization of supercomplexes was specific to cardiac tissue and could be recapitulated in cardiomyocytes derived from BTHS patients. Moreover, our analyses demonstrate a cardiac-specific loss of succinate dehydrogenase (SDH), an enzyme linking the respiratory chain with the tricarboxylic acid cycle. As a similar defect of SDH is apparent in patient cell-derived cardiomyocytes, we conclude that these defects represent a molecular basis for the cardiac pathology in Barth syndrome. PMID:26697888

  9. 17 beta-hydroxysteroid dehydrogenase activity in canine pancreas

    The mitochondrial fraction of the dog pancreas showed NAD(H)-dependent enzyme activity of 17 beta-hydroxysteroid dehydrogenase. The enzyme catalyzes oxidoreduction between androstenedione and testosterone. The apparent Km value of the enzyme for androstenedione was 9.5 +/- 0.9 microM, the apparent Vmax was determined as 0.4 nmol mg-1 min-1, and the optimal pH was 6.5. In phosphate buffer, pH 7.0, maximal rate of androstenedione reduction was observed at 37 degrees C. The oxidation of testosterone by the enzyme proceeded at the same rate as the reduction of the androstenedione at a pH of 6.8-7.0. The apparent Km value and the optimal pH of the enzyme for testosterone were 3.5 +/- 0.5 microM and 7.5, respectively

  10. Lactate dehydrogenase (LDH isoenzymes patterns in ocular tumours

    Singh Rajendra

    1991-01-01

    Full Text Available Estimation of lactate dehydrogenase (LDH isoenzymes in the serum and aqueous humor was carried out in 15 cases of benign ocular tumour, 15 cases of malignant tumor and 15 normal cases. Cases of both sexes aged between 1 year and 75 years were included. LDH, isoenzymes specially LDH4 and LDH5 are higher and LDH1 and LDH2 lower in sera of patients with malignant tumor specially retinoblastoma as compared to benign tumor cases and control cases. LDH isoenzymes in aqueous humor are significantly higher and show a characteristic pattern in retinoblastoma cases, the concentration was presumably too low in the control, malignant tumor other than retinoblastoma and benign tumor cases as its fractionation was not possible.

  11. Evaluation of Serum Lactate Dehydrogenase Activity in a Virtual Environment

    V.M.T. Trindade

    2013-05-01

    Full Text Available Introduction: Lactate dehydrogenase is a citosolic enzyme involved in reversible transformation of pyruvate to lactate. It participates in anaerobic glycolysis of skeletal muscle and red blood cells, in liver gluconeogenesis and in aerobic metabolism of heart muscle. The determination of its activity helps in the diagnosis of various diseases, because it is increased in serum of patients suffering from myocardial infarction, acute hepatitis, muscular dystrophy and cancer. This paper presents a learning object, mediated by computer, which contains the simulation of the laboratory determination serum lactate dehydrogenase activity measured by the spectrophotometric method, based in the decrease of absorbance at 340 nm. Materials and Methods: Initially, pictures and videos were obtained recording the procedure of the methodology. The most representative images were selected, edited and inserted into an animation developed with the aid of the tool Adobe ® Flash ® CS3. The validation of the object was performed by the students of Biochemistry I (Pharmacy-UFRGS from the second semester of 2009 and both of 2010. Results and Discussion: The analysis of students' answers revealed that 80% attributed the excellence of the navigation program, the display format and to aid in learning. Conclusion: Therefore, this software can be considered an adequate teaching resource as well as an innovative support in the construction of theoretical and practical knowledge of Biochemistry. Available at: http://www6.ufrgs.br/gcoeb/LDH

  12. Pyruvate Dehydrogenase Kinase as a Novel Therapeutic Target in Oncology

    Gopinath eSutendra

    2013-03-01

    Full Text Available Current drug development in oncology is non-selective as it typically focuses on pathways essential for the survival of all dividing cells. The unique metabolic profile of cancer, which is characterized by increased glycolysis and suppressed mitochondrial glucose oxidation provides cancer cells with a proliferative advantage, conducive with apoptosis resistance and even increased angiogenesis. Recent evidence suggests that targeting the cancer-specific metabolic and mitochondrial remodeling may offer selectivity in cancer treatment. Pyruvate dehydrogenase kinase (PDK is a mitochondrial enzyme that is activated in a variety of cancers and results in the selective inhibition of pyruvate dehydrogenase (PDH, a complex of enzymes that converts cytosolic pyruvate to mitochondrial acetyl-CoA, the substrate for the Krebs’ cycle. Inhibition of PDK with either small interfering RNAs or the orphan drug dichloroacetate (DCA shifts the metabolism of cancer cells from glycolysis to glucose oxidation and reverses the suppression of mitochondria-dependent apoptosis. In addition, this therapeutic strategy increases the production of diffusible Krebs’ cycle intermediates and mitochondria-derived reactive oxygen species (mROS, activating p53 or inhibiting pro-proliferative and pro-angiogenic transcription factors like nuclear factor of activated T-cells (NFAT and hypoxia-inducible factor 1α (HIF1α. These effects result in decreased tumor growth and angiogenesis in a variety of cancers with high selectivity. In a small but mechanistic clinical trial in patients with glioblastoma, a highly aggressive and vascular form of brain cancer, DCA decreased tumor angiogenesis and tumor growth, suggesting that metabolic targeting therapies can be translated directly to patients. Therefore, reversing the mitochondrial suppression with metabolic-modulating drugs, like PDK inhibitors holds promise in the rapidly expanding field of metabolic oncology.

  13. Immunohistochemical analysis of aldehyde dehydrogenase isoforms and their association with estrogen-receptor status and disease progression in breast cancer

    Opdenaker, Lynn M; Arnold, Kimberly M; Pohlig, Ryan T; Padmanabhan, Jayasree S; Flynn, Daniel C; Sims-Mourtada, Jennifer

    2014-01-01

    In many types of tumors, especially breast tumors, aldehyde dehydrogenase (ALDH) activity has been used to identify cancer stem-like cells within the tumor. The presence and quantity of these cells are believed to predict the response of tumors to chemotherapy. Therefore, identification and eradication of these cells would be necessary to cure the patient. However, there are 19 different ALDH isoforms that could contribute to the enzyme activity. ALDH1A1 and ALDH1A3 are among the isoforms mostly responsible for the increased ALDH activity observed in these stem-like cells, although the main isoforms vary in different tissues and tumor types. In the study reported here, we attempted to determine if ALDH1A1 or ALDH1A3, specifically, correlate with tumor stage, grade, and hormone-receptor status in breast-cancer patients. While there was no significant correlation between ALDH1A1 and any of the parameters tested, we were able to identify a positive correlation between ALDH1A3 and tumor stage in triple-negative cancers. In addition, ALDH1A3 was negatively correlated with estrogen-receptor status. Our data suggest that ALDH1A3 could be utilized as a marker to identify stem-like cells within triple-negative tumors. PMID:25540596

  14. Catalytic properties of Sepharose-bound L-alanine dehydrogenase from Bacillus cereus.

    Mureşan, L; Vancea, D; Presecan, E; Porumb, H; Lascu, I; Oargă, M; Matinca, D; Abrudan, I; Bârzu, O

    1983-02-15

    (1) L-Alanine dehydrogenase from Bacillus cereus was purified by a two-step chromatographic procedure involving Cibacron-Blue 3G-A Sepharose 4B-CL, and Sepharose 6B-CL, and immobilized on CNBr-activated Sepharose 4B. (2) Following immobilization via two of the six subunits, L-alanine dehydrogenase retained 66% of the specific activity of the soluble enzyme. The affinity of the immobilized enzyme for NH4+, pyruvate and L-alanine, was not different to that of the soluble form. The Km of the Sepharose-bound L-alanine dehydrogenase for pyridine coenzymes was 6-8-times higher than in the soluble case. (3) The stability of L-alanine dehydrogenase towards urea or thermal denaturation was increased by immobilization. (4) The incubation at 37 degrees C for 24 h of the immobilized L-alanine dehydrogenase with 3 M NH4Cl/NH4OH buffer (pH 9) released 70% of the enzyme. The specific activity and the affinity of the 'solubilized' L-alanine dehydrogenase for the pyridine coenzymes was the same as that obtained with the original, soluble L-alanine dehydrogenase. PMID:6404304

  15. Hexose-6-phosphate dehydrogenase contributes to skeletal muscle homeostasis independent of 11β-hydroxysteroid dehydrogenase type 1.

    Semjonous, Nina M

    2011-01-01

    Glucose-6-phosphate (G6P) metabolism by the enzyme hexose-6-phosphate dehydrogenase (H6PDH) within the sarcoplasmic reticulum lumen generates nicotinamide adenine dinucleotide phosphate (reduced) to provide the redox potential for the enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) to activate glucocorticoid (GC). H6PDH knockout (KO) mice have a switch in 11β-HSD1 activity, resulting in GC inactivation and hypothalamic-pituitary-adrenal axis activation. Importantly, H6PDHKO mice develop a type II fiber myopathy with abnormalities in glucose metabolism and activation of the unfolded protein response (UPR). GCs play important roles in muscle physiology, and therefore, we have examined the importance of 11β-HSD1 and GC metabolism in mediating aspects of the H6PDHKO myopathy. To achieve this, we examined 11β-HSD1\\/H6PDH double-KO (DKO) mice, in which 11β-HSD1 mediated GC inactivation is negated. In contrast to H6PDHKO mice, DKO mice GC metabolism and hypothalamic-pituitary-adrenal axis set point is similar to that observed in 11β-HSD1KO mice. Critically, in contrast to 11β-HSD1KO mice, DKO mice phenocopy the salient features of the H6PDHKO, displaying reduced body mass, muscle atrophy, and vacuolation of type II fiber-rich muscle, fasting hypoglycemia, increased muscle glycogen deposition, and elevated expression of UPR genes. We propose that muscle G6P metabolism through H6PDH may be as important as changes in the redox environment when considering the mechanism underlying the activation of the UPR and the ensuing myopathy in H6PDHKO and DKO mice. These data are consistent with an 11β-HSD1-independent function for H6PDH in which sarcoplasmic reticulum G6P metabolism and nicotinamide adenine dinucleotide phosphate-(oxidized)\\/nicotinamide adenine dinucleotide phosphate (reduced) redox status are important for maintaining muscle homeostasis.

  16. Dimerization and enzymatic activity of fungal 17β-hydroxysteroid dehydrogenase from the short-chain dehydrogenase/reductase superfamily

    Kristan Katja

    2005-12-01

    Full Text Available Abstract Background 17β-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus (17β-HSDcl is a member of the short-chain dehydrogenase/reductase (SDR superfamily. SDR proteins usually function as dimers or tetramers and 17β-HSDcl is also a homodimer under native conditions. Results We have investigated here which secondary structure elements are involved in the dimerization of 17β-HSDcl and examined the importance of dimerization for the enzyme activity. Sequence similarity with trihydroxynaphthalene reductase from Magnaporthe grisea indicated that Arg129 and His111 from the αE-helices interact with the Asp121, Glu117 and Asp187 residues from the αE and αF-helices of the neighbouring subunit. The Arg129Asp and His111Leu mutations both rendered 17β-HSDcl monomeric, while the mutant 17β-HSDcl-His111Ala was dimeric. Circular dichroism spectroscopy analysis confirmed the conservation of the secondary structure in both monomers. The three mutant proteins all bound coenzyme, as shown by fluorescence quenching in the presence of NADP+, but both monomers showed no enzymatic activity. Conclusion We have shown by site-directed mutagenesis and structure/function analysis that 17β-HSDcl dimerization involves the αE and αF helices of both subunits. Neighbouring subunits are connected through hydrophobic interactions, H-bonds and salt bridges involving amino acid residues His111 and Arg129. Since the substitutions of these two amino acid residues lead to inactive monomers with conserved secondary structure, we suggest dimerization is a prerequisite for catalysis. A detailed understanding of this dimerization could lead to the development of compounds that will specifically prevent dimerization, thereby serving as a new type of inhibitor.

  17. Differential expression of aldehyde dehydrogenase 1a1 (ALDH1 in normal ovary and serous ovarian tumors

    Penumatsa Krishna

    2010-12-01

    Full Text Available Abstract Background We showed there are specific ALDH1 autoantibodies in ovarian autoimmune disease and ovarian cancer, suggesting a role for ALDH1 in ovarian pathology. However, there is little information on the ovarian expression of ALDH1. Therefore, we compared ALDH1 expression in normal ovary and benign and malignant ovarian tumors to determine if ALDH1 expression is altered in ovarian cancer. Since there is also recent interest in ALDH1 as a cancer stem cell (CSC marker, we assessed co-expression of ALDH1 with CSC markers in order to determine if ALDH1 is a potential CSC marker in ovarian cancer. Methods mRNA and protein expression were compared in normal human ovary and serous ovarian tumors using quantitative Reverse-Transcriptase PCR, Western blot (WB and semi-quantitative immunohistochemistry (IHC. ALDH1 enzyme activity was confirmed in primary ovarian cells by flow cytometry (FC using ALDEFLUOR assay. Results ALDH1 mRNA expression was significantly reduced (p Conclusions Total ALDH1 expression is significantly reduced in malignant ovarian tumors while it is relatively unchanged in benign tumors compared to normal ovary. Thus, ALDH1 expression in the ovary does not appear to be similar to breast, lung or colon cancer suggesting possible functional differences in these cancers. Significance These observations suggest that reduced ALDH1 expression is associated with malignant transformation in ovarian cancer and provides a basis for further study of the mechanism of ALDH1 in this process.

  18. A new dawn for plant mitochondrial NAD(P)H dehydrogenases

    Møller, I.M.

    2002-01-01

    The expression of complex I and two homologues of bacterial and yeast NADH dehydrogenases, NDA and NDB, have been studied in potato leaf mitochondria. The mRNA level of NDA is completely light dependent and shows a diurnal rhythm with a sharp maximum just after dawn. NDA protein quantity and inte...... internal rotenone-insensitive NADH dehydrogenase activity are also light dependent. These findings suggest that NDA has a role in photorespiration and might be identical to the previously unidentified internal rotenone-insensitive NADH dehydrogenase....

  19. Purification of yeast alcohol dehydrogenase by using immobilized metal affinity cryogels

    Akduman, Begüm [Chemistry Department, Adnan Menderes University, Aydın (Turkey); Uygun, Murat [Koçarlı Vocational and Training School, Adnan Menderes University, Aydın (Turkey); Uygun, Deniz Aktaş, E-mail: daktas@adu.edu.tr [Chemistry Department, Adnan Menderes University, Aydın (Turkey); Akgöl, Sinan [Biochemistry Department, Ege University, İzmir (Turkey); Denizli, Adil [Chemistry Department, Hacettepe University, Ankara (Turkey)

    2013-12-01

    In this study, poly(2-hydroxyethyl methacrylate–glycidylmethacrylate) [poly(HEMA–GMA)] cryogels were prepared by radical cryocopolymerization of HEMA with GMA as a functional comonomer and N,N′-methylene-bisacrylamide (MBAAm) as a crosslinker. Iminodiacetic acid (IDA) functional groups were attached via ring opening of the epoxy group on the poly(HEMA–GMA) cryogels and then Zn(II) ions were chelated with these structures. Characterization of cryogels was performed by FTIR, SEM, EDX and swelling studies. These cryogels have interconnected pores of 30–50 μm size. The equilibrium swelling degree of Zn(II) chelated poly(HEMA–GMA)-IDA cryogels was approximately 600%. Zn(II) chelated poly(HEMA–GMA)-IDA cryogels were used in the adsorption of alcohol dehydrogenase from aqueous solutions and adsorption was performed in continuous system. The effects of pH, alcohol dehydrogenase concentration, temperature, and flow rate on adsorption were investigated. The maximum amount of alcohol dehydrogenase adsorption was determined to be 9.94 mg/g cryogel at 1.0 mg/mL alcohol dehydrogenase concentration and in acetate buffer at pH 5.0 with a flow rate of 0.5 mL/min. Desorption of adsorbed alcohol dehydrogenase was carried out by using 1.0 M NaCI at pH 8.0 phosphate buffer and desorption yield was found to be 93.5%. Additionally, these cryogels were used for purification of alcohol dehydrogenase from yeast with a single-step. The purity of desorbed alcohol dehydrogenase was shown by silver-stained SDS–PAGE. This purification process can successfully be used for the purification of alcohol dehydrogenase from unclarified yeast homogenates and this work is the first report about the usage of the cryogels for purification of alcohol dehydrogenase. - Highlights: • Poly(HEMA–GMA) cryogels were synthesized by radical cryocopolymerization technique. • Prepared cryogels were functionalized with IDA, then Zn(II) ions were chelated to the cryogel. • Zn(II) chelated poly

  20. Purification of yeast alcohol dehydrogenase by using immobilized metal affinity cryogels

    In this study, poly(2-hydroxyethyl methacrylate–glycidylmethacrylate) [poly(HEMA–GMA)] cryogels were prepared by radical cryocopolymerization of HEMA with GMA as a functional comonomer and N,N′-methylene-bisacrylamide (MBAAm) as a crosslinker. Iminodiacetic acid (IDA) functional groups were attached via ring opening of the epoxy group on the poly(HEMA–GMA) cryogels and then Zn(II) ions were chelated with these structures. Characterization of cryogels was performed by FTIR, SEM, EDX and swelling studies. These cryogels have interconnected pores of 30–50 μm size. The equilibrium swelling degree of Zn(II) chelated poly(HEMA–GMA)-IDA cryogels was approximately 600%. Zn(II) chelated poly(HEMA–GMA)-IDA cryogels were used in the adsorption of alcohol dehydrogenase from aqueous solutions and adsorption was performed in continuous system. The effects of pH, alcohol dehydrogenase concentration, temperature, and flow rate on adsorption were investigated. The maximum amount of alcohol dehydrogenase adsorption was determined to be 9.94 mg/g cryogel at 1.0 mg/mL alcohol dehydrogenase concentration and in acetate buffer at pH 5.0 with a flow rate of 0.5 mL/min. Desorption of adsorbed alcohol dehydrogenase was carried out by using 1.0 M NaCI at pH 8.0 phosphate buffer and desorption yield was found to be 93.5%. Additionally, these cryogels were used for purification of alcohol dehydrogenase from yeast with a single-step. The purity of desorbed alcohol dehydrogenase was shown by silver-stained SDS–PAGE. This purification process can successfully be used for the purification of alcohol dehydrogenase from unclarified yeast homogenates and this work is the first report about the usage of the cryogels for purification of alcohol dehydrogenase. - Highlights: • Poly(HEMA–GMA) cryogels were synthesized by radical cryocopolymerization technique. • Prepared cryogels were functionalized with IDA, then Zn(II) ions were chelated to the cryogel. • Zn(II) chelated poly

  1. Crystal structure of Saccharomyces cerevisiae 6-phosphogluconate dehydrogenase Gnd1

    Zhou Cong-Zhao

    2007-06-01

    Full Text Available Abstract Background As the third enzyme of the pentose phosphate pathway, 6-phosphogluconate dehydrogenase (6PGDH is the main generator of cellular NADPH. Both thioredoxin reductase and glutathione reductase require NADPH as the electron donor to reduce oxidized thioredoxin or glutathione (GSSG. Since thioredoxin and GSH are important antioxidants, it is not surprising that 6PGDH plays a critical role in protecting cells from oxidative stress. Furthermore the activity of 6PGDH is associated with several human disorders including cancer and Alzheimer's disease. The 3D structural investigation would be very valuable in designing small molecules that target this enzyme for potential therapeutic applications. Results The crystal structure of 6-phosphogluconate dehydrogenase (6PGDH/Gnd1 from Saccharomyces cerevisiae has been determined at 2.37 Å resolution by molecular replacement. The overall structure of Gnd1 is a homodimer with three domains for each monomer, a Rossmann fold NADP+ binding domain, an all-α helical domain contributing the majority to hydrophobic interaction between the two subunits and a small C-terminal domain penetrating the other subunit. In addition, two citrate molecules occupied the 6PG binding pocket of each monomer. The intact Gnd1 had a Km of 50 ± 9 μM for 6-phosphogluconate and of 35 ± 6 μM for NADP+ at pH 7.5. But the truncated mutants without the C-terminal 35, 39 or 53 residues of Gnd1 completely lost their 6PGDH activity, despite remaining the homodimer in solution. Conclusion The overall tertiary structure of Gnd1 is similar to those of 6PGDH from other species. The substrate and coenzyme binding sites are well conserved, either from the primary sequence alignment, or from the 3D structural superposition. Enzymatic activity assays suggest a sequential mechanism of catalysis, which is in agreement with previous studies. The C-terminal domain of Gnd1 functions as a hook to further tighten the dimer, but it is not

  2. A simple method for the rapid determination of the stereospecificity of NAD-dependent dehydrogenases applied to mammalian IMP dehydrogenase and bacterial NADH peroxidase.

    Cooney, D; Hamel, E; Cohen, M; Kang, G J; Dalal, M; Marquez, V

    1987-11-01

    The stereospecificity of IMP dehydrogenase (IMP:NAD+ oxidoreductase, EC 1.1.1.205) from two different sources was determined. The enzyme preparations were obtained from murine lymphoblasts and from Escherichia coli. Both enzymes transferred the 2-3H of IMP to the pro-S position of carbon atom C-4 of the nicotinamide ring in NAD. Thus, B-sided stereospecificity is common to the enzyme from two very different species. In addition, the studies described here demonstrate that alcohol dehydrogenase and NADH peroxidase, used as auxiliary enzymes, in combination with a microdistillation procedure, should permit rapid determination of the stereospecificity of any NAD-dependent dehydrogenase for which the appropriate tritiated substrate is available. PMID:2889473

  3. Kernicterus by glucose-6-phosphate dehydrogenase deficiency: a case report and review of the literature

    Cossio de Gurrola Gladys

    2008-05-01

    Full Text Available Abstract Introduction Glucose-6-phosphate dehydrogenase deficiency is an X-linked recessive disease that causes acute or chronic hemolytic anemia and potentially leads to severe jaundice in response to oxidative agents. This deficiency is the most common human innate error of metabolism, affecting more than 400 million people worldwide. Case presentation Here, we present the first documented case of kernicterus in Panama, in a glucose-6-phosphate dehydrogenase-deficient newborn clothed in naphthalene-impregnated garments, resulting in reduced psychomotor development, neurosensory hypoacousia, absence of speech and poor reflex of the pupil to light. Conclusion Mutational analysis revealed the glucose-6-phosphate dehydrogenase Mediterranean polymorphic variant, which explained the development of kernicterus after exposition of naphthalene. As the use of naphthalene in stored clothes is a common practice, glucose-6-phosphate dehydrogenase testing in neonatal screening could prevent severe clinical consequences.

  4. Structural Biology of Proteins of the Multi-enzyme Assembly Human Pyruvate Dehydrogenase Complex

    2003-01-01

    Objectives and research challenges of this effort include: 1. Need to establish Human Pyruvate Dehydrogenase Complex protein crystals; 2. Need to test value of microgravity for improving crystal quality of Human Pyruvate Dehydrogenase Complex protein crystals; 3. Need to improve flight hardware in order to control and understand the effects of microgravity on crystallization of Human Pyruvate Dehydrogenase Complex proteins; 4. Need to integrate sets of national collaborations with the restricted and specific requirements of flight experiments; 5. Need to establish a highly controlled experiment in microgravity with a rigor not yet obtained; 6. Need to communicate both the rigor of microgravity experiments and the scientific value of results obtained from microgravity experiments to the national community; and 7. Need to advance the understanding of Human Pyruvate Dehydrogenase Complex structures so that scientific and commercial advance is identified for these proteins.

  5. Glucose-6-Phosphate Dehydrogenase deficiency presented with convulsion: a rare case

    Alparslan Merdin

    2014-03-01

    Full Text Available Red blood cells carry oxygen in the body and Glucose-6-Phosphate Dehydrogenase protects these cells from oxidative chemicals. If there is a lack of Glucose-6-Phosphate Dehydrogenase, red blood cells can go acute hemolysis. Convulsion is a rare presentation for acute hemolysis due to Glucose-6-Phosphate Dehydrogenase deficiency. Herein, we report a case report of a Glucose-6-Phosphate Dehydrogenase deficiency diagnosed patient after presentation with convulsion. A 70 year-old woman patient had been hospitalized because of convulsion and fatigue. She has not had similar symptoms before. She had ingested fava beans in the last two days. Her hypophyseal and brain magnetic resonance imaging were normal. Blood transfusion was performed and the patient recovered.

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

    Zhang, Rongzhen; Xu, Yan; Xiao, Rong

    2015-12-01

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

  7. Induction of glutamate dehydrogenase in the ovine fetal liver by dexamethasone infusion during late gestation

    M. Timmerman (Michelle); R.B. Wilkening; T.R. Regnault

    2003-01-01

    textabstractGlucocorticoids near term are known to upregulate many important enzyme systems prior to birth. Glutamate dehydrogenase (GDH) is a mitochondrial enzyme that catalyzes both the reversible conversion of ammonium nitrogen into organic nitrogen (glutamate production) and th

  8. Asp295 Stabilizes the Active-Site Loop Structure of Pyruvate Dehydrogenase, Facilitating Phosphorylation of Ser292 by Pyruvate Dehydrogenase-Kinase

    Hirani, Tripty A.; Alejandro Tovar-Méndez; Miernyk, Ján A.; Randall, Douglas D.

    2011-01-01

    We have developed an in vitro system for detailed analysis of reversible phosphorylation of the plant mitochondrial pyruvate dehydrogenase complex, comprising recombinant Arabidopsis thalianaα2β2-heterotetrameric pyruvate dehydrogenase (E1) plus A. thaliana E1-kinase (AtPDK). Upon addition of MgATP, Ser292, which is located within the active-site loop structure of E1α, is phosphorylated. In addition to Ser292, Asp295 and Gly297 are highly conserved in the E1α active-site loop sequences. Mutat...

  9. Nuclear lactate dehydrogenase modulates histone modification in human hepatocytes

    Highlights: • Nuclear LDH is up-regulated under oxidative stress. • SIRT1 is co-immunoprecipitated bound to nuclear LDH. • Nuclear LDH is involved in histone deacetylation and epigenetics. - Abstract: It is becoming increasingly apparent that the nucleus harbors metabolic enzymes that affect genetic transforming events. Here, we describe a nuclear isoform of lactate dehydrogenase (nLDH) and its ability to orchestrate histone deacetylation by controlling the availability of nicotinamide adenine dinucleotide (NAD+), a key ingredient of the sirtuin-1 (SIRT1) deacetylase system. There was an increase in the expression of nLDH concomitant with the presence of hydrogen peroxide (H2O2) in the culture medium. Under oxidative stress, the NAD+ generated by nLDH resulted in the enhanced deacetylation of histones compared to the control hepatocytes despite no discernable change in the levels of SIRT1. There appeared to be an intimate association between nLDH and SIRT1 as these two enzymes co-immunoprecipitated. The ability of nLDH to regulate epigenetic modifications by manipulating NAD+ reveals an intricate link between metabolism and the processing of genetic information

  10. Phosphorylation of the pyruvate dehydrogenase complex isolated from Ascaris suum

    Thissen, J.; Komuniecki, R.

    1987-05-01

    The pyruvate dehydrogenase complex (PDC) from body wall muscle of the porcine nematode, Ascaris suum, plays a pivotal role in anaerobic mitochondrial metabolism. As in mammalian mitochondria, PDC activity is inhibited by the phosphorylation of the ..cap alpha..PDH subunit, catalyzed by an associated PDH/sub a/ kinase. However, in contrast to PDC's isolated from all other eukaryotic sources, phosphorylation decreases the mobility of the ..cap alpha..PDH subunit on SDS-PAGE and permits the separation of the phosphorylated and nonphosphorylated ..cap alpha..PDH's. Phosphorylation and the inactivation of the Ascaris PDC correspond directly, and the additional phosphorylation that occurs after complete inactivation in mammalian PDC's is not observed. The purified ascarid PDC incorporates 10 nmoles /sup 32/P/mg P. Autoradiography of the radiolabeled PDC separated by SDS-PAGE yields a band which corresponds to the phosphorylated ..cap alpha..PDH and a second, faint band which is present only during the first three minutes of PDC inactivation, intermediate between the phosphorylated and nonphosphorylated ..cap alpha..PDH subunit. Tryptic digests of the /sup 32/P-PDC yields one major phosphopeptide, when separated by HPLC, and its amino acid sequence currently is being determined.

  11. Phosphorylation of the pyruvate dehydrogenase complex isolated from Ascaris suum

    The pyruvate dehydrogenase complex (PDC) from body wall muscle of the porcine nematode, Ascaris suum, plays a pivotal role in anaerobic mitochondrial metabolism. As in mammalian mitochondria, PDC activity is inhibited by the phosphorylation of the αPDH subunit, catalyzed by an associated PDH/sub a/ kinase. However, in contrast to PDC's isolated from all other eukaryotic sources, phosphorylation decreases the mobility of the αPDH subunit on SDS-PAGE and permits the separation of the phosphorylated and nonphosphorylated αPDH's. Phosphorylation and the inactivation of the Ascaris PDC correspond directly, and the additional phosphorylation that occurs after complete inactivation in mammalian PDC's is not observed. The purified ascarid PDC incorporates 10 nmoles 32P/mg P. Autoradiography of the radiolabeled PDC separated by SDS-PAGE yields a band which corresponds to the phosphorylated αPDH and a second, faint band which is present only during the first three minutes of PDC inactivation, intermediate between the phosphorylated and nonphosphorylated αPDH subunit. Tryptic digests of the 32P-PDC yields one major phosphopeptide, when separated by HPLC, and its amino acid sequence currently is being determined

  12. Alcoholism and alcohol drinking habits predicted from alcohol dehydrogenase genes

    Tolstrup, Janne Schurmann; Nordestgaard, Børge Grønne; Rasmussen, Søren;

    2008-01-01

    Alcohol drinking habits and alcoholism are partly genetically determined. Alcohol is degraded primarily by alcohol dehydrogenase (ADH) wherein genetic variation that affects the rate of alcohol degradation is found in ADH1B and ADH1C. It is biologically plausible that these variations may...... be associated with alcohol drinking habits and alcoholism. By genotyping 9080 white men and women from the general population, we found that men and women with ADH1B slow vs fast alcohol degradation drank more alcohol and had a higher risk of everyday drinking, heavy drinking, excessive drinking...... and of alcoholism. For example, the weekly alcohol intake was 9.8 drinks (95% confidence interval (CI): 9.1-11) among men with the ADH1B.1/1 genotype compared to 7.5 drinks (95% CI: 6.4-8.7) among men with the ADH1B.1/2 genotype, and the odds ratio (OR) for heavy drinking was 3.1 (95% CI: 1.7-5.7) among men...

  13. Alcoholism and alcohol drinking habits predicted from alcohol dehydrogenase genes

    Tolstrup, J.S.; Nordestgaard, Børge; Rasmussen, S.;

    2008-01-01

    Alcohol is degraded primarily by alcohol dehydrogenase (ADH) wherein genetic variation that affects the rate of alcohol degradation is found in ADH1B and ADH1C. It is biologically plausible that these variations may be associated with alcohol drinking habits and alcoholism. By genotyping 9080 white...... men and women from the general population, we found that men and women with ADH1B slow vs fast alcohol degradation drank more alcohol and had a higher risk of everyday drinking, heavy drinking, excessive drinking and of alcoholism. For example, the weekly alcohol intake was 9.8 drinks (95% confidence......, individuals with ADH1C slow vs fast alcohol degradation had a higher risk of heavy and excessive drinking. For example, the OR for heavy drinking was 1.4 (95% CI: 1.1-1.8) among men with the ADH1C.1/2 genotype and 1.4 (95% CI: 1.0-1.9) among men with the ADH1B.2/2 genotype, compared with men with the ADH1C.1...

  14. Aldehyde dehydrogenase inhibition as a pathogenic mechanism in Parkinson disease.

    Fitzmaurice, Arthur G; Rhodes, Shannon L; Lulla, Aaron; Murphy, Niall P; Lam, Hoa A; O'Donnell, Kelley C; Barnhill, Lisa; Casida, John E; Cockburn, Myles; Sagasti, Alvaro; Stahl, Mark C; Maidment, Nigel T; Ritz, Beate; Bronstein, Jeff M

    2013-01-01

    Parkinson disease (PD) is a neurodegenerative disorder particularly characterized by the loss of dopaminergic neurons in the substantia nigra. Pesticide exposure has been associated with PD occurrence, and we previously reported that the fungicide benomyl interferes with several cellular processes potentially relevant to PD pathogenesis. Here we propose that benomyl, via its bioactivated thiocarbamate sulfoxide metabolite, inhibits aldehyde dehydrogenase (ALDH), leading to accumulation of the reactive dopamine metabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL), preferential degeneration of dopaminergic neurons, and development of PD. This hypothesis is supported by multiple lines of evidence. (i) We previously showed in mice the metabolism of benomyl to S-methyl N-butylthiocarbamate sulfoxide, which inhibits ALDH at nanomolar levels. We report here that benomyl exposure in primary mesencephalic neurons (ii) inhibits ALDH and (iii) alters dopamine homeostasis. It induces selective dopaminergic neuronal damage (iv) in vitro in primary mesencephalic cultures and (v) in vivo in a zebrafish system. (vi) In vitro cell loss was attenuated by reducing DOPAL formation. (vii) In our epidemiology study, higher exposure to benomyl was associated with increased PD risk. This ALDH model for PD etiology may help explain the selective vulnerability of dopaminergic neurons in PD and provide a potential mechanism through which environmental toxicants contribute to PD pathogenesis. PMID:23267077

  15. SERUM VALUES OF ALKALINE PHOSPHATASE AND LACTATE DEHYDROGENASE IN OSTEOSARCOMA

    ZUMÁRRAGA, JUAN PABLO; BAPTISTA, ANDRÉ MATHIAS; ROSA, LUIS PABLO DE LA; CAIERO, MARCELO TADEU; CAMARGO, OLAVO PIRES DE

    2016-01-01

    ABSTRACT Objective: To study the relationship between the pre and post chemotherapy (CT) serum levels of alkaline phosphatase (AP) and lactate dehydrogenase (LDH), and the percentage of tumor necrosis (TN) found in specimens after the pre surgical CT in patients with osteosarcoma. Methods: Series of cases with retrospective evaluation of patients diagnosed with osteosarcoma. Participants were divided into two groups according to serum values of both enzymes. The values of AP and LDH were obtained before and after preoperative CT. The percentage of tumor necrosis (TN) of surgical specimens of each patient was also included. Results: One hundred and thirty seven medical records were included from 1990 to 2013. Both the AP as LDH decreased in the patients studied, being the higher in pre CT than post CT. The average LHD decrease was 795.12U/L and AP decrease was 437.40 U/L. The average TN was 34.10 %. There was no statistically significant correlation between the serums values and the percentage of tumoral necrosis. Conclusion: The serum levels values of AP and LDH are not good predictors for the chemotherapy-induced necrosis in patients with osteosarcoma. Level of Evidence IV, Case Series. PMID:27217815

  16. Novel inhibitors of mitochondrial sn-glycerol 3-phosphate dehydrogenase.

    Adam L Orr

    Full Text Available Mitochondrial sn-glycerol 3-phosphate dehydrogenase (mGPDH is a ubiquinone-linked enzyme in the mitochondrial inner membrane best characterized as part of the glycerol phosphate shuttle that transfers reducing equivalents from cytosolic NADH into the mitochondrial electron transport chain. Despite the widespread expression of mGPDH and the availability of mGPDH-null mice, the physiological role of this enzyme remains poorly defined in many tissues, likely because of compensatory pathways for cytosolic regeneration of NAD⁺ and mechanisms for glycerol phosphate metabolism. Here we describe a novel class of cell-permeant small-molecule inhibitors of mGPDH (iGP discovered through small-molecule screening. Structure-activity analysis identified a core benzimidazole-phenyl-succinamide structure as being essential to inhibition of mGPDH while modifications to the benzimidazole ring system modulated both potency and off-target effects. Live-cell imaging provided evidence that iGPs penetrate cellular membranes. Two compounds (iGP-1 and iGP-5 were characterized further to determine potency and selectivity and found to be mixed inhibitors with IC₅₀ and K(i values between ∼1-15 µM. These novel mGPDH inhibitors are unique tools to investigate the role of glycerol 3-phosphate metabolism in both isolated and intact systems.

  17. Detailed kinetics and regulation of mammalian 2-oxoglutarate dehydrogenase

    Dash Ranjan K

    2011-09-01

    Full Text Available Abstract Background Mitochondrial 2-oxoglutarate (α-ketoglutarate dehydrogenase complex (OGDHC, a key regulatory point of tricarboxylic acid (TCA cycle, plays vital roles in multiple pathways of energy metabolism and biosynthesis. The catalytic mechanism and allosteric regulation of this large enzyme complex are not fully understood. Here computer simulation is used to test possible catalytic mechanisms and mechanisms of allosteric regulation of the enzyme by nucleotides (ATP, ADP, pH, and metal ion cofactors (Ca2+ and Mg2+. Results A model was developed based on an ordered ter-ter enzyme kinetic mechanism combined with con-formational changes that involve rotation of one lipoic acid between three catalytic sites inside the enzyme complex. The model was parameterized using a large number of kinetic data sets on the activity of OGDHC, and validated by comparison of model predictions to independent data. Conclusions The developed model suggests a hybrid rapid-equilibrium ping-pong random mechanism for the kinetics of OGDHC, consistent with previously reported mechanisms, and accurately describes the experimentally observed regulatory effects of cofactors on the OGDHC activity. This analysis provides a single consistent theoretical explanation for a number of apparently contradictory results on the roles of phosphorylation potential, NAD (H oxidation-reduction state ratio, as well as the regulatory effects of metal ions on ODGHC function.

  18. Yeast cell-based analysis of human lactate dehydrogenase isoforms.

    Mohamed, Lulu Ahmed; Tachikawa, Hiroyuki; Gao, Xiao-Dong; Nakanishi, Hideki

    2015-12-01

    Human lactate dehydrogenase (LDH) has attracted attention as a potential target for cancer therapy and contraception. In this study, we reconstituted human lactic acid fermentation in Saccharomyces cerevisiae, with the goal of constructing a yeast cell-based LDH assay system. pdc null mutant yeast (mutated in the endogenous pyruvate decarboxylase genes) are unable to perform alcoholic fermentation; when grown in the presence of an electron transport chain inhibitor, pdc null strains exhibit a growth defect. We found that introduction of the human gene encoding LDHA complemented the pdc growth defect; this complementation depended on LDHA catalytic activity. Similarly, introduction of the human LDHC complemented the pdc growth defect, even though LDHC did not generate lactate at the levels seen with LDHA. In contrast, the human LDHB did not complement the yeast pdc null mutant, although LDHB did generate lactate in yeast cells. Expression of LDHB as a red fluorescent protein (RFP) fusion yielded blebs in yeast, whereas LDHA-RFP and LDHC-RFP fusion proteins exhibited cytosolic distribution. Thus, LDHB exhibits several unique features when expressed in yeast cells. Because yeast cells are amenable to genetic analysis and cell-based high-throughput screening, our pdc/LDH strains are expected to be of use for versatile analyses of human LDH. PMID:26126931

  19. Undetected Toxicity Risk in Pharmacogenetic Testing for Dihydropyrimidine Dehydrogenase

    Felicia Stefania Falvella

    2015-04-01

    Full Text Available Fluoropyrimidines, the mainstay agents for the treatment of colorectal cancer, alone or as a part of combination therapies, cause severe adverse reactions in about 10%–30% of patients. Dihydropyrimidine dehydrogenase (DPD, a key enzyme in the catabolism of 5-fluorouracil, has been intensively investigated in relation to fluoropyrimidine toxicity, and several DPD gene (DPYD polymorphisms are associated with decreased enzyme activity and increased risk of fluoropyrimidine-related toxicity. In patients carrying non-functional DPYD variants (c.1905+1G>A, c.1679T>G, c.2846A>T, fluoropyrimidines should be avoided or reduced according to the patients’ homozygous or heterozygous status, respectively. For other common DPYD variants (c.496A>G, c.1129-5923C>G, c.1896T>C, conflicting data are reported and their use in clinical practice still needs to be validated. The high frequency of DPYD polymorphism and the lack of large prospective trials may explain differences in studies’ results. The epigenetic regulation of DPD expression has been recently investigated to explain the variable activity of the enzyme. DPYD promoter methylation and its regulation by microRNAs may affect the toxicity risk of fluoropyrimidines. The studies we reviewed indicate that pharmacogenetic testing is promising to direct personalised dosing of fluoropyrimidines, although further investigations are needed to establish the role of DPD in severe toxicity in patients treated for colorectal cancer.

  20. Lactate Dehydrogenase in Hepatocellular Carcinoma: Something Old, Something New

    Faloppi, Luca; Bianconi, Maristella; Memeo, Riccardo; Casadei Gardini, Andrea; Giampieri, Riccardo; Bittoni, Alessandro; Andrikou, Kalliopi; Del Prete, Michela; Cascinu, Stefano; Scartozzi, Mario

    2016-01-01

    Hepatocellular carcinoma (HCC) is the most common primary liver tumour (80–90%) and represents more than 5.7% of all cancers. Although in recent years the therapeutic options for these patients have increased, clinical results are yet unsatisfactory and the prognosis remains dismal. Clinical or molecular criteria allowing a more accurate selection of patients are in fact largely lacking. Lactic dehydrogenase (LDH) is a glycolytic key enzyme in the conversion of pyruvate to lactate under anaerobic conditions. In preclinical models, upregulation of LDH has been suggested to ensure both an efficient anaerobic/glycolytic metabolism and a reduced dependence on oxygen under hypoxic conditions in tumour cells. Data from several analyses on different tumour types seem to suggest that LDH levels may be a significant prognostic factor. The role of LDH in HCC has been investigated by different authors in heterogeneous populations of patients. It has been tested as a potential biomarker in retrospective, small, and nonfocused studies in patients undergoing surgery, transarterial chemoembolization (TACE), and systemic therapy. In the major part of these studies, high LDH serum levels seem to predict a poorer outcome. We have reviewed literature in this setting trying to resume basis for future studies validating the role of LDH in this disease. PMID:27314036

  1. Expression of Plasmodium falciparum lactate dehydrogenase in Escherichia coli.

    Bzik, D J; Fox, B A; Gonyer, K

    1993-05-01

    A Plasmodium falciparum gene is described which encodes lactate dehydrogenase activity (P. falciparum LDH). The P. falciparum LDH gene contains no introns and is present in a single copy on chromosome 13. P. falciparum LDH was expressed in all asexual blood stages as a 1.6-kb mRNA. The predicted 316 amino acid protein coding region of P. falciparum LDH was inserted into the prokaryotic expression vector pKK223-3 and a 33-kDa protein having LDH activity was synthesized in Escherichia coli. P. falciparum LDH primary structure displays high amino acid similarity (50-57%) to vertebrate and bacterial LDH, but lacks the amino terminal extension observed in all vertebrate LDH. The majority of amino acid residues implicated in substrate and coenzyme binding and catalysis of other LDH are well conserved in P. falciparum LDH. However, several notable differences in amino acid composition were observed. P. falciparum LDH contained several distinctive single amino acid insertions and deletions compared to other LDH enzymes, and most remarkably, it contained a novel insertion of 5 amino acids within the conserved mobile loop region near arginine residue 109, a residue which is known to make contact with pyruvate in the ternary complex of other LDH. These results suggest that novel features of P. falciparum LDH primary structure may be correlated with previously characterized and distinctive kinetic, biochemical, immunochemical, and electrophoretic properties of P. falciparum LDH. PMID:8515777

  2. Nuclear lactate dehydrogenase modulates histone modification in human hepatocytes

    Castonguay, Zachary; Auger, Christopher; Thomas, Sean C.; Chahma, M’hamed; Appanna, Vasu D., E-mail: vappanna@laurentian.ca

    2014-11-07

    Highlights: • Nuclear LDH is up-regulated under oxidative stress. • SIRT1 is co-immunoprecipitated bound to nuclear LDH. • Nuclear LDH is involved in histone deacetylation and epigenetics. - Abstract: It is becoming increasingly apparent that the nucleus harbors metabolic enzymes that affect genetic transforming events. Here, we describe a nuclear isoform of lactate dehydrogenase (nLDH) and its ability to orchestrate histone deacetylation by controlling the availability of nicotinamide adenine dinucleotide (NAD{sup +}), a key ingredient of the sirtuin-1 (SIRT1) deacetylase system. There was an increase in the expression of nLDH concomitant with the presence of hydrogen peroxide (H{sub 2}O{sub 2}) in the culture medium. Under oxidative stress, the NAD{sup +} generated by nLDH resulted in the enhanced deacetylation of histones compared to the control hepatocytes despite no discernable change in the levels of SIRT1. There appeared to be an intimate association between nLDH and SIRT1 as these two enzymes co-immunoprecipitated. The ability of nLDH to regulate epigenetic modifications by manipulating NAD{sup +} reveals an intricate link between metabolism and the processing of genetic information.

  3. Structural and Kinetic Studies of Formate Dehydrogenase from Candida boidinii.

    Guo, Qi; Gakhar, Lokesh; Wickersham, Kyle; Francis, Kevin; Vardi-Kilshtain, Alexandra; Major, Dan T; Cheatum, Christopher M; Kohen, Amnon

    2016-05-17

    The structure of formate dehydrogenase from Candida boidinii (CbFDH) is of both academic and practical interests. First, this enzyme represents a unique model system for studies on the role of protein dynamics in catalysis, but so far these studies have been limited by the availability of structural information. Second, CbFDH and its mutants can be used in various industrial applications (e.g., CO2 fixation or nicotinamide recycling systems), and the lack of structural information has been a limiting factor in commercial development. Here, we report the crystallization and structural determination of both holo- and apo-CbFDH. The free-energy barrier for the catalyzed reaction was computed and indicates that this structure indeed represents a catalytically competent form of the enzyme. Complementing kinetic examinations demonstrate that the recombinant CbFDH has a well-organized reactive state. Finally, a fortuitous observation has been made: the apoenzyme crystal was obtained under cocrystallization conditions with a saturating concentration of both the cofactor (NAD(+)) and inhibitor (azide), which has a nanomolar dissociation constant. It was found that the fraction of the apoenzyme present in the solution is less than 1.7 × 10(-7) (i.e., the solution is 99.9999% holoenzyme). This is an extreme case where the crystal structure represents an insignificant fraction of the enzyme in solution, and a mechanism rationalizing this phenomenon is presented. PMID:27100912

  4. Group X Aldehyde Dehydrogenases of Pseudomonas aeruginosa PAO1 Degrade Hydrazones

    Taniyama, Kosuke; Itoh, Hideomi; Takuwa, Atsushi; Sasaki, Yasuyuki; Yajima, Shunsuke; Toyofuku, Masanori; Nomura, Nobuhiko; Takaya, Naoki

    2012-01-01

    Hydrazones are natural and synthetic compounds containing a C=N-N moiety. Here we found that the opportunistic pathogen Pseudomonas aeruginosa PAO1 produced NAD+- or NADP+-dependent hydrazone dehydrogenase (HDH), which converts hydrazones to the corresponding hydrazides and acids rather than to the simple hydrolytic product aldehydes. Gene cloning indicated that the HDH is part of the group X aldehyde dehydrogenase (ALDH) family, which is distributed among bacteria, although the physiological...

  5. Kernicterus by glucose-6-phosphate dehydrogenase deficiency: a case report and review of the literature

    Cossio de Gurrola Gladys; Araúz Juan; Durán Elfilda; Aguilar-Medina Maribel; Ramos-Payán Rosalío; García-Magallanes Noemí; Pacheco Gerardo; Arámbula Meraz Eliakym

    2008-01-01

    Abstract Introduction Glucose-6-phosphate dehydrogenase deficiency is an X-linked recessive disease that causes acute or chronic hemolytic anemia and potentially leads to severe jaundice in response to oxidative agents. This deficiency is the most common human innate error of metabolism, affecting more than 400 million people worldwide. Case presentation Here, we present the first documented case of kernicterus in Panama, in a glucose-6-phosphate dehydrogenase-deficient newborn clothed in nap...

  6. Cloning and Expression of a Xylitol-4-Dehydrogenase Gene from Pantoea ananatis

    Aarnikunnas, J. S.; Pihlajaniemi, A.; Palva, A; Leisola, M.; Nyyssölä, A.

    2006-01-01

    The Pantoea ananatis ATCC 43072 mutant strain is capable of growing with xylitol as the sole carbon source. The xylitol-4-dehydrogenase (XDH) catalyzing the oxidation of xylitol to l-xylulose was isolated from the cell extract of this strain. The N-terminal amino acid sequence of the purified protein was determined, and an oligonucleotide deduced from this peptide sequence was used to isolate the xylitol-4-dehydrogenase gene (xdh) from a P. ananatis gene library. Nucleotide sequence analysis ...

  7. Optimization of production, purification and lyophilisation of cellobiose dehydrogenase by Sclerotium rolfsii

    Fischer, Christin; Krause, Annett; Kleinschmidt, Thomas

    2014-01-01

    Background The enzyme cellobiose dehydrogenase (CDH) can be used to oxidize lactose to lactobionic acid. As Sclerotium rolfsii is known to be a good producer of CDH, the aim of this paper was to simplify its production and secondly to systematically study its purification aiming for a high yield. Two preservation methods (freezing and freeze-drying) and the influence of several protectants were investigated. Results Production of cellobiose dehydrogenase was optimized leading to a more simpli...

  8. Pyruvate Dehydrogenase Kinase 4 Promotes Vascular Calcification via SMAD1/5/8 Phosphorylation

    Sun Joo Lee; Ji Yun Jeong; Chang Joo Oh; Sungmi Park; Joon-Young Kim; Han-Jong Kim; Nam Doo Kim; Young-Keun Choi; Ji-Yeon Do; Younghoon Go; Chae-Myung Ha; Je-Yong Choi; Seung Huh; Nam Ho Jeoung; Ki-Up Lee

    2015-01-01

    Vascular calcification, a pathologic response to defective calcium and phosphate homeostasis, is strongly associated with cardiovascular mortality and morbidity. In this study, we have observed that pyruvate dehydrogenase kinase 4 (PDK4) is upregulated and pyruvate dehydrogenase complex phosphorylation is increased in calcifying vascular smooth muscle cells (VSMCs) and in calcified vessels of patients with atherosclerosis, suggesting that PDK4 plays an important role in vascular calcification...

  9. Search for Human Lactate Dehydrogenase A Inhibitors Using Structure-Based Modeling

    Nilov, D.; Prokhorova, E.; Švedas, V.

    2015-01-01

    The human lactate dehydrogenase isoform A plays an important role in the anaerobic metabolism of tumour cells and therefore constitutes an attractive target in the oncology field. Full-atom models of lactate dehydrogenase A (in complex with NADH and in the apo form) have been generated to enable structure-based design of novel inhibitors competing with pyruvate and NADH. The structural criteria for the selection of potential inhibitors were established, and virtual screening of a library of l...

  10. Soil Dehydrogenases as an Indicator of Contamination of the Environment with Petroleum Products

    Kaczyńska, Grażyna; Borowik, Agata; Wyszkowska, Jadwiga

    2015-01-01

    The aim of the research was to compare the effects of various petroleum products, biodiesel, diesel oil, fuel oil and unleaded petrol on soil dehydrogenases, and to evaluate biostimulation with compost and urea in the restoration of homeostasis of the soil contaminated with these products. The obtained results allowed for defining the weight of dehydrogenases in monitoring of the environment subjected to pressure from petroleum hydrocarbons. The studies were carried out under laboratory condi...

  11. Identification and functional characterization of isocitrate dehydrogenase 1 (IDH1) mutations in thyroid cancer

    Murugan, Avaniyapuram Kannan; Bojdani, Ermal; Xing, Mingzhao

    2010-01-01

    Mutations in the genes for isocitrate dehydrogenase 1 (IDH1) and isocitrate dehydrogenase 2 (IDH2) have been recently identified in glioblastoma. In the present study, we investigated IDH1 and IDH2 mutations in follicular thyroid cancer (FTC) and anaplastic thyroid cancer (ATC), with the latter, like glioblastoma, having a rapidly aggressive and lethal clinical course. By direct genomic DNA sequencing, we analyzed exon 4 of the IDH1 and IDH2 genes that harbored the mutation hot spots codon 13...

  12. Inhibiting sperm pyruvate dehydrogenase complex and its E3 subunit, dihydrolipoamide dehydrogenase affects fertilization in Syrian hamsters.

    Archana B Siva

    Full Text Available BACKGROUND/AIMS: The importance of sperm capacitation for mammalian fertilization has been confirmed in the present study via sperm metabolism. Involvement of the metabolic enzymes pyruvate dehydrogenase complex (PDHc and its E3 subunit, dihydrolipoamide dehydrogenase (DLD in hamster in vitro fertilization (IVF via in vitro sperm capacitation is being proposed through regulation of sperm intracellular lactate, pH and calcium. METHODOLOGY AND PRINCIPAL FINDINGS: Capacitated hamster spermatozoa were allowed to fertilize hamster oocytes in vitro which were then assessed for fertilization, microscopically. PDHc/DLD was inhibited by the use of the specific DLD-inhibitor, MICA (5-methoxyindole-2-carboxylic acid. Oocytes fertilized with MICA-treated (MT [and thus PDHc/DLD-inhibited] spermatozoa showed defective fertilization where 2nd polar body release and pronuclei formation were not observed. Defective fertilization was attributable to capacitation failure owing to high lactate and low intracellular pH and calcium in MT-spermatozoa during capacitation. Moreover, this defect could be overcome by alkalinizing spermatozoa, before fertilization. Increasing intracellular calcium in spermatozoa pre-IVF and in defectively-fertilized oocytes, post-fertilization rescued the arrest seen, suggesting the role of intracellular calcium from either of the gametes in fertilization. Parallel experiments carried out with control spermatozoa capacitated in medium with low extracellular pH or high lactate substantiated the necessity of optimal sperm intracellular lactate levels, intracellular pH and calcium during sperm capacitation, for proper fertilization. CONCLUSIONS: This study confirms the importance of pyruvate/lactate metabolism in capacitating spermatozoa for successful fertilization, besides revealing for the first time the importance of sperm PDHc/ DLD in fertilization, via the modulation of sperm intracellular lactate, pH and calcium during capacitation. In

  13. The Crystal Structure of Aquifex aeolicus Prephenate Dehydrogenase Reveals the Mode of Tyrosine Inhibition

    Sun, Warren; Shahinas, Dea; Bonvin, Julie; Hou, Wenjuan; Kimber, Matthew S.; Turnbull, Joanne; Christendat, Dinesh; (Guelph); (Toronto); (ConU)

    2009-08-14

    TyrA proteins belong to a family of dehydrogenases that are dedicated to l-tyrosine biosynthesis. The three TyrA subclasses are distinguished by their substrate specificities, namely the prephenate dehydrogenases, the arogenate dehydrogenases, and the cyclohexadienyl dehydrogenases, which utilize prephenate, l-arogenate, or both substrates, respectively. The molecular mechanism responsible for TyrA substrate selectivity and regulation is unknown. To further our understanding of TyrA-catalyzed reactions, we have determined the crystal structures of Aquifex aeolicus prephenate dehydrogenase bound with NAD(+) plus either 4-hydroxyphenylpyuvate, 4-hydroxyphenylpropionate, or l-tyrosine and have used these structures as guides to target active site residues for site-directed mutagenesis. From a combination of mutational and structural analyses, we have demonstrated that His-147 and Arg-250 are key catalytic and binding groups, respectively, and Ser-126 participates in both catalysis and substrate binding through the ligand 4-hydroxyl group. The crystal structure revealed that tyrosine, a known inhibitor, binds directly to the active site of the enzyme and not to an allosteric site. The most interesting finding though, is that mutating His-217 relieved the inhibitory effect of tyrosine on A. aeolicus prephenate dehydrogenase. The identification of a tyrosine-insensitive mutant provides a novel avenue for designing an unregulated enzyme for application in metabolic engineering.

  14. Pyruvate dehydrogenase kinase inhibition: Reversing the Warburg effect in cancer therapy

    Hayden Bell

    2016-06-01

    Full Text Available The poor efficacy of many cancer chemotherapeutics, which are often non-selective and highly toxic, is attributable to the remarkable heterogeneity and adaptability of cancer cells. The Warburg effect describes the up regulation of glycolysis as the main source of adenosine 5’-triphosphate in cancer cells, even under normoxic conditions, and is a unique metabolic phenotype of cancer cells. Mitochondrial suppression is also observed which may be implicated in apoptotic suppression and increased funneling of respiratory substrates to anabolic processes, conferring a survival advantage. The mitochondrial pyruvate dehydrogenase complex is subject to meticulous regulation, chiefly by pyruvate dehydrogenase kinase. At the interface between glycolysis and the tricarboxylic acid cycle, the pyruvate dehydrogenase complex functions as a metabolic gatekeeper in determining the fate of glucose, making pyruvate dehydrogenase kinase an attractive candidate in a bid to reverse the Warburg effect in cancer cells. The small pyruvate dehydrogenase kinase inhibitor dichloroacetate has, historically, been used in conditions associated with lactic acidosis but has since gained substantial interest as a potential cancer chemotherapeutic. This review considers the Warburg effect as a unique phenotype of cancer cells in-line with the history of and current approaches to cancer therapies based on pyruvate dehydrogenase kinase inhibition with particular reference to dichloroacetate and its derivatives.

  15. Immunohistochemical analysis of aldehyde dehydrogenase isoforms and their association with estrogen-receptor status and disease progression in breast cancer

    Opdenaker LM

    2014-12-01

    Full Text Available Lynn M Opdenaker,1,2 Kimberly M Arnold,1,3 Ryan T Pohlig,3,4 Jayasree S Padmanabhan,1 Daniel C Flynn,1,3 Jennifer Sims-Mourtada1–3 1Center for Translational Cancer Research, Helen F Graham Cancer Center, Christiana Care Health Services, Inc., Newark, Delaware, USA; 2Department of Biological Sciences, 3Department of Medical Laboratory Sciences, 4Biostatistics Core Facility, University of Delaware, Newark, Delaware, USA Abstract: In many types of tumors, especially breast tumors, aldehyde dehydrogenase (ALDH activity has been used to identify cancer stem-like cells within the tumor. The presence and quantity of these cells are believed to predict the response of tumors to chemotherapy. Therefore, identification and eradication of these cells would be necessary to cure the patient. However, there are 19 different ALDH isoforms that could contribute to the enzyme activity. ALDH1A1 and ALDH1A3 are among the isoforms mostly responsible for the increased ALDH activity observed in these stem-like cells, although the main isoforms vary in different tissues and tumor types. In the study reported here, we attempted to determine if ALDH1A1 or ALDH1A3, specifically, correlate with tumor stage, grade, and hormone-receptor status in breast-cancer patients. While there was no significant correlation between ALDH1A1 and any of the parameters tested, we were able to identify a positive correlation between ALDH1A3 and tumor stage in triple-negative cancers. In addition, ALDH1A3 was negatively correlated with estrogen-receptor status. Our data suggest that ALDH1A3 could be utilized as a marker to identify stem-like cells within triple-negative tumors. Keywords: breast tumor, ALDH, ALDH1A1, ALDH1A3, stem-like cells, triple-negative cancer

  16. Role of pyruvate dehydrogenase complex in traumatic brain injury and Measurement of pyruvate dehydrogenase enzyme by dipstick test

    Sharma Pushpa

    2009-01-01

    Full Text Available Objectives: The present study was designed to investigate the role of a mitochondrial enzyme pyruvate dehydrogenase (PDH on the severity of brain injury, and the effects of pyruvate treatment in rats with traumatic brain injury (TBI. Materials and Methods: We examined rats subjected to closed head injury using a fluid percussion device, and treated with sodium pyruvate (antioxidant and substrate for PDH enzyme. At 72 h post injury, blood was analyzed for blood gases, acid-base status, total PDH enzyme using a dipstick test and malondialdehyde (MDA levels as a marker of oxidative stress. Brain homogenates from right hippocampus (injured area were analyzed for PDH content, and immunostained hippocampus sections were used to determine the severity of gliosis and PDH E1-∞ subunit. Results: Our data demonstrate that TBI causes a significant reduction in PDH enzyme, disrupt-acid-base balance and increase oxidative stress in blood. Also, lower PDH enzyme in blood is related to the increased gliosis and loss of its PDH E1-∞ subunit PDH in brain tissue, and these effects of TBI were prevented by pyruvate treatment. Conclusion: Lower PDH enzyme levels in blood are related to the global oxidative stress, increased gliosis in brain, and severity of brain injury following TBI. These effects can be prevented by pyruvate through the protection of PDH enzyme and its subunit E-1.

  17. Autocrine function of aldehyde dehydrogenase 1 as a determinant of diet- and sex-specific differences in visceral adiposity.

    Yasmeen, Rumana; Reichert, Barbara; Deiuliis, Jeffrey; Yang, Fangping; Lynch, Alisha; Meyers, Joseph; Sharlach, Molly; Shin, Sangsu; Volz, Katharina S; Green, Kari B; Lee, Kichoon; Alder, Hansjuerg; Duester, Gregg; Zechner, Rudolf; Rajagopalan, Sanjay; Ziouzenkova, Ouliana

    2013-01-01

    Mechanisms for sex- and depot-specific fat formation are unclear. We investigated the role of retinoic acid (RA) production by aldehyde dehydrogenase 1 (Aldh1a1, -a2, and -a3), the major RA-producing enzymes, on sex-specific fat depot formation. Female Aldh1a1(-/-) mice, but not males, were resistant to high-fat (HF) diet-induced visceral adipose formation, whereas subcutaneous fat was reduced similarly in both groups. Sexual dimorphism in visceral fat (VF) was attributable to elevated adipose triglyceride lipase (Atgl) protein expression localized in clusters of multilocular uncoupling protein 1 (Ucp1)-positive cells in female Aldh1a1(-/-) mice compared with males. Estrogen decreased Aldh1a3 expression, limiting conversion of retinaldehyde (Rald) to RA. Rald effectively induced Atgl levels via nongenomic mechanisms, demonstrating indirect regulation by estrogen. Experiments in transgenic mice expressing an RA receptor response element (RARE-lacZ) revealed HF diet-induced RARE activation in VF of females but not males. In humans, stromal cells isolated from VF of obese subjects also expressed higher levels of Aldh1 enzymes compared with lean subjects. Our data suggest that an HF diet mediates VF formation through a sex-specific autocrine Aldh1 switch, in which Rald-mediated lipolysis in Ucp1-positive visceral adipocytes is replaced by RA-mediated lipid accumulation. Our data suggest that Aldh1 is a potential target for sex-specific antiobesity therapy. PMID:22933113

  18. Glucose-6-phosphate-dehydrogenase deficiency and its correlation with other risk factors in jaundiced newborns in Southern Brazil

    Clarissa Gutirrez Carvalho; Simone Martins Castro; Ana Paula Santin; Carina Zaleski; Felipe Gutirrez Carvalho; Roberto Giugliani

    2011-01-01

    Objective:To evaluate the correlation between glucose-6-phosphate-dehydrogenase (G6PD) deficiency and neonatal jaundice.Methods: Prospective, observational case-control study was conducted on490 newborns admitted to Hospital de Clínicas de Porto Alegre for phototherapy, who all experienced35 or more weeks of gestation, from March to December2007. Enzymatic screening ofG6PD activity was performed, followed byPCR.Results:There was prevalence of4.6% and a boy-girl ratio of3:1 in jaundiced newborns. No jaundiced neonate withABO incompatibility presented G6PD deficiency, and no Mediterranean mutation was found. A higher proportion of deficiency was observed in Afro-descendants. There was no association withUGT1A1 variants. Conclusions:G6PD deficiency is not related to severe hyperbilirubinemia and considering the high miscegenation in this area of Brazil, other gene interactions should be investigated.

  19. NAD(P)H dehydrogenase, quinone 1 (NQO1), protects melanin-producing cells from cytotoxicity of rhododendrol.

    Okubo, Ayaka; Yasuhira, Shinji; Shibazaki, Masahiko; Takahashi, Kazuhiro; Akasaka, Toshihide; Masuda, Tomoyuki; Maesawa, Chihaya

    2016-05-01

    Rhododendrol (RD) is a potent tyrosinase inhibitor that is metabolized to RD-quinone by tyrosinase, which may underlie the cytotoxicity of RD and leukoderma of the skin that may result. We have examined how forced expression of the NAD(P)H quinone dehydrogenase, quinone 1 (NQO1), a major quinone-reducing enzyme in cytosol, affects the survival of RD-treated cells. We found that treatment of the mouse melanoma cell line B16BL6 or normal human melanocytes with carnosic acid, a transcriptional inducer of the NQO1 gene, notably suppressed the cell killing effect of RD. This effect was mostly abolished by ES936, a highly specific NQO1 inhibitor. Moreover, conditional overexpression of the human NQO1 transgene in B16BL6 led to an expression-dependent increase of cell survival after RD treatment. Our results suggest that NQO1 attenuates the cytotoxicity of RD and/or its metabolites. PMID:26847926

  20. Human Aldehyde Dehydrogenase Genes: Alternatively-Spliced Transcriptional Variants and Their Suggested Nomenclature

    Black, William J.; Stagos, Dimitrios; Marchitti, Satori A.; Nebert, Daniel W.; Tipton, Keith F.; Bairoch, Amos; Vasiliou, Vasilis

    2011-01-01

    OBJECTIVE The human aldehyde dehydrogenase (ALDH) gene superfamily consists of 19 genes encoding enzymes critical for NAD(P)+-dependent oxidation of endogenous and exogenous aldehydes, including drugs and environmental toxicants. Mutations in ALDH genes are the molecular basis of several disease states (e.g. Sjögren-Larsson syndrome, pyridoxine-dependent seizures, and type II hyperprolinemia) and may contribute to the etiology of complex diseases such as cancer and Alzheimer’s disease. The aim of this nomenclature update was to identify splice transcriptional variants principally for the human ALDH genes. METHODS Data-mining methods were used to retrieve all human ALDH sequences. Alternatively-spliced transcriptional variants were determined based upon: a) criteria for sequence integrity and genomic alignment; b) evidence of multiple independent cDNA sequences corresponding to a variant sequence; and c) if available, empirical evidence of variants from the literature. RESULTS AND CONCLUSION Alternatively-spliced transcriptional variants and their encoded proteins exist for most of the human ALDH genes; however, their function and significance remain to be established. When compared with the human genome, rat and mouse include an additional gene, Aldh1a7, in the ALDH1A subfamily. In order to avoid confusion when identifying splice variants in various genomes, nomenclature guidelines for the naming of such alternative transcriptional variants and proteins are recommended herein. In addition, a web database (www.aldh.org) has been developed to provide up-to-date information and nomenclature guidelines for the ALDH superfamily. PMID:19823103

  1. Cloning and expression of bacterial genes coding amino acid dehydrogenases (oxidoreductases)

    Full text: The synthesis of 15N-labeled amino acids from the corresponding α-ketoacids can be accomplished in vitro using bacterial NAD-dependent amino acid dehydrogenases. The example of alanine dehydrogenase (AlaDH) and leucine dehydrogenase (LeuDH) will be presented here. Both enzymes belong to NAD dependent oxidoreductase family. AlaDH or L-alanine NAD-oxidoreductase (EC 1.4.1.1) promotes the reversible oxidative deamination of L-alanine to pyruvate (pyruvic acid). LeuDH or L-leucine NAD-oxidoreductase (EC 1.4.1.9) catalyses the reversible oxidative deamination of many related L-amino acids to corresponding α-ketoacids. The bacterial genes encoding AlaDH from Bacillus subtilis and LeuDH from Bacillus stearothermophilus were cloned separately in pET21b vector, and overexpressed in Escherichia coli BL21(DE3) strain. The [15N]L-alanine was synthesized by reductive amination of pyruvate, in the presence of 15NH4Cl, NADH, AlaDH and glucose dehydrogenase. The [15N]L-leucine, [15N]L-isoleucine, [15N]L-norleucine, [15N]L-valine and [15N]L-norvaline were produced in the same conditions using LeuDH, as a catalyst, and α- ketoisocaproate, DL-α-keto-β-methyl-n-valerate, α-ketocaproate, α-ketoisovalerate and α-ketovalerate, respectively, as substrates. In all cases, the reaction mixtures included glucose dehydrogenase for NADH regeneration with glucose as electron donor. The NADH renewal is more convenient with glucose dehydrogenase than other methods described before using formate dehydrogenase or alcohol dehydrogenase. The glucose dehydrogenase is very active and do not inhibit 15N-labeled amino acid synthesis. As determined by mass spectroscopy, the 15N-labeled amino acids were synthesized with yields between 60% and 95%. Our results demonstrate the usefulness of recombinant amino acid dehydrogenases for in vitro synthesis of 15N-labeled amino acids. (author)

  2. Alcohol dehydrogenase and aldehyde dehydrogenase gene polymorphisms, alcohol intake and the risk of colorectal cancer in the European Prospective Investigation into Cancer and Nutrition study

    Ferrari, P.; McKay, J. D.; Jenab, M.;

    2012-01-01

    BACKGROUND/OBJECTIVES: Heavy alcohol drinking is a risk factor of colorectal cancer (CRC), but little is known on the effect of polymorphisms in the alcohol-metabolizing enzymes, alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) on the alcohol-related risk of CRC in Caucasian populati......BACKGROUND/OBJECTIVES: Heavy alcohol drinking is a risk factor of colorectal cancer (CRC), but little is known on the effect of polymorphisms in the alcohol-metabolizing enzymes, alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) on the alcohol-related risk of CRC in Caucasian...... populations.SUBJECTS/METHODS: A nested case-control study (1269 cases matched to 2107controls by sex, age, study centre and date of blood collection) was conducted within the European Prospective Investigation into Cancer and Nutrition (EPIC) to evaluate the impact of rs1229984 (ADH1B), rs1573496 (ADH7......) and rs441 (ALDH2) polymorphisms on CRC risk. Using the wild-type variant of each polymorphism as reference category, CRC risk estimates were calculated using conditional logistic regression, with adjustment for matching factors.RESULTS: Individuals carrying one copy of the rs1229984(A) (ADH1B) allele...

  3. Characterization of aldehyde dehydrogenase isozymes in ovarian cancer tissues and sphere cultures

    Aldehyde dehydrogenases belong to a superfamily of detoxifying enzymes that protect cells from carcinogenic aldehydes. Of the superfamily, ALDH1A1 has gained most attention because current studies have shown that its expression is associated with human cancer stem cells. However, ALDH1A1 is only one of the 19 human ALDH subfamilies currently known. The purpose of the present study was to determine if the expression and activities of other major ALDH isozymes are associated with human ovarian cancer and ovarian cancer sphere cultures. Immunohistochemistry was used to delineate ALDH isozyme localization in clinical ovarian tissues. Western Blot analyses were performed on lysates prepared from cancer cell lines and ovarian cancer spheres to confirm the immunohistochemistry findings. Quantitative reverse transcription-polymerase chain reactions were used to measure the mRNA expression levels. The Aldefluor® assay was used to measure ALDH activity in cancer cells from the four tumor subtypes. Immunohistochemical staining showed significant overexpression of ALDH1A3, ALDH3A2, and ALDH7A1 isozymes in ovarian tumors relative to normal ovarian tissues. The expression and activity of ALDH1A1 is tumor type-dependent, as seen from immunohistochemisty, Western blot analysis, and the Aldefluor® assay. The expression was elevated in the mucinous and endometrioid ovarian epithelial tumors than in serous and clear cell tumors. In some serous and most clear cell tumors, ALDH1A1 expression was found in the stromal fibroblasts. RNA expression of all studied ALDH isozymes also showed higher expression in endometrioid and mucinous tumors than in the serous and clear cell subtypes. The expression of ALDH enzymes showed tumor type-dependent induction in ovarian cancer cells growing as sphere suspensions in serum-free medium. The results of our study indicate that ALDH enzyme expression and activity may be associated with specific cell types in ovarian tumor tissues and vary according to

  4. Furosemide and 11beta-hydroxysteroid dehydrogenase activity, in man.

    Palermo, M; Armanini, D; Shackleton, C H L; Sorba, G; Cossu, M; Roitman, E; Scaroni, C; Delitala, G

    2002-09-01

    Mineralocorticoid receptors possess the same affinity for aldosterone and for cortisol and preferential binding of aldosterone is modulated by the 11 beta-hydroxysteroid dehydrogenase (11 beta-OHSD) enzyme, which converts cortisol to its inactive metabolite cortisone. Several endogenous or exogenous compounds able to inhibit the enzyme have been described and, as a consequence, produce the syndrome of apparent mineralocorticoid excess (AME) characterized by hypertension, hypokalemia, volume repletion and suppression of the renin-angiotensin-aldosterone system. High doses of furosemide, a diuretic that works in the luminal surface of the thick ascending limb of Henle's loop, have been reported to inhibit 11 beta-OHSD activity to the same extent as licorice in vivo and in vitro, in rat. The aim of our study was to verify the effect of the drug on 11 beta-OHSD activity in man at the doses currently used in clinical practice. We tested the activity of 11 beta-OHSD following both acute and protracted administration of furosemide. In the acute study, the drug was administered at low (40 mg i.v. in bolo) and high doses (infusion of 10 mg/kg bw i.v for six hours); the protracted furosemide administration consisted in 50 mg/day for 20 days, by mouth. The ratios between the cortisol metabolites tetrahydrocortisol plus allo-tetrahydrocortisol to tetra-hydrocortisone and urinary free cortisol to urinary free cortisone were used to measure the activity of 11 beta-OHSD. Urinary cortisol, cortisone and their metabolites were tested by a gas-chromatographic/mass spectrometric method. Neither acute nor prolonged administration of furosemide did affect the activity of 11 beta-OHSD although the drug was able to modify plasma aldosterone and PRA secretion and to determine hypokalemia. Our results suggest that furosemide does not play a significant role in 11 beta-OHSD modulation in humans, at least at the dosage used in clinical practice. PMID:12373630

  5. Function of C-terminal hydrophobic region in fructose dehydrogenase

    Fructose dehydrogenase (FDH) catalyzes oxidation of D-fructose into 2-keto-D-fructose and is one of the enzymes allowing a direct electron transfer (DET)-type bioelectrocatalysis. FDH is a heterotrimeric membrane-bound enzyme (subunit I, II, and III) and subunit II has a C terminal hydrophobic region (CHR), which was expected to play a role in anchoring to membranes from the amino acid sequence. We have constructed a mutated FDH lacking of CHR (ΔchrFDH). Contrary to the expected function of CHR, ΔchrFDH is expressed in the membrane fraction, and subunit I/III subcomplex (ΔcFDH) is also expressed in a similar activity level but in the soluble fraction. In addition, the enzyme activity of the purified ΔchrFDH is about one twentieth of the native FDH. These results indicate that CHR is concerned with the binding between subunit I(/III) and subunit II and then with the enzyme activity. ΔchrFDH has clear DET activity that is larger than that expected from the solution activity, and the characteristics of the catalytic wave of ΔchrFDH are very similar to those of FDH. The deletion of CHR seems to increase the amounts of the enzyme with the proper orientation for the DET reaction at electrode surfaces. Gel filtration chromatography coupled with urea treatment shows that the binding in ΔchrFDH is stronger than that in FDH. It can be considered that the rigid binding between subunit I(/III) and II without CHR results in a conformation different from the native one, which leads to the decrease in the enzyme activity in solution

  6. Screening and Characterization of Proline Dehydrogenase Flavoenzyme Producing Pseudomonas Entomophila

    H Shahbaz- Mohammadi

    2011-12-01

    Full Text Available Background and Objectives: Proline dehydrogenase (ProDH; 1.5.99.8 plays an important role in specific determination of plasma proline level in biosensor and diagnostic kits. The goal of this research was to isolate and characterize ProDH enzyme from Iranian soil microorganisms.Materials and Methods: Screening of L-proline degradative enzymes from soil samples was carried out employing enrichment culture techniques. The isolate was characterized by biochemical reactions and specific PCR amplification. The target ProDH was purified and the effects of pH and temperature on the activity and stability were also tested.Results: Among the 250 isolates recovered from 40 soil samples, only one strain characterized as Pseudomonas entomophila displayed the highest enzyme activity toward L-proline (350 U/l than others. The enzyme of interest was identified as a ProDH and had Km value of 32 mM for L-proline. ProDH exhibited its best activity at temperature range of 25 to 35°C and its highest activity was achieved at 30°C. It was almost stable at temperatures between 25-30°C for 2 hours. The optimum pH activity of ProDH reaction was 8.5 and its activity was stable in pH range of 8.0-9.0 upto 24 hours. The enzyme was purified with a yield of 8.5% and a purification factor of 37.7. The molecular mass of the purified ProDH was about 40 kDa, and determined to be a monomeric protein."nConclusion: This is the first report concerning the ProDH production by a P. entomophila bacterium isolated from soil sample.

  7. Urinary Bladder Paragangliomas: Analysis of Succinate Dehydrogenase and Outcome.

    Gupta, Sounak; Zhang, Jun; Rivera, Michael; Erickson, Lori A

    2016-09-01

    Paragangliomas of the urinary bladder can arise sporadically or as a part of hereditary syndromes including those with underlying mutations in the succinate dehydrogenase (SDH) genes, which serve as tumor suppressors. SDH deficiency can be screened for by absence of immunohistochemical detection of SDHB. In this study of 11 cases, clinical follow-up was available for 9/11 cases. The cases were reviewed and graded based on the grading system for adrenal pheochromocytomas and paragangliomas (GAPP) criteria. Immunohistochemistry was performed for Ki67 and SDHB. Proliferative index was calculated by quantification of Ki67-positive cells at hot spots. The medical record was accessed for documentation of germline SDH mutations. Urinary bladder paragangliomas had a female predilection (8/11 cases), and 5/11 cases exhibited metastatic behavior. Patients with metastatic disease tended to be younger (mean age 43 vs 49 years), have larger lesions (5.8 vs 1.5 cm), and presented with catecholamine excess (4/4 vs 2/6 patients with non-metastatic lesions). Patients with metastatic disease had a higher mean Ki67 proliferation rate (4.9 vs 1.3 %) and GAPP score (mean of 5.8 vs 3.8) (p = 0.01). IHC for SDHB expression revealed loss of expression in 2/6 cases of non-metastatic paragangliomas compared to 4/5 patients with metastatic paragangliomas. Interestingly, of these four patients, two had a documented mutation of SDHB, one patient had a SDHC mutation, and another patient had a history of familial disease without mutation analysis being performed. Our study, suggests that SDH loss was suggestive of metastatic behavior in addition to younger age at diagnosis, larger tumor size, and higher Ki67 proliferation rate and catecholamine type. PMID:27262318

  8. Erythrocyte glucose-6-phosphate dehydrogenase from Brazilian opossum Didelphis marsupialis

    Barretto O.C. de O.

    2006-01-01

    Full Text Available In a comparative study of erythrocyte metabolism of vertebrates, the specific activity of glucose-6-phosphate dehydrogenase (G6PD of the Brazilian opossum Didelphis marsupialis in a hemolysate was shown to be high, 207 ± 38 IU g-1 Hb-1 min-1 at 37ºC, compared to the human erythrocyte activity of 12 ± 2 IU g-1 Hb-1 min-1 at 37ºC. The apparent high specific activity of the mixture led us to investigate the physicochemical properties of the opossum enzyme. We report that reduced glutathione (GSH in the erythrocytes was only 50% higher than in human erythrocytes, a value lower than expected from the high G6PD activity since GSH is maintained in a reduced state by G6PD activity. The molecular mass, determined by G-200 Sephadex column chromatography at pH 8.0, was 265 kDa, which is essentially the same as that of human G6PD (260 kDa. The Michaelis-Menten constants (Km: 55 µM for glucose-6-phosphate and nicotinamide adenine dinucleotide phosphate (Km: 3.3 µM were similar to those of the human enzyme (Km: 50-70 and Km: 2.9-4.4, respectively. A 450-fold purification of the opossum enzyme was achieved and the specific activity of the purified enzyme, 90 IU/mg protein, was actually lower than the 150 IU/mg protein observed for human G6PD. We conclude that G6PD after purification from the hemolysate of D. marsupialis does not have a high specific activity. Thus, it is quite probable that the red cell hyperactivity reported may be explained by increased synthesis of G6PD molecules per unit of hemoglobin or to reduced inactivation in the RBC hemolysate.

  9. Erythrocyte glucose-6-phosphate dehydrogenase from Brazilian opossum Didelphis marsupialis.

    Barretto, O C de O; Oshiro, M; Oliveira, R A G; Fedullo, J D L; Nonoyama, K

    2006-05-01

    In a comparative study of erythrocyte metabolism of vertebrates, the specific activity of glucose-6-phosphate dehydrogenase (G6PD) of the Brazilian opossum Didelphis marsupialis in a hemolysate was shown to be high, 207 +/- 38 IU g-1 Hb-1 min-1 at 37 degrees C, compared to the human erythrocyte activity of 12 +/- 2 IU g-1 Hb-1 min-1 at 37 degrees C. The apparent high specific activity of the mixture led us to investigate the physicochemical properties of the opossum enzyme. We report that reduced glutathione (GSH) in the erythrocytes was only 50% higher than in human erythrocytes, a value lower than expected from the high G6PD activity since GSH is maintained in a reduced state by G6PD activity. The molecular mass, determined by G-200 Sephadex column chromatography at pH 8.0, was 265 kDa, which is essentially the same as that of human G6PD (260 kDa). The Michaelis-Menten constants (Km: 55 microM) for glucose-6-phosphate and nicotinamide adenine dinucleotide phosphate (Km: 3.3 microM) were similar to those of the human enzyme (Km: 50-70 and Km: 2.9-4.4, respectively). A 450-fold purification of the opossum enzyme was achieved and the specific activity of the purified enzyme, 90 IU/mg protein, was actually lower than the 150 IU/mg protein observed for human G6PD. We conclude that G6PD after purification from the hemolysate of D. marsupialis does not have a high specific activity. Thus, it is quite probable that the red cell hyperactivity reported may be explained by increased synthesis of G6PD molecules per unit of hemoglobin or to reduced inactivation in the RBC hemolysate. PMID:16648898

  10. Triazaspirodimethoxybenzoyls as Selective Inhibitors of Mycobacterial Lipoamide Dehydrogenase

    Bryk, Ruslana; Arango, Nancy; Venugopal, Aditya; Warren, J. David; Park, Yun-Hee; Patel, Mulchand S.; Lima, Christopher D.; Nathan, Carl (Weill-Med); (SKI); (SUNYB)

    2010-06-25

    Mycobacterium tuberculosis (Mtb) remains the leading single cause of death from bacterial infection. Here we explored the possibility of species-selective inhibition of lipoamide dehydrogenase (Lpd), an enzyme central to Mtb's intermediary metabolism and antioxidant defense. High-throughput screening of combinatorial chemical libraries identified triazaspirodimethoxybenzoyls as high-nanomolar inhibitors of Mtb's Lpd that were noncompetitive versus NADH, NAD{sup +}, and lipoamide and >100-fold selective compared to human Lpd. Efficacy required the dimethoxy and dichlorophenyl groups. The structure of an Lpd-inhibitor complex was resolved to 2.42 {angstrom} by X-ray crystallography, revealing that the inhibitor occupied a pocket adjacent to the Lpd NADH/NAD{sup +} binding site. The inhibitor did not overlap with the adenosine moiety of NADH/NAD{sup +} but did overlap with positions predicted to bind the nicotinamide rings in NADH and NAD{sup +} complexes. The dimethoxy ring occupied a deep pocket adjacent to the FAD flavin ring where it would block coordination of the NADH nicotinamide ring, while the dichlorophenyl group occupied a more exposed pocket predicted to coordinate the NAD{sup +} nicotinamide. Several residues that are not conserved between the bacterial enzyme and its human homologue were predicted to contribute both to inhibitor binding and to species selectivity, as confirmed for three residues by analysis of the corresponding mutant Mtb Lpd proteins. Thus, nonconservation of residues lining the electron-transfer tunnel in Mtb Lpd can be exploited for development of species-selective Lpd inhibitors.