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Sample records for boidinii formate dehydrogenase

  1. Whole-cell bioreduction of aromatic α-keto esters using Candida tenuis xylose reductase and Candida boidinii formate dehydrogenase co-expressed in Escherichia coli

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    Egger Sigrid

    2008-12-01

    Full Text Available Abstract Background Whole cell-catalyzed biotransformation is a clear process option for the production of chiral alcohols via enantioselective reduction of precursor ketones. A wide variety of synthetically useful reductases are expressed heterologously in Escherichia coli to a high level of activity. Therefore, this microbe has become a prime system for carrying out whole-cell bioreductions at different scales. The limited capacity of central metabolic pathways in E. coli usually requires that reductase coenzyme in the form of NADPH or NADH be regenerated through a suitable oxidation reaction catalyzed by a second NADP+ or NAD+ dependent dehydrogenase that is co-expressed. Candida tenuis xylose reductase (CtXR was previously shown to promote NADH dependent reduction of aromatic α-keto esters with high Prelog-type stereoselectivity. We describe here the development of a new whole-cell biocatalyst that is based on an E. coli strain co-expressing CtXR and formate dehydrogenase from Candida boidinii (CbFDH. The bacterial system was evaluated for the synthesis of ethyl R-4-cyanomandelate under different process conditions and benchmarked against a previously described catalyst derived from Saccharomyces cerevisiae expressing CtXR. Results Gene co-expression from a pETDuet-1 vector yielded about 260 and 90 units of intracellular CtXR and CbFDH activity per gram of dry E. coli cell mass (gCDW. The maximum conversion rate (rS for ethyl 4-cyanobenzoylformate by intact or polymyxin B sulphate-permeabilized cells was similar (2 mmol/gCDWh, suggesting that the activity of CbFDH was partly rate-limiting overall. Uncatalyzed ester hydrolysis in substrate as well as inactivation of CtXR and CbFDH in the presence of the α-keto ester constituted major restrictions to the yield of alcohol product. Using optimized reaction conditions (100 mM substrate; 40 gCDW/L, we obtained ethyl R-4-cyanomandelate with an enantiomeric excess (e.e. of 97.2% in a yield of 82

  2. Co-expression of formate dehydrogenase form Candida boidinii and (R)-specific carbonyl reductase form Candida parapsilosis CCTCC M203011 in Escherichia coli%(R)-专一性羰基还原酶与甲酸脱氢酶基因在大肠杆菌中的共表达

    Institute of Scientific and Technical Information of China (English)

    孙莹; 张荣珍; 徐岩

    2008-01-01

    [目的]通过研究-专一性拨基还原酶和甲酸脱氢酶基因在大肠杆菌中的共表达,解决较高底物浓度下不对称转化反应的辅酶限制性问题.[方法]分别以近平滑假丝酵母(candida parapsitosis CCTCC M203011)和博伊丁假丝酵母(Candida boidinii)基因组为模板,采用PCR方法扩增得到(R伏)-专一性拨基还原酶基因(rcr)和甲酸脱氢酶基因(fdh),克隆到共表达载体pETDuetTM-1中进行表达.共表达质粒 pETDuet-rcr-fdh转化稀有密码子优化型菌株E.coli Rosetta,获得重组菌E.coli Rosetta/pETDuet-rcr-fdh.结果 在30℃ 条件下,经1mmol/L IPTG诱导表达8h后,SDS-PAGE结果表明-专一性拨基还原酶和甲酸脱氢酶均有明显的表达,其相对分子质量分别为37kDa和40kDa.以高浓度(6glL)2一经基苯乙酮为底物时,0.19重组菌细胞催化产生(R)-苯基乙二醇,产物光学纯度为100% e.e.,产率为85.9%.与无甲酸脱氢酶参与辅酶再生循环的重组菌E.coli Rosetta/pETDuet-rrc;相比,产物光学纯度和产率分别提高了1.3和2.7倍.讨论 该重组菌的构建为基因工程法生物合成(R)-苯基乙二醇的工业应用奠定了基础.

  3. Characterization of a flavinogenic mutant of methanol yeast Candida boidinii and its extracellular secretion of riboflavin.

    Science.gov (United States)

    Suryadi, H; Yoshida, N; Yamada-Onodera, K; Katsuragi, T; Tani, Y

    2000-01-01

    A flavinogenic mutant was derived from Candida boidinii by mutagenesis. The mutant was smaller than the wild type, did not grow on a minimal medium, and required l-tryptophan, l-leucine, inositol, and nicotinate for growth. The mutant was defective in the oxidative pentose phosphate pathway, lacking glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. The specific activities of the transaldolase and transketolase of the mutant were higher than those of the wild type. These high activities might direct the flux of the carbon source to the nonoxidative pathway with formation of a large amount of pentose phosphates, increasing riboflavin synthesis. Under microaerobic conditions at 25 degrees C, 90 mg/l riboflavin was obtained. PMID:16232817

  4. Microbial surface displaying formate dehydrogenase and its application in optical detection of formate.

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    Liu, Aihua; Feng, Ruirui; Liang, Bo

    2016-09-01

    In the present work, NAD(+)-dependent formate dehydrogenase (FDH), encoded by fdh gene from Candida boidinii was successfully displayed on Escherichia coli cell surface using ice nucleation protein (INP) from Pseudomonas borealis DL7 as an anchoring protein. Localization of matlose binding protein (MBP)-INP-FDH fusion protein on the E. coli cell surface was characterized by SDS-PAGE and enzymatic activity assay. FDH activity was monitored through the oxidation of formate catalyzed by cell-surface-displayed FDH with its cofactor NAD(+), and the production of NADH can be detected spectrometrically at 340nm. After induction for 24h in Luria-Bertani medium containing isopropyl-β-d-thiogalactopyranoside, over 80% of MBP-INP-FDH fusion protein present on the surface of E. coli cells. The cell-surface-displayed FDH showed optimal temperature of 50°C and optimal pH of 9.0. Additionally, the cell-surface-displayed FDH retained its original enzymatic activity after incubation at 4°C for one month with the half-life of 17days at 40°C and 38h at 50°C. The FDH activity could be inhibited to different extents by some transition metal ions and anions. Moreover, the E. coli cells expressing FDH showed different tolerance to solvents. The recombinant whole cell exhibited high formate specificity. Finally, the E. coli cell expressing FDH was used to assay formate with a wide linear range of 5-700μM and a low limit of detection of 2μM. It is anticipated that the genetically engineered cells may have a broad application in biosensors, biofuels and cofactor regeneration system. PMID:27444330

  5. Phosphorylation of formate dehydrogenase in potato tuber mitochondria

    DEFF Research Database (Denmark)

    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...

  6. Host cell and expression engineering for development of an E. coli ketoreductase catalyst: Enhancement of formate dehydrogenase activity for regeneration of NADH

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    Mädje Katharina

    2012-01-01

    Full Text Available Abstract Background Enzymatic NADH or NADPH-dependent reduction is a widely applied approach for the synthesis of optically active organic compounds. The overall biocatalytic conversion usually involves in situ regeneration of the expensive NAD(PH. Oxidation of formate to carbon dioxide, catalyzed by formate dehydrogenase (EC 1.2.1.2; FDH, presents an almost ideal process solution for coenzyme regeneration that has been well established for NADH. Because isolated FDH is relatively unstable under a range of process conditions, whole cells often constitute the preferred form of the biocatalyst, combining the advantage of enzyme protection in the cellular environment with ease of enzyme production. However, the most prominent FDH used in biotransformations, the enzyme from the yeast Candida boidinii, is usually expressed in limiting amounts of activity in the prime host for whole cell biocatalysis, Escherichia coli. We therefore performed expression engineering with the aim of enhancing FDH activity in an E. coli ketoreductase catalyst. The benefit resulting from improved NADH regeneration capacity is demonstrated in two transformations of technological relevance: xylose conversion into xylitol, and synthesis of (S-1-(2-chlorophenylethanol from o-chloroacetophenone. Results As compared to individual expression of C. boidinii FDH in E. coli BL21 (DE3 that gave an intracellular enzyme activity of 400 units/gCDW, co-expression of the FDH with the ketoreductase (Candida tenuis xylose reductase; XR resulted in a substantial decline in FDH activity. The remaining FDH activity of only 85 U/gCDW was strongly limiting the overall catalytic activity of the whole cell system. Combined effects from increase in FDH gene copy number, supply of rare tRNAs in a Rosetta strain of E. coli, dampened expression of the ketoreductase, and induction at low temperature (18°C brought up the FDH activity threefold to a level of 250 U/gCDW while reducing the XR activity by

  7. Interconversion between formate and hydrogen carbonate by tungsten-containing formate dehydrogenase-catalyzed mediated bioelectrocatalysis

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    Kento Sakai

    2015-09-01

    Full Text Available We have focused on the catalytic properties of tungsten-containing formate dehydrogenase (FoDH1 from Methylobacterium extorquens AM1 to construct a bioelectrochemical interconversion system between formate (HCOO− and hydrogen carbonate (HCO3−. FoDH1 catalyzes both of the HCOO oxidation and the HCO3− reduction with several artificial dyes. The bi-molecular reaction rate constants between FoDH1 and the artificial electron acceptors and NAD+ (as the natural electron acceptor show the property called a linear free energy relationship (LFER, indicating that FoDH1 would have no specificity to NAD+. Similar LFER is also observed for the catalytic reduction of HCO3−. The reversible reaction between HCOO− and HCO3− through FoDH1 has been realized on cyclic voltammetry by using methyl viologen (MV as a mediator and by adjusting pH from the thermodynamic viewpoint. Potentiometric measurements have revealed that the three redox couples, MV2+/MV·−+, HCOO−/HCO3−, FoDH1 (ox/red, reach an equilibrium in the bulk solution when the two-way bioelectrocatalysis proceeds in the presence of FoDH1 and MV. The steady-state voltammograms with two-way bioelectrocatalytic properties are interpreted on a simple model by considering the solution equilibrium.

  8. Lactate dehydrogenase has no control on lactate production but has a strong negative control on formate production in Lactococcus lactis

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    Andersen, H.W.; Pedersen, M.B.; Hammer, Karin;

    2001-01-01

    enhanced in the strain deleted for lactate dehydrogenase. What is more surprising is that the enzyme had a strong negative control (C- LDH(F1)J=-1.3) on the flux to formate at the wild-type level of lactate dehydrogenase. Furthermore, we showed that L. lactis has limited excess of capacity of lactate...

  9. Peroxisomal assembly : membrane proliferation precedes the induction of the abundant matrix proteins in the methylotrophic yeast Candida boidinii

    NARCIS (Netherlands)

    Veenhuis, Marten; Goodman, Joel M.

    1990-01-01

    Peroxisomes are massively induced when methylotrophic yeasts are cultured in medium containing methanol. These organelles contain enzymes that catalyze the initial steps of methanol assimilation. In Candida boidinii, a methylotrophic yeast, the peroxisomal matrix (internal compartment) is composed a

  10. 'Rod and ring' formation from IMP dehydrogenase is regulated through the one-carbon metabolic pathway.

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    Calise, S John; Purich, Daniel L; Nguyen, Thuy; Saleem, Dania A; Krueger, Claire; Yin, Joyce D; Chan, Edward K L

    2016-08-01

    'Rods and rings' (RRs) are conserved, non-membrane-bound intracellular polymeric structures composed, in part, of inosine monophosphate dehydrogenase (IMPDH), a key enzyme leading to GMP and GTP biosynthesis. RR formation is induced by IMPDH inhibitors as well as glutamine deprivation. They also form upon treatment of cells with glutamine synthetase inhibitors. We now report that depriving cells of serine and glycine promotes RR formation, and we have traced these effects to dihydrofolate reductase (DHFR) and serine hydroxymethyltransferase-2 (SHMT2), pivotal enzymes in one-carbon metabolism and nucleotide biosynthesis. RR assembly is likewise induced upon DHFR inhibition by methotrexate or aminopterin as well as siRNA-mediated knockdown of DHFR or SHMT2. Because RR assembly occurs when guanine nucleotide biosynthesis is inhibited, and because RRs rapidly disassemble after the addition of guanine nucleotide precursors, RR formation might be an adaptive homeostatic mechanism, allowing IMPDH to sense changes in the one-carbon folate pathway. PMID:27343244

  11. Corynebacterium glutamicum harbours a molybdenum cofactor-dependent formate dehydrogenase which alleviates growth inhibition in the presence of formate.

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    Witthoff, Sabrina; Eggeling, Lothar; Bott, Michael; Polen, Tino

    2012-09-01

    Here, we show that Corynebacterium glutamicum ATCC 13032 co-metabolizes formate when it is grown with glucose as the carbon and energy source. CO(2) measurements during bioreactor cultivation and use of (13)C-labelled formate demonstrated that formate is almost completely oxidized to CO(2). The deletion of fdhF (cg0618), annotated as formate dehydrogenase (FDH) and located in a cluster of genes conserved in the family Corynebacteriaceae, prevented formate utilization. Similarly, deletion of fdhD (cg0616) resulted in the inability to metabolize formate and deletion of cg0617 markedly reduced formate utilization. These results illustrated that all three gene products are required for FDH activity. Growth studies with molybdate and tungstate indicated that the FDH from C. glutamicum ATCC 13032 is a molybdenum-dependent enzyme. The presence of 100 mM formate caused a 25 % lowered growth rate during cultivation of C. glutamicum ATCC 13032 wild-type in glucose minimal medium. This inhibitory effect was increased in the strains lacking FDH activity. Our data demonstrate that C. glutamicum ATCC 13032 possesses an FDH with a currently unknown electron acceptor. The presence of the FDH might help the soil bacterium C. glutamicum ATCC 13032 to alleviate growth retardation caused by formate, which is ubiquitously present in the environment. PMID:22767548

  12. Integrated Process of Arabinose Biopurification and Xylitol Fermentation Based on the Diverse Action of Candida boidinii

    OpenAIRE

    Fehér, C.; Gazsó, Z.; Gál, B.; Kontra, A.; Barta, Z.; Réczey, K.

    2016-01-01

    Hemicellulosic hydrolysates of agro-residues are promising raw materials for xylitol and arabinose production through biotechnological methods. Two-step acidic fractionation of corn fibre was developed to produce a glucose- and arabinose-rich hydrolysate and a xylose-rich hydrolysate. An integrated process of arabinose biopurification on the glucose- and arabinose-rich hydrolysate and xylitol fermentation on the xylose-rich hydrolysate using Candida boidinii NCAIM Y.01308 was introduced, i...

  13. Crystal structures of complexes of NAD+-dependent formate dehydrogenase from methylotrophic bacterium Pseudomonas sp. 101 with formate

    International Nuclear Information System (INIS)

    Formate dehydrogenase (FDH) from the methylotrophic bacterium Pseudomonas sp. 101 catalyzes oxidation of formate to NI2 with the coupled reduction of nicotinamide adenine dinucleotide (NAD+). The three-dimensional structures of the apo form (the free enzyme) and the holo form (the ternary FDH-NAD+-azide complex) of FDH have been established earlier. In the present study, the structures of FDH complexes with formate are solved at 2.19 and 2.28 A resolution by the molecular replacement method and refined to the R factors of 22.3 and 20.5%, respectively. Both crystal structures contain four protein molecules per asymmetric unit. These molecules form two dimers identical to the dimer of the apo form of FDH. Two possible formatebinding sites are found in the active site of the FDH structure. In the complexes the sulfur atom of residue Cys354 exists in the oxidized state

  14. High-frequency transformation of a methylotrophic yeast, Candida boidinii, with autonomously replicating plasmids which are also functional in Saccharomyces cerevisiae.

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    Sakai, Y.; Goh, T K; Tani, Y

    1993-01-01

    We have developed a transformation system which uses autonomous replicating plasmids for a methylotrophic yeast, Candida boidinii. Two autonomous replication sequences, CARS1 and CARS2, were newly cloned from the genome of C. boidinii. Plasmids having both a CARS fragment and the C. boidinii URA3 gene transformed C. boidinii ura3 cells to Ura+ phenotype at frequencies of up to 10(4) CFU/micrograms of DNA. From Southern blot analysis, CARS plasmids seemed to exist in polymeric forms as well as...

  15. Light driven CO2 fixation by using cyanobacterial photosystem I and NADPH-dependent formate dehydrogenase.

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    Masaki Ihara

    Full Text Available The ultimate goal of this research is to construct a new direct CO2 fixation system using photosystems in living algae. Here, we report light-driven formate production from CO2 by using cyanobacterial photosystem I (PS I. Formate, a chemical hydrogen carrier and important industrial material, can be produced from CO2 by using the reducing power and the catalytic function of formate dehydrogenase (FDH. We created a bacterial FDH mutant that experimentally switched the cofactor specificity from NADH to NADPH, and combined it with an in vitro-reconstituted cyanobacterial light-driven NADPH production system consisting of PS I, ferredoxin (Fd, and ferredoxin-NADP(+-reductase (FNR. Consequently, light-dependent formate production under a CO2 atmosphere was successfully achieved. In addition, we introduced the NADPH-dependent FDH mutant into heterocysts of the cyanobacterium Anabaena sp. PCC 7120 and demonstrated an increased formate concentration in the cells. These results provide a new possibility for photo-biological CO2 fixation.

  16. Stability and activity of alcohol dehydrogenases in W/O-microemulsions: enantioselective reduction including cofactor regeneration.

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    Orlich, B; Berger, H; Lade, M; Schomäcker, R

    2000-12-20

    Microemulsions provide an interesting alternative to classical methods for the conversion of less water-soluble substrates by alcohol dehydrogenase, but until now stability and activity were too low for economically useful processes. The activity and stability of the enzymes are dependent on the microemulsion composition, mostly the water and the surfactant concentration. Therefore, it is necessary to know the exact phase behavior of a given microemulsion reaction system and the corresponding enzyme behavior therein. Because of their economic and ecologic suitability polyethoxylated fatty alcohols were investigated concerning their phase behavior and their compatibility with enzymes in ternary mixtures. The phase behavior of Marlipal O13-60 (C13EO6 in industrial quality)/cyclohexane/water and its effect on the activity and stability of alcohol dehydrogenase from Yeast (YADH) and horse liver (HLADH) and the carbonyl reductase from Candida parapsilosis (CPCR) is presented in this study. Beside the macroscopic phase behavior of the reaction system, the viscosity of the system indicates structural changes of aggregates in the microemulsion. The changes of the enzyme activities with the composition are discussed on the basis of transitions from reverse micelles to swollen reverse micelles and finally, the transition to the phase separation. The formate dehydrogenase from Candida boidinii was used for the NADH-regeneration during reduction reactions. While the formate dehydrogenase did not show any kinetic effect on the microemulsion composition, the other enzymes show significant changes of activity and stability varying the water or surfactant concentration of the microemulsion. Under certain conditions, stability could be maintained with HLADH for several weeks. Successful experiments with semi-batch processes including cofactor regeneration and product separation were performed.

  17. Efficient production of (R-2-hydroxy-4-phenylbutyric acid by using a coupled reconstructed D-lactate dehydrogenase and formate dehydrogenase system.

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    Binbin Sheng

    Full Text Available (R-2-hydroxy-4-phenylbutyric acid [(R-HPBA] is a key precursor for the production of angiotensin-converting enzyme inhibitors. However, the product yield and concentration of reported (R-HPBA synthetic processes remain unsatisfactory.The Y52L/F299Y mutant of NAD-dependent D-lactate dehydrogenase (D-nLDH in Lactobacillus bulgaricus ATCC 11842 was found to have high bio-reduction activity toward 2-oxo-4-phenylbutyric acid (OPBA. The mutant D-nLDHY52L/F299Y was then coexpressed with formate dehydrogenase in Escherichia coli BL21 (DE3 to construct a novel biocatalyst E. coli DF. Thus, a novel bio-reduction process utilizing whole cells of E. coli DF as the biocatalyst and formate as the co-substrate for cofactor regeneration was developed for the production of (R-HPBA from OPBA. The biocatalysis conditions were then optimized.Under the optimum conditions, 73.4 mM OPBA was reduced to 71.8 mM (R-HPBA in 90 min. Given its high product enantiomeric excess (>99% and productivity (47.9 mM h(-1, the constructed coupling biocatalysis system is a promising alternative for (R-HPBA production.

  18. Dimerization interface of 3-hydroxyacyl-CoA dehydrogenase tunes the formation of its catalytic intermediate.

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    Yingzhi Xu

    Full Text Available 3-Hydroxyacyl-CoA dehydrogenase (HAD, EC 1.1.1.35 is a homodimeric enzyme localized in the mitochondrial matrix, which catalyzes the third step in fatty acid β-oxidation. The crystal structures of human HAD and subsequent complexes with cofactor/substrate enabled better understanding of HAD catalytic mechanism. However, numerous human diseases were found related to mutations at HAD dimerization interface that is away from the catalytic pocket. The role of HAD dimerization in its catalytic activity needs to be elucidated. Here, we solved the crystal structure of Caenorhabditis elegans HAD (cHAD that is highly conserved to human HAD. Even though the cHAD mutants (R204A, Y209A and R204A/Y209A with attenuated interactions on the dimerization interface still maintain a dimerization form, their enzymatic activities significantly decrease compared to that of the wild type. Such reduced activities are in consistency with the reduced ratios of the catalytic intermediate formation. Further molecular dynamics simulations results reveal that the alteration of the dimerization interface will increase the fluctuation of a distal region (a.a. 60-80 that plays an important role in the substrate binding. The increased fluctuation decreases the stability of the catalytic intermediate formation, and therefore the enzymatic activity is attenuated. Our study reveals the molecular mechanism about the essential role of the HAD dimerization interface in its catalytic activity via allosteric effects.

  19. Reduction of Carbon Dioxide by a Molybdenum-Containing Formate Dehydrogenase: A Kinetic and Mechanistic Study.

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    Maia, Luisa B; Fonseca, Luis; Moura, Isabel; Moura, José J G

    2016-07-20

    Carbon dioxide accumulation is a major concern for the ecosystems, but its abundance and low cost make it an interesting source for the production of chemical feedstocks and fuels. However, the thermodynamic and kinetic stability of the carbon dioxide molecule makes its activation a challenging task. Studying the chemistry used by nature to functionalize carbon dioxide should be helpful for the development of new efficient (bio)catalysts for atmospheric carbon dioxide utilization. In this work, the ability of Desulfovibrio desulfuricans formate dehydrogenase (Dd FDH) to reduce carbon dioxide was kinetically and mechanistically characterized. The Dd FDH is suggested to be purified in an inactive form that has to be activated through a reduction-dependent mechanism. A kinetic model of a hysteretic enzyme is proposed to interpret and predict the progress curves of the Dd FDH-catalyzed reactions (initial lag phase and subsequent faster phase). Once activated, Dd FDH is able to efficiently catalyze, not only the formate oxidation (kcat of 543 s(-1), Km of 57.1 μM), but also the carbon dioxide reduction (kcat of 46.6 s(-1), Km of 15.7 μM), in an overall reaction that is thermodynamically and kinetically reversible. Noteworthy, both Dd FDH-catalyzed formate oxidation and carbon dioxide reduction are completely inactivated by cyanide. Current FDH reaction mechanistic proposals are discussed and a different mechanism is here suggested: formate oxidation and carbon dioxide reduction are proposed to proceed through hydride transfer and the sulfo group of the oxidized and reduced molybdenum center, Mo(6+)═S and Mo(4+)-SH, are suggested to be the direct hydride acceptor and donor, respectively. PMID:27348246

  20. Characterization of inosine monophosphate dehydrogenase from Staphylococcus aureus ATCC12600 and its involvement in biofilm formation

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    S. Yeswanth

    2013-10-01

    Full Text Available Background: In Staphylococcus aureus purine metabolism plays a crucial role in the formation of biofilm which is a key pathogenic factor. The present study is aimed in the characterization of inosine monophosphate dehydrogenase (IMPDH from Staphylococcus aureus ATCC 12600. Methods: IMPDH gene was amplified using primers designed from IMPDH gene sequence of S. aureus reported in the database. Then polymerase chain reaction (PCR product was cloned in the Sma I site of M13mp18 and expressed in Escherichia coli JM109. The recombinant IMPDH (rIMPDH was overexpressed with 1 mM isopropyl beta-D-1- thiogalactopyranoside (IPTG; Michaelis constant (Km, maximum enzyme velocity (Vmax and catalytic constant (Kcat of expressed IMPDH were determined. Results: The enzyme kinetics of IMPDH grown under aerobic conditions showed a Km of 43.71±1.56 µM, Vmax of 0.247±0.84/µM/mg/min and Kcat of 2.74±0.015/min while in anaerobic conditions the kinetics showed Km of 42.81±3.154/ µM, Vmax of 0.378±0.036 µM/mg/min and Kcat of 4.78±0.021 /min, indicating elevated levels of IMPDH activity under anaerobic conditions. Three-folds increased activity in the presence of 1 mM adenosine triphosphate (ATP correlated with biofilm formation. The kinetics of pure rIMPDH were close to the native IMPDH of S. aureus ATCC12600 and the enzyme showed single band in sodium dodecyl sulphate polyacrylamide gel electrophoresis with a molecular weight of 53 KDa. Conclusions: Elevated activity of IMPDH was observed in S. aureus grown under anaerobic conditions and this was correlated with the biofilm formation indicating the linkage between purine metabolism and pathogenesis.

  1. The xylose reductase/xylitol dehydrogenase/xylulokinase ratio affects product formation in recombinant xylose-utilising Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Eliasson, Anna; Hofmeyr, J.H.S.; Pedler, S.;

    2001-01-01

    Data simulations based on a kinetic model implied that under simplified simulation conditions a 1:greater than or equal to 10:greater than or equal to4 relation of the xylose reductase (XR)/xylitol dehydrogenase (XDH)/xylulokinase (XK) ratio was optimal in minimising xylitol formation during xylose...... utilisation in yeast. The steady-state level of the intermediary xylitol depended also, to a great extent, on the NADH and NAD(+) concentrations. Anaerobic xylose utilisation was investigated for three different recombinant. XR-, XDH- and XK-expressing Saccharomyces cerevisiae strains, TMB 3002, TMB 3003 and...... TMB 3004, to verify the model predictions. Overexpression of XK was found to be necessary for ethanol formation from xylose. Furthermore, the xylitol formation decreased with decreasing XR/XDH ratio, while the ethanol formation increased. Of the three strains, TMB 3004, which was the strain with a XR...

  2. Structural basis for double cofactor specificity in a new formate dehydrogenase from the acidobacterium Granulicella mallensis MP5ACTX8.

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    Fogal, Stefano; Beneventi, Elisa; Cendron, Laura; Bergantino, Elisabetta

    2015-11-01

    Formate dehydrogenases (FDHs) are considered particularly useful enzymes in biocatalysis when the regeneration of the cofactor NAD(P)H is required, that is, in chiral synthesis with dehydrogenases. Their utilization is however limited to the recycling of NAD(+), since all (apart one) of the FDHs characterized so far are strictly specific for this cofactor, and this is a major drawback for their otherwise wide applicability. Despite the many attempts performed to modify cofactor specificity by protein engineering different NAD(+)-dependent FDHs, in the general practice, glucose or phosphite dehydrogenases are chosen for the recycling of NADP(+). We report on the functional and structural characterization of a new FDH, GraFDH, identified by mining the genome of the extremophile prokaryote Granulicella mallensis MP5ACTX8. The new enzyme displays a valuable stability in the presence of many organic cosolvents as well as double cofactor specificity, with NADP(+) preferred over NAD(+) at acidic pH values, at which it also shows the highest stability. The quite low affinities for both cofactors as well as for the substrate formate indicate, however, that the native enzyme requires optimization to be applied as biocatalytic tool. We also determined the crystal structure of GraFDH both as apoprotein and as holoprotein, either in complex with NAD(+) or NADP(+). Noticeably, the latter represents the first structure of an FDH enzyme in complex with NADP(+). This fine picture of the structural determinants involved in cofactor selectivity will possibly boost protein engineering of the new enzyme or other homolog FDHs in view of their biocatalytic exploitation for NADP(+) recycling. PMID:26104866

  3. Formation of functional heterodimers by isozymes 1 and 2 of pyruvate dehydrogenase kinase.

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    Boulatnikov, Igor; Popov, Kirill M

    2003-02-21

    Pyruvate dehydrogenase kinase (PDK) is a mitochondrial enzyme responsible for regulation of the pyruvate dehydrogenase complex and, consequently, aerobic oxidation of carbohydrate fuels in general. In mammals, there are four genetically and biochemically distinct forms of PDK that are expressed in a tissue-specific manner (PDK1, PDK2, PDK3, and PDK4). These protein kinases have been shown to function as dimers, but the possibility of heterodimerization between various isozyme subunits has not yet been investigated. Here, we demonstrate that two members of the PDK family, PDK1 and PDK2, form heterodimeric species when coexpressed in the same Escherichia coli cell. The heterodimeric kinase produced in vivo was purified to near homogeneity by affinity chromatography. The purified kinase was stable and was not subjected to reassortment of the subunits. The heterodimeric kinase was catalytically active and was clearly distinct from homodimeric PDK1 or PDK2 with respect to kinetic parameters, site specificity and regulation. These data strongly suggest that heterodimerization between PDK1 and PDK2 adds another level of diversity to this protein family in addition to that which arises from gene multiplicity. PMID:12573248

  4. Recombinant formate dehydrogenase from Arabidopsis thaliana: Preparation, crystal growth in microgravity, and preliminary X-ray diffraction study

    Energy Technology Data Exchange (ETDEWEB)

    Shabalin, I. G., E-mail: inna@ns.crys.ras.ru [Russian Academy of Sciences, Shubnikov Institute of Crystallography (Russian Federation); Serov, A. E.; Skirgello, O. E. [Moscow State University, Faculty of Chemistry (Russian Federation); Timofeev, V. I.; Samygina, V. R. [Russian Academy of Sciences, Shubnikov Institute of Crystallography (Russian Federation); Popov, V. O. [Russian Academy of Sciences, Bach Institute of Biochemistry (Russian Federation); Tishkov, V. I. [Moscow State University, Faculty of Chemistry (Russian Federation); Kuranova, I. P. [Russian Academy of Sciences, Shubnikov Institute of Crystallography (Russian Federation)

    2010-09-15

    Crystals of high-purity recombinant NAD{sup +}-dependent formate dehydrogenase from the higher plant Arabidopsis thaliana (AraFDH) were grown in microgravity in the Modul'-1 protein crystallization apparatus on the International Space Station. The space-grown crystals have larger sizes than those grown on Earth. X-ray diffraction data suitable for determining the three-dimensional structure were collected from the space-grown crystals to a resolution of 1.22 A using an X-ray synchrotron source. The crystals belong to sp. gr. P4{sub 3}2{sub 1}2; the unit-cell parameters are a = b = 107.865 A, c = 71.180 A, {alpha} = {beta} = {gamma} = 90 deg.

  5. Recombinant formate dehydrogenase from Arabidopsis thaliana: Preparation, crystal growth in microgravity, and preliminary X-ray diffraction study

    International Nuclear Information System (INIS)

    Crystals of high-purity recombinant NAD+-dependent formate dehydrogenase from the higher plant Arabidopsis thaliana (AraFDH) were grown in microgravity in the Modul'-1 protein crystallization apparatus on the International Space Station. The space-grown crystals have larger sizes than those grown on Earth. X-ray diffraction data suitable for determining the three-dimensional structure were collected from the space-grown crystals to a resolution of 1.22 A using an X-ray synchrotron source. The crystals belong to sp. gr. P43212; the unit-cell parameters are a = b = 107.865 A, c = 71.180 A, α = β = γ = 90 deg.

  6. Peroxisomes induced in Candida boidinii by methanol, oleic acid and D-alanine vary in metabolic function but share common integral membrane proteins

    NARCIS (Netherlands)

    Goodman, Joel M.; Trapp, Steven B.; Hwang, Harold; Veenhuis, Marten

    1990-01-01

    Peroxisomes massively proliferate in the methylotrophic yeast Candida boidinii when cultured on methanol as the only carbon and energy source. These organelles contain enzymes that catalyze the initial reactions of methanol utilization. The membranes contain abundant proteins of unknown function; th

  7. De Novo Assembly of Candida sojae and Candida boidinii Genomes, Unexplored Xylose-Consuming Yeasts with Potential for Renewable Biochemical Production.

    Science.gov (United States)

    Borelli, Guilherme; José, Juliana; Teixeira, Paulo José Pereira Lima; Dos Santos, Leandro Vieira; Pereira, Gonçalo Amarante Guimarães

    2016-01-01

    Candida boidinii and Candida sojae yeasts were isolated from energy cane bagasse and plague-insects. Both have fast xylose uptake rate and produce great amounts of xylitol, which are interesting features for food and 2G ethanol industries. Because they lack published genomes, we have sequenced and assembled them, offering new possibilities for gene prospection. PMID:26769937

  8. Proliferation and metabolic significance of peroxisomes in Candida boidinii during growth on D-alanine or oleic acid as the sole carbon source

    NARCIS (Netherlands)

    Sulter, G.J.; Waterham, H.R.; Goodman, J.M.; Veenhuis, M.

    1990-01-01

    We have studied the induction of peroxisomes in the methylotrophic yeast Candida boidinii by D-alanine and oleic acid. The organism was able to utilize each of these compounds as the sole carbon source and grew with growth rates of µ = 0.20 h-1 (on D-alanine) or µ = 0.43 h-1 (on oleic acid). Growth

  9. Cloning, expression and characterization of glycerol dehydrogenase involved in 2,3-butanediol formation in Serratia marcescens H30.

    Science.gov (United States)

    Zhang, Liaoyuan; Xu, Quanming; Peng, Xiaoqian; Xu, Boheng; Wu, Yuehao; Yang, Yulong; Sun, Shujing; Hu, Kaihui; Shen, Yaling

    2014-09-01

    The meso-2,3-butanediol dehydrogenase (meso-BDH) from S. marcescens H30 is responsible for converting acetoin into 2,3-butanediol during sugar fermentation. Inactivation of the meso-BDH encoded by budC gene does not completely abolish 2,3-butanediol production, which suggests that another similar enzyme involved in 2,3-butanediol formation exists in S. marcescens H30. In the present study, a glycerol dehydrogenase (GDH) encoded by gldA gene from S. marcescens H30 was expressed in Escherichia coli BL21(DE3), purified and characterized for its properties. In vitro conversion indicated that the purified GDH could catalyze the interconversion of (3S)-acetoin/meso-2,3-butanediol and (3R)-acetoin/(2R,3R)-2,3-butanediol. (2S,3S)-2,3-Butanediol was not a substrate for the GDH at all. Kinetic parameters of the GDH enzyme showed lower K m value and higher catalytic efficiency for (3S/3R)-acetoin in comparison to those for (2R,3R)-2,3-butanediol and meso-2,3-butanediol, implying its physiological role in favor of 2,3-butanediol formation. Maximum activity for reduction of (3S/3R)-acetoin and oxidations of meso-2,3-butanediol and glycerol was observed at pH 8.0, while it was pH 7.0 for diacetyl reduction. The enzyme exhibited relative high thermotolerance with optimum temperature of 60 °C in the oxidation-reduction reactions. Over 60 % of maximum activity was retained at 70 °C. Additionally, the GDH activity was significantly enhanced for meso-2,3-BD oxidation in the presence of Fe(2+) and for (3S/3R)-acetoin reduction in the presence of Mn(2+), while several cations inhibited its activity, particularly Fe(2+) and Fe(3+) for (3S/3R)-acetoin reduction. The properties provided potential application for single configuration production of acetoin and 2,3-butanediol .

  10. Altered Fermentative Metabolism in Chlamydomonas reinhardtii Mutants Lacking Pyruvate Formate Lyase and Both Pyruvate Formate Lyase and Alcohol Dehydrogenase

    Energy Technology Data Exchange (ETDEWEB)

    Catalanotti, C.; Dubini, A.; Subramanian, V.; Yang, W. Q.; Magneschi, L.; Mus, F.; Seibert, M.; Posewitz, M. C.; Grossman, A. R.

    2012-02-01

    Chlamydomonas reinhardtii, a unicellular green alga, often experiences hypoxic/anoxic soil conditions that activate fermentation metabolism. We isolated three Chlamydomonas mutants disrupted for the pyruvate formate lyase (PFL1) gene; the encoded PFL1 protein catalyzes a major fermentative pathway in wild-type Chlamydomonas cells. When the pfl1 mutants were subjected to dark fermentative conditions, they displayed an increased flux of pyruvate to lactate, elevated pyruvate decarboxylation, ethanol accumulation, diminished pyruvate oxidation by pyruvate ferredoxin oxidoreductase, and lowered H2 production. The pfl1-1 mutant also accumulated high intracellular levels of lactate, succinate, alanine, malate, and fumarate. To further probe the system, we generated a double mutant (pfl1-1 adh1) that is unable to synthesize both formate and ethanol. This strain, like the pfl1 mutants, secreted lactate, but it also exhibited a significant increase in the levels of extracellular glycerol, acetate, and intracellular reduced sugars and a decrease in dark, fermentative H2 production. Whereas wild-type Chlamydomonas fermentation primarily produces formate and ethanol, the double mutant reroutes glycolytic carbon to lactate and glycerol. Although the metabolic adjustments observed in the mutants facilitate NADH reoxidation and sustained glycolysis under dark, anoxic conditions, the observed changes could not have been predicted given our current knowledge of the regulation of fermentation metabolism.

  11. Mixed disulfide formation at Cys141 leads to apparent unidirectional attenuation of Aspergillus niger NADP-glutamate dehydrogenase activity.

    Directory of Open Access Journals (Sweden)

    Adhish S Walvekar

    Full Text Available NADP-Glutamate dehydrogenase from Aspergillus niger (AnGDH exhibits sigmoid 2-oxoglutarate saturation. Incubation with 2-hydroxyethyl disulfide (2-HED, the disulfide of 2-mercaptoethanol resulted in preferential attenuation of AnGDH reductive amination (forward activity but with a negligible effect on oxidative deamination (reverse activity, when monitored in the described standard assay. Such a disulfide modified AnGDH displaying less than 1.0% forward reaction rate could be isolated after 2-HED treatment. This unique forward inhibited GDH form (FIGDH, resembling a hypothetical 'one-way' active enzyme, was characterized. Kinetics of 2-HED mediated inhibition and protein thiol titrations suggested that a single thiol group is modified in FIGDH. Two site-directed cysteine mutants, C141S and C415S, were constructed to identify the relevant thiol in FIGDH. The forward activity of C141S alone was insensitive to 2-HED, implicating Cys141 in FIGDH formation. It was observed that FIGDH displayed maximal reaction rate only after a pre-incubation with 2-oxoglutarate and NADPH. In addition, compared to the native enzyme, FIGDH showed a four fold increase in K0.5 for 2-oxoglutarate and a two fold increase in the Michaelis constants for ammonium and NADPH. With no change in the GDH reaction equilibrium constant, the FIGDH catalyzed rate of approach to equilibrium from reductive amination side was sluggish. Altered kinetic properties of FIGDH at least partly account for the observed apparent loss of forward activity when monitored under defined assay conditions. In sum, although Cys141 is catalytically not essential, its covalent modification provides a striking example of converting the biosynthetic AnGDH into a catabolic enzyme.

  12. Specific and sustainable bioelectro-reduction of carbon dioxide to formate on a novel enzymatic cathode.

    Science.gov (United States)

    Zhang, Lijuan; Liu, Junyi; Ong, Jacky; Li, Sam Fong Yau

    2016-11-01

    To specifically convert waste CO2 into renewable chemicals, enzymatic electrosynthesis (EES) of formate from CO2 reduction was investigated in a bioelectrochemical system (BES). A novel cathode with immobilized enzyme and electropolymerized mediator-regenerator was fabricated for such bioelectrocatalytic EES. Formate dehydrogenase from Candida boidinii (CbFDH) was set as a new model enzyme in BES. Modified Nafion micelles with appropriate pore size were found to be suitable for immobilization of CbFDH and protection of its enzymatic activity and lifetime at optimal pH of 6.0. The enzymatic electrosynthesis activity of immobilized CbFDH was characterized systematically. Quite a small overpotential was required in the bioelectrochemical EES reaction. A two-electron transfer process was confirmed in the CbFDH-catalyzed reduction of bicarbonate to formate. With electro-polymerized neutral red (PolyNR) as a NADH (mediator)-regenerator, efficient formate production could be achieved at a maximum rate of 159.89 mg L(-1) h(-1) under poised potential of -0.80 V (vs. SHE). The immobilized CbFDH and electropolymerized PolyNR on an enzymatic cathode contributed greatly to sustainable EES, giving energy-rich formate as the only catalysis product. PMID:27501309

  13. The Molybdenum Active Site of Formate Dehydrogenase Is Capable of Catalyzing C-H Bond Cleavage and Oxygen Atom Transfer Reactions.

    Science.gov (United States)

    Hartmann, Tobias; Schrapers, Peer; Utesch, Tillmann; Nimtz, Manfred; Rippers, Yvonne; Dau, Holger; Mroginski, Maria Andrea; Haumann, Michael; Leimkühler, Silke

    2016-04-26

    Formate dehydrogenases (FDHs) are capable of performing the reversible oxidation of formate and are enzymes of great interest for fuel cell applications and for the production of reduced carbon compounds as energy sources from CO2. Metal-containing FDHs in general contain a highly conserved active site, comprising a molybdenum (or tungsten) center coordinated by two molybdopterin guanine dinucleotide molecules, a sulfido and a (seleno-)cysteine ligand, in addition to a histidine and arginine residue in the second coordination sphere. So far, the role of these amino acids in catalysis has not been studied in detail, because of the lack of suitable expression systems and the lability or oxygen sensitivity of the enzymes. Here, the roles of these active site residues is revealed using the Mo-containing FDH from Rhodobacter capsulatus. Our results show that the cysteine ligand at the Mo ion is displaced by the formate substrate during the reaction, the arginine has a direct role in substrate binding and stabilization, and the histidine elevates the pKa of the active site cysteine. We further found that in addition to reversible formate oxidation, the enzyme is further capable of reducing nitrate to nitrite. We propose a mechanistic scheme that combines both functionalities and provides important insights into the distinct mechanisms of C-H bond cleavage and oxygen atom transfer catalyzed by formate dehydrogenase. PMID:27054466

  14. Mice deficient in 11beta-hydroxysteroid dehydrogenase type 1 lack bone marrow adipocytes, but maintain normal bone formation

    DEFF Research Database (Denmark)

    Justesen, Jeannette; Mosekilde, Lis; Holmes, Megan;

    2004-01-01

    Glucocorticoids (GCs) exert potent, but poorly characterized, effects on the skeleton. The cellular activity of GCs is regulated at a prereceptor level by 11beta-hydroxysteroid dehydrogenases (11betaHSDs). The type 1 isoform, which predominates in bone, functions as a reductase in intact cells...

  15. Molybdenum-containing membrane-bound formate dehydrogenase isolated from Citrobacter sp. S-77 having high stability against oxygen, pH, and temperature.

    Science.gov (United States)

    Nguyen, Nga T; Yatabe, Takeshi; Yoon, Ki-Seok; Ogo, Seiji

    2014-10-01

    Membrane-bound formate dehydrogenase (FDH) was purified to homogeneity from a facultative anaerobic bacterium Citrobacter sp. S-77. The FDH from Citrobacter sp. S-77 (FDHS77) was a monomer with molecular mass of approximately 150 kDa. On SDS-PAGE, the purified FDHS77 showed as three different protein bands with molecular mass of approximately 95, 87, and 32 kDa, respectively. Based on the N-terminal amino acid sequence analysis, the sequence alignments observed for the 87 kDa protein band were identical to that of the large subunit of 95 kDa, indicating that the purified FDHS77 consisted of two subunits; a 95 kDa large subunit and a 32 kDa small subunit. The purified FDHS77 in this purification did not contain a heme b subunit, but the FDHS77 showed significant activity for formate oxidation, determined by the Vmax of 30.4 U/mg using benzyl viologen as an electron acceptor. The EPR and ICP-MS spectra indicate that the FDHS77 is a molybdenum-containing enzyme, displaying a remarkable O2-stability along with thermostability and pH resistance. This is the first report of the purification and characterization of a FDH from Citrobacter species.

  16. Altered Fermentative Metabolism in Chlamydomonas reinhardtii Mutants Lacking Pyruvate Formate Lyase and Both Pyruvate Formate Lyase and Alcohol Dehydrogenase[W

    Science.gov (United States)

    Catalanotti, Claudia; Dubini, Alexandra; Subramanian, Venkataramanan; Yang, Wenqiang; Magneschi, Leonardo; Mus, Florence; Seibert, Michael; Posewitz, Matthew C.; Grossman, Arthur R.

    2012-01-01

    Chlamydomonas reinhardtii, a unicellular green alga, often experiences hypoxic/anoxic soil conditions that activate fermentation metabolism. We isolated three Chlamydomonas mutants disrupted for the pyruvate formate lyase (PFL1) gene; the encoded PFL1 protein catalyzes a major fermentative pathway in wild-type Chlamydomonas cells. When the pfl1 mutants were subjected to dark fermentative conditions, they displayed an increased flux of pyruvate to lactate, elevated pyruvate decarboxylation, ethanol accumulation, diminished pyruvate oxidation by pyruvate ferredoxin oxidoreductase, and lowered H2 production. The pfl1-1 mutant also accumulated high intracellular levels of lactate, succinate, alanine, malate, and fumarate. To further probe the system, we generated a double mutant (pfl1-1 adh1) that is unable to synthesize both formate and ethanol. This strain, like the pfl1 mutants, secreted lactate, but it also exhibited a significant increase in the levels of extracellular glycerol, acetate, and intracellular reduced sugars and a decrease in dark, fermentative H2 production. Whereas wild-type Chlamydomonas fermentation primarily produces formate and ethanol, the double mutant reroutes glycolytic carbon to lactate and glycerol. Although the metabolic adjustments observed in the mutants facilitate NADH reoxidation and sustained glycolysis under dark, anoxic conditions, the observed changes could not have been predicted given our current knowledge of the regulation of fermentation metabolism. PMID:22353371

  17. Glucose-6-phosphate dehydrogenase

    Science.gov (United States)

    ... 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 ...

  18. Examining the anti-candidal activity of 10 selected Indian herbs and investigating the effect of Lawsonia inermis extract on germ tube formation, protease, phospholipase, and aspartate dehydrogenase enzyme activity in Candida albicans

    Science.gov (United States)

    Ravichandran, Sripathy; Muthuraman, Sundararaman

    2016-01-01

    Objective: The objective of the study is to identify potential anti-candidal agents from natural resources and elucidate the effect of Lawsonia inermis extract on major virulent factors of Candida albicans. Materials and Methods: Plants, the most abundant and readily available resource of diverse bioactives, were chosen for the anti-candidal screening study. Ten different plants that were proven to have antimicrobial activity but not explored much for anti-candidal activity were chosen for this study. Ethyl acetate extract of these plant leaves were tested for the anti-candidal activity. Extracts with good anti-candidal activity were further screened for its effect in C. albicans germ tube formation and enzyme (protease, phospholipase, and aspartate dehydrogenase) activity. Results: Among 10 plants screened, L. inermis extract showed complete inhibition of C. albicans. On further evaluation, this extract completely inhibited C. albicans germ tube formation in serum until the end of incubation period (3 h). This extract also exhibited dose-dependent inhibitory activity against two major virulent enzymes of C. albicans, proteases (27–33%) and phospholipases (44.5%). In addition to it, this extract completely inhibited both the isoforms of constitutive candidal enzyme aspartate dehydrogenase, thereby affecting amino acid biosynthesis. Conclusion: Thus, this study confirms the anti-candidal potential of L. inermis and hence can be considered further for development of anti-candidal drug. PMID:26997722

  19. A role for tungsten in the biology of Campylobacter jejuni: tungstate stimulates formate dehydrogenase activity and is transported via an ultra-high affinity ABC system distinct from the molybdate transporter.

    Science.gov (United States)

    Smart, Jonathan P; Cliff, Matthew J; Kelly, David J

    2009-11-01

    The food-borne pathogen Campylobacter jejuni possesses no known tungstoenzymes, yet encodes two ABC transporters (Cj0300-0303 and Cj1538-1540) homologous to bacterial molybdate (ModABC) uptake systems and the tungstate transporter (TupABC) of Eubacterium acidaminophilum respectively. The actual substrates and physiological role of these transporters were investigated. Tryptophan fluorescence spectroscopy and isothermal titration calorimetry of the purified periplasmic binding proteins of each system revealed that while Cj0303 is unable to discriminate between molybdate and tungstate (K(D) values for both ligands of 4-8 nM), Cj1540 binds tungstate with a K(D) of 1.0 +/- 0.2 pM; 50 000-fold more tightly than molybdate. Induction-coupled plasma mass spectroscopy of single and double mutants showed that this large difference in affinity is reflected in a lower cellular tungsten content in a cj1540 (tupA) mutant compared with a cj0303c (modA) mutant. Surprisingly, formate dehydrogenase (FDH) activity was decreased approximately 50% in the tupA strain, and supplementation of the growth medium with tungstate significantly increased FDH activity in the wild type, while inhibiting known molybdoenzymes. Our data suggest that C. jejuni possesses a specific, ultra-high affinity tungstate transporter that supplies tungsten for incorporation into FDH. Furthermore, possession of two MoeA paralogues may explain the formation of both molybdopterin and tungstopterin in this bacterium. PMID:19818021

  20. Glucose-6-phosphate dehydrogenase deficiency

    Science.gov (United States)

    ... 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 ...

  1. Pyruvate Dehydrogenase Kinase 4

    OpenAIRE

    Cadoudal, Thomas; Distel, Emilie; Durant, Sylvie; Fouque, Françoise; Blouin, Jean-Marc; Collinet, Martine; Bortoli, Sylvie; Forest, Claude; Benelli, Chantal

    2008-01-01

    OBJECTIVE—Pyruvate dehydrogenase complex (PDC) serves as the metabolic switch between glucose and fatty acid utilization. PDC activity is inhibited by PDC kinase (PDK). PDC shares the same substrate, i.e., pyruvate, as glyceroneogenesis, a pathway controlling fatty acid release from white adipose tissue (WAT). Thiazolidinediones activate glyceroneogenesis. We studied the regulation by rosiglitazone of PDK2 and PDK4 isoforms and tested the hypothesis that glyceroneogenesis could be controlled ...

  2. Application of NAD-dependent polyol dehydrogenases for enzymatic mannitol/sorbitol production with coenzyme regeneration.

    Science.gov (United States)

    Parmentier, S; Arnaut, F; Soetaert, W; Vandamme, E J

    2003-01-01

    D-Mannitol and D-sorbitol were produced enzymatically from D-fructose using NAD-dependent polyol dehydrogenases. For the production of D-mannitol the Leuconostoc mesenteroides mannitol dehydrogenase could be used. Gluconobacter oxydans cell extract contained however both mannitol and sorbitol dehydrogenase. When this cell extract was used, the reduction of D-fructose resulted in a mixture of D-sorbitol and D-mannitol. To determine the optimal bioconversion conditions the polyol dehydrogenases were characterized towards pH- and temperature-optimum and -stability. As a compromise between enzyme activity and stability, the bioconversion reactions were performed at pH 6.5 and 25 degrees C. Since the polyol dehydrogenases are NADH-dependent, an efficient coenzyme regeneration was needed. Regeneration of NADH was accomplished by formate dehydrogenase-mediated oxidation of formate into CO2.

  3. Dehydrogenase isoenzyme polymorphism in genus Prunus, subgenus Cerasus

    Directory of Open Access Journals (Sweden)

    Čolić Slavica

    2012-01-01

    Full Text Available Dehydrogenase polymorphism was studied in 36 sour cherry (Prunus cerasus L., sweet cherry (Prunus avuim L., mahaleb (Prunus mahaleb L., ground cherry (Prunus fruticosa Pall., duke cherry (Prunus gondounii Redh., Japanese flowering cherry (Prunus serrulata Lindl. and four iterspecific hybrids (standard cherry rootstocks ‘Gisela 5’, ‘Gisela 6’, ‘Max Ma’ and ‘Colt’. Inner bark of one-year-old shoots, in dormant stage, was used for enzyme extraction. Vertical PAGE was used for isoenzyme analysis: alcohol dehydrogenase (ADH, formate dehydrogenase (FDH, glutamate dehydrogenase (GDH, isocitrate dehydrogenaze (IDH, malate dehydrogenase (MDH, phosphogluconate dehydrogenase (PGD, and shikimate dehydrogenase (SDH. All studied systems were polymorphic at 10 loci: Adh -1 (3 genotypes and Adh-2 (5 genotypes, Fdh-1 (2 genotypes, Gdh-1 (3 genotypes, Idh-1 (4 genotypes i Idh -2 (5 genotypes, Mdh-1 (3 genotypes, Pgd-1 (4 genotypes, Sdh-1 (1 genotype i Sdh-2 (3 genotypes. Cluster analysis was used to construct dendrogram on which four groups of similar genotypes were separated. Obtained results indicate that studied enzyme systems can be used for determination of genus Prunus, subgenus Cerasus. Among studied enzyme systems ADH, IDH and SDH were the most polymorphic and most useful to identify genetic variability. Polymorphism of FDH and GDH in genus Prunus, subgenus Cerasus was described first time in this work. First results for dehydrogenase variability of Oblačinska indicate that polymorphism of loci Idh-2 and Sdh-2 can be useful for discrimination of different clones.

  4. Glucose-6-Phosphate Dehydrogenase Deficiency Overview

    Science.gov (United States)

    ... 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 ...

  5. Sorbitol dehydrogenase is a zinc enzyme.

    OpenAIRE

    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...

  6. Specific biotinylation of IMP dehydrogenase

    OpenAIRE

    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.

  7. Formation of In Vitro Mixed-Species Biofilms by Lactobacillus pentosus and Yeasts Isolated from Spanish-Style Green Table Olive Fermentations.

    Science.gov (United States)

    León-Romero, Ángela; Domínguez-Manzano, Jesús; Garrido-Fernández, Antonio; Arroyo-López, Francisco Noé; Jiménez-Díaz, Rufino

    2015-11-13

    The present work details the in vitro interactions between Lactobacillus pentosus and yeast strains isolated from table olive processing to form mixed biofilms. Among the different pairs assayed, the strongest biofilms were obtained from L. pentosus and Candida boidinii strain cocultures. However, biofilm formation was inhibited in the presence of d-(+)-mannose. In addition, biofilm formation by C. boidinii monoculture was stimulated in the absence of cell-cell contact with L. pentosus. Scanning electron microscopy revealed that a sort of "sticky" material formed by the yeasts contributed to substrate adherence. Hence, the data obtained in this work suggest that yeast-lactobacilli biofilms may be favored by the presence of a specific mate of yeast and L. pentosus, and that more than one mechanism might be implicated in the biofilm formation. This knowledge will help in the design of appropriate mixed starter cultures of L. pentosus-yeast species pairs that are able to improve the quality and safety of Spanish-style green table olive processing.

  8. Formation of In Vitro Mixed-Species Biofilms by Lactobacillus pentosus and Yeasts Isolated from Spanish-Style Green Table Olive Fermentations

    Science.gov (United States)

    León-Romero, Ángela; Domínguez-Manzano, Jesús; Garrido-Fernández, Antonio; Arroyo-López, Francisco Noé

    2015-01-01

    The present work details the in vitro interactions between Lactobacillus pentosus and yeast strains isolated from table olive processing to form mixed biofilms. Among the different pairs assayed, the strongest biofilms were obtained from L. pentosus and Candida boidinii strain cocultures. However, biofilm formation was inhibited in the presence of d-(+)-mannose. In addition, biofilm formation by C. boidinii monoculture was stimulated in the absence of cell-cell contact with L. pentosus. Scanning electron microscopy revealed that a sort of “sticky” material formed by the yeasts contributed to substrate adherence. Hence, the data obtained in this work suggest that yeast-lactobacilli biofilms may be favored by the presence of a specific mate of yeast and L. pentosus, and that more than one mechanism might be implicated in the biofilm formation. This knowledge will help in the design of appropriate mixed starter cultures of L. pentosus-yeast species pairs that are able to improve the quality and safety of Spanish-style green table olive processing. PMID:26567305

  9. Enzymic and structural studies on Drosophila alcohol dehydrogenase and other short-chain dehydrogenases/reductases

    NARCIS (Netherlands)

    Smilda, T; Kamminga, AH; Reinders, P; Baron, W; Vlieg, JETV; Beintema, JJ

    2001-01-01

    Enzymic and structural studies on Drosophila alcohol dehydrogenases and other short-chain dehydrogenases/reductases (SDRs) are presented. Like alcohol dehydrogenases from other Drosophila species, the enzyme from D, simulans is more active on secondary than on primary alcohols, although ethanol is i

  10. 21 CFR 862.1670 - Sorbitol dehydrogenase test system.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Sorbitol dehydrogenase test system. 862.1670... Systems § 862.1670 Sorbitol dehydrogenase test system. (a) Identification. A sorbitol dehydrogenase test system is a device intended to measure the activity of the enzyme sorbitol dehydrogenase in...

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

    NARCIS (Netherlands)

    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

  12. Microbial alcohol dehydrogenases: identification, characterization and engineering

    NARCIS (Netherlands)

    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

  13. Transcriptional Regulation of Pyruvate Dehydrogenase Kinase

    OpenAIRE

    Ji Yun Jeong; Nam Ho Jeoung; Keun-Gyu Park; In-Kyu Lee

    2012-01-01

    The pyruvate dehydrogenase complex (PDC) activity is crucial to maintains blood glucose and ATP levels, which largely depends on the phosphorylation status by pyruvate dehydrogenase kinase (PDK) isoenzymes. Although it has been reported that PDC is phosphorylated and inactivated by PDK2 and PDK4 in metabolically active tissues including liver, skeletal muscle, heart, and kidney during starvation and diabetes, the precise mechanisms by which expression of PDK2 and PDK4 are transcriptionally re...

  14. Screening of aspartate dehydrogenase of bacteria

    OpenAIRE

    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...

  15. Phosphorylation site on yeast pyruvate dehydrogenase complex

    International Nuclear Information System (INIS)

    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

  16. A New Biochemical Way for Conversion of CO2 to Methanol via Dehydrogenases Encapsulated in SiO2 Matrix

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    CO2 is converted to methanol through an enzymatic approach using formate dehydro- genase (FateDH), formaldehyde dehydrogenase (FaldDH) and alcohol dehydrogenase (ADH) co- encapsulated in silica gel prepared by modified sol-gel process as catalysts, TEOS as precursor, NADH as an electron donor. The highest yield of methanol was up to 92.1% under 37℃, pH7.0 and 0.3Mpa.

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

    Science.gov (United States)

    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

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

    International Nuclear Information System (INIS)

    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)

  19. Detection of Recombinant Protein Expression of Formate Dehydrogenase in Single Living Escherichia Coli Cell by Laser Tweezers Raman Spectroscopy%单细胞激光拉曼光谱检测重组大肠杆菌细胞表达甲酸脱氢酶

    Institute of Scientific and Technical Information of China (English)

    卢明倩; 董蓉; 温顺华; 张韦; 王巧贞; 黄庶识; 陈丽梅

    2012-01-01

    甲酸脱氢酶(FDH,EC1.2.1.2)在工业生产中有重要的应用价值,工业上应用的FDH可以通过构建高水平表达重组FDH蛋白的基因工程菌生产,用分子生物学的方法检测重组蛋白的高效表达和积累操作繁杂,耗时耗力且需要破碎细胞.为了寻找一种简单快速,不需破碎细胞,且能实时检测FDH重组蛋白在基因工程菌中表达情况的方法,本研究应用单细胞激光拉曼光谱分析技术(LTRS),研究IPTG诱导不同时间后甲酸脱氢酶重组蛋白(FDH)在大肠杆菌细胞中的表达水平.结果表明,FDH的特征峰1004,1355,1455和1667 cm-1随着IPTG诱导时间的延长而增强,说明在诱导培养过程中FDH重组蛋白在重组大肠杆菌细胞中表达并积累,这4个峰强的增加值所反映的FDH表达量与SDS-PAGE电泳分析结果一致.实验结果证明,LTRS是快速有效检测单个大肠杆菌活细胞体内重组FDH实时表达的一种非入侵方法.%The detection of the expression of formate dehydrogenase (FDH) recombination proteins in E. coli by molecular methods is time-consuming and hard-working and needs to destruct E. coli cells. To explore a simple and rapid method without cell destruction to detect the real-time expression of FDH recombination proteins in E. coli, Laser Tweezers Raman spectroscopy (LTRS) was used to investigate the recombinant protein expression of formate dehydrogenase (FDH) in the single living E. coli cell at different culture times following the induction with isopropyl thiogalactoside (IPTG). The result showed that the characteristic peaks corresponding to the recombinant FDH protein were gradually enhanced with an in increase in IPTG induction time, indicating the expression and the accumulation of FDH protein in recombinant E. coli cells. This result is consistent with that obtained by the analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This evidence confirms that LTRS is an effectively method

  20. Staphylococcus epidermidis: metabolic adaptation and biofilm formation in response to different oxygen concentrations.

    Science.gov (United States)

    Uribe-Alvarez, Cristina; Chiquete-Félix, Natalia; Contreras-Zentella, Martha; Guerrero-Castillo, Sergio; Peña, Antonio; Uribe-Carvajal, Salvador

    2016-02-01

    Staphylococcus epidermidis has become a major health hazard. It is necessary to study its metabolism and hopefully uncover therapeutic targets. Cultivating S. epidermidis at increasing oxygen concentration [O2] enhanced growth, while inhibiting biofilm formation. Respiratory oxidoreductases were differentially expressed, probably to prevent reactive oxygen species formation. Under aerobiosis, S. epidermidis expressed high oxidoreductase activities, including glycerol-3-phosphate dehydrogenase, pyruvate dehydrogenase, ethanol dehydrogenase and succinate dehydrogenase, as well as cytochromes bo and aa3; while little tendency to form biofilms was observed. Under microaerobiosis, pyruvate dehydrogenase and ethanol dehydrogenase decreased while glycerol-3-phosphate dehydrogenase and succinate dehydrogenase nearly disappeared; cytochrome bo was present; anaerobic nitrate reductase activity was observed; biofilm formation increased slightly. Under anaerobiosis, biofilms grew; low ethanol dehydrogenase, pyruvate dehydrogenase and cytochrome bo were still present; nitrate dehydrogenase was the main terminal electron acceptor. KCN inhibited the aerobic respiratory chain and increased biofilm formation. In contrast, methylamine inhibited both nitrate reductase and biofilm formation. The correlation between the expression and/or activity or redox enzymes and biofilm-formation activities suggests that these are possible therapeutic targets to erradicate S. epidermidis.

  1. Affinity chromatography of bacterial lactate dehydrogenases.

    Science.gov (United States)

    Kelly, N; Delaney, M; O'Carra, P

    1978-06-01

    The affinity system used was the immobilized oxamate derivative previously used to purify mammalian lactate dehydrogenases. The bacterial dehydrogenases specific for the L-stereoisomer of lactate behaved in the same way as the mammalian enzymes, binding strongly in the presence of NADH. The D-lactate-specific enzymes, however, did not show any biospecific affinity for this gel. The L-specific enzymes could be purified to homogeneity in one affinity-chromatographic step. The D-specific enzymes could be efficiently separated from the L-specific ones and could then be further purified on an immobilized NAD derivative. The mechanism of activation of the lactate dehydrogenase from Streptococcus faecalis by fructose 1,6-bisphosphate was investigated by using the immobilized oxamate gel. PMID:666726

  2. Inducible xylitol dehydrogenases in enteric bacteria.

    OpenAIRE

    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...

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

  5. Microbial alcohol dehydrogenases: identification, characterization and engineering

    OpenAIRE

    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...

  6. Optimization of Adsorptive Immobilization of Alcohol Dehydrogenases

    NARCIS (Netherlands)

    Trivedi, Archana; Heinemann, Matthias; Spiess, Antje C.; Daussmann, Thomas; Büchs, Jochen

    2005-01-01

    In this work, a systematic examination of various parameters of adsorptive immobilization of alcohol dehydrogenases (ADHs) on solid support is performed and the impact of these parameters on immobilization efficiency is studied. Depending on the source of the enzymes, these parameters differently in

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

    OpenAIRE

    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...

  8. Characterization of xylitol dehydrogenase from Debaryomyces hansenii

    Energy Technology Data Exchange (ETDEWEB)

    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.

  9. Increasing anaerobic acetate consumption and ethanol yields in Saccharomyces cerevisiae with NADPH-specific alcohol dehydrogenase.

    Science.gov (United States)

    Henningsen, Brooks M; Hon, Shuen; Covalla, Sean F; Sonu, Carolina; Argyros, D Aaron; Barrett, Trisha F; Wiswall, Erin; Froehlich, Allan C; Zelle, Rintze M

    2015-12-01

    Saccharomyces cerevisiae has recently been engineered to use acetate, a primary inhibitor in lignocellulosic hydrolysates, as a cosubstrate during anaerobic ethanolic fermentation. However, the original metabolic pathway devised to convert acetate to ethanol uses NADH-specific acetylating acetaldehyde dehydrogenase and alcohol dehydrogenase and quickly becomes constrained by limited NADH availability, even when glycerol formation is abolished. We present alcohol dehydrogenase as a novel target for anaerobic redox engineering of S. cerevisiae. Introduction of an NADPH-specific alcohol dehydrogenase (NADPH-ADH) not only reduces the NADH demand of the acetate-to-ethanol pathway but also allows the cell to effectively exchange NADPH for NADH during sugar fermentation. Unlike NADH, NADPH can be freely generated under anoxic conditions, via the oxidative pentose phosphate pathway. We show that an industrial bioethanol strain engineered with the original pathway (expressing acetylating acetaldehyde dehydrogenase from Bifidobacterium adolescentis and with deletions of glycerol-3-phosphate dehydrogenase genes GPD1 and GPD2) consumed 1.9 g liter(-1) acetate during fermentation of 114 g liter(-1) glucose. Combined with a decrease in glycerol production from 4.0 to 0.1 g liter(-1), this increased the ethanol yield by 4% over that for the wild type. We provide evidence that acetate consumption in this strain is indeed limited by NADH availability. By introducing an NADPH-ADH from Entamoeba histolytica and with overexpression of ACS2 and ZWF1, we increased acetate consumption to 5.3 g liter(-1) and raised the ethanol yield to 7% above the wild-type level.

  10. Benzaldehyde dehydrogenase from chitosan-treated Sorbus aucuparia cell cultures.

    Science.gov (United States)

    Gaid, Mariam M; Sircar, Debabrata; Beuerle, Till; Mitra, Adinpunya; Beerhues, Ludger

    2009-09-01

    Cell cultures of Sorbus aucuparia respond to the addition of chitosan with the accumulation of the biphenyl phytoalexin aucuparin. The carbon skeleton of this inducible defense compound is formed by biphenyl synthase (BIS) from benzoyl-CoA and three molecules of malonyl-CoA. The formation of benzoyl-CoA proceeds via benzaldehyde as an intermediate. Benzaldehyde dehydrogenase (BD), which converts benzaldehyde into benzoic acid, was detected in cell-free extracts from S. aucuparia cell cultures. BD and BIS were induced by chitosan treatment. The preferred substrate for BD was benzaldehyde (K(m)=49 microM). Cinnamaldehyde and various hydroxybenzaldehydes were relatively poor substrates. BD activity was strictly dependent on the presence of NAD(+) as a cofactor (K(m)=67 microM).

  11. Dihydrodiol dehydrogenase and polycyclic aromatic hydrocarbon metabolism

    Energy Technology Data Exchange (ETDEWEB)

    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.

  12. Characterization of 10-Hydroxygeraniol Dehydrogenase from Catharanthus roseus Reveals Cascaded Enzymatic Activity in Iridoid Biosynthesis

    OpenAIRE

    Ramakrishnan Krithika; Prabhakar Lal Srivastava; Bajaj Rani; Kolet, Swati P.; Manojkumar Chopade; Mantri Soniya; Hirekodathakallu V. Thulasiram

    2015-01-01

    Catharanthus roseus [L.] is a major source of the monoterpene indole alkaloids (MIAs), which are of significant interest due to their therapeutic value. These molecules are formed through an intermediate, cis-trans-nepetalactol, a cyclized product of 10-oxogeranial. One of the key enzymes involved in the biosynthesis of MIAs is an NAD(P)+ dependent oxidoreductase system, 10-hydroxygeraniol dehydrogenase (Cr10HGO), which catalyses the formation of 10-oxogeranial from 10-hydroxygeraniol via 10-...

  13. Variation of transition-state structure as a function of the nucleotide in reactions catalyzed by dehydrogenases. 1. Liver alcohol dehydrogenase with benzyl alcohol and yeast aldehyde dehydrogenase with benzaldehyde.

    Science.gov (United States)

    Scharschmidt, M; Fisher, M A; Cleland, W W

    1984-11-01

    Primary intrinsic deuterium and 13C isotope effects have been determined for liver (LADH) and yeast (YADH) alcohol dehydrogenases with benzyl alcohol as substrate and for yeast aldehyde dehydrogenase (ALDH) with benzaldehyde as substrate. These values have also been determined for LADH as a function of changing nucleotide substrate. As the redox potential of the nucleotide changes from -0.320 V with NAD to -0.258 V with acetylpyridine-NAD, the product of primary and secondary deuterium isotope effects rises from 4 toward 6.5, while the primary 13C isotope effect drops from 1.025 to 1.012, suggesting a trend from a late transition state with NAD to one that is more symmetrical. The values of Dk (again the product of primary and secondary isotope effects) and 13k for YADH with NAD are 7 and 1.023, suggesting for this very slow reaction a more stretched, and thus symmetrical, transition state. With ALDH and NAD, the primary 13C isotope effect on the hydride transfer step lies in the range 1.3-1.6%, and the alpha-secondary deuterium isotope effect on the same step is at least 1.22, but 13C isotope effects on formation of the thiohemiacetal intermediate and on the addition of water to the thio ester intermediate are less than 1%. On the basis of the relatively large 13C isotope effects, we conclude that carbon motion is involved in the hydride transfer steps of dehydrogenase reactions.

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

    Directory of Open Access Journals (Sweden)

    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

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

    OpenAIRE

    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. ...

  16. Molecular genetic analysis of human alcohol dehydrogenase

    OpenAIRE

    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...

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

    DEFF Research Database (Denmark)

    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...

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

    Directory of Open Access Journals (Sweden)

    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.

  19. Cloning of the rat pyruvate dehydrogenase kinase 4 gene promoter: activation of pyruvate dehydrogenase kinase 4 by the peroxisome proliferator-activated receptor gamma coactivator.

    Science.gov (United States)

    Ma, Ke; Zhang, Yi; Elam, Marshall B; Cook, George A; Park, Edwards A

    2005-08-19

    The pyruvate dehydrogenase complex catalyzes the conversion of pyruvate to acetyl-CoA in mitochondria and is a key regulatory enzyme in the metabolism of glucose to acetyl-CoA. Phosphorylation of pyruvate dehydrogenase by the pyruvate dehydrogenase kinases (PDK) inhibits pyruvate dehydrogenase complex activity. There are four PDK isoforms, and expression of PDK4 and PDK2 genes is elevated in starvation and diabetes, allowing glucose to be conserved while fatty acid oxidation is increased. In these studies we have investigated the transcriptional mechanisms by which the expression of the PDK4 gene is increased. The peroxisome proliferator-activated receptor gamma coactivator (PGC-1alpha) stimulates the expression of genes involved in hepatic gluconeogenesis and mitochondrial fatty acid oxidation. We have found that PGC-1alpha will induce the expression of both the PDK2 and PDK4 genes in primary rat hepatocytes and ventricular myocytes. We cloned the promoter for the rat PDK4 gene. Hepatic nuclear factor 4 (HNF4), which activates many genes in the liver, will induce PDK4 expression. Although HNF4 and PGC-1alpha interact to stimulate several genes encoding gluconeogenic enzymes, the induction of PDK4 does not involve interactions of PGC-1alpha with HNF4. Using the chromatin immunoprecipitation assay, we have demonstrated that HNF4 and PGC-1alpha are associated with the PDK4 gene in vivo. Our data suggest that by inducing PDK genes PGC-1alpha will direct pyruvate away from metabolism into acetyl-CoA and toward the formation of oxaloacetate and into the gluconeogenic pathway. PMID:15967803

  20. The structure of retinal dehydrogenase type II at 2.7 A resolution: implications for retinal specificity.

    Science.gov (United States)

    Lamb, A L; Newcomer, M E

    1999-05-11

    Retinoic acid, a hormonally active form of vitamin A, is produced in vivo in a two step process: retinol is oxidized to retinal and retinal is oxidized to retinoic acid. Retinal dehydrogenase type II (RalDH2) catalyzes this last step in the production of retinoic acid in the early embryo, possibly producing this putative morphogen to initiate pattern formation. The enzyme is also found in the adult animal, where it is expressed in the testis, lung, and brain among other tissues. The crystal structure of retinal dehydrogenase type II cocrystallized with nicotinamide adenine dinucleotide (NAD) has been determined at 2.7 A resolution. The structure was solved by molecular replacement using the crystal structure of a mitochondrial aldehyde dehydrogenase (ALDH2) as a model. Unlike what has been described for the structures of two aldehyde dehydrogenases involved in the metabolism of acetaldehyde, the substrate access channel is not a preformed cavity into which acetaldehyde can readily diffuse. Retinal dehydrogenase appears to utilize a disordered loop in the substrate access channel to discriminate between retinaldehyde and short-chain aldehydes.

  1. Transcriptional Regulation of Pyruvate Dehydrogenase Kinase

    Directory of Open Access Journals (Sweden)

    Ji Yun Jeong

    2012-10-01

    Full Text Available The pyruvate dehydrogenase complex (PDC activity is crucial to maintains blood glucose and ATP levels, which largely depends on the phosphorylation status by pyruvate dehydrogenase kinase (PDK isoenzymes. Although it has been reported that PDC is phosphorylated and inactivated by PDK2 and PDK4 in metabolically active tissues including liver, skeletal muscle, heart, and kidney during starvation and diabetes, the precise mechanisms by which expression of PDK2 and PDK4 are transcriptionally regulated still remains unclear. Insulin represses the expression of PDK2 and PDK4 via phosphorylation of FOXO through PI3K/Akt signaling pathway. Several nuclear hormone receptors activated due to fasting or increased fat supply, including peroxisome proliferator-activated receptors, glucocorticoid receptors, estrogen-related receptors, and thyroid hormone receptors, also participate in the up-regulation of PDK2 and PDK4; however, the endogenous ligands that bind those nuclear receptors have not been identified. It has been recently suggested that growth hormone, adiponectin, epinephrine, and rosiglitazone also control the expression of PDK4 in tissue-specific manners. In this review, we discuss several factors involved in the expressional regulation of PDK2 and PDK4, and introduce current studies aimed at providing a better understanding of the molecular mechanisms that underlie the development of metabolic diseases such as diabetes.

  2. Transcriptional regulation of pyruvate dehydrogenase kinase.

    Science.gov (United States)

    Jeong, Ji Yun; Jeoung, Nam Ho; Park, Keun-Gyu; Lee, In-Kyu

    2012-10-01

    The pyruvate dehydrogenase complex (PDC) activity is crucial to maintains blood glucose and ATP levels, which largely depends on the phosphorylation status by pyruvate dehydrogenase kinase (PDK) isoenzymes. Although it has been reported that PDC is phosphorylated and inactivated by PDK2 and PDK4 in metabolically active tissues including liver, skeletal muscle, heart, and kidney during starvation and diabetes, the precise mechanisms by which expression of PDK2 and PDK4 are transcriptionally regulated still remains unclear. Insulin represses the expression of PDK2 and PDK4 via phosphorylation of FOXO through PI3K/Akt signaling pathway. Several nuclear hormone receptors activated due to fasting or increased fat supply, including peroxisome proliferator-activated receptors, glucocorticoid receptors, estrogen-related receptors, and thyroid hormone receptors, also participate in the up-regulation of PDK2 and PDK4; however, the endogenous ligands that bind those nuclear receptors have not been identified. It has been recently suggested that growth hormone, adiponectin, epinephrine, and rosiglitazone also control the expression of PDK4 in tissue-specific manners. In this review, we discuss several factors involved in the expressional regulation of PDK2 and PDK4, and introduce current studies aimed at providing a better understanding of the molecular mechanisms that underlie the development of metabolic diseases such as diabetes. PMID:23130316

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

    NARCIS (Netherlands)

    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

  4. INFLUENCE OF SELECTED PHARMACEUTICALS ON ACTIVATED SLUDGE DEHYDROGENASE ACTIVITY

    Directory of Open Access Journals (Sweden)

    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%.

  5. Enantioselective Synthesis of Vicinal (R,R)-Diols by Saccharomyces cerevisiae Butanediol Dehydrogenase.

    Science.gov (United States)

    Calam, Eduard; González-Roca, Eva; Fernández, M Rosario; Dequin, Sylvie; Parés, Xavier; Virgili, Albert; Biosca, Josep A

    2016-03-01

    Butanediol dehydrogenase (Bdh1p) from Saccharomyces cerevisiae belongs to the superfamily of the medium-chain dehydrogenases and reductases and converts reversibly R-acetoin and S-acetoin to (2R,3R)-2,3-butanediol and meso-2,3-butanediol, respectively. It is specific for NAD(H) as a coenzyme, and it is the main enzyme involved in the last metabolic step leading to (2R,3R)-2,3-butanediol in yeast. In this study, we have used the activity of Bdh1p in different forms-purified enzyme, yeast extracts, permeabilized yeast cells, and as a fusion protein (with yeast formate dehydrogenase, Fdh1p)-to transform several vicinal diketones to the corresponding diols. We have also developed a new variant of the delitto perfetto methodology to place BDH1 under the control of the GAL1 promoter, resulting in a yeast strain that overexpresses butanediol dehydrogenase and formate dehydrogenase activities in the presence of galactose and regenerates NADH in the presence of formate. While the use of purified Bdh1p allows the synthesis of enantiopure (2R,3R)-2,3-butanediol, (2R,3R)-2,3-pentanediol, (2R,3R)-2,3-hexanediol, and (3R,4R)-3,4-hexanediol, the use of the engineered strain (as an extract or as permeabilized cells) yields mixtures of the diols. The production of pure diol stereoisomers has also been achieved by means of a chimeric fusion protein combining Fdh1p and Bdh1p. Finally, we have determined the selectivity of Bdh1p toward the oxidation/reduction of the hydroxyl/ketone groups from (2R,3R)-2,3-pentanediol/2,3-pentanedione and (2R,3R)-2,3-hexanediol/2,3-hexanedione. In conclusion, Bdh1p is an enzyme with biotechnological interest that can be used to synthesize chiral building blocks. A scheme of the favored pathway with the corresponding intermediates is proposed for the Bdh1p reaction.

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

    Directory of Open Access Journals (Sweden)

    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.

  7. Stability of immobilized yeast alcohol dehydrogenase

    Energy Technology Data Exchange (ETDEWEB)

    Ooshima, H.; Genko, Y.; Harano, Y.

    1981-12-01

    The effects of substrate on stabilities of native (NA) and three kinds of immobilized yeast alcohol dehydrogenase (IMA), namely PGA (the carrier; porous glass), SEA (agarose gel) prepared covalently, and AMA (anion-exchange resin) prepared ionically, were studied. The following results were obtained. 1) The deactivations of NA and IMA free from the substrate or in the presence of ethanol obey the first-order kinetics, whereas, in the presence of butyraldehyde, their deactivation behaviors are explained on the basis of coexistence of two components of YADHs, namely the labile E1 and the comparatively stable E2, with different first-order deactivation constants. (2) A few attempts for stabilization of IMA were carried out from the viewpoint of the effects of crosslinkages among the subunits of YADH for PGA and the multibonding between the carrier and enzyme for SEA. The former is effective for the stabilization, whereas the latter is not. (Refs. 19).

  8. Interactions between heparinoids and alcohol dehydrogenase.

    Science.gov (United States)

    Paulíková, H; Valusová, E; Antalík, M

    1997-07-01

    The interaction between polysulfated polysaecharides (low-molecular-weight heparin LMWH, dextran sulfate DS and pentosan sulfate PS) and yeast alcohol dehydrogenase (YADH) was investigated. The fluorescence and UV spectra of YADH after adding the tested polysaccharides have confirmed the interaction between the enzyme and these compounds. Kinetic studies have shown that LMWH, DS and PS are inhibitors of YADH (mixed type with respect to NAD). The most potent inhibitor is PS (ID50=37.5 ng/ml, Ki=0.6 muM). The inhibition effect depends on the ionic strength (the inhibition decreased by about 50% in the presence of 100 mM Na2SO4) and pH value (the inhibition decreased at pH>7). The results indicate that the inhibition effect of these polyanions is caused by their electrostatic interactions with the NAD-binding region of YADH.

  9. Optimization of adsorptive immobilization of alcohol dehydrogenases.

    Science.gov (United States)

    Trivedi, Archana; Heinemann, Matthias; Spiess, Antje C; Daussmann, Thomas; Büchs, Jochen

    2005-04-01

    In this work, a systematic examination of various parameters of adsorptive immobilization of alcohol dehydrogenases (ADHs) on solid support is performed and the impact of these parameters on immobilization efficiency is studied. Depending on the source of the enzymes, these parameters differently influence the immobilization efficiency, expressed in terms of residual activity and protein loading. Residual activity of 79% was achieved with ADH from bakers' yeast (YADH) after optimizing the immobilization parameters. A step-wise drying process has been found to be more effective than one-step drying. A hypothesis of deactivation through bubble nucleation during drying of the enzyme/glass bead suspension at low drying pressure (300% residual activity was found after drying. Hyperactivation of the enzyme is probably caused by structural changes in the enzyme molecule during the drying process. ADH from Thermoanaerobacter species (ADH T) is found to be stable under drying conditions (>15 kPa) in contrast to LBADH and YADH.

  10. Untangling the glutamate dehydrogenase allosteric nightmare.

    Science.gov (United States)

    Smith, Thomas J; Stanley, Charles A

    2008-11-01

    Glutamate dehydrogenase (GDH) is found in all living organisms, but only animal GDH is regulated by a large repertoire of metabolites. More than 50 years of research to better understand the mechanism and role of this allosteric network has been frustrated by its sheer complexity. However, recent studies have begun to tease out how and why this complex behavior evolved. Much of GDH regulation probably occurs by controlling a complex ballet of motion necessary for catalytic turnover and has evolved concomitantly with a long antenna-like feature of the structure of the enzyme. Ciliates, the 'missing link' in GDH evolution, might have created the antenna to accommodate changing organelle functions and was refined in humans to, at least in part, link amino acid catabolism with insulin secretion.

  11. Pyruvate dehydrogenase and 3-fluoropyruvate: chemical competence of 2-acetylthiamin pyrophosphate as an acetyl group donor to dihydrolipoamide

    International Nuclear Information System (INIS)

    The pyruvate dehydrogenase component (E1) of the pyruvate dehydrogenase complex catalyzes the decomposition of 3-fluoropyruvate to CO2, fluoride anion, and acetate. Acetylthiamin pyrophosphate (acetyl-TPP) is an intermediate in this reaction. Incubation of the pyruvate dehydrogenase complex with 3-fluoro[1,2-14]pyruvate, TPP, coenzyme A (CoASH), and either NADH or pyruvate as reducing systems leads to the formation of [14]acetyl-CoA. In this reaction the acetyl group of acetyl-TPP is partitioned by transfer to both CoASH (87 +/- 2%) and water (13 +/- 2%). When the E1 component is incubated with 3-fluoro[1,2-14]pyruvate, TPP, and dihydrolipoamide, [14]acetyldihydrolipoamide is produced. The formation of [14C]acetyldihydrolipoamide was examined as a function of dihydrolipoamide concentration (0.25-16 mM). A plot of the extent of acetyl group partitioning to dihydrolipoamide as a function of 1/[dihydrolipoamide] showed 95 +/- 2% acetyl group transfer to dihydrolipoamide when dihydrolipoamide concentration was extrapolated to infinity. It is concluded that acetyl-TPP is chemically competent as an intermediate for the pyruvate dehydrogenase complex catalyzed oxidative decarboxylation of pyruvate

  12. The structure and allosteric regulation of glutamate dehydrogenase.

    Science.gov (United States)

    Li, Ming; Li, Changhong; Allen, Aron; Stanley, Charles A; Smith, Thomas J

    2011-09-01

    Glutamate dehydrogenase (GDH) has been extensively studied for more than 50 years. Of particular interest is the fact that, while considered by most to be a 'housekeeping' enzyme, the animal form of GDH is heavily regulated by a wide array of allosteric effectors and exhibits extensive inter-subunit communication. While the chemical mechanism for GDH has remained unchanged through epochs of evolution, it was not clear how or why animals needed to evolve such a finely tuned form of this enzyme. As reviewed here, recent studies have begun to elucidate these issues. Allosteric regulation first appears in the Ciliates and may have arisen to accommodate evolutionary changes in organelle function. The occurrence of allosteric regulation appears to be coincident with the formation of an 'antenna' like feature rising off the tops of the subunits that may be necessary to facilitate regulation. In animals, this regulation further evolved as GDH became integrated into a number of other regulatory pathways. In particular, mutations in GDH that abrogate GTP inhibition result in dangerously high serum levels of insulin and ammonium. Therefore, allosteric regulation of GDH plays an important role in insulin homeostasis. Finally, several compounds have been identified that block GDH-mediated insulin secretion that may be to not only find use in treating these insulin disorders but to kill tumors that require glutamine metabolism for cellular energy.

  13. Effect of 15-hydroxyprostaglandin dehydrogenase inhibitor on wound healing.

    Science.gov (United States)

    Seo, Seung Yong; Han, Song-Iy; Bae, Chun Sik; Cho, Hoon; Lim, Sung Chul

    2015-06-01

    PGE2 is an important mediator of wound healing. It is degraded and inactivated by 15-hydroxyprostaglandin dehydrogenase (15-PGDH). Various growth factors, type IV collagen, TIMP-2 and PGE2 are important mediators of inflammation involving wound healing. Overproduction of TGF-β and suppression of PGE2 are found in excessive wound scarring. If we make the condition downregulating growth factors and upregulating PGE2, the wound will have a positive effect which results in little scar formation after healing. TD88 is a 15-PGDH inhibitor based on thiazolinedione structure. We evaluated the effect of TD88 on wound healing. In 10 guinea pigs (4 control and 6 experimental groups), we made four 1cm diameter-sized circular skin defects on each back. TD88 and vehicle were applicated on the wound twice a day for 4 days in the experimental and control groups, respectively. Tissue samples were harvested for qPCR and histomorphometric analyses on the 2nd and 4th day after treatment. Histomorphometric analysis showed significant reepithelization in the experimental group. qPCR analysis showed significant decrease of PDGF, CTGF and TIMP-2, but significant increase of type IV collagen in the experimental group. Taken together TD88 could be a good effector on wound healing, especially in the aspects of prevention of scarring.

  14. Structure of a bifunctional alcohol dehydrogenase involved in bioethanol generation in Geobacillus thermoglucosidasius.

    Science.gov (United States)

    Extance, Jonathan; Crennell, Susan J; Eley, Kirstin; Cripps, Roger; Hough, David W; Danson, Michael J

    2013-10-01

    Bifunctional alcohol/aldehyde dehydrogenase (ADHE) enzymes are found within many fermentative microorganisms. They catalyse the conversion of an acyl-coenzyme A to an alcohol via an aldehyde intermediate; this is coupled to the oxidation of two NADH molecules to maintain the NAD(+) pool during fermentative metabolism. The structure of the alcohol dehydrogenase (ADH) domain of an ADHE protein from the ethanol-producing thermophile Geobacillus thermoglucosidasius has been determined to 2.5 Å resolution. This is the first structure to be reported for such a domain. In silico modelling has been carried out to generate a homology model of the aldehyde dehydrogenase domain, and this was subsequently docked with the ADH-domain structure to model the structure of the complete ADHE protein. This model suggests, for the first time, a structural mechanism for the formation of the large multimeric assemblies or `spirosomes' that are observed for this ADHE protein and which have previously been reported for ADHEs from other organisms.

  15. Biospecific affinity chromatographic purification of octopine dehydrogenase from molluscs.

    Science.gov (United States)

    Mulcahy, P; Griffin, T; O'Carra, P

    1997-02-01

    The development of a biospecific affinity chromatographic method for the purification of octopine dehydrogenase from molluscs is described. The method utilizes immobilized NAD+ derivatives in conjunction with soluble specific substrates to promote binding. Using this method, octopine dehydrogenase has been purified to electrophoretic homogeneity in a single chromatographic step from three different marine invertebrate sources [the queen scallop, Chlamys opercularis (adductor muscle), the great scallop, Pecten maximus (adductor muscle), and the squid Loligo vulgaris (mantle muscle)]. However, the system is not applicable to the purification of octopine dehydrogenase from some other marine invertebrate sources investigated (the mussel Mytilus edulis and the topshell Monodonta lineata). PMID:9116492

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

    Directory of Open Access Journals (Sweden)

    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

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

    Science.gov (United States)

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

    2008-01-01

    /xylitol dehydrogenase strain and the xylose isomerase strain, respectively. Conclusion The combination of the xylose reductase/xylitol dehydrogenase pathway and the bacterial arabinose isomerase pathway resulted in both higher pentose sugar uptake and higher overall ethanol production than the combination of the xylose isomerase pathway and the bacterial arabinose isomerase pathway. Moreover, the flux through the bacterial arabinose pathway did not increase when combined with the xylose isomerase pathway. This suggests that the low activity of the bacterial arabinose pathway cannot be ascribed to arabitol formation via the xylose reductase enzyme. PMID:18947407

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

    Directory of Open Access Journals (Sweden)

    Hahn-Hägerdal Bärbel

    2008-10-01

    for the xylose reductase/xylitol dehydrogenase strain and the xylose isomerase strain, respectively. Conclusion The combination of the xylose reductase/xylitol dehydrogenase pathway and the bacterial arabinose isomerase pathway resulted in both higher pentose sugar uptake and higher overall ethanol production than the combination of the xylose isomerase pathway and the bacterial arabinose isomerase pathway. Moreover, the flux through the bacterial arabinose pathway did not increase when combined with the xylose isomerase pathway. This suggests that the low activity of the bacterial arabinose pathway cannot be ascribed to arabitol formation via the xylose reductase enzyme.

  19. A novel glutamate dehydrogenase from bovine brain: purification and characterization.

    Science.gov (United States)

    Lee, J; Kim, S W; Cho, S W

    1995-08-01

    A soluble form of novel glutamate dehydrogenase has been purified from bovine brain. The preparation was homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and composed of six identical subunits having a subunit size of 57,500 Da. The biochemical properties of glutamate dehydrogenase such as N-terminal amino acids sequences, kinetic parameters, amino acids analysis, and optimum pH were examined in both reductive amination of alpha-ketoglutarate and oxidative deamination of glutamate. N-terminal amino acid sequences of the bovine brain enzyme showed the significant differences in the first 5 amino acids compared to other glutamate dehydrogenases from various sources. These results indicate that glutamate dehydrogenase isolated from bovine brain is a novel polypeptide.

  20. Targeting isocitrate dehydrogenase (IDH) in cancer.

    Science.gov (United States)

    Fujii, Takeo; Khawaja, Muhammad Rizwan; DiNardo, Courtney D; Atkins, Johnique T; Janku, Filip

    2016-05-01

    Isocitrate dehydrogenase (IDH) is an essential enzyme for cellular respiration in the tricarboxylic acid (TCA) cycle. Recurrent mutations in IDH1 or IDH2 are prevalent in several cancers including glioma, acute myeloid leukemia (AML), cholangiocarcinoma and chondrosarcoma. The mutated IDH1 and IDH2 proteins have a gain-of-function, neomorphic activity, catalyzing the reduction of α-ketoglutarate (α-KG) to 2-hydroxyglutarate (2-HG) by NADPH. Cancer-associated IDH mutations block normal cellular differentiation and promote tumorigenesis via the abnormal production of the oncometabolite 2-HG. High levels of 2-HG have been shown to inhibit α-KG dependent dioxygenases, including histone and deoxyribonucleic acid (DNA) demethylases, which play a key role in regulating the epigenetic state of cells. Current targeted inhibitors of IDH1 (AG120, IDH305), IDH2 (AG221), and pan-IDH1/2 (AG881) selectively inhibit mutant IDH protein and induce cell differentiation in in vitro and in vivo models. Preliminary results from phase I clinical trials with IDH inhibitors in patients with advanced hematologic malignancies have demonstrated an objective response rate ranging from 31% to 40% with durable responses (>1 year) observed. Furthermore, the IDH inhibitors have demonstrated early signals of activity in solid tumors with IDH mutations, including cholangiocarcinomas and low grade gliomas. PMID:27355333

  1. Lactic dehydrogenase and cancer: an overview.

    Science.gov (United States)

    Gallo, Monica; Sapio, Luigi; Spina, Annamaria; Naviglio, Daniele; Calogero, Armando; Naviglio, Silvio

    2015-01-01

    Despite the intense scientific efforts made, there are still many tumors that are difficult to treat and the percentage of patient survival in the long-term is still too low. Thus, new approaches to the treatment of cancer are needed. Cancer is a highly heterogeneous and complex disease, whose development requires a reorganization of cell metabolism. Most tumor cells downregulate mitochondrial oxidative phosphorylation and increase the rate of glucose consumption and lactate release, independently of oxygen availability (Warburg effect). This metabolic rewiring is largely believed to favour tumor growth and survival, although the underlying molecular mechanisms are not completely understood. Importantly, the correlation between the aerobic glycolysis and cancer is widely regarded as a useful biochemical basis for the development of novel anticancer strategies. Among the enzymes involved in glycolysis, lactate dehydrogenase (LDH) is emerging as a very attractive target for possible pharmacological approaches in cancer therapy. This review addresses the state of the art and the perspectives concerning LDH both as a useful diagnostic marker and a relevant molecular target in cancer therapy and management.

  2. COMPUTATIONAL STUDIES OF THE KINETIC ISOTOPE EFFECT INMETHYLAMINE DEHYDROGENASE

    OpenAIRE

    Kopec-Harding, Kamilla Rosa

    2012-01-01

    There is currently experimental evidence of hydrogen tunnelling in over 20 different enzymes include yeast alcohol dehydrogenase (YADH), morphinone reductase (MR) and methylamine dehydrogenase (MADH). Various models have been used to describe hydrogen tunnelling in enzymes including the static barrier model, the vibrationally enhanced ground state tunnelling model (VEGST) and the environmentally coupled tunnelling model (ECT). Despite some differences in these models, there is a general cons...

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

    Science.gov (United States)

    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

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

    OpenAIRE

    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...

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

    OpenAIRE

    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...

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

    OpenAIRE

    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...

  7. Expression of a Heterologous Glutamate Dehydrogenase Gene in Lactococcus lactis Highly Improves the Conversion of Amino Acids to Aroma Compounds

    OpenAIRE

    Rijnen, Liesbeth; Courtin, Pascal; Gripon, Jean-Claude; Yvon, Mireille

    2000-01-01

    The first step of amino acid degradation in lactococci is a transamination, which requires an α-keto acid as the amino group acceptor. We have previously shown that the level of available α-keto acid in semihard cheese is the first limiting factor for conversion of amino acids to aroma compounds, since aroma formation is greatly enhanced by adding α-ketoglutarate to cheese curd. In this study we introduced a heterologous catabolic glutamate dehydrogenase (GDH) gene into Lactococcus lactis so ...

  8. 2-Oxoglutarate dehydrogenase is a more significant source of O2(·-)/H2O2 than pyruvate dehydrogenase in cardiac and liver tissue.

    Science.gov (United States)

    Mailloux, Ryan J; Gardiner, Danielle; O'Brien, Marisa

    2016-08-01

    Pyruvate dehydrogenase (Pdh) and 2-oxoglutarate dehydrogenase (Ogdh) are vital for Krebs cycle metabolism and sources of reactive oxygen species (ROS). O2(·-)/H2O2 formation by Pdh and Ogdh from porcine heart were compared when operating under forward or reverse electron transfer conditions. Comparisons were also conducted with liver and cardiac mitochondria. During reverse electron transfer (RET) from NADH, purified Ogdh generated ~3-3.5× more O2(·-)/H2O2 in comparison to Pdh when metabolizing 0.5-10µM NADH. Under forward electron transfer (FET) conditions Ogdh generated ~2-4× more O2(·-)/H2O2 than Pdh. In both liver and cardiac mitochondria, Ogdh displayed significantly higher rates of ROS formation when compared to Pdh. Ogdh was also a significant source of ROS in liver mitochondria metabolizing 50µM and 500µM pyruvate or succinate. Finally, we also observed that DTT directly stimulated O2(·-)/H2O2 formation by purified Pdh and Ogdh and in cardiac or liver mitochondria in the absence of substrates and cofactors. Taken together, Ogdh is a more potent source of ROS than Pdh in liver and cardiac tissue. Ogdh is also an important ROS generator regardless of whether pyruvate or succinate serve as the sole source of carbon. Our observations provide insight into the ROS generating capacity of either complex in cardiac and liver tissue. The evidence presented herein also indicates DTT, a reductant that is routinely added to biological samples, should be avoided when assessing mitochondrial O2(·-)/H2O2 production. PMID:27394173

  9. Genetics Home Reference: phosphoglycerate dehydrogenase deficiency

    Science.gov (United States)

    ... Serine is a part of chemical messengers called neurotransmitters that transmit signals in the nervous system . Proteins ... serine likely prevents the production of proteins and neurotransmitters in the brain and impairs the formation of ...

  10. Site-directed mutagenesis of aldehyde dehydrogenase-2 suggests three distinct pathways of nitroglycerin biotransformation.

    Science.gov (United States)

    Wenzl, M Verena; Beretta, Matteo; Griesberger, Martina; Russwurm, Michael; Koesling, Doris; Schmidt, Kurt; Mayer, Bernd; Gorren, Antonius C F

    2011-08-01

    To elucidate the mechanism underlying reduction of nitroglycerin (GTN) to nitric oxide (NO) by mitochondrial aldehyde dehydrogenase (ALDH2), we generated mutants of the enzyme lacking the cysteines adjacent to reactive Cys302 (C301S and C303S), the glutamate that participates as a general base in aldehyde oxidation (E268Q) or combinations of these residues. The mutants were characterized regarding acetaldehyde dehydrogenation, GTN-triggered enzyme inactivation, GTN denitration, NO formation, and soluble guanylate cyclase activation. Lack of the cysteines did not affect dehydrogenase activity but impeded GTN denitration, aggravated GTN-induced enzyme inactivation, and increased NO formation. A triple mutant lacking the cysteines and Glu268 catalyzed sustained formation of superstoichiometric amounts of NO and exhibited slower rates of inactivation. These results suggest three alternative pathways for the reaction of ALDH2 with GTN, all involving formation of a thionitrate/sulfenyl nitrite intermediate at Cys302 as the initial step. In the first pathway, which predominates in the wild-type enzyme and reflects clearance-based GTN denitration, the thionitrate apparently reacts with one of the adjacent cysteine residues to yield nitrite and a protein disulfide. The predominant reaction catalyzed by the single and double cysteine mutants requires Glu268 and results in irreversible enzyme inactivation. Finally, combined lack of the cysteines and Glu268 shifts the reaction toward formation of the free NO radical, presumably through homolytic cleavage of the sulfenyl nitrite intermediate. Although the latter reaction accounts for less than 10% of total turnover of GTN metabolism catalyzed by wild-type ALDH2, it is most likely essential for vascular GTN bioactivation.

  11. A "stripping" ligand tactic for use with the kinetic locking-on strategy: its use in the resolution and bioaffinity chromatographic purification of NAD(+)-dependent dehydrogenases.

    Science.gov (United States)

    O'Flaherty, M; O'Carra, P; McMahon, M; Mulcahy, P

    1999-08-01

    The kinetic locking-on strategy utilizes soluble analogues of the target enzymes' specific substrate to promote selective adsorption of individual NAD(+)-dependent dehydrogenases on their complementary immobilized cofactor derivative. Application of this strategy to the purification of NAD(+)-dependent dehydrogenases from crude extracts has proven that it can yield bioaffinity systems capable of producing one-chromatographic-step purifications with yields approaching 100%. However, in some cases the purified enzyme preparation was found to be contaminated with other proteins weakly bound to the immobilized cofactor derivative through binary complex formation and/or nonspecific interactions, which continuously "dribbled" off the matrix during the chromatographic procedure. The fact that this problem can be overcome by including a short pulse of 5'-AMP (stripping ligand) in the irrigant a couple of column volumes prior to the discontinuation of the specific substrate analogue (locking-on ligand) is clear from the results presented in this report. The general effectiveness of this auxiliary tactic has been assessed using model studies and through incorporation into an actual purification from a crude cellular extract. The results confirm the usefulness of the stripping-ligand tactic for the resolution and purification of NAD(+)-dependent dehydrogenases when using the locking-on strategy. These studies have been carried out using bovine liver glutamate dehydrogenase (GDH, EC 1.4.1.3), yeast alcohol dehydrogenase (YADH, EC 1.1.1.1), porcine heart mitochondrial malate dehydrogenase (mMDH, EC 1.1.1.37), and bovine heart L-lactate dehydrogenase (l-LDH, EC 1.1.1.27).

  12. A "stripping" ligand tactic for use with the kinetic locking-on strategy: its use in the resolution and bioaffinity chromatographic purification of NAD(+)-dependent dehydrogenases.

    Science.gov (United States)

    O'Flaherty, M; O'Carra, P; McMahon, M; Mulcahy, P

    1999-08-01

    The kinetic locking-on strategy utilizes soluble analogues of the target enzymes' specific substrate to promote selective adsorption of individual NAD(+)-dependent dehydrogenases on their complementary immobilized cofactor derivative. Application of this strategy to the purification of NAD(+)-dependent dehydrogenases from crude extracts has proven that it can yield bioaffinity systems capable of producing one-chromatographic-step purifications with yields approaching 100%. However, in some cases the purified enzyme preparation was found to be contaminated with other proteins weakly bound to the immobilized cofactor derivative through binary complex formation and/or nonspecific interactions, which continuously "dribbled" off the matrix during the chromatographic procedure. The fact that this problem can be overcome by including a short pulse of 5'-AMP (stripping ligand) in the irrigant a couple of column volumes prior to the discontinuation of the specific substrate analogue (locking-on ligand) is clear from the results presented in this report. The general effectiveness of this auxiliary tactic has been assessed using model studies and through incorporation into an actual purification from a crude cellular extract. The results confirm the usefulness of the stripping-ligand tactic for the resolution and purification of NAD(+)-dependent dehydrogenases when using the locking-on strategy. These studies have been carried out using bovine liver glutamate dehydrogenase (GDH, EC 1.4.1.3), yeast alcohol dehydrogenase (YADH, EC 1.1.1.1), porcine heart mitochondrial malate dehydrogenase (mMDH, EC 1.1.1.37), and bovine heart L-lactate dehydrogenase (l-LDH, EC 1.1.1.27). PMID:10425164

  13. Interaction of carbohydrates with alcohol dehydrogenase: Effect on enzyme activity.

    Science.gov (United States)

    Jadhav, Swati B; Bankar, Sandip B; Granström, Tom; Ojamo, Heikki; Singhal, Rekha S; Survase, Shrikant A

    2015-09-01

    Alcohol dehydrogenase was covalently conjugated with three different oxidized carbohydrates i.e., glucose, starch and pectin. All the carbohydrates inhibited the enzyme. The inhibition was studied with respect to the inhibition rate constant, involvement of thiol groups in the binding, and structural changes in the enzyme. The enzyme activity decreased to half of its original activity at the concentration of 2 mg/mL of pectin, 4 mg/mL of glucose and 10 mg/mL of starch within 10 min at pH 7. This study showed oxidized pectin to be a potent inhibitor of alcohol dehydrogenase followed by glucose and starch. Along with the aldehyde-amino group interaction, thiol groups were also involved in the binding between alcohol dehydrogenase and carbohydrates. The structural changes occurring on binding of alcohol dehydrogenase with oxidized carbohydrates was also confirmed by fluorescence spectrophotometry. Oxidized carbohydrates could thus be used as potential inhibitors of alcohol dehydrogenase.

  14. [Features of glutamate dehydrogenase in fetal and adult rumen tissue].

    Science.gov (United States)

    Kalachniuk, H I; Fomenko, I S; Kalachniuk, L H; Kavai, Sh; Marounek, M; Savka, O H

    2001-01-01

    Glutamate dehydrogenase (GDH) from rumen mucosa of cow fetus, liver and two forms from mucosa (bacterial and tissue) of the adult animal were partly purified and characterized. The activity of the bacterial glutamate dehydrogenase was shown to depend on qualities of a biomass of microbes, adhered on surface of rumen mucosa. All enzymes from tissues (GDHTRF, TRC, TLC), revealed the hypersensibility to increase in the concentration medium of Zn2+, guanosine triphosphate (GTP), acting here in a role of negative modulators, and also adenosine monophosphate (AMP) and leucine, which acted as activators. However, in the same concentrations these effectors do not influence the activity of the bacterial glutamate dehydrogenase. And if all tissues enzymes are highly specific to coenzyme NADH, the bacterial ones almost in 3 times is more active at NADPH use. PMID:11642036

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

  17. The activity of alcohol dehydrogenase (ADH) isoenzymes and aldehyde dehydrogenase (ALDH) in the sera of patients with brain cancer.

    Science.gov (United States)

    Jelski, Wojciech; Laniewska-Dunaj, Magdalena; Orywal, Karolina; Kochanowicz, Jan; Rutkowski, Robert; Szmitkowski, Maciej

    2014-12-01

    Human brain tissue contains various alcohol dehydrogenase (ADH) isoenzymes and possess also aldehyde dehydrogenase (ALDH) activity. In our last experiments we have shown that ADH and ALDH are present also in the brain tumour cells. Moreover the activities of total ADH and class I isoenzymes were significantly higher in cancer tissue than healthy cells. It can suggests that these changes may be reflected by enzyme activity in the serum of patients with brain cancer. Serum samples were taken for routine biochemical investigation from 62 patients suffering from brain cancer (36 glioblastoma, 26 meningioma). For the measurement of the activity of class I and II ADH isoenzymes and ALDH activity, the fluorometric methods were used. The total ADH activity and activity of class III and IV isoenzymes were measured by the photometric method. A statistically significant increase of class I alcohol dehydrogenase isoenzymes was found in the sera of patients with brain cancer. The median activity of this class isoenzyme in the patients group increased about 24 % in the comparison to the control level. The total alcohol dehydrogenase activity was also significantly higher (26 %) among patients with brain tumour than healthy ones. The activities of other tested ADH isoenzymes and total ALDH were unchanged. The increase of the activity of total ADH and class I alcohol dehydrogenase isoenzyme in the sera of patients with brain cancer seems to be caused by the release of this isoenzyme from tumour's cells.

  18. Crystal structure of homoisocitrate dehydrogenase from Schizosaccharomyces pombe

    Energy Technology Data Exchange (ETDEWEB)

    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

  19. Cloning, characterization and functional expression of Taenia solium 17 beta-hydroxysteroid dehydrogenase.

    Science.gov (United States)

    Aceves-Ramos, A; de la Torre, P; Hinojosa, L; Ponce, A; García-Villegas, R; Laclette, J P; Bobes, R J; Romano, M C

    2014-07-01

    The 17β-hydroxysteroid dehydrogenases (17β-HSD) are key enzymes involved in the formation (reduction) and inactivation (oxidation) of sex steroids. Several types have been found in vertebrates including fish, as well as in invertebrates like Caenorhabditis elegans, Ciona intestinalis and Haliotis diversicolor supertexta. To date limited information is available about this enzyme in parasites. We showed previously that Taenia solium cysticerci are able to synthesize sex steroid hormones in vitro when precursors are provided in the culture medium. Here, we identified a T. solium 17β-HSD through in silico blast searches in the T. solium genome database. This coding sequence was amplified by RT-PCR and cloned into the pcDNA 3.1(+) expression vector. The full length cDNA contains 957bp, corresponding to an open reading frame coding for 319 aa. The highest identity (84%) at the protein level was found with the Echinococcus multilocularis 17β-HSD although significant similarities were also found with other invertebrate and vertebrate 17β-HSD sequences. The T. solium Tsol-17βHSD belongs to the short-chain dehydrogenase/reductase (SDR) protein superfamily. HEK293T cells transiently transfected with Tsol17β-HSD induced expression of Tsol17β-HSD that transformed 3H-androstenedione into testosterone. In contrast, 3H-estrone was not significantly transformed into estradiol. In conclusion, T. solium cysticerci express a 17β-HSD that catalyzes the androgen reduction. The enzyme belongs to the short chain dehydrogenases/reductase family and shares motifs and activity with the type 3 enzyme of some other species.

  20. Properties of Lactate Dehydrogenase in a Psychrophilic Marine Bacterium

    OpenAIRE

    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...

  1. Purification and characterization of 3-isopropylmalate dehydrogenase from Thiobacillus thiooxidans.

    Science.gov (United States)

    Kawaguchi, H; Inagaki, K; Matsunami, H; Nakayama, Y; Tano, T; Tanaka, H

    2000-01-01

    3-Isopropylmalate dehydrogenase was purified to homogeneity from the acidophilic autotroph Thiobacillus thiooxidans. The native enzyme was a dimer of molecular weight 40,000. The apparent K(m) values for 3-isopropylmalate and NAD+ were estimated to be 0.13 mM and 8.7 mM, respectively. The optimum pH for activity was 9.0 and the optimum temperature was 65 degrees C. The properties of the enzyme were similar to those of the Thiobacillus ferrooxidans enzyme, expect for substrate specificity. T. thiooxidans 3-isopropylmalate dehydrogenase could not utilize malate as a substrate.

  2. Prostaglandin dehydrogenase and the initiation of labor.

    Science.gov (United States)

    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

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

    International Nuclear Information System (INIS)

    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.)

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

    Institute of Scientific and Technical Information of China (English)

    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.

  5. Interaction of glutaric aciduria type 1-related glutaryl-CoA dehydrogenase with mitochondrial matrix proteins.

    Directory of Open Access Journals (Sweden)

    Jessica Schmiesing

    Full Text Available Glutaric aciduria type 1 (GA1 is an inherited neurometabolic disorder caused by mutations in the GCDH gene encoding glutaryl-CoA dehydrogenase (GCDH, which forms homo- and heteromeric complexes in the mitochondrial matrix. GA1 patients are prone to the development of encephalopathic crises which lead to an irreversible disabling dystonic movement disorder. The clinical and biochemical manifestations of GA1 vary considerably and lack correlations to the genotype. Using an affinity chromatography approach we report here for the first time on the identification of mitochondrial proteins interacting directly with GCDH. Among others, dihydrolipoamide S-succinyltransferase (DLST involved in the formation of glutaryl-CoA, and the β-subunit of the electron transfer flavoprotein (ETFB serving as electron acceptor, were identified as GCDH binding partners. We have adapted the yellow fluorescent protein-based fragment complementation assay and visualized the oligomerization of GCDH as well as its direct interaction with DLST and ETFB in mitochondria of living cells. These data suggest that GCDH is a constituent of multimeric mitochondrial dehydrogenase complexes, and the characterization of their interrelated functions may provide new insights into the regulation of lysine oxidation and the pathophysiology of GA1.

  6. New complexes containing the internal alternative NADH dehydrogenase (Ndi1) in mitochondria of Saccharomyces cerevisiae.

    Science.gov (United States)

    Matus-Ortega, M G; Cárdenas-Monroy, C A; Flores-Herrera, O; Mendoza-Hernández, G; Miranda, M; González-Pedrajo, B; Vázquez-Meza, H; Pardo, J P

    2015-10-01

    Mitochondria of Saccharomyces cerevisiae lack the respiratory complex I, but contain three rotenone-insensitive NADH dehydrogenases distributed on both the external (Nde1 and Nde2) and internal (Ndi1) surfaces of the inner mitochondrial membrane. These enzymes catalyse the transfer of electrons from NADH to ubiquinone without the translocation of protons across the membrane. Due to the high resolution of the Blue Native PAGE (BN-PAGE) technique combined with digitonin solubilization, several bands with NADH dehydrogenase activity were observed on the gel. The use of specific S. cerevisiae single and double mutants of the external alternative elements (ΔNDE1, ΔNDE2, ΔNDE1/ΔNDE2) showed that the high and low molecular weight complexes contained the Ndi1. Some of the Ndi1 associations took place with complexes III and IV, suggesting the formation of respirasome-like structures. Complex II interacted with other proteins to form a high molecular weight supercomplex with a molecular mass around 600 kDa. We also found that the majority of the Ndi1 was in a dimeric form, which is in agreement with the recently reported three-dimensional structure of the protein.

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

    OpenAIRE

    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...

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

    OpenAIRE

    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...

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

    NARCIS (Netherlands)

    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

  10. Purification and characterization of xylitol dehydrogenase from Fusarium oxysporum

    DEFF Research Database (Denmark)

    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...

  11. 21 CFR 862.1420 - Isocitric dehydrogenase test system.

    Science.gov (United States)

    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...

  12. 21 CFR 862.1440 - Lactate dehydrogenase test system.

    Science.gov (United States)

    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...

  13. 21 CFR 862.1380 - Hydroxybutyric dehydrogenase test system.

    Science.gov (United States)

    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...

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

    Science.gov (United States)

    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

  15. Alcohol consumption, alcohol dehydrogenase 3 polymorphism, and colorectal adenomas

    NARCIS (Netherlands)

    Tiemersma, E.W.; Wark, P.A.; Ocké, M.C.; Bunschoten, A.; Otten, M.H.; Kok, F.J.; Kampman, E.

    2003-01-01

    Alcohol is a probable risk factor with regard to colorectal neoplasm and is metabolized to the carcinogen acetaldehyde by the genetically polymorphic alcohol dehydrogenase 3 (ADH3) enzyme. We evaluated whether the association between alcohol and colorectal adenomas is modified by ADH3 polymorphism.

  16. Effects of aerobic training on pyruvate dehydrogenase and pyruvate dehydrogenase kinase in human skeletal muscle.

    Science.gov (United States)

    LeBlanc, Paul J; Peters, Sandra J; Tunstall, Rebecca J; Cameron-Smith, David; Heigenhauser, George J F

    2004-06-01

    This study examined the effects of short- and long-term aerobic training on the stable up-regulation of pyruvate dehydrogenase (PDH) and PDH kinase (PDK) in human skeletal muscle. We hypothesized that 8 weeks, but not 1 week, of aerobic training would increase total PDH (PDHt) and PDK activities compared to pretraining, and this would be detectable at the level of gene transcription (mRNA) and/or gene translation (protein). Resting muscle biopsies were taken before and after 1 and 8 weeks of aerobic cycle exercise training. PDHt and PDK activities, and their respective protein and mRNA expression, did not differ after 1 week of aerobic training. PDHt activity increased 31% after 8 weeks and this may be partially due to a 1.3-fold increase in PDH-E(1)alpha protein expression. PDK activity approximately doubled after 8 weeks of aerobic training and this was attributed to a 1.3-fold increase in PDK2 isoform protein expression. Similar to 1 week, no changes were observed at the mRNA level after 8 weeks of training. These findings suggest that aerobically trained human skeletal muscle has an increased maximal capacity to utilize carbohydrates, evident by increased PDHt, but increased metabolic control sensitivity to pyruvate through increased contribution of PDK2 to total PDK activity. PMID:15020699

  17. Acyl-CoA Dehydrogenase 9 Is Required for the Biogenesis of Oxidative Phosphorylation Complex I

    NARCIS (Netherlands)

    J. Nouws; L. Nijtmans; S.M. Houten; M. Brand; M. Huynen; H. Venselaar; S. Hoefs; J. Gloerich; J. Kronick; T. Hutchin; P. Willems; R. Rodenburg; R. Wanders; L. van den Heuvel; J. Smeitink; R.O. Vogel

    2010-01-01

    Acyl-CoA dehydrogenase 9 (ACAD9) is a recently identified member of the acyl-CoA dehydrogenase family. It closely resembles very long-chain acyl-CoA dehydrogenase (VLCAD), involved in mitochondria! (3 oxidation of long-chain fatty acids. Contrary to its previously proposed involvement in fatty acid

  18. Crystal structure of quinone-dependent alcohol dehydrogenase from Pseudogluconobacter saccharoketogenes. A versatile dehydrogenase oxidizing alcohols and carbohydrates.

    Science.gov (United States)

    Rozeboom, Henriëtte J; Yu, Shukun; Mikkelsen, Rene; Nikolaev, Igor; Mulder, Harm J; Dijkstra, Bauke W

    2015-12-01

    The quinone-dependent alcohol dehydrogenase (PQQ-ADH, E.C. 1.1.5.2) from the Gram-negative bacterium Pseudogluconobacter saccharoketogenes IFO 14464 oxidizes primary alcohols (e.g. ethanol, butanol), secondary alcohols (monosaccharides), as well as aldehydes, polysaccharides, and cyclodextrins. The recombinant protein, expressed in Pichia pastoris, was crystallized, and three-dimensional (3D) structures of the native form, with PQQ and a Ca(2+) ion, and of the enzyme in complex with a Zn(2+) ion and a bound substrate mimic were determined at 1.72 Å and 1.84 Å resolution, respectively. PQQ-ADH displays an eight-bladed β-propeller fold, characteristic of Type I quinone-dependent methanol dehydrogenases. However, three of the four ligands of the Ca(2+) ion differ from those of related dehydrogenases and they come from different parts of the polypeptide chain. These differences result in a more open, easily accessible active site, which explains why PQQ-ADH can oxidize a broad range of substrates. The bound substrate mimic suggests Asp333 as the catalytic base. Remarkably, no vicinal disulfide bridge is present near the PQQ, which in other PQQ-dependent alcohol dehydrogenases has been proposed to be necessary for electron transfer. Instead an associated cytochrome c can approach the PQQ for direct electron transfer.

  19. Biochemical characterization of a bifunctional acetaldehyde-alcohol dehydrogenase purified from a facultative anaerobic bacterium Citrobacter sp. S-77.

    Science.gov (United States)

    Tsuji, Kohsei; Yoon, Ki-Seok; Ogo, Seiji

    2016-03-01

    Acetaldehyde-alcohol dehydrogenase (ADHE) is a bifunctional enzyme consisting of two domains of an N-terminal acetaldehyde dehydrogenase (ALDH) and a C-terminal alcohol dehydrogenase (ADH). The enzyme is known to be important in the cellular alcohol metabolism. However, the role of coenzyme A-acylating ADHE responsible for ethanol production from acetyl-CoA remains uncertain. Here, we present the purification and biochemical characterization of an ADHE from Citrobacter sp. S-77 (ADHE(S77)). Interestingly, the ADHE(S77) was unable to be solubilized from membrane with detergents either 1% Triton X-100 or 1% Sulfobetaine 3-12. However, the enzyme was easily dissociated from membrane by high-salt buffers containing either 1.0 M NaCl or (NH(4))(2)SO(4) without detergents. The molecular weight of a native protein was estimated as approximately 400 kDa, consisting of four identical subunits of 96.3 kDa. Based on the specific activity and kinetic analysis, the ADHES77 tended to have catalytic reaction towards acetaldehyde elimination rather than acetaldehyde formation. Our experimental observation suggests that the ADHES77 may play a pivotal role in modulating intracellular acetaldehyde concentration.

  20. 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

    DEFF Research Database (Denmark)

    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...

  1. Non-thermal effect of a ceramics radiation on a yeast glucose-6-phosphate dehydrogenase

    International Nuclear Information System (INIS)

    Non-thermal effect of a ceramics radiation on glucose-6-phosphate dehydrogenase has been investigated using the enzyme, glucose-6-phosphate and NADP+ separately irradiated at 10 degrees C by a ceracompo R plate and a ceramics un-sewed cloth (sheet). The Km for glucose-6-phosphate was increased 20% after 6 h of irradiation by the plate, but the Vmax/Km was decreased 24. After 3 h of irradiation by the sheet, the Km was increased 17%, but after 6 h of irradiation it was decreased 11%. The 3 h of irradiation by the sheet slightly increased both enthalpy and entropy changes of the reaction, but the 6 h of irradiation significantly decreased them. Both thermodynamic parameters in the activated state were increased by the sheet irradiation. The promotion energy for both formations of the enzyme-substrate and their activated complex depended on enthalpy. The different effects of two ceramics radiators on G6PDH activity were discussed

  2. Inhibition of glyceraldehyde-3-phosphate dehydrogenase by peptide and protein peroxides generated by singlet oxygen attack

    DEFF Research Database (Denmark)

    Morgan, Philip E; Dean, Roger T; Davies, Michael Jonathan

    2002-01-01

    inhibited by these peroxides in the absence of added Fe2+-EDTA. The presence of this metal-ion complex enhanced the inhibition observed with these enzymes consistent with the occurrence of radical-mediated reactions. Overall, these studies demonstrate that singlet oxygen-mediated damage to an initial target...... protein can result in selective subsequent damage to other proteins, as evidenced by loss of enzymatic activity, via the formation and subsequent reactions of protein peroxides. These reactions may be important in the development of cellular dysfunction as a result of photo-oxidation.......Reaction of certain peptides and proteins with singlet oxygen (generated by visible light in the presence of rose bengal dye) yields long-lived peptide and protein peroxides. Incubation of these peroxides with glyceraldehyde-3-phosphate dehydrogenase, in the absence of added metal ions, results...

  3. Single amino-acid substitution in the N-terminal arm altered the tetramer stability of rat muscle lactate dehydrogenase A

    Institute of Scientific and Technical Information of China (English)

    YUAN; Chong; (

    2001-01-01

    [1]Price, N. C., Assembly of multi-subunit structures, in Mechanisms of Protein Folding (ed. Pain, R. H.), New York: Oxford University Press, 1994, 160-193.[2]Casal, J. I., Ahern, T. J., Davenport, R. C. et al., Subunit interface of triosephosphate isomerase: Site-directed mutagenesis and characterization of the altered enzyme, Biochemistry, 1987, 26: 1258-1264.[3]Chakerian, A. E., Matthews, K. S., Characterization of mutations in oligomerization domain of lac repressor protein, J. Biol. Chem., 1991, 266: 22206-22214.[4]Mandelman, D., Schwarz, F. P., Li, H. Y. et al., The role of quaternary interactions on the stability and activity of ascorbate peroxidase, Protein Sci., 1998, 7: 2089-2098.[5]Thomas, M. C., Ballantine, S. P., Bethell, S. S. et al., Single amino acid substitutions disrupt tetramer formation in the dihydroneopterin aldolase enzyme of Pneumocystis carinii, Biochemistry, 1998, 37: 11629-11636.[6]Holbrook, J. J., Liljas, A., Steindel, S. J. et al., Lactate dehydrogenase, in The Enzymes (ed. Boyer, P. D.), Vol. 11, 3rd ed., New York: Academic Press, 1975, 191-292.[7]Zettlmeissl, G., Rudolph, R., Jaenicke, R., Reconstitution of lactic dehydrogenase after acid dissociation, Eur. J. Biochem., 1981, 121: 169-175.[8]Zettlmeissl, G., Rudolph, R., Jaenicke, R., Rate-determining folding and association reactions on the reconstitution pathway of porcine skeletal muscle lactic dehydrogenase after denaturation by guanidine hydrochloride, Biochemistry, 1982, 21: 3946-3950.[9]Hermann, R., Jaenicke, R., Rudolph, R., Analysis of the reconstitution of oligomeric enzymes by cross-linking with glutaraldehyde: Kinetics of reassociation of lactic dehydrogenase, Biochemistry, 1981, 20: 5195-5201.[10]Jaenicke, R., Folding and association of protein, Prog. Biophys. Mol. Biol., 1987, 49: 117-237.[11]Opitz, U., Rudolph, R., Jaenicke, R. et al., Proteolytic dimeric of porcine muscle lactate dehydrogenase: Characterization, folding, and

  4. The catalytic reaction mechanism of drosophilid alcohol dehydrogenases

    Directory of Open Access Journals (Sweden)

    Imin Wushur

    2015-03-01

    Full Text Available The present review describes the current knowledge about the reaction mechanism of drosophilid alcohol dehydrogenases (DADH, a member of the short chain dehydrogenase/reductase (SDR superfamily. Included is the binding order of the substrates to the enzyme, rate limiting steps, stereochemistry of the reaction, active site topology, role of important amino acids and water molecules in the reaction and pH dependence of kinetic coefficients. We focus on the contribution from steady state kinetics where alternative substrates, dead end and product inhibitors, isotopes and mutated DADHs have been used as well as on the contributions from X-ray crystallography, NMR and theoretical calculations. Furthermore, we also raise some open questions in order to fully understand the reaction mechanism of this enzyme.

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

    Science.gov (United States)

    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

  6. Direct Observation of Correlated Interdomain Motion in Alcohol Dehydrogenase

    OpenAIRE

    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...

  7. Optic neuropathy in a patient with pyruvate dehydrogenase deficiency

    Energy Technology Data Exchange (ETDEWEB)

    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.)

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

    OpenAIRE

    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 ...

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

    OpenAIRE

    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...

  10. The pivotal role of pyruvate dehydrogenase kinases in metabolic flexibility

    OpenAIRE

    Zhang, Shuai; Hulver, Matthew W.; McMillan, Ryan P.; Cline, Mark A.; Gilbert, Elizabeth R

    2014-01-01

    Abstract Metabolic flexibility is the capacity of a system to adjust fuel (primarily glucose and fatty acids) oxidation based on nutrient availability. The ability to alter substrate oxidation in response to nutritional state depends on the genetically influenced balance between oxidation and storage capacities. Competition between fatty acids and glucose for oxidation occurs at the level of the pyruvate dehydrogenase complex (PDC). The PDC is normally active in most tissues in the fed state,...

  11. R-lipoic acid inhibits mammalian pyruvate dehydrogenase kinase.

    Science.gov (United States)

    Korotchkina, Lioubov G; Sidhu, Sukhdeep; Patel, Mulchand S

    2004-10-01

    The four pyruvate dehydrogenase kinase (PDK) and two pyruvate dehydrogenase phosphatase (PDP) isoenzymes that are present in mammalian tissues regulate activity of the pyruvate dehydrogenase complex (PDC) by phosphorylation/dephosphorylation of its pyruvate dehydrogenase (E1) component. The effect of lipoic acids on the activity of PDKs and PDPs was investigated in purified proteins system. R-lipoic acid, S-lipoic acid and R-dihydrolipoic acid did not significantly affect activities of PDPs and at the same time inhibited PDKs to different extents (PDK1>PDK4 approximately PDK2>PDK3 for R-LA). Since lipoic acids inhibited PDKs activity both when reconstituted in PDC and in the presence of E1 alone, dissociation of PDK from the lipoyl domains of dihydrolipoamide acetyltransferase in the presence of lipoic acids is not a likely explanation for inhibition. The activity of PDK1 towards phosphorylation sites 1, 2 and 3 of E1 was decreased to the same extent in the presence of R-lipoic acid, thus excluding protection of the E1 active site by lipoic acid from phosphorylation. R-lipoic acid inhibited autophosphorylation of PDK2 indicating that it exerted its effect on PDKs directly. Inhibition of PDK1 by R-lipoic acid was not altered by ADP but was decreased in the presence of pyruvate which itself inhibits PDKs. An inhibitory effect of lipoic acid on PDKs would result in less phosphorylation of E1 and hence increased PDC activity. This finding provides a possible mechanism for a glucose (and lactate) lowering effect of R-lipoic acid in diabetic subjects. PMID:15512796

  12. A guide to 17beta-hydroxysteroid dehydrogenases.

    Science.gov (United States)

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

    Science.gov (United States)

    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

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

    OpenAIRE

    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 ...

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

    OpenAIRE

    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...

  16. Encapsulation of Alcohol Dehydrogenase in Mannitol by Spray Drying

    OpenAIRE

    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...

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

    OpenAIRE

    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 (...

  18. An efficient ribitol-specific dehydrogenase from Enterobacter aerogenes.

    Science.gov (United States)

    Singh, Ranjitha; Singh, Raushan; Kim, In-Won; Sigdel, Sujan; Kalia, Vipin C; Kang, Yun Chan; Lee, Jung-Kul

    2015-05-01

    An NAD(+)-dependent ribitol dehydrogenase from Enterobacter aerogenes KCTC 2190 (EaRDH) was cloned and successfully expressed in Escherichia coli. The complete 729-bp gene was amplified, cloned, expressed, and subsequently purified in an active soluble form using nickel affinity chromatography. The enzyme had an optimal pH and temperature of 11.0 and 45°C, respectively. Among various polyols, EaRDH exhibited activity only toward ribitol, with Km, Vmax, and kcat/Km values of 10.3mM, 185Umg(-1), and 30.9s(-1)mM(-1), respectively. The enzyme showed strong preference for NAD(+) and displayed no detectable activity with NADP(+). Homology modeling and sequence analysis of EaRDH, along with its biochemical properties, confirmed that EaRDH belongs to the family of NAD(+)-dependent ribitol dehydrogenases, a member of short-chain dehydrogenase/reductase (SCOR) family. EaRDH showed the highest activity and unique substrate specificity among all known RDHs. Homology modeling and docking analysis shed light on the molecular basis of its unusually high activity and substrate specificity.

  19. Delineation of an in vivo inhibitor for Aspergillus glutamate dehydrogenase.

    Science.gov (United States)

    Choudhury, Rajarshi; Noor, Shahid; Varadarajalu, Lakshmi Prabha; Punekar, Narayan S

    2008-01-01

    NADP-glutamate dehydrogenase (NADP-GDH) along with glutamine synthetase plays a pivotal role in ammonium assimilation. Specific inhibitors were valuable in defining the importance of glutamine synthetase in nitrogen metabolism. Selective in vivo inhibition of NADP-GDH has so far been an elusive desideratum. Isophthalate, a potent in vitro inhibitor of Aspergillus niger NADP-GDH [Noor S, Punekar NS. Allosteric NADP-glutamate dehydrogenase from aspergilli: purification, characterization and implications for metabolic regulation at the carbon-nitrogen interface. Microbiology 2005;151:1409-19], was evaluated for its efficacy in vivo. Dimethyl ester of isophthalate (DMIP), but not isophthalate, inhibited A. niger growth on agar as well as in liquid culture. This was ascribed to the inability of isophthalate to enter fungal mycelia. Subsequent to DMIP addition however, intracellular isophthalate could be demonstrated. Apart from NAD-GDH, no other enzyme including NAD-glutamate synthase was inhibited by isophthalate. A cross-over at NADP-GDH step of metabolism was observed as a direct consequence of isophthalate (formed in vivo from DMIP) inhibiting this enzyme. Addition of ammonium to DMIP-treated A. niger mycelia resulted in intensive vacuolation, retraction of cytoplasm and autolysis. Taken together, these results implicate glutamate dehydrogenase and NADP-GDH in particular, as a key target of in vivo isophthalate inhibition during ammonium assimilation. PMID:22578865

  20. Genetic improvement of Escherichia coli for ethanol production: chromosomal integration of Zymomonas mobilis genes encoding pyruvate decarboxylase and alcohol dehydrogenase II.

    OpenAIRE

    Ohta, K.; Beall, D S; Mejia, J P; Shanmugam, K. T.; Ingram, L O

    1991-01-01

    Zymomonas mobilis genes for pyruvate decarboxylase (pdc) and alcohol dehydrogenase II (adhB) were integrated into the Escherichia coli chromosome within or near the pyruvate formate-lyase gene (pfl). Integration improved the stability of the Z. mobilis genes in E. coli, but further selection was required to increase expression. Spontaneous mutants were selected for resistance to high level of chloramphenicol that also expressed high levels of the Z. mobilis genes. Analogous mutants were selec...

  1. Potent inhibition of aldehyde dehydrogenase-2 by diphenyleneiodonium: focus on nitroglycerin bioactivation.

    Science.gov (United States)

    Neubauer, Regina; Neubauer, Andrea; Wölkart, Gerald; Schwarzenegger, Christine; Lang, Barbara; Schmidt, Kurt; Russwurm, Michael; Koesling, Doris; Gorren, Antonius C F; Schrammel, Astrid; Mayer, Bernd

    2013-09-01

    Aldehyde dehydrogenase-2 (ALDH2) catalyzes vascular bioactivation of the antianginal drug nitroglycerin (GTN) to yield nitric oxide (NO) or a related species that activates soluble guanylate cyclase (sGC), resulting in cGMP-mediated vasodilation. Accordingly, established ALDH2 inhibitors attenuate GTN-induced vasorelaxation in vitro and in vivo. However, the ALDH2 hypothesis has not been reconciled with early studies demonstrating potent inhibition of the GTN response by diphenyleneiodonium (DPI), a widely used inhibitor of flavoproteins, in particular NADPH oxidases. We addressed this issue and investigated the effects of DPI on GTN-induced relaxation of rat aortic rings and the function of purified ALDH2. DPI (0.3 µM) inhibited the high affinity component of aortic relaxation to GTN without affecting the response to NO, indicating that the drug interfered with GTN bioactivation. Denitration and bioactivation of 1-2 µM GTN, assayed as 1,2-glycerol dinitrate formation and activation of purified sGC, respectively, were inhibited by DPI with a half-maximally active concentration of about 0.2 µM in a GTN-competitive manner. Molecular modeling indicated that DPI binds to the catalytic site of ALDH2, and this was confirmed by experiments showing substrate-competitive inhibition of the dehydrogenase and esterase activities of the enzyme. Our data identify ALDH2 as highly sensitive target of DPI and explain inhibition of GTN-induced relaxation by this drug observed previously. In addition, the data provide new evidence for the essential role of ALDH2 in GTN bioactivation and may have implications to other fields of ALDH2 research, such as hepatic ethanol metabolism and cardiac ischemia/reperfusion injury.

  2. Characterization of testis-specific isoenzyme of human pyruvate dehydrogenase.

    Science.gov (United States)

    Korotchkina, Lioubov G; Sidhu, Sukhdeep; Patel, Mulchand S

    2006-04-01

    Pyruvate dehydrogenase (PDH), the first component of the human pyruvate dehydrogenase complex, has two isoenzymes, somatic cell-specific PDH1 and testis-specific PDH2 with 87% sequence identity in the alpha subunit of alpha(2) beta(2) PDH. The presence of functional testis-specific PDH2 is important for sperm cells generating nearly all their energy from carbohydrates via pyruvate oxidation. Kinetic and regulatory properties of recombinant human PDH2 and PDH1 were compared in this study. Site-specific phosphorylation/dephosphorylation of the three phosphorylation sites by four PDH kinases (PDK1-4) and two PDH phosphatases (PDP1-2) were investigated by substituting serines with alanine or glutamate in PDHs. PDH2 was found to be very similar to PDH1 as follows: (i) in specific activities and kinetic parameters as determined by the pyruvate dehydrogenase complex assay; (ii) in thermostability at 37 degrees C; (iii) in the mechanism of inactivation by phosphorylation of three sites; and (iv) in the phosphorylation of sites 1 and 2 by PDK3. In contrast, the differences for PDH2 were indicated as follows: (i) by a 2.4-fold increase in binding affinity for the PDH-binding domain of dihydrolipoamide acetyltransferase as measured by surface plasmon resonance; (ii) by possible involvement of Ser-264 (site 1) of PDH2 in catalysis as evident by its kinetic behavior; and (iii) by the lower activities of PDK1, PDK2, and PDK4 as well as PDP1 and PDP2 toward PDH2. These differences between PDH2 and PDH1 are less than expected from substitution of 47 amino acids in each PDH2 alpha subunit. The multiple substitutions may have compensated for any drastic alterations in PDH2 structure thereby preserving its kinetic and regulatory characteristics largely similar to that of PDH1. PMID:16436377

  3. Identification, Cloning, and Characterization of l-Phenylserine Dehydrogenase from Pseudomonas syringae NK-15

    Directory of Open Access Journals (Sweden)

    Sakuko Ueshima

    2010-01-01

    Full Text Available The gene encoding d-phenylserine dehydrogenase from Pseudomonas syringae NK-15 was identified, and a 9,246-bp nucleotide sequence containing the gene was sequenced. Six ORFs were confirmed in the sequenced region, four of which were predicted to form an operon. A homology search of each ORF predicted that orf3 encoded l-phenylserine dehydrogenase. Hence, orf3 was cloned and overexpressed in Escherichia coli cells and recombinant ORF3 was purified to homogeneity and characterized. The purified ORF3 enzyme showed l-phenylserine dehydrogenase activity. The enzymological properties and primary structure of l-phenylserine dehydrogenase (ORF3 were quite different from those of d-phenylserine dehydrogenase previously reported. l-Phenylserine dehydrogenase catalyzed the NAD+-dependent oxidation of the β-hydroxyl group of l-β-phenylserine. l-Phenylserine and l-threo-(2-thienylserine were good substrates for l-phenylserine dehydrogenase. The genes encoding l-phenylserine dehydrogenase and d-phenylserine dehydrogenase, which is induced by phenylserine, are located in a single operon. The reaction products of both enzymatic reactions were 2-aminoacetophenone and CO2.

  4. Structural determinants of stereospecificity in yeast alcohol dehydrogenase.

    OpenAIRE

    Weinhold, E G; Glasfeld, A; Ellington, A D; Benner, S A

    1991-01-01

    Replacing Leu-182 by Ala in yeast alcohol dehydrogenase (YADH; alcohol:NAD+ oxidoreductase, EC 1.1.1.1) yields a mutant that retains 34% of its kcat value and makes one stereochemical "mistake" every 850,000 turnovers (instead of approximately 1 error every 7,000,000,000 turnovers in native YADH) in its selection of the 4-Re hydrogen of NADH. Half of the decrease in stereochemical fidelity comes from an increase in the rate of transfer of the 4-Si hydrogen of NADH. The mutant also accepts 5-m...

  5. Functional characterization of a vanillin dehydrogenase in Corynebacterium glutamicum

    OpenAIRE

    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...

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

    DEFF Research Database (Denmark)

    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...

  7. Genetic Control of Malate Dehydrogenase Isozymes in Maize

    OpenAIRE

    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...

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

    DEFF Research Database (Denmark)

    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...... of 266 amino acid residues with a calculated molecular mass of 28,426 Da. The gene was overexpressed in E. coli BL21(DE3) and the protein was purified as an active soluble form using glutathione S-transferase affinity chromatography. The molecular mass of the purified enzyme was estimated...

  9. Essential histidine residue in 3-ketosteroid-Δ1-dehydrogenase

    OpenAIRE

    Matsushita, Hiroyuki; Itagaki, Eiji; 板垣, 英治

    1992-01-01

    The variation with pH of kinetic parameters was examined for 3-ketosteroid-Δ1-dehydrogenase from Nocardia corallina. The V(max)/K(m) profile for 4-androstenedione indicates that activity is lost upon protonation of a cationic acid-type group with a pK value of 7.7. The enzyme was inactivated by diethylpyrocarbonate at pH 7.4 and the inactivation was substantially prevented by androstadienedione. Analyses of reactivation with neutral hydroxylamine, pH variation, and spectral changes of the ina...

  10. In vitro hydrogen production by glucose dehydrogenase and hydrogenase

    Energy Technology Data Exchange (ETDEWEB)

    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.

  11. Inactivation of Lactate Dehydrogenase from Pig Heart by o-Phthalaldehyde

    Institute of Scientific and Technical Information of China (English)

    郑延斌; 王政; 陈宝玉; 王希成

    2003-01-01

    Treatment of lactate dehydrogenase (LDH) with o-phthalaldehyde resulted in a time-dependent loss of enzyme activity.The inactivation followed pseudo first-order kinetics over a wide range of the inhibitor.The second-order rate constant for the inactivation of LDH was estimated to be 1.52 (mol/L)-1·s-1.The modified enzyme showed a characteristic fluorescence emission spectrum with a maximum at 405 nm upon excitation at 337 nm, consistent with the formation of isoindole derivatives by the cross-linking of proximal cysteine and lysine residues.The loss of enzyme activity was concomitant with the increase in absorbance at 337 nm.Stoichiometric study of the reaction showed that complete loss of activity was accompanied by formation of approximately four moles of isoindole derivatives per mole of LDH subunits.One of the substrates, NADH, partially prevented the enzyme from reacting with o-phthalaldehyde, whereas the other substrate, pyruvate, did not provide any protection.Protection experiments suggest that one of the cysteine-lysine pairs modified by o-phthalaldehyde is near the NADH binding site of LDH.

  12. Characterization of 10-hydroxygeraniol dehydrogenase from Catharanthus roseus reveals cascaded enzymatic activity in iridoid biosynthesis.

    Science.gov (United States)

    Krithika, Ramakrishnan; Srivastava, Prabhakar Lal; Rani, Bajaj; Kolet, Swati P; Chopade, Manojkumar; Soniya, Mantri; Thulasiram, Hirekodathakallu V

    2015-01-01

    Catharanthus roseus [L.] is a major source of the monoterpene indole alkaloids (MIAs), which are of significant interest due to their therapeutic value. These molecules are formed through an intermediate, cis-trans-nepetalactol, a cyclized product of 10-oxogeranial. One of the key enzymes involved in the biosynthesis of MIAs is an NAD(P)(+) dependent oxidoreductase system, 10-hydroxygeraniol dehydrogenase (Cr10HGO), which catalyses the formation of 10-oxogeranial from 10-hydroxygeraniol via 10-oxogeraniol or 10-hydroxygeranial. This work describes the cloning and functional characterization of Cr10HGO from C. roseus and its role in the iridoid biosynthesis. Substrate specificity studies indicated that, Cr10HGO has good activity on substrates such as 10-hydroxygeraniol, 10-oxogeraniol or 10-hydroxygeranial over monohydroxy linear terpene derivatives. Further it was observed that incubation of 10-hydroxygeraniol with Cr10HGO and iridoid synthase (CrIDS) in the presence of NADP(+) yielded a major metabolite, which was characterized as (1R, 4aS, 7S, 7aR)-nepetalactol by comparing its retention time, mass fragmentation pattern, and co-injection studies with that of the synthesized compound. These results indicate that there is concerted activity of Cr10HGO with iridoid synthase in the formation of (1R, 4aS, 7S, 7aR)-nepetalactol, an important intermediate in iridoid biosynthesis. PMID:25651761

  13. The bifunctional aldehyde-alcohol dehydrogenase controls ethanol and acetate production in Entamoeba histolytica under aerobic conditions.

    Science.gov (United States)

    Pineda, Erika; Encalada, Rusely; Olivos-García, Alfonso; Néquiz, Mario; Moreno-Sánchez, Rafael; Saavedra, Emma

    2013-01-16

    By applying metabolic control analysis and inhibitor titration we determined the degree of control (flux control coefficient) of pyruvate:ferredoxin oxidoreductase (PFOR) and bifunctional aldehyde-alcohol dehydrogenase (ADHE) over the fluxes of fermentative glycolysis of Entamoeba histolytica subjected to aerobic conditions. The flux-control coefficients towards ethanol and acetate formation determined for PFOR titrated with diphenyleneiodonium were 0.07 and 0.09, whereas for ADHE titrated with disulfiram were 0.33 and -0.19, respectively. ADHE inhibition induced significant accumulation of glycolytic intermediates and lower ATP content. These results indicate that ADHE exerts significant flux-control on the carbon end-product formation of amoebas subjected to aerobic conditions. PMID:23201265

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

    Science.gov (United States)

    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

  15. Crystal structure of a chimaeric bacterial glutamate dehydrogenase

    Energy Technology Data Exchange (ETDEWEB)

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

    2016-05-23

    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+versusNADP+, 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 fromClostridium symbiosum. Here, the crystal structure of a chimaeric clostridial/Escherichia colienzyme 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 parentE. colidomain II, although there are subtle differences in catalytic activity.

  16. Differing roles of pyruvate dehydrogenase kinases during mouse oocyte maturation.

    Science.gov (United States)

    Hou, Xiaojing; Zhang, Liang; Han, Longsen; Ge, Juan; Ma, Rujun; Zhang, Xuesen; Moley, Kelle; Schedl, Tim; Wang, Qiang

    2015-07-01

    Pyruvate dehydrogenase kinases (PDKs) modulate energy homeostasis in multiple tissues and cell types, under various nutrient conditions, through phosphorylation of the α subunit (PDHE1α, also known as PDHA1) of the pyruvate dehydrogenase (PDH) complex. However, the roles of PDKs in meiotic maturation are currently unknown. Here, by undertaking knockdown and overexpression analysis of PDK paralogs (PDK1-PDK4) in mouse oocytes, we established the site-specificity of PDKs towards the phosphorylation of three serine residues (Ser232, Ser293 and Ser300) on PDHE1α. We found that PDK3-mediated phosphorylation of Ser293-PDHE1α results in disruption of meiotic spindle morphology and chromosome alignment and decreased total ATP levels, probably through inhibition of PDH activity. Unexpectedly, we discovered that PDK1 and PDK2 promote meiotic maturation, as their knockdown disturbs the assembly of the meiotic apparatus, without significantly altering ATP content. Moreover, phosphorylation of Ser232-PDHE1α was demonstrated to mediate PDK1 and PDK2 action in meiotic maturation, possibly through a mechanism that is distinct from PDH inactivation. These findings reveal that there are divergent roles of PDKs during oocyte maturation and indicate a new mechanism controlling meiotic structure. PMID:25991547

  17. Crystal structure of a chimaeric bacterial glutamate dehydrogenase.

    Science.gov (United States)

    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. Engineering of pyranose dehydrogenase for increased oxygen reactivity.

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    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.

  1. Glutamate dehydrogenase from pumpkin cotyledons: characterization and isoenzymes.

    Science.gov (United States)

    Chou, K H; Splittstoesser, W E

    1972-04-01

    Glutamate dehydrogenase from pumpkin (Cucurbita moschata Pior. cultivar Dickinson Field) cotyledons was found in both soluble and particulate fractions with the bulk of the activity in the soluble fraction. Both enzymes used NAD(H) and NADP(H) but NAD(H) was favored. The enzymes were classified as glutamate-NAD oxidoreductase, deaminating (EC 1.4.1.3). Both enzymes were heat stable, had a pH optimum for reductive amination of 8.0, and were inhibited by high concentrations of NH(4) (+) or alpha-ketoglutarate. The soluble enzyme was more sensitive to NH(4) (+) inhibition and was activated by metal ions after ammonium sulfate fractionation while the solubilized particulate enzyme was not. Inhibition by ethylenediaminetetraacetate was restored by several divalent ions and inhibition by p-hydroxymercuribenzoate was reversed by glutathione. Particulate glutamate dehydrogenase showed a greater activity with NADP. The molecular weights of the enzymes are 250,000. Separation of the enzymes by disc gel electrophoresis showed that during germination the soluble isoenzymes increased from 1 to 7 in number, while only one particulate isoenzyme was found at any time. This particulate isoenzyme was identical with one of the soluble isoenzymes. A number of methods indicated that the soluble isoenzymes were not simply removed from the particulate fraction and that true isoenzymes were found.

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

    Science.gov (United States)

    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. Structural basis for cellobiose dehydrogenase action during oxidative cellulose degradation

    Science.gov (United States)

    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

  4. The pivotal role of pyruvate dehydrogenase kinases in metabolic flexibility.

    Science.gov (United States)

    Zhang, Shuai; Hulver, Matthew W; McMillan, Ryan P; Cline, Mark A; Gilbert, Elizabeth R

    2014-01-01

    Metabolic flexibility is the capacity of a system to adjust fuel (primarily glucose and fatty acids) oxidation based on nutrient availability. The ability to alter substrate oxidation in response to nutritional state depends on the genetically influenced balance between oxidation and storage capacities. Competition between fatty acids and glucose for oxidation occurs at the level of the pyruvate dehydrogenase complex (PDC). The PDC is normally active in most tissues in the fed state, and suppressing PDC activity by pyruvate dehydrogenase (PDH) kinase (PDK) is crucial to maintain energy homeostasis under some extreme nutritional conditions in mammals. Conversely, inappropriate suppression of PDC activity might promote the development of metabolic diseases. This review summarizes PDKs' pivotal role in control of metabolic flexibility under various nutrient conditions and in different tissues, with emphasis on the best characterized PDK4. Understanding the regulation of PDC and PDKs and their roles in energy homeostasis could be beneficial to alleviate metabolic inflexibility and to provide possible therapies for metabolic diseases, including type 2 diabetes (T2D). PMID:24520982

  5. Toxicity of Nitrification Inhibitors on Dehydrogenase Activity in Soils

    Directory of Open Access Journals (Sweden)

    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.

  6. The oxidative pentose phosphate pathway in the haloarchaeon Haloferax volcanii involves a novel type of glucose-6-phosphate dehydrogenase--The archaeal Zwischenferment.

    Science.gov (United States)

    Pickl, Andreas; Schönheit, Peter

    2015-04-28

    The oxidative pentose phosphate pathway (OPPP), catalyzing the oxidation of glucose-6-phosphate to ribulose-5-phosphate is ubiquitous in eukarya and bacteria but has not yet been reported in archaea. In haloarchaea a putative 6-phosphogluconate dehydrogenase (6PGDH) is annotated, whereas a gene coding for glucose-6-phosphate dehydrogenase (Glc6PDH) could not be identified. Here we report the purification and characterization of a novel type of Glc6PDH in Haloferax volcanii that is not related to bacterial and eukaryal Glc6PDHs and the encoding gene is designated as azf (archaeal zwischenferment). Further, recombinant H. volcanii 6PGDH was characterized. Deletion mutant analyses indicate that both, Glc6PDH and 6PGDH, are functionally involved in pentose phosphate formation in vivo. This is the first report on the operation of the OPPP in the domain of archaea.

  7. Structural studies on metal-containing enzymes. T4 endonuclease VII and D. gigas formate dehydrogenase

    NARCIS (Netherlands)

    Raaijmakers, H.C.A.

    2001-01-01

    Many biological processes require metal ions, and many of these metal-ion functions involve metalloproteins. The metal ions in metalloproteins are often critical to the protein's function, structure, or stability. This thesis focuses on two of these proteins, bacteriophage T4 endonuclease VII (EndoV

  8. Formate dehydrogenases and hydrogenases in syntrophic propionate-oxidizing communities : gene analysis and transcritional profiling

    NARCIS (Netherlands)

    Worm, P.

    2010-01-01

    Many places on earth are without oxygen (anaerobic) such as rice paddy fields, swamps and sediments of freshwater lakes and oceans. When oxygen, nitrate or other electron acceptors are not present, organic material is degraded to carbon dioxide and methane by mixed microbial species that each hav

  9. Active site of Zn2+-dependent sn-glycerol-1-phosphate dehydrogenase from Aeropyrum pernix K1

    Directory of Open Access Journals (Sweden)

    Jin-Suk Han

    2005-01-01

    Full Text Available The enzyme sn-glycerol-1-phosphate dehydrogenase (Gro1PDH, EC 1.1.1.261 is key to the formation of the enantiomeric configuration of the glycerophosphate backbone (sn-glycerol-1-phosphate of archaeal ether lipids. This enzyme catalyzes the reversible conversion between dihydroxyacetone phosphate and glycerol-1-phosphate. To date, no information about the active site and catalytic mechanism of this enzyme has been reported. Using the sequence and structural information for glycerol dehydrogenase, we constructed six mutants (D144N, D144A, D191N, H271A, H287A and D191N/H271A of Gro1PDH from Aeropyrum pernix K1 and examined their characteristics to clarify the active site of this enzyme. The enzyme was found to be a zinc-dependent metalloenzyme, containing one zinc ion for every monomer protein that was essential for activity. Site-directed mutagenesis of D144 increased the activity of the enzyme. Mutants D144N and D144A exhibited low affinity for the substrates and higher activity than the wild type, but their affinity for the zinc ion was the same as that of the wild type. Mutants D191N, H271A and H287A had a low affinity for the zinc ion and a low activity compared with the wild type. The double mutation, D191N/ H271A, had no enzyme activity and bound no zinc. From these results, it was clarified that residues D191, H271 and H287 participate in the catalytic activity of the enzyme by binding the zinc ion, and that D144 has an effect on substrate binding. The structure of the active site of Gro1PDH from A. pernix K1 seems to be similar to that of glycerol dehydrogenase, despite the differences in substrate specificity and biological role.

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

    OpenAIRE

    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...

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

    OpenAIRE

    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...

  12. The diagnostic value of alcohol dehydrogenase (ADH) isoenzymes and aldehyde dehydrogenase (ALDH) measurement in the sera of gastric cancer patients.

    Science.gov (United States)

    Jelski, Wojciech; Orywal, Karolina; Laniewska, Magdalena; Szmitkowski, Maciej

    2010-12-01

    Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) are present in gastric cancer cells (GC). Moreover, the activity of total ADH and class IV isoenzymes is significantly higher in cancer tissue than in healthy mucosa. The activity of these enzymes in cancer cells is probably reflected in the sera and could thus be helpful for diagnostics of gastric cancer. The aim of this study was to investigate a potential role of ADH and ALDH as tumor markers for gastric cancer. We defined diagnostic sensitivity, specificity, predictive value for positive and negative results, and receiver-operating characteristics (ROC) curve for tested enzymes. Serum samples were taken from 168 patients with gastric cancer before treatment and from 168 control subjects. Total ADH activity and class III and IV isoenzymes were measured by photometric but ALDH activity and ADH I and II by the fluorometric method, with class-specific fluorogenic substrates. There was significant increase in the activity of ADH IV isoenzyme and ADH total in the sera of gastric cancer patients compared to the control. The diagnostic sensitivity for ADH IV was 73%, specificity 79%, positive and negative predictive values were 81 and 72% respectively. Area under ROC curve for ADH IV was 0.67. The results suggest a potential role for ADH IV as marker of gastric cancer.

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

    Science.gov (United States)

    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. Structural basis for the dysfunctioning of human 2-oxo acid dehydrogenase complexes

    NARCIS (Netherlands)

    Hengeveld, A.F.; Kok, de A.

    2002-01-01

    2-oxo acid dehydrogenase complexes are a ubiquitous family of multienzyme systems that catalyse the oxidative decarboxylation of various 2-oxo acid substrates. They play a key role in the primary energy metabolism: in glycolysis (pyruvate dehydrogenase complex), the citric acid cycle (2-oxoglutarate

  15. 21 CFR 864.7360 - Erythrocytic glucose-6-phosphate dehydrogenase assay.

    Science.gov (United States)

    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...

  16. The lactate dehydrogenase of the icefish heart: biochemical adaptations to hypoxia tolerance.

    Science.gov (United States)

    Feller, G; Pauly, J P; Smal, A; O'Carra, P; Gerday, C

    1991-09-20

    Cardiac lactate dehydrogenase from the hemoglobin- and myoglobin-free antarctic icefish has been purified by affinity chromatography. Structural and kinetic properties of the enzyme were found close or identical to those of its skeletal muscle counterpart and other M-type lactate dehydrogenases. A model involving a dual oxidative-anaerobic metabolism of the icefish heart is proposed. PMID:1911860

  17. Isolation, characterization and evaluation of the Pichia pastoris sorbitol dehydrogenase promoter for expression of heterologous proteins.

    Science.gov (United States)

    Periyasamy, Sankar; Govindappa, Nagaraj; Sreenivas, Suma; Sastry, Kedarnath

    2013-11-01

    Sorbitol is used as a non-repressive carbon source to develop fermentation process for Mut(s) recombinant clones obtained using the AOX1 promoter in Pichia pastoris. Sorbitol dehydrogenase is an enzyme in the carbohydrate metabolism that catalyzes reduction of D-fructose into D-sorbitol in the presence of NADH. The small stretch of 211bps upstream region of sorbitol dehydrogenase coding gene has all the promoter elements like CAAT box, GC box, etc. It is able to promote protein production under repressive as well as non-repressive carbon sources. In this study, the strength of the sorbitol dehydrogenase promoter was evaluated by expression of two heterologous proteins: human serum albumin and erythrina trypsin inhibitor. Sorbitol dehydrogenase promoter allowed constitutive expression of recombinant proteins in all carbon sources that were tested to grow P. pastoris and showed activity similar to GAP promoter. The sorbitol dehydrogenase promoter was active in all the growth phases of the P. pastoris.

  18. Krebs cycle metabolite profiling for identification and stratification of pheochromocytomas/paragangliomas due to succinate dehydrogenase deficiency

    NARCIS (Netherlands)

    Richter, S; Peitzsch, M.; Rapizzi, E.; Lenders, J.W.M.; Qin, N.; Cubas, A.A. de; Schiavi, F.; Rao, J.U.; Beuschlein, F.; Quinkler, M.; Timmers, H.J.L.M.; Opocher, G.; Mannelli, M.; Pacak, K.; Robledo, M.; Eisenhofer, G.

    2014-01-01

    CONTEXT: Mutations of succinate dehydrogenase A/B/C/D genes (SDHx) increase susceptibility to development of pheochromocytomas and paragangliomas (PPGLs), with particularly high rates of malignancy associated with SDHB mutations. OBJECTIVE: We assessed whether altered succinate dehydrogenase product

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

    DEFF Research Database (Denmark)

    Yao, Shuo; Just Mikkelsen, Marie

    2010-01-01

    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...... 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...

  20. The oxidation of yeast alcohol dehydrogenase-1 by hydrogen peroxide in vitro.

    Science.gov (United States)

    Men, Lijie; Wang, Yinsheng

    2007-01-01

    Yeast alcohol dehydrogenase (YADH) plays an important role in the conversion of alcohols to aldehydes or ketones. YADH-1 is a zinc-containing protein, and it accounts for the major part of ADH activity in growing baker's yeast. To gain insight into how oxidative modification of the enzyme affects its function, we exposed YADH-1 to hydrogen peroxide in vitro and assessed the oxidized protein by LC-MS/MS analysis of proteolytic cleavage products of the protein and by measurements of enzymatic activity, zinc release, and thiol/thiolate loss. The results illustrated that Cys43 and Cys153, which reside at the active site of the protein, could be selectively oxidized to cysteine sulfinic acid (Cys-SO2H) and cysteine sulfonic acid (Cys-SO3H). In addition, H2O2 induced the formation of three disulfide bonds: Cys43-Cys153 in the catalytic domain, Cys103-Cys111 in the noncatalytic zinc center, and Cys276-Cys277. Therefore, our results support the notion that the oxidation of cysteine residues in the zinc-binding domain of proteins can go beyond the formation of disulfide bond(s); the formation of Cys-SO2H and Cys-SO3H is also possible. Furthermore, most methionines could be oxidized to methionine sulfoxides. Quantitative measurement results revealed that, among all the cysteine residues, Cys43 was the most susceptible to H2O2 oxidation, and the major oxidation products of this cysteine were Cys-SO2H and Cys-SO3H. The oxidation of Cys43 might be responsible for the inactivation of the enzyme upon H2O2 treatment.

  1. Crystal structure of the NADP-dependent mannitol dehydrogenase from Cladosporium herbarum: Implications for oligomerisation and catalysis.

    Science.gov (United States)

    Nüss, Dorota; Goettig, Peter; Magler, Iris; Denk, Ursula; Breitenbach, Michael; Schneider, Peter B; Brandstetter, Hans; Simon-Nobbe, Birgit

    2010-08-01

    The ascomycete Cladosporium herbarum is a prominent fungal inducer of Type I allergy. The only major allergen identified so far is Cla h 8, a NADP-dependent mannitol dehydrogenase (MtDH). MtDH, a cytoplasmic protein of 28.5kDa, belongs to the Short chain Dehydrogenases/Reductases (SDR), acting as a NADP-dependent oxidoreductase. In this study, we found that C. herbarum MtDH can exist as monomers, dimers and tetramers in solution and, correspondingly, forms tetramers and higher oligomers in two crystal structures. Additionally, we identified a unique adaptive binding site for the metal ions Na(+) and Zn(2+) that were distinguished by an anomalous dispersion experiment. A Translation-Libration-Screw analysis confirmed the stabilising effect of Zn(2+) for the tetrameric assembly. Moreover, the zinc containing structure explains the mode of MtDH multimerisation by metal bridging of the tetramers. The formation of oligomers and higher multimers of MtDH provides a missing link to its allergenic properties. Based on the well defined active site region and a comparative analysis with related structures, we can also clarify the atypical enzymatic properties of MtDH by two alternative binding modes of the substrate to the active site.

  2. Identification of the 11 beta-hydroxysteroid dehydrogenase type 1 mRNA and protein in human mononuclear leukocytes.

    Science.gov (United States)

    Fiore, C; Nardi, A; Dalla Valle, L; Pellati, D; Krozowski, Z; Colombo, L; Armanini, D

    2009-10-01

    The enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) catalyzes the interconversion between inactive 11-ketoglucocorticoids and their active 11beta-hydroxy derivatives, such as cortisol and corticosterone. We have investigated the expression of 11beta-HSD1 in freshly isolated human peripheral mononuclear leukocytes (MNL). The presence of 11beta-HSD1 mRNA was demonstrated in total RNA by RT-PCR using specific primers designed on the 4th and 5th exons of the human 11beta-HSD1 gene. Fragments of the expected size were consistently detected on agarose gels, and sequencing showed complete identity with the corresponding sequence deposited in GenBank. The occurrence of 11beta-HSD1 protein was established by Western immunoblot analysis with a specific polyclonal antibody. Enzyme oxo-reductase activity was investigated by incubating 12 samples of MNL isolated from from 8 subjects with [3H]cortisone and formation of cortisol was established only in 4 subjects (yield range: 0.15-1.3%) after acetylation and TLC, blank subtraction and correction for losses. 18beta-Glycyrrhetinic acid, an inhibitor of 11 beta-HSD1, reduced cortisol production below detection limit. Dehydrogenase activity could not be demonstrated. It is suggested that, although enzyme activity of 11beta-HSD1 in circulating MNL is low, it is apparently ready for enhancement after MNL migration to sites of inflammation. PMID:19235128

  3. Fusarium graminearum pyruvate dehydrogenase kinase 1 (FgPDK1) Is Critical for Conidiation, Mycelium Growth, and Pathogenicity

    Science.gov (United States)

    Gao, Tao; Chen, Jian; Shi, Zhiqi

    2016-01-01

    Pyruvate dehydrogenase kinase (PDK) is an important mitochondrial enzyme that blocks the production of acetyl-CoA by selectively inhibiting the activity of pyruvate dehydrogenase (PDH) through phosphorylation. PDK is an effectively therapeutic target in cancer cells, but the physiological roles of PDK in phytopathogens are largely unknown. To address these gaps, a PDK gene (FgPDK1) was isolated from Fusarium graminearum that is an economically important pathogen infecting cereals. The deletion of FgPDK1 in F. graminearum resulted in the increase in PDH activity, coinciding with several phenotypic defects, such as growth retardation, failure in perithecia and conidia production, and increase in pigment formation. The ΔFgPDK1 mutants showed enhanced sensitivity to osmotic stress and cell membrane-damaging agent. Physiological detection indicated that reactive oxygen species (ROS) accumulation and plasma membrane damage (indicated by PI staining, lipid peroxidation, and electrolyte leakage) occurred in ΔFgPDK1 mutants. The deletion of FgPDK1 also prohibited the production of deoxynivalenol (DON) and pathogenicity of F. graminearum, which may resulted from the decrease in the expression of Tri6. Taken together, this study firstly identified the vital roles of FgPDK1 in the development of phytopathogen F. graminearum, which may provide a potentially novel clue for target-directed development of agricultural fungicides. PMID:27341107

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

    Science.gov (United States)

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

    Science.gov (United States)

    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

  6. IMP Dehydrogenase: Structural Schizophrenia and an Unusual Base

    Energy Technology Data Exchange (ETDEWEB)

    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.

  7. Lactate dehydrogenase (LDH isoenzymes patterns in ocular tumours

    Directory of Open Access Journals (Sweden)

    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.

  8. The reaction of choline dehydrogenase with some electron acceptors.

    Science.gov (United States)

    Barrett, M C; Dawson, A P

    1975-12-01

    1. The choline dehydrogenase (EC 1.1.99.1) WAS SOLUBILIZED FROM ACETONE-DRIED POWDERS OF RAT LIVER MITOCHONDRIA BY TREATMENT WITH Naja naja venom. 2. The kinetics of the reaction of enzyme with phenazine methosulphate and ubiquinone-2 as electron acceptors were investigated. 3. With both electron acceptors the reaction mechanism appears to involve a free, modified-enzyme intermediate. 4. With some electron acceptors the maximum velocity of the reaction is independent of the nature of the acceptor. With phenazine methosulphate and ubiquinone-2 as acceptors the Km value for choline is also independent of the nature of the acceptor molecule. 5. The mechanism of the Triton X-100-solubilized enzyme is apparently the smae as that for the snake venom solubilized enzyme.

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

    Science.gov (United States)

    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

  10. Lactate dehydrogenase assay for assessment of polycation cytotoxicity

    DEFF Research Database (Denmark)

    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...

  11. Cytokinin oxidase or dehydrogenase? Mechanism of cytokinin degradation in cereals

    DEFF Research Database (Denmark)

    Galuszka, P.; Frebort, I.; Sebela, M.;

    2001-01-01

    An enzyme degrading cytokinins with isoprenoid side chain, previously named cytokinin oxidase, was purified to near homogeneity from wheat and barley grains. New techniques were developed for the enzyme activity assay and staining on native electrophoretic gels to identify the protein. The purified...... wheat enzyme is a monomer 60 kDa, its N-terminal amino-acid sequence shows similarity to hypothetical cytokinin oxidase genes from Arabidopsis thaliana, but not to the enzyme from maize. N-6-isopentenyl-2-(2-hydroxyethylamino)-9-methyladenine is the best substrate from all the cytokinins tested....... Interestingly, oxygen was not required and hydrogen peroxide not produced during the catalytic reaction, so the enzyme behaves as a dehydrogenase rather than an oxidase. This was confirmed by the ability of the enzyme to transfer electrons to artificial electron acceptors, such as phenazine methosulfate and 2...

  12. Involvement of snapdragon benzaldehyde dehydrogenase in benzoic acid biosynthesis.

    Science.gov (United States)

    Long, Michael C; Nagegowda, Dinesh A; Kaminaga, Yasuhisa; Ho, Kwok Ki; Kish, Christine M; Schnepp, Jennifer; Sherman, Debra; Weiner, Henry; Rhodes, David; Dudareva, Natalia

    2009-07-01

    Benzoic acid (BA) is an important building block in a wide spectrum of compounds varying from primary metabolites to secondary products. Benzoic acid biosynthesis from L-phenylalanine requires shortening of the propyl side chain by two carbons, which can occur via a beta-oxidative pathway or a non-beta-oxidative pathway, with benzaldehyde as a key intermediate. The non-beta-oxidative route requires benzaldehyde dehydrogenase (BALDH) to convert benzaldehyde to BA. Using a functional genomic approach, we identified an Antirrhinum majus (snapdragon) BALDH, which exhibits 40% identity to bacterial BALDH. Transcript profiling, biochemical characterization of the purified recombinant protein, molecular homology modeling, in vivo stable isotope labeling, and transient expression in petunia flowers reveal that BALDH is capable of oxidizing benzaldehyde to BA in vivo. GFP localization and immunogold labeling studies show that this biochemical step occurs in the mitochondria, raising a question about the role of subcellular compartmentalization in BA biosynthesis.

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

    Directory of Open Access Journals (Sweden)

    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.

  14. Inhibitor-bound structures of human pyruvate dehydrogenase kinase 4.

    Science.gov (United States)

    Kukimoto-Niino, Mutsuko; Tokmakov, Alexander; Terada, Takaho; Ohbayashi, Naomi; Fujimoto, Takako; Gomi, Sumiko; Shiromizu, Ikuya; Kawamoto, Masaki; Matsusue, Tomokazu; Shirouzu, Mikako; Yokoyama, Shigeyuki

    2011-09-01

    The mitochondrial pyruvate dehydrogenase complex (PDC) catalyzes the oxidative decarboxylation of pyruvate to acetyl-CoA. PDC activity is tightly regulated by four members of a family of pyruvate dehydrogenase kinase isoforms (PDK1-4), which phosphorylate and inactivate PDC. Recently, the development of specific inhibitors of PDK4 has become an especially important focus for the pharmaceutical management of diabetes and obesity. In this study, crystal structures of human PDK4 complexed with either AMPPNP, ADP or the inhibitor M77976 were determined. ADP-bound PDK4 has a slightly wider active-site cleft and a more disordered ATP lid compared with AMPPNP-bound PDK4, although both forms of PDK4 assume open conformations with a wider active-site cleft than that in the closed conformation of the previously reported ADP-bound PDK2 structure. M77976 binds to the ATP-binding pocket of PDK4 and causes local conformational changes with complete disordering of the ATP lid. M77976 binding also leads to a large domain rearrangement that further expands the active-site cleft of PDK4 compared with the ADP- and AMPPNP-bound forms. Biochemical analyses revealed that M77976 inhibits PDK4 with increased potency compared with the previously characterized PDK inhibitor radicicol. Thus, the present structures demonstrate for the first time the flexible and dynamic aspects of PDK4 in the open conformation and provide a basis for the development of novel inhibitors targeting the nucleotide-binding pocket of PDK4. PMID:21904029

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

    Science.gov (United States)

    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

  16. Lactate dehydrogenase concentration in nasal wash fluid indicates severity of rhinovirus-induced wheezy bronchitis in preschool children.

    Science.gov (United States)

    Cangiano, Giulia; Proietti, Elena; Kronig, Marie Noelle; Kieninger, Elisabeth; Sadeghi, Christine D; Gorgievski, Meri; Barbani, Maria Teresa; Midulla, Fabio; Tapparel, Caroline; Kaiser, Laurent; Alves, Marco P; Regamey, Nicolas

    2014-12-01

    The clinical course of rhinovirus (RV)-associated wheezing illnesses is difficult to predict. We measured lactate dehydrogenase concentrations, RV load, antiviral and proinflammatory cytokines in nasal washes obtained from 126 preschool children with RV wheezy bronchitis. lactate dehydrogenase values were inversely associated with subsequent need for oxygen therapy. lactate dehydrogenase may be a useful biomarker predicting disease severity in RV wheezy bronchitis.

  17. Crystal structure of (R)-3-hydroxybutyryl-CoA dehydrogenase PhaB from Ralstonia eutropha.

    Science.gov (United States)

    Kim, Jieun; Chang, Jeong Ho; Kim, Eun-Jung; Kim, Kyung-Jin

    2014-01-17

    (R)-3-hydroxybutyryl-CoA dehydrogenase PhaB from Ralstonia eutropha H16 (RePhaB) is an enzyme that catalyzes the NADPH-dependent reduction of acetoacetyl-CoA, an intermediate of polyhydroxyalkanoates (PHA) synthetic pathways. Polymeric PHA is used to make bioplastics, implant biomaterials, and biofuels. Here, we report the crystal structures of RePhaB apoenzyme and in complex with either NADP(+) or acetoacetyl-CoA, which provide the catalytic mechanism of the protein. RePhaB contains a Rossmann fold and a Clamp domain for binding of NADP(+) and acetoacetyl-CoA, respectively. The NADP(+)-bound form of RePhaB structure reveals that the protein has a unique cofactor binding mode. Interestingly, in the RePhaB structure in complex with acetoacetyl-CoA, the conformation of the Clamp domain, especially the Clamp-lid, undergoes a large structural change about 4.6 Å leading to formation of the substrate pocket. These structural observations, along with the biochemical experiments, suggest that movement of the Clamp-lid enables the substrate binding and ensures the acetoacetyl moiety is located near to the nicotinamide ring of NADP(+). PMID:24211201

  18. Mitochondrial isocitrate dehydrogenase is inactivated upon oxidation and reactivated by thioredoxin-dependent reduction in Arabidopsis

    Directory of Open Access Journals (Sweden)

    Keisuke eYoshida

    2014-09-01

    Full Text Available Regulation of mitochondrial metabolism is essential for ensuring cellular growth and maintenance in plants. Based on redox-proteomics analysis, several proteins involved in diverse mitochondrial reactions have been identified as potential redox-regulated proteins. NAD+-dependent isocitrate dehydrogenase (IDH, a key enzyme in the tricarboxylic acid cycle, is one such candidate. In this study, we investigated the redox regulation mechanisms of IDH by biochemical procedures. In contrast to mammalian and yeast counterparts reported to date, recombinant IDH in Arabidopsis mitochondria did not show adenylate-dependent changes in enzymatic activity. Instead, IDH was inactivated by oxidation treatment and partially reactivated by subsequent reduction. Functional IDH forms a heterodimer comprising regulatory (IDH-r and catalytic (IDH-c subunits. IDH-r was determined to be the target of oxidative modifications forming an oligomer via intermolecular disulfide bonds. Mass spectrometric analysis combined with tryptic digestion of IDH-r indicated that Cys128 and Cys216 are involved in intermolecular disulfide bond formation. Furthermore, we showed that mitochondria-localized o-type thioredoxin (Trx-o promotes the reduction of oxidized IDH-r. These results suggest that IDH-r is susceptible to oxidative stress, and Trx-o serves to convert oxidized IDH-r to the reduced form that is necessary for active IDH complex.

  19. Preparation of novel silica-coated alginate gel beads for efficient encapsulation of yeast alcohol dehydrogenase.

    Science.gov (United States)

    Xu, Song-Wei; Lu, Yang; Li, Jian; Zhang, Yu-Fei; Jiang, Zhong-Yi

    2007-01-01

    Biomimetic formation has undoubtedly inspired the preparation of novel organic-inorganic hybrid composites. In this study, silica-coated alginate gel beads were prepared by coating the surface of alginate gel beads with silica film derived from tetramethoxysilane (TMOS). The composition and structure of the silica film were characterized by FT-IR and SEM equipped with EDX. The swelling behavior of silica-coated alginate gel beads was studied to be more stable against swelling than that of alginate gel beads. The results showed that silica-coated alginate gel beads exhibited appropriate diffusion property. The effective diffusion coefficient (D(e)) of NADH in silica-coated alginate beads was 1.76 x 10(-10) m2/s, while the effective diffusion coefficient in alginate beads was 1.84 x 10(-10) m2/s. The model enzyme yeast alcohol dehydrogenase (YADH) was encapsulated in silica-coated alginate and pure alginate beads, respectively. Enzyme leakage of YADH in alginate gel beads was determined to be 32%, while the enzyme leakage in silica-coated alginate gel beads was as low as 11%. Furthermore, the relative activity of YADH in alginate gel beads decreased almost to zero after 10 recycles, while the relative activity of YADH in silica-coated alginate gel beads was 81.3%. The recycling stability of YADH in silica-coated alginate gel beads was found to be increased significantly mainly due to the effective inhibition of enzyme leakage by compact silica film.

  20. Heterologous expression of Pycnoporus cinnabarinus cellobiose dehydrogenase in Pichia pastoris and involvement in saccharification processes

    Directory of Open Access Journals (Sweden)

    Bey Mathieu

    2011-12-01

    Full Text Available Abstract Background Cellobiose dehydrogenase (CDH is an extracellular hemoflavoenzyme produced by lignocellulose-degrading fungi including Pycnoporus cinnabarinus. We investigated the cellulolytic system of P. cinnabarinus, focusing on the involvement of CDH in the deconstruction of lignocellulosic biomass. Results First, P. cinnabarinus growth conditions were optimized for CDH production. Following growth under cellulolytic conditions, the main components secreted were cellulases, xylanases and CDH. To investigate the contribution of P. cinnabarinus secretome in saccharification processes, the Trichoderma reesei enzymatic cocktail was supplemented with the P. cinnabarinus secretome. A significant enhancement of the degradation of wheat straw was observed with (i the production of a large amount of gluconic acid, (ii increased hemicellulose degradation, and (iii increased overall degradation of the lignocellulosic material. P. cinnabarinus CDH was heterologously expressed in Pichia pastoris to obtain large amounts of pure enzyme. In a bioreactor, the recombinant CDH (rCDH expression level reached 7800 U/L. rCDH exhibited values of biochemical parameters similar to those of the natural enzyme, and was able to bind cellulose despite the absence of a carbohydrate-binding module (CBM. Following supplementation of purified rCDH to T. reesei enzymatic cocktail, formation of gluconic acid and increased hemicellulose degradation were observed, thus confirming the previous results observed with P. cinnabarinus secretome. Conclusions We demonstrate that CDH offers an attractive tool for saccharification process enhancement due to gluconic acid production from raw lignocellulosic material.

  1. Silencing of mitochondrial NADP{sup +}-dependent isocitrate dehydrogenase gene enhances glioma radiosensitivity

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Sung Youl [School of Life Sciences and Biotechnology, College of Natural Sciences, Kyungpook National University, Taegu (Korea, Republic of); Yoo, Young Hyun [Mitochondria Hub Regulation Center, Dong-A University College of Medicine, Busan (Korea, Republic of); Park, Jeen-Woo, E-mail: parkjw@knu.ac.kr [School of Life Sciences and Biotechnology, College of Natural Sciences, Kyungpook National University, Taegu (Korea, Republic of)

    2013-04-05

    Highlights: •Silencing of the IDPm gene enhances IR-induced autophagy in glioma cells. •Autophagy inhibition augmented apoptosis of irradiated glioma cells. •Results offer a redox-active therapeutic strategy for the treatment of cancer. -- Abstract: Reactive oxygen species (ROS) levels are elevated in organisms that have been exposed to ionizing radiation and are protagonists in the induction of cell death. Recently, we demonstrated that the control of mitochondrial redox balance and the cellular defense against oxidative damage are primary functions of mitochondrial NADP{sup +}-dependent isocitrate dehydrogenase (IDPm) via the supply of NADPH for antioxidant systems. In the present study, we report an autophagic response to ionizing radiation in A172 glioma cells transfected with small interfering RNA (siRNA) targeting the IDPm gene. Autophagy in A172 transfectant cells was associated with enhanced autophagolysosome formation and GFP–LC3 punctuation/aggregation. Furthermore, we found that the inhibition of autophagy by chloroquine augmented apoptotic cell death of irradiated A172 cells transfected with IDPm siRNA. Taken together, our data suggest that autophagy functions as a survival mechanism in A172 cells against ionizing radiation-induced apoptosis and the sensitizing effect of IDPm siRNA and autophagy inhibitor on the ionizing radiation-induced apoptotic cell death of glioma cells offers a novel redox-active therapeutic strategy for the treatment of cancer.

  2. Novel protein-protein interactions between Entamoeba histolyticad-phosphoglycerate dehydrogenase and phosphoserine aminotransferase.

    Science.gov (United States)

    Mishra, Vibhor; Kumar, Ashutosh; Ali, Vahab; Nozaki, Tomoyoshi; Zhang, Kam Y J; Bhakuni, Vinod

    2012-08-01

    Physical interactions between d-phosphoglycerate dehydrogenase (EhPGDH) and phosphoserine aminotransferase (EhPSAT) from an enteric human parasite Entamoeba histolytica was observed by pull-down assay, gel filtration chromatography, chemical cross-linking, emission anisotropy, molecular docking and molecular dynamic simulations. The protein-protein complex had a 1:1 stochiometry with a dissociation constant of 3.453 × 10(-7) M. Ionic interactions play a significant role in complex formation and stability. Analysis of the energy minimized average simulated model of the protein complex show that the nucleotide binding domain of EhPGDH specifically interacts with EhPSAT. Denaturation studies suggest that the nucleotide binding domain (Nbd) and substrate binding domain (Sbd) of EhPGDH are independent folding/unfolding units. Thus the Nbd-EhPGDH was separately cloned over-expressed and purified to homogeneity. Fluorescence anisotropy study show that the purified Nbd interacts with EhPSAT. Forward enzyme catalyzed reaction for the EhPGDH-PSAT complex showed efficient Km values for 3-phosphoglyceric acid as compared to only EhPGDH suggesting a possibility of substrate channelling in the protein complex. PMID:22386871

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

    Energy Technology Data Exchange (ETDEWEB)

    Deng, Xiaoyi; Gujjar, Ramesh; El Mazouni, Farah; Kaminsky, Werner; Malmquist, Nicholas A.; Goldsmith, Elizabeth J.; Rathod, Pradipsinh K.; Phillips, Margaret A.; (UWASH); (UTSMC)

    2010-01-20

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    LENUS (Irish Health Repository)

    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.

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

    Science.gov (United States)

    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

  7. Affinity chromatography of nicotinamide-adenine dinucleotide-linked dehydrogenases on immobilized derivatives of the dinucleotide.

    Science.gov (United States)

    Barry, S; O'Carra, P

    1973-12-01

    1. Three established methods for immobilization of ligands through primary amino groups promoted little or no attachment of NAD(+) through the 6-amino group of the adenine residue. Two of these methods (coupling to CNBr-activated agarose and to carbodi-imide-activated carboxylated agarose derivatives) resulted instead in attachment predominantly through the ribosyl residues. Other immobilized derivatives were prepared by azolinkage of NAD(+) (probably through the 8 position of the adenine residue) to a number of different spacer-arm-agarose derivatives. 2. The effectiveness of these derivatives in the affinity chromatography of a variety of NAD-linked dehydrogenases was investigated, applying rigorous criteria to distinguish general or non-specific adsorption effects from truly NAD-specific affinity (bio-affinity). The ribosyl-attached NAD(+) derivatives displayed negligible bio-affinity for any of the NAD-linked dehydrogenases tested. The most effective azo-linked derivative displayed strong bio-affinity for glycer-aldehyde 3-phosphate dehydrogenase, weaker bio-affinity for lactate dehydrogenase and none at all for malate dehydrogenase, although these three enzymes have very similar affinities for soluble NAD(+). Alcohol dehydrogenase and xanthine dehydrogenase were subject to such strong non-specific interactions with the hydrocarbon spacer-arm assembly that any specific affinity was completely eclipsed. 3. It is concluded that, in practice, the general effectiveness of a general ligand may be considerably distorted and attenuated by the nature of the immobilization linkage. However, this attenuation can result in an increase in specific effectiveness, allowing dehydrogenases to be separated from one another in a manner unlikely to be feasible if the general effectiveness of the ligand remained intact. 4. The bio-affinity of the various derivatives for lactate dehydrogenase is correlated with the known structure of the NAD(+)-binding site of this enzyme. Problems

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

    Science.gov (United States)

    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

  9. 4-dihydrotrisporin-dehydrogenase, an enzyme of the sex hormone pathway of Mucor mucedo: purification, cloning of the corresponding gene, and developmental expression.

    Science.gov (United States)

    Wetzel, Jana; Scheibner, Olaf; Burmester, Anke; Schimek, Christine; Wöstemeyer, Johannes

    2009-01-01

    The NADP-dependent 4-dihydrotrisporin-dehydrogenase is a (-) mating-type-specific enzyme in the pathway from beta-carotene to trisporic acid. This substance and its isomers and derivatives represent the general system of sexual communication in zygomycetes. The (-) mating type of Mucor mucedo was stimulated by trisporic acid and the enzyme was purified by ion exchange and affinity chromatography. Several peptides of the 26-kDa protein, digested with trypsin, were sequenced by mass spectrometry. Oligonucleotides based on protein sequence data were used for PCR amplification of genomic DNA. The primary PCR fragment was sequenced and the complete gene, TSP2, was isolated. A labeled TSP2 hybridization probe detects a single-copy gene in the genome of M. mucedo. Northern blot analysis with RNAs from different growth stages reveals that the expression of the gene depends on the developmental stage of the mycelium in both mating types of M. mucedo. At the enzyme level, activity is found exclusively in the (-) mating type. However, renaturation of proteins in sodium dodecyl sulfate-containing gels revealed the TSP2 gene product in both mating types. Analyzing the protein sequence places the enzyme in the short chain dehydrogenase superfamily. Thus, it has an evolutionary origin distinct from that of the previously isolated 4-dihydromethyltrisporate dehydrogenase, which belongs to the aldo/keto reductase superfamily. Apart from the TSP2 genes in the three sequenced zygomycetous genomes (Phycomyces blakesleeanus, Rhizopus oryzae, and Mucor circinelloides), the closest relative is the Myxococcus xanthus CsgA gene product, which is also a short chain dehydrogenase, involved in C signaling and fruiting body formation.

  10. 4-Dihydrotrisporin-Dehydrogenase, an Enzyme of the Sex Hormone Pathway of Mucor mucedo: Purification, Cloning of the Corresponding Gene, and Developmental Expression▿

    Science.gov (United States)

    Wetzel, Jana; Scheibner, Olaf; Burmester, Anke; Schimek, Christine; Wöstemeyer, Johannes

    2009-01-01

    The NADP-dependent 4-dihydrotrisporin-dehydrogenase is a (−) mating-type-specific enzyme in the pathway from β-carotene to trisporic acid. This substance and its isomers and derivatives represent the general system of sexual communication in zygomycetes. The (−) mating type of Mucor mucedo was stimulated by trisporic acid and the enzyme was purified by ion exchange and affinity chromatography. Several peptides of the 26-kDa protein, digested with trypsin, were sequenced by mass spectrometry. Oligonucleotides based on protein sequence data were used for PCR amplification of genomic DNA. The primary PCR fragment was sequenced and the complete gene, TSP2, was isolated. A labeled TSP2 hybridization probe detects a single-copy gene in the genome of M. mucedo. Northern blot analysis with RNAs from different growth stages reveals that the expression of the gene depends on the developmental stage of the mycelium in both mating types of M. mucedo. At the enzyme level, activity is found exclusively in the (−) mating type. However, renaturation of proteins in sodium dodecyl sulfate-containing gels revealed the TSP2 gene product in both mating types. Analyzing the protein sequence places the enzyme in the short chain dehydrogenase superfamily. Thus, it has an evolutionary origin distinct from that of the previously isolated 4-dihydromethyltrisporate dehydrogenase, which belongs to the aldo/keto reductase superfamily. Apart from the TSP2 genes in the three sequenced zygomycetous genomes (Phycomyces blakesleeanus, Rhizopus oryzae, and Mucor circinelloides), the closest relative is the Myxococcus xanthus CsgA gene product, which is also a short chain dehydrogenase, involved in C signaling and fruiting body formation. PMID:18931040

  11. Formate hydrogenlyase in the hyperthermophilic archaeon, Thermococcus litoralis

    Directory of Open Access Journals (Sweden)

    Rákhely Gábor

    2008-06-01

    Full Text Available Abstract Background Thermococcus litoralis is a heterotrophic facultative sulfur dependent hyperthermophilic Archaeon, which was isolated from a shallow submarine thermal spring. It has been successfully used in a two-stage fermentation system, where various keratinaceous wastes of animal origin were converted to biohydrogen. In this system T. litoralis performed better than its close relative, P. furiosus. Therefore, new alternative enzymes involved in peptide and hydrogen metabolism were assumed in T. litoralis. Results An about 10.5 kb long genomic region was isolated and sequenced from Thermococcus litoralis. In silico analysis revealed that the region contained a putative operon consisting of eight genes: the fdhAB genes coding for a formate dehydrogenase and the mhyCDEFGH genes encoding a [NiFe] hydrogenase belonging to the group of the H2-evolving, energy-conserving, membrane-bound hydrogenases. Reverse transcription linked quantitative Real-Time PCR and Western blotting experiments showed that the expression of the fdh-mhy operon was up-regulated during fermentative growth on peptides and down-regulated in cells cultivated in the presence of sulfur. Immunoblotting and protein separation experiments performed on cell fractions indicated that the formate dehydrogenase part of the complex is associated to the membrane-bound [NiFe] hydrogenase. Conclusion The formate dehydrogenase together with the membrane-bound [NiFe] hydrogenase formed a formate hydrogenlyase (formate dehydrogenase coupled hydrogenase, FDH-MHY complex. The expression data suggested that its physiological role is linked to the removal of formate likely generated during anaerobic peptide fermentation.

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

    Energy Technology Data Exchange (ETDEWEB)

    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

  13. Importance of glucose-6-phosphate dehydrogenase (G6PDH) for vanillin tolerance in Saccharomyces cerevisiae.

    Science.gov (United States)

    Nguyen, Trinh Thi My; Kitajima, Sakihito; Izawa, Shingo

    2014-09-01

    Vanillin is derived from lignocellulosic biomass and, as one of the major biomass conversion inhibitors, inhibits yeast growth and fermentation. Vanillin was recently shown to induce the mitochondrial fragmentation and formation of mRNP granules such as processing bodies and stress granules in Saccharomyces cerevisiae. Furfural, another major biomass conversion inhibitor, also induces oxidative stress and is reduced in an NAD(P)H-dependent manner to its less toxic alcohol derivative. Therefore, the pentose phosphate pathway (PPP), through which most NADPH is generated, plays a role in tolerance to furfural. Although vanillin also induces oxidative stress and is reduced to vanillyl alcohol in a NADPH-dependent manner, the relationship between vanillin and PPP has not yet been investigated. In the present study, we examined the importance of glucose-6-phosphate dehydrogenase (G6PDH), which catalyzes the rate-limiting NADPH-producing step in PPP, for yeast tolerance to vanillin. The growth of the null mutant of G6PDH gene (zwf1Δ) was delayed in the presence of vanillin, and vanillin was efficiently reduced in the culture of wild-type cells but not in the culture of zwf1Δ cells. Furthermore, zwf1Δ cells easily induced the activation of Yap1, an oxidative stress responsive transcription factor, mitochondrial fragmentation, and P-body formation with the vanillin treatment, which indicated that zwf1Δ cells were more susceptible to vanillin than wild type cells. These findings suggest the importance of G6PDH and PPP in the response of yeast to vanillin.

  14. Gimeracil, an inhibitor of dihydropyrimidine dehydrogenase, inhibits the early step in homologous recombination

    International Nuclear Information System (INIS)

    Gimeracil (5-chloro-2, 4-dihydroxypyridine) is an inhibitor of dihydropyrimidine dehydrogenase (DPYD), which degrades pyrimidine including 5-fluorouracil in the blood. Gimeracil was originally added to an oral fluoropyrimidine derivative S-1 to yield prolonged 5-fluorouracil concentrations in serum and tumor tissues. We have already reported that gimeracil had radiosensitizing effects by partially inhibiting homologous recombination (HR) in the repair of DNA double strand breaks. We investigated the mechanisms of gimeracil radiosensitization. Comet assay and radiation-induced focus formation of various kinds of proteins involved in HR was carried out. Small interfering RNA (siRNA) for DPYD were transfected to HeLa cells to investigate the target protein for radiosensitization with gimeracil. SCneo assay was carried out to examine whether DPYD depletion by siRNA inhibited HR repair of DNA double strand breaks. Tail moments in neutral comet assay increased in gimeracil-treated cells. Gimeracil restrained the formation of foci of Rad51 and replication protein A (RPA), whereas it increased the number of foci of Nbs1, Mre11, Rad50, and FancD2. When HeLa cells were transfected with the DPYD siRNA before irradiation, the cells became more radiosensitive. The degree of radiosensitization by transfection of DPYD siRNA was similar to that of gimeracil. Gimeracil did not sensitize DPYD-depleted cells. Depletion of DPYD by siRNA significantly reduced the frequency of neopositive clones in SCneo assay. Gimeracil partially inhibits the early step in HR. It was found that DPYD is the target protein for radiosensitization by gimeracil. The inhibitors of DPYD, such as gimeracil, could enhance the efficacy of radiotherapy through partial suppression of HR-mediated DNA repair. (author)

  15. Aldehyde dehydrogenase activity selects for the holoclone phenotype in prostate cancer cells

    International Nuclear Information System (INIS)

    Highlights: ► Isolated ALDHHi PC3 cells preferentially form primitive holoclone-type colonies. ► Primitive holoclone colonies are predominantly ALDHLo but contain rare ALDHHi cells. ► Holoclone-forming cells are not restricted to the ALDHHi population. ► ALDH phenotypic plasticity occurs in PC3 cells (ALDHLo to ALDHHi and vice versa). ► ALDHHi cells are observed but very rare in PC3 spheroids grown in stem cell medium. -- Abstract: Aldehyde dehydrogenase 1 (ALDH) activity is considered to be a marker of cancer stem cells (CSCs) in many tumour models, since these cells are more proliferative and tumourigenic than ALDHLo cells in experimental models. However it is unclear whether all CSC-like cells are within the ALDHHi population, or whether all ALDHHi cells are highly proliferative and tumourigenic. The ability to establish a stem cell hierarchy in vitro, whereby sub-populations of cells have differing proliferative and differentiation capacities, is an alternate indication of the presence of stem cell-like populations within cell lines. In this study, we have examined the interaction between ALDH status and the ability to establish a stem cell hierarchy in PC3 prostate cancer cells. We demonstrate that PC3 cells contain a stem cell hierarchy, and isolation of ALDHHi cells enriches for the most primitive holoclone population, however holoclone formation is not restricted to ALDHHi cells. In addition, we show that ALDH activity undergoes phenotypic plasticity, since the ALDHLo population can develop ALDHHi populations comparable to parental cells within 2 weeks in culture. Furthermore, we show that the majority of ALDHHi cells are found within the least primitive paraclone population, which is circumvented by culturing PC3 cells as spheroids in defined medium favouring stem cell characteristics. Although ALDHHi status enriches for holoclone formation, this activity may be mediated by a minority of ALDHHi cells.

  16. Induction of mitochondrial reactive oxygen species production by GSH mediated S-glutathionylation of 2-oxoglutarate dehydrogenase.

    Science.gov (United States)

    Mailloux, Ryan J; Craig Ayre, D; Christian, Sherri L

    2016-08-01

    2-Oxoglutarate dehydrogenase (Ogdh) is an important mitochondria redox sensor that can undergo S-glutathionylation following an increase in H2O2 levels. Although S-glutathionylation is required to protect Ogdh from irreversible oxidation while simultaneously modulating its activity it remains unknown if glutathione can also modulate reactive oxygen species (ROS) production by the complex. We report that reduced (GSH) and oxidized (GSSG) glutathione control O2(∙-)/H2O2 formation by Ogdh through protein S-glutathionylation reactions. GSSG (1mM) induced a modest decrease in Ogdh activity which was associated with a significant decrease in O2(∙-)/H2O2 formation. GSH had the opposite effect, amplifying O2(∙-)/H2O2 formation by Ogdh. Incubation of purified Ogdh in 2.5mM GSH led to significant increase in O2(∙-)/H2O2 formation which also lowered NADH production. Inclusion of enzymatically active glutaredoxin-2 (Grx2) in reaction mixtures reversed the GSH-mediated amplification of O2(∙-)/H2O2 formation. Similarly pre-incubation of permeabilized liver mitochondria from mouse depleted of GSH showed an approximately ~3.5-fold increase in Ogdh-mediated O2(∙-)/H2O2 production that was matched by a significant decrease in NADH formation which could be reversed by Grx2. Taken together, our results demonstrate GSH and GSSG modulate ROS production by Ogdh through S-glutathionylation of different subunits. This is also the first demonstration that GSH can work in the opposite direction in mitochondria-amplifying ROS formation instead of quenching it. We propose that this regulatory mechanism is required to modulate ROS emission from Ogdh in response to variations in glutathione redox buffering capacity. PMID:26928132

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

    Directory of Open Access Journals (Sweden)

    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

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

    Science.gov (United States)

    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.

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

    NARCIS (Netherlands)

    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

  20. Stellar formation

    CERN Document Server

    Reddish, V C

    1978-01-01

    Stellar Formation brings together knowledge about the formation of stars. In seeking to determine the conditions necessary for star formation, this book examines questions such as how, where, and why stars form, and at what rate and with what properties. This text also considers whether the formation of a star is an accident or an integral part of the physical properties of matter. This book consists of 13 chapters divided into two sections and begins with an overview of theories that explain star formation as well as the state of knowledge of star formation in comparison to stellar structure

  1. Structural determinants of stereospecificity in yeast alcohol dehydrogenase.

    Science.gov (United States)

    Weinhold, E G; Glasfeld, A; Ellington, A D; Benner, S A

    1991-10-01

    Replacing Leu-182 by Ala in yeast alcohol dehydrogenase (YADH; alcohol:NAD+ oxidoreductase, EC 1.1.1.1) yields a mutant that retains 34% of its kcat value and makes one stereochemical "mistake" every 850,000 turnovers (instead of approximately 1 error every 7,000,000,000 turnovers in native YADH) in its selection of the 4-Re hydrogen of NADH. Half of the decrease in stereochemical fidelity comes from an increase in the rate of transfer of the 4-Si hydrogen of NADH. The mutant also accepts 5-methylnicotinamide adenine dinucleotide, a cofactor analog not accepted by native YADH. The stereospecificity of the mutant is lower still with analogs of NADH where the carboxamide group of the nicotinamide ring is replaced by groups with weaker hydrogen bonding potential. For example, with thio-NADH, the mutant enzyme makes 1 stereochemical "mistake" every 450 turnovers. Finally, the double mutant T157S/L182A, in which Thr-157 is replaced by Ser and Leu-182 is replaced by Ala, also shows decreased stereochemical fidelity. These results suggest that Si transfer in the mutant enzymes arises from NADH bound in a syn conformation in the active site and that this binding is not obstructed in native YADH by side chains essential for catalysis.

  2. RECIPIENT PRETRANSPLANT INOSINE MONOPHOSPHATE DEHYDROGENASE ACTIVITY IN NONMYELOABLATIVE HCT

    Science.gov (United States)

    Bemer, Meagan J.; Risler, Linda J.; Phillips, Brian R.; Wang, Joanne; Storer, Barry E.; Sandmaier, Brenda M.; Duan, Haichuan; Raccor, Brianne S.; Boeckh, Michael J.; McCune, Jeannine S.

    2014-01-01

    Mycophenolic acid, the active metabolite of mycophenolate mofetil (MMF), inhibits inosine monophosphate dehydrogenase (IMPDH) activity. IMPDH is the rate-limiting enzyme involved in de novo synthesis of guanosine nucleotides and catalyzes the oxidation of inosine 5’- monophosphate (IMP) to xanthosine 5’-monophosphate (XMP). We developed a highly sensitive liquid chromatography–mass spectrometry method to quantitate XMP concentrations in peripheral blood mononuclear cells (PMNC) isolated from the recipient pretransplant and used this method to determine IMPDH activity in 86 nonmyeloablative allogeneic hematopoietic cell transplantation (HCT) patients. The incubation procedure and analytical method yielded acceptable within-sample and within-individual variability. Considerable between-individual variability was observed (12.2-fold). Low recipient pretransplant IMPDH activity was associated with increased day +28 donor T-cell chimerism, more acute graft-versus-host disease (GVHD), lower neutrophil nadirs, and more cytomegalovirus reactivation, but not with chronic GVHD, relapse, non-relapse mortality, or overall mortality. We conclude that quantitation of the recipient’s pretransplant IMPDH activity in PMNC lysate could provide a useful biomarker to evaluate a recipient’s sensitivity to MMF, but confirmatory studies are needed. Further trials should be conducted to confirm our findings and to optimize postgrafting immunosuppression in nonmyeloablative HCT recipients. PMID:24923537

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

    Science.gov (United States)

    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

  4. Functional characterization of a vanillin dehydrogenase in Corynebacterium glutamicum.

    Science.gov (United States)

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

    2015-01-27

    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 ~51 kDa, whereas the apparent native Mr values revealed by gel filtration chromatography were 49.5, 92.3, 159.0 and 199.2 kDa, indicating the presence of dimeric, trimeric and tetrameric forms. Moreover, the enzyme showed its highest level of activity toward vanillin at pH 7.0 and 30°C, and interestingly, it could utilize NAD(+) and NADP(+) as coenzymes with similar efficiency and showed no obvious difference toward NAD(+) and NADP(+). In addition to vanillin, this enzyme exhibited catalytic activity toward a broad range of substrates, including p-hydroxybenzaldehyde, 3,4-dihydroxybenzaldehyde, o-phthaldialdehyde, cinnamaldehyde, syringaldehyde and benzaldehyde. Conserved catalytic residues or putative cofactor interactive sites were identified based on sequence alignment and comparison with previous studies, and the function of selected residues were verified by site-directed mutagenesis analysis. Finally, the vdh deletion mutant partially lost its ability to grow on vanillin, indicating the presence of alternative VDH(s) in Corynebacterium glutamicum. Taken together, this study contributes to understanding the VDH diversity from bacteria and the aromatic metabolism pathways in C. glutamicum.

  5. Phenotypic and Neuropathological Characterization of Fetal Pyruvate Dehydrogenase Deficiency.

    Science.gov (United States)

    Pirot, Nathalie; Crahes, Marie; Adle-Biassette, Homa; Soares, Anais; Bucourt, Martine; Boutron, Audrey; Carbillon, Lionel; Mignot, Cyril; Trestard, Laetitia; Bekri, Soumeya; Laquerrière, Annie

    2016-03-01

    To distinguish pyruvate dehydrogenase deficiency (PDH) from other antenatal neurometabolic disorders thereby improving prenatal diagnosis, we describe imaging findings, clinical phenotype, and brain lesions in fetuses from 3 families with molecular characterization of this condition. Neuropathological analysis was performed in 4 autopsy cases from 3 unrelated families with subsequent biochemical and molecular confirmation of PDH complex deficiency. In 2 families there were mutations in the PDHA1 gene; in the third family there was a mutation in the PDHB gene. All fetuses displayed characteristic craniofacial dysmorphism of varying severity, absence of visceral lesions, and associated encephaloclastic and developmental supra- and infratentorial lesions. Neurodevelopmental abnormalities included microcephaly, migration abnormalities (pachygyria, polymicrogyria, periventricular nodular heterotopias), and cerebellar and brainstem hypoplasia with hypoplastic dentate nuclei and pyramidal tracts. Associated clastic lesions included asymmetric leukomalacia, reactive gliosis, large pseudocysts of germinolysis, and basal ganglia calcifications. The diagnosis of PDH deficiency should be suspected antenatally with the presence of clastic and neurodevelopmental lesions and a relatively characteristic craniofacial dysmorphism. Postmortem examination is essential for excluding other closely related entities, thereby allowing for biochemical and molecular confirmation. PMID:26865159

  6. Phosphoglycerate Dehydrogenase: Potential Therapeutic Target and Putative Metabolic Oncogene

    Directory of Open Access Journals (Sweden)

    Cheryl K. Zogg

    2014-01-01

    Full Text Available Exemplified by cancer cells’ preference for glycolysis, for example, the Warburg effect, altered metabolism in tumorigenesis has emerged as an important aspect of cancer in the past 10–20 years. Whether due to changes in regulatory tumor suppressors/oncogenes or by acting as metabolic oncogenes themselves, enzymes involved in the complex network of metabolic pathways are being studied to understand their role and assess their utility as therapeutic targets. Conversion of glycolytic intermediate 3-phosphoglycerate into phosphohydroxypyruvate by the enzyme phosphoglycerate dehydrogenase (PHGDH—a rate-limiting step in the conversion of 3-phosphoglycerate to serine—represents one such mechanism. Forgotten since classic animal studies in the 1980s, the role of PHGDH as a potential therapeutic target and putative metabolic oncogene has recently reemerged following publication of two prominent papers near-simultaneously in 2011. Since that time, numerous studies and a host of metabolic explanations have been put forward in an attempt to understand the results observed. In this paper, I review the historic progression of our understanding of the role of PHGDH in cancer from the early work by Snell through its reemergence and rise to prominence, culminating in an assessment of subsequent work and what it means for the future of PHGDH.

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

    DEFF Research Database (Denmark)

    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...

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

    DEFF Research Database (Denmark)

    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...

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

    Science.gov (United States)

    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

  10. 11β-Hydroxysteroid Dehydrogenase 2 in Preeclampsia

    Directory of Open Access Journals (Sweden)

    Katarzyna Kosicka

    2016-01-01

    Full Text Available Preeclampsia is a serious medical problem affecting the mother and her child and influences their health not only during the pregnancy, but also many years after. Although preeclampsia is a subject of many research projects, the etiology of the condition remains unclear. One of the hypotheses related to the etiology of preeclampsia is the deficiency in placental 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2, the enzyme which in normal pregnancy protects the fetus from the excess of maternal cortisol. The reduced activity of the enzyme was observed in placentas from pregnancies complicated with preeclampsia. That suggests the overexposure of the developing child to maternal cortisol, which in high levels exerts proapoptotic effects and reduces fetal growth. The fetal growth restriction due to the diminished placental 11β-HSD2 function may be supported by the fact that preeclampsia is often accompanied with fetal hypotrophy. The causes of the reduced function of 11β-HSD2 in placental tissue are still discussed. This paper summarizes the phenomena that may affect the activity of the enzyme at various steps on the way from the gene to the protein.

  11. Glucose-6-phosphate dehydrogenase deficiency: the added value of cytology.

    Science.gov (United States)

    Roelens, Marie; Dossier, Claire; Fenneteau, Odile; Couque, Nathalie; Da Costa, Lydie

    2016-06-01

    We report the case of a 2 year-old boy hospitalized into the emergency room for influenza pneumonia infection. The evolution was marked by a respiratory distress syndrome, a severe hemolytic anemia, associated with thrombocytopenia and kidney failure. First, a diagnosis of hemolytic uremic syndrome (HUS) has been judiciously suggested due to the classical triad: kidney failure, hemolytic anemia and thrombocytopenia. But, strikingly, blood smears do not exhibit schizocytes, but instead ghosts and hemighosts, some characteristic features of a glucose-6-phosphate dehydrogenase deficiency. Our hypothesis has been confirmed by enzymatic dosage and molecular biology. The unusual initial aplastic feature of this anemia could be the result of a transient erythroblastopenia due to the viral agent, at the origin of the G6PD crisis on a background of a major erythrocyte anti-oxydant enzyme defect. This case of G6PD defect points out the continuously importance of the cytology, which was able to redirect the diagnosis by the hemighost and ghost detection. PMID:27101632

  12. Glutamate dehydrogenase: structure, allosteric regulation, and role in insulin homeostasis.

    Science.gov (United States)

    Li, Ming; Li, Changhong; Allen, Aron; Stanley, Charles A; Smith, Thomas J

    2014-01-01

    Glutamate dehydrogenase (GDH) is a homohexameric enzyme that catalyzes the reversible oxidative deamination of L-glutamate to 2-oxoglutarate. Only in the animal kingdom is this enzyme heavily allosterically regulated by a wide array of metabolites. The major activators are ADP and leucine and inhibitors include GTP, palmitoyl CoA, and ATP. Spontaneous mutations in the GTP inhibitory site that lead to the hyperinsulinism/hyperammonemia (HHS) syndrome have shed light as to why mammalian GDH is so tightly regulated. Patients with HHS exhibit hypersecretion of insulin upon consumption of protein and concomitantly extremely high levels of ammonium in the serum. The atomic structures of four new inhibitors complexed with GDH complexes have identified three different allosteric binding sites. Using a transgenic mouse model expressing the human HHS form of GDH, at least three of these compounds blocked the dysregulated form of GDH in pancreatic tissue. EGCG from green tea prevented the hyper-response to amino acids in whole animals and improved basal serum glucose levels. The atomic structure of the ECG-GDH complex and mutagenesis studies is directing structure-based drug design using these polyphenols as a base scaffold. In addition, all of these allosteric inhibitors are elucidating the atomic mechanisms of allostery in this complex enzyme.

  13. The structure and allosteric regulation of mammalian glutamate dehydrogenase.

    Science.gov (United States)

    Li, Ming; Li, Changhong; Allen, Aron; Stanley, Charles A; Smith, Thomas J

    2012-03-15

    Glutamate dehydrogenase (GDH) is a homohexameric enzyme that catalyzes the reversible oxidative deamination of l-glutamate to 2-oxoglutarate. Only in the animal kingdom is this enzyme heavily allosterically regulated by a wide array of metabolites. The major activators are ADP and leucine, while the most important inhibitors include GTP, palmitoyl CoA, and ATP. Recently, spontaneous mutations in the GTP inhibitory site that lead to the hyperinsulinism/hyperammonemia (HHS) syndrome have shed light as to why mammalian GDH is so tightly regulated. Patients with HHS exhibit hypersecretion of insulin upon consumption of protein and concomitantly extremely high levels of ammonium in the serum. The atomic structures of four new inhibitors complexed with GDH complexes have identified three different allosteric binding sites. Using a transgenic mouse model expressing the human HHS form of GDH, at least three of these compounds were found to block the dysregulated form of GDH in pancreatic tissue. EGCG from green tea prevented the hyper-response to amino acids in whole animals and improved basal serum glucose levels. The atomic structure of the ECG-GDH complex and mutagenesis studies is directing structure-based drug design using these polyphenols as a base scaffold. In addition, all of these allosteric inhibitors are elucidating the atomic mechanisms of allostery in this complex enzyme.

  14. Nuclear lactate dehydrogenase modulates histone modification in human hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    OpenAIRE

    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...

  16. The Carboxy-Terminal Tail of Pyruvate Dehydrogenase Kinase 2 Is Required for the Kinase Activity†

    OpenAIRE

    Klyuyeva, Alla; Tuganova, Alina; Popov, Kirill M.

    2005-01-01

    Pyruvate dehydrogenase kinase 2 (PDK2) is a prototypical mitochondrial protein kinase that regulates the activity of the pyruvate dehydrogenase complex. Recent structural studies have established that PDK2 consists of a catalytic core built of the B and K domains and the relatively long amino and carboxyl tails of unknown function. Here, we show that the carboxy-terminal truncation variants of PDK2 display a greatly diminished capacity for phosphorylation of holo-PDC. This effect is due large...

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

    OpenAIRE

    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...

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

    OpenAIRE

    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 ...

  19. The metabolism of fatty alcohols in lipid nanoparticles by alcohol dehydrogenase.

    Science.gov (United States)

    Dong, X; Mumper, R J

    2006-09-01

    Fatty alcohols are commonly used in lipid-based drug delivery systems including parenteral emulsions and solid lipid nanoparticles (NPs). The purpose of these studies was to determine whether horse liver alcohol dehydrogenase (HLADH), a NAD-dependent enzyme, could metabolize the fatty alcohols within the NPs and thus serve as a mechanism to degrade these NPs in the body. Solid nanoparticles (endogenous alcohol dehydrogenase enzyme systems.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  1. Decrease in nicotinamide adenine dinucleotide dehydrogenase is related to skin pigmentation.

    Science.gov (United States)

    Nakama, Mitsuo; Murakami, Yuhko; Tanaka, Hiroshi; Nakata, Satoru

    2012-03-01

    Skin pigmentation is caused by various physical and chemical factors. It might also be influenced by changes in the physiological function of skin with aging. Nicotinamide adenine dinucleotide (NADH) dehydrogenase is an enzyme related to the mitochondrial electron transport system and plays a key role in cellular energy production. It has been reported that the functional decrease in this system causes Parkinson's disease. Another study reports that the amount of NADH dehydrogenase in heart and skeletal muscle decreases with aging. A similar decrease in the skin would probably affect its physiological function. However, no reports have examined the age-related change in levels of NADH dehydrogenase in human skin. In this study, we investigated this change and its effect on skin pigmentation using cultured human epidermal keratinocytes. The mRNA expression of NDUFA1, NDUFB7, and NDUFS2, subunits of NADH dehydrogenase, and its activity were significantly decreased in late passage keratinocytes compared to early passage cells. Conversely, the mRNA expression of melanocyte-stimulating cytokines, interleukin-1 alpha and endothelin 1, was increased in late passage cells. On the other hand, the inhibition of NADH dehydrogenase upregulated the mRNA expression of melanocyte-stimulating cytokines. Moreover, the level of NDUFB7 mRNA was lower in pigmented than in nonpigmented regions of skin in vivo. These results suggest the decrease in NADH dehydrogenase with aging to be involved in skin pigmentation.

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

    Directory of Open Access Journals (Sweden)

    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.

  3. Galaxy formation

    International Nuclear Information System (INIS)

    Galaxy formation is at the forefront of observation and theory in cosmology. An improved understanding is essential for improving our knowledge both of the cosmological parameters, of the contents of the universe, and of our origins. In these lectures intended for graduate students, galaxy formation theory is reviewed and confronted with recent observational issues. In lecture 1, the following topics are presented: star formation considerations, including IMF, star formation efficiency and star formation rate, the origin of the galaxy luminosity function, and feedback in dwarf galaxies. In lecture 2, we describe formation of disks and massive spheroids, including the growth of supermassive black holes, negative feedback in spheroids, the AGN-star formation connection, star formation rates at high redshift and the baryon fraction in galaxies.

  4. Galaxy formation

    CERN Document Server

    Silk, Joseph; Dvorkin, Irina

    2015-01-01

    Galaxy formation is at the forefront of observation and theory in cosmology. An improved understanding is essential for improving our knowledge both of the cosmological parameters, of the contents of the universe, and of our origins. In these lectures intended for graduate students, galaxy formation theory is reviewed and confronted with recent observational issues. In Lecture 1, the following topics are presented: star formation considerations, including IMF, star formation efficiency and star formation rate, the origin of the galaxy luminosity function, and feedback in dwarf galaxies. In Lecture 2, we describe formation of disks and massive spheroids, including the growth of supermassive black holes, negative feedback in spheroids, the AGN-star formation connection, star formation rates at high redshift and the baryon fraction in galaxies.

  5. Soil formation.

    NARCIS (Netherlands)

    Breemen, van N.; Buurman, P.

    1998-01-01

    Soil Formation deals with qualitative and quantitative aspects of soil formation (or pedogenesis) and the underlying chemical, biological, and physical processes. The starting point of the text is the process - and not soil classification. Effects of weathering and new formation of minerals, mobilis

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

    Directory of Open Access Journals (Sweden)

    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.

  7. The alcohol dehydrogenase system in the xylose-fermenting yeast Candida maltosa.

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    Yuping Lin

    Full Text Available BACKGROUND: The alcohol dehydrogenase (ADH system plays a critical role in sugar metabolism involving in not only ethanol formation and consumption but also the general "cofactor balance" mechanism. Candida maltosa is able to ferment glucose as well as xylose to produce a significant amount of ethanol. Here we report the ADH system in C. maltosa composed of three microbial group I ADH genes (CmADH1, CmADH2A and CmADH2B, mainly focusing on its metabolic regulation and physiological function. METHODOLOGY/PRINCIPAL FINDINGS: Genetic analysis indicated that CmADH2A and CmADH2B tandemly located on the chromosome could be derived from tandem gene duplication. In vitro characterization of enzymatic properties revealed that all the three CmADHs had broad substrate specificities. Homo- and heterotetramers of CmADH1 and CmADH2A were demonstrated by zymogram analysis, and their expression profiles and physiological functions were different with respect to carbon sources and growth phases. Fermentation studies of ADH2A-deficient mutant showed that CmADH2A was directly related to NAD regeneration during xylose metabolism since CmADH2A deficiency resulted in a significant accumulation of glycerol. CONCLUSIONS/SIGNIFICANCE: Our results revealed that CmADH1 was responsible for ethanol formation during glucose metabolism, whereas CmADH2A was glucose-repressed and functioned to convert the accumulated ethanol to acetaldehyde. To our knowledge, this is the first demonstration of function separation and glucose repression of ADH genes in xylose-fermenting yeasts. On the other hand, CmADH1 and CmADH2A were both involved in ethanol formation with NAD regeneration to maintain NADH/NAD ratio in favor of producing xylitol from xylose. In contrast, CmADH2B was expressed at a much lower level than the other two CmADH genes, and its function is to be further confirmed.

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

    Science.gov (United States)

    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

  9. 11 beta-Hydroxysteroid dehydrogenase activity in hypothalamic obesity.

    Science.gov (United States)

    Tiosano, Dov; Eisentein, Israel; Militianu, Daniela; Chrousos, George P; Hochberg, Ze'ev

    2003-01-01

    After extensive suprasellar operations for hypothalamic tumor removal, some patients develop Cushing-like morbid obesity while they receive replacement doses of glucocorticoids. In this study, we examined the hypothesis that target tissue conversion of inactive 11-ketosteroids to active 11 beta-OH glucocorticoids might explain the obesity of some patients with hypothalamic lesions. Toward this aim, we studied 10 patients with hypothalamic obesity and secondary adrenal insufficiency and 6 control Addisonian patients while they were on glucocorticoid replacement therapy. Pituitary hormone deficiencies were replaced when medically indicated. Twenty-four-hour urine was collected after a single oral dose of 12 mg/m(2) hydrocortisone acetate. The ratios of free and conjugated cortisol (F) to cortisone (E) and their metabolites, [tetrahydrocortisol (THF)+5 alpha THF]/tetrahyrdocortisone (THE), dihydrocortisols/dihydrocortisones, cortols/cortolones, and (F+E)/(THF+THE+5 alpha THF), were considered to represent 11 beta-hydroxysteroid dehydrogenase (HSD) activity. The 11-OH/11-oxo ratios were significantly higher in the urine of patients with hypothalamic obesity. The 11-OH/11-oxo ratios, however, did not correlate with the degree of obesity, yet a significant correlation was found between conjugated F/E and the ratio of visceral fat to sc fat measured by computerized tomography at the umbilical level. The consequence of increased 11 beta-HSD1 activity and the shift of the interconversion toward cortisol may contribute to the effects of the latter in adipose tissue. We propose that deficiency of hypothalamic messengers after surgical injury induces a paracrine/autocrine effect of enhanced glucocorticoid activity due to up-regulated 11 beta-HSD1 activity. PMID:12519880

  10. Blending foundry sands with soil: Effect on dehydrogenase activity.

    Science.gov (United States)

    Dungan, Robert S; Kukier, Urzsula; Lee, Brad

    2006-03-15

    Each year U.S. foundries landfill several million tons of sand that can no longer be used to make metalcasting molds and cores. A possible use for these materials is as an ingredient in manufactured soils; however, potentially harmful metals and resin binders (used to make cores) may adversely impact the soil microbial community. In this study, the dehydrogenase activity (DHA) of soil amended with molding sand (clay-coated sand known as "green sand") or core sands at 10%, 30%, and 50% (dry wt.) was determined. The green sands were obtained from iron, aluminum, and brass foundries; the core sands were made with phenol-formaldehyde or furfuryl alcohol based resins. Overall, incremental additions of these sands resulted in a decrease in the DHA which lasted throughout the 12-week experimental period. A brass green sand, which contained high concentrations of Cu, Pb, and Zn, severely impacted the DHA. By week 12 no DHA was detected in the 30% and 50% treatments. In contrast, the DHA in soil amended with an aluminum green sand was 2.1 times higher (all blending ratios), on average, at week 4 and 1.4 times greater (30% and 50% treatments only) than the controls by week 12. In core sand-amended soil, the DHA results were similar to soils amended with aluminum and iron green sands. Increased activity in some treatments may be a result of the soil microorganisms utilizing the core resins as a carbon source. The DHA assay is a sensitive indicator of environmental stress caused by foundry sand constituents and may be useful to assess which foundry sands are suitable for beneficial use in the environment. PMID:15975632

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

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    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.

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

    International Nuclear Information System (INIS)

    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

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

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    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.

  14. Structural basis of cooperativity in human UDP-glucose dehydrogenase.

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    Venkatachalam Rajakannan

    Full Text Available BACKGROUND: UDP-glucose dehydrogenase (UGDH is the sole enzyme that catalyzes the conversion of UDP-glucose to UDP-glucuronic acid. The product is used in xenobiotic glucuronidation in hepatocytes and in the production of proteoglycans that are involved in promoting normal cellular growth and migration. Overproduction of proteoglycans has been implicated in the progression of certain epithelial cancers, while inhibition of UGDH diminished tumor angiogenesis in vivo. A better understanding of the conformational changes occurring during the UGDH reaction cycle will pave the way for inhibitor design and potential cancer therapeutics. METHODOLOGY: Previously, the substrate-bound of UGDH was determined to be a symmetrical hexamer and this regular symmetry is disrupted on binding the inhibitor, UDP-α-D-xylose. Here, we have solved an alternate crystal structure of human UGDH (hUGDH in complex with UDP-glucose at 2.8 Å resolution. Surprisingly, the quaternary structure of this substrate-bound protein complex consists of the open homohexamer that was previously observed for inhibitor-bound hUGDH, indicating that this conformation is relevant for deciphering elements of the normal reaction cycle. CONCLUSION: In all subunits of the present open structure, Thr131 has translocated into the active site occupying the volume vacated by the absent active water and partially disordered NAD+ molecule. This conformation suggests a mechanism by which the enzyme may exchange NADH for NAD+ and repolarize the catalytic water bound to Asp280 while protecting the reaction intermediates. The structure also indicates how the subunits may communicate with each other through two reaction state sensors in this highly cooperative enzyme.

  15. Short-chain dehydrogenases/reductases in cyanobacteria.

    Science.gov (United States)

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

    2012-03-01

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

  16. Maize cytokinin dehydrogenase isozymes are localized predominantly to the vacuoles.

    Science.gov (United States)

    Zalabák, David; Johnová, Patricie; Plíhal, Ondřej; Šenková, Karolina; Šamajová, Olga; Jiskrová, Eva; Novák, Ondřej; Jackson, David; Mohanty, Amitabh; Galuszka, Petr

    2016-07-01

    The maize genome encompasses 13 genes encoding for cytokinin dehydrogenase isozymes (CKXs). These enzymes are responsible for irreversible degradation of cytokinin plant hormones and thus, contribute regulating their levels. Here, we focus on the unique aspect of CKXs: their diverse subcellular distribution, important in regulating cytokinin homeostasis. Maize CKXs were tagged with green fluorescent protein (GFP) and transiently expressed in maize protoplasts. Most of the isoforms, namely ZmCKX1, ZmCKX2, ZmCKX4a, ZmCKX5, ZmCKX6, ZmCKX8, ZmCKX9, and ZmCKX12, were associated with endoplasmic reticulum (ER) several hours after transformation. GFP-fused CKXs were observed to accumulate in putative prevacuolar compartments. To gain more information about the spatiotemporal localization of the above isoforms, we prepared stable expression lines of all ZmCKX-GFP fusions in Arabidopsis thaliana Ler suspension culture. All the ER-associated isoforms except ZmCKX1 and ZmCKX9 were found to be targeted primarily to vacuoles, suggesting that ER-localization is a transition point in the intracellular secretory pathway and vacuoles serve as these isoforms' final destination. ZmCKX9 showed an ER-like localization pattern similar to those observed in the transient maize assay. Apoplastic localization of ZmCKX1 was further confirmed and ZmCKX10 showed cytosolic/nuclear localization due to the absence of the signal peptide sequence as previously reported. Additionally, we prepared GFP-fused N-terminal signal deletion mutants of ZmCKX2 and ZmCKX9 and clearly demonstrated that the localization pattern of these mutant forms was cytosolic/nuclear. This study provides the first complex model for spatiotemporal localization of the key enzymes of the cytokinin degradation/catabolism in monocotyledonous plants. PMID:27031423

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

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    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.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

  20. Origin and evolution of medium chain alcohol dehydrogenases.

    Science.gov (United States)

    Jörnvall, Hans; Hedlund, Joel; Bergman, Tomas; Kallberg, Yvonne; Cederlund, Ella; Persson, Bengt

    2013-02-25

    Different lines of alcohol dehydrogenases (ADHs) have separate superfamily origins, already recognized but now extended and re-evaluated by re-screening of the latest databank update. The short-chain form (SDR) is still the superfamily with most abundant occurrence, most multiple divergence, most prokaryotic emphasis, and most non-complicated architecture. This pattern is compatible with an early appearance at the time of the emergence of prokaryotic cellular life. The medium-chain form (MDR) is also old but second in terms of all the parameters above, and therefore compatible with a second emergence. However, this step appears seemingly earlier than previously considered, and may indicate sub-stages of early emergences at the increased resolution available from the now greater number of data entries. The Zn-MDR origin constitutes a third stage, possibly compatible with the transition to oxidative conditions on earth. Within all these three lines, repeated enzymogeneses gave the present divergence. MDR-ADH origin(s), at a fourth stage, may also be further resolved in multiple or extended modes, but the classical liver MDR-ADH of the liver type can still be traced to a gene duplication ~550 MYA (million years ago), at the early vertebrate radiation, compatible with the post-eon-shift, "Cambrian explosion". Classes and isozymes correspond to subsequent and recent duplicatory events, respectively. They illustrate a peculiar pattern with functional and emerging evolutionary distinctions between parent and emerging lines, suggesting a parallelism between duplicatory and mutational events, now also visible at separate sub-stages. Combined, all forms show distinctive patterns at different levels and illustrate correlations with global events. They further show that simple molecular observations on patterns, multiplicities and occurrence give much information, suggesting common divergence rules not much disturbed by horizontal gene transfers after the initial origins. PMID

  1. Residues that influence coenzyme preference in the aldehyde dehydrogenases.

    Science.gov (United States)

    González-Segura, Lilian; Riveros-Rosas, Héctor; Julián-Sánchez, Adriana; Muñoz-Clares, Rosario A

    2015-06-01

    To find out the residues that influence the coenzyme preference of aldehyde dehydrogenases (ALDHs), we reviewed, analyzed and correlated data from their known crystal structures and amino-acid sequences with their published kinetic parameters for NAD(P)(+). We found that the conformation of the Rossmann-fold loops participating in binding the adenosine ribose is very conserved among ALDHs, so that coenzyme specificity is mainly determined by the nature of the residue at position 195 (human ALDH2 numbering). Enzymes with glutamate or proline at 195 prefer NAD(+) because the side-chains of these residues electrostatically and/or sterically repel the 2'-phosphate group of NADP(+). But contrary to the conformational rigidity of proline, the conformational flexibility of glutamate may allow NADP(+)-binding in some enzymes by moving the carboxyl group away from the 2'-phosphate group, which is possible if a small neutral residue is located at position 224, and favored if the residue at position 53 interacts with Glu195 in a NADP(+)-compatible conformation. Of the residues found at position 195, only glutamate interacts with the NAD(+)-adenosine ribose; glutamine and histidine cannot since their side-chain points are opposite to the ribose, probably because the absence of the electrostatic attraction by the conserved nearby Lys192, or its electrostatic repulsion, respectively. The shorter side-chains of other residues-aspartate, serine, threonine, alanine, valine, leucine, or isoleucine-are distant from the ribose but leave room for binding the 2'-phosphate group. Generally, enzymes having a residue different from Glu bind NAD(+) with less affinity, but they can also bind NADP(+) even sometimes with higher affinity than NAD(+), as do enzymes containing Thr/Ser/Gln195. Coenzyme preference is a variable feature within many ALDH families, consistent with being mainly dependent on a single residue that apparently has no other structural or functional roles, and therefore can

  2. Heterologous overexpression of Glomerella cingulata FAD-dependent glucose dehydrogenase in Escherichia coli and Pichia pastoris

    Science.gov (United States)

    2011-01-01

    Background FAD dependent glucose dehydrogenase (GDH) currently raises enormous interest in the field of glucose biosensors. Due to its superior properties such as high turnover rate, substrate specificity and oxygen independence, GDH makes its way into glucose biosensing. The recently discovered GDH from the ascomycete Glomerella cingulata is a novel candidate for such an electrochemical application, but also of interest to study the plant-pathogen interaction of a family of wide-spread, crop destroying fungi. Heterologous expression is a necessity to facilitate the production of GDH for biotechnological applications and to study its physiological role in the outbreak of anthracnose caused by Glomerella (anamorph Colletotrichum) spp. Results Heterologous expression of active G. cingulata GDH has been achieved in both Escherichia coli and Pichia pastoris, however, the expressed volumetric activity was about 4800-fold higher in P. pastoris. Expression in E. coli resulted mainly in the formation of inclusion bodies and only after co-expression with molecular chaperones enzymatic activity was detected. The fed-batch cultivation of a P. pastoris transformant resulted in an expression of 48,000 U L-1 of GDH activity (57 mg L-1). Recombinant GDH was purified by a two-step purification procedure with a yield of 71%. Comparative characterization of molecular and catalytic properties shows identical features for the GDH expressed in P. pastoris and the wild-type enzyme from its natural fungal source. Conclusions The heterologous expression of active GDH was greatly favoured in the eukaryotic host. The efficient expression in P. pastoris facilitates the production of genetically engineered GDH variants for electrochemical-, physiological- and structural studies. PMID:22151971

  3. Biosilica-coated kappa-carrageenan microspheres for yeast alcohol dehydrogenase encapsulation.

    Science.gov (United States)

    Jiang, Yan; Jiang, Yan-Jun; Zhang, Yu-Fei; Li, Jian; Zhang, Lei; Jiang, Zhong-Yi

    2007-01-01

    In this study, a novel polysaccharide/inorganic hybrid biocomposite was prepared through biomineralization-inspired process for efficient immobilization of yeast alcohol dehydrogenase (YADH). YADH-encapsulated kappa-carrageenan microspheres (KCM) were first coated with chitosan, which was used to guide and catalyze the biomimetic formation of silica, and then coated with silica derived from tetraethoxysiliane (TEOS). Scanning electron microscopy (SEM) equipped with a energy dispersive X-ray spectrometer (EDX) was employed to investigate the composition and thickness of silica film. Compared with KCM, the silica-coated kappa-carrageenan microspheres (SKCM) containing YADH exhibited improved anti-swelling and catalytic properties. Enzyme leakage of YADH in KCM was detected to be 63.9% after 1 h, while the enzyme leakage in SKCM was as low as 18.2%. The KCM adsorbed water promptly and after 1 h maximum water uptake can be as high as 1576 wt%, while the water uptake of SKCM was only 143 wt% even after 48 h. The optimum pH and temperature for encapsulated YADH were pH 6.5 and 25 degrees C, which were just the same as those for free YADH, but the encapsulated YADH exhibited broader pH and temperature ranges for high activity. Furthermore, the relative activity of YADH in KCM declined almost to zero after 8 recycles, while the relative activity of YADH in SKCM still maintained more than 50%. The significantly increased recycling stability of YADH in SKCM may be attributed to the effective inhibition of enzyme leakage by the compact biosilica layer.

  4. Pituitary Adenoma With Paraganglioma/Pheochromocytoma (3PAs) and Succinate Dehydrogenase Defects in Humans and Mice

    Science.gov (United States)

    Xekouki, Paraskevi; Szarek, Eva; Bullova, Petra; Giubellino, Alessio; Quezado, Martha; Mastroyannis, Spyridon A.; Mastorakos, Panagiotis; Wassif, Christopher A.; Raygada, Margarita; Rentia, Nadia; Dye, Louis; Cougnoux, Antony; Koziol, Deloris; Sierra, Maria de La Luz; Lyssikatos, Charalampos; Belyavskaya, Elena; Malchoff, Carl; Moline, Jessica; Eng, Charis; Maher, Louis James; Pacak, Karel; Lodish, Maya

    2015-01-01

    Context: Germline mutations in genes coding succinate dehydrogenase (SDH) subunits A, B, C, and D have been identified in familial paragangliomas (PGLs)/pheochromocytomas (PHEOs) and other tumors. We described a GH-secreting pituitary adenoma (PA) caused by SDHD mutation in a patient with familial PGLs. Additional patients with PAs and SDHx defects have since been reported. Design: We studied 168 patients with unselected sporadic PA and with the association of PAs, PGLs, and/or pheochromocytomas, a condition we named the 3P association (3PAs) for SDHx germline mutations. We also studied the pituitary gland and hormonal profile of Sdhb+/− mice and their wild-type littermates at different ages. Results: No SDHx mutations were detected among sporadic PA, whereas three of four familial cases were positive for a mutation (75%). Most of the SDHx-deficient PAs were either prolactinomas or somatotropinomas. Pituitaries of Sdhb+/− mice older than 12 months had an increased number mainly of prolactin-secreting cells and several ultrastructural abnormalities such as intranuclear inclusions, altered chromatin nuclear pattern, and abnormal mitochondria. Igf-1 levels of mutant mice tended to be higher across age groups, whereas Prl and Gh levels varied according to age and sex. Conclusion: The present study confirms the existence of a new association that we termed 3PAs. It is due mostly to germline SDHx defects, although sporadic cases of 3PAs without SDHx defects also exist. Using Sdhb+/− mice, we provide evidence that pituitary hyperplasia in SDHx-deficient cells may be the initial abnormality in the cascade of events leading to PA formation. PMID:25695889

  5. Characterization of the major dehydrogenase related to d-lactic acid synthesis in Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293.

    Science.gov (United States)

    Li, Ling; Eom, Hyun-Ju; Park, Jung-Mi; Seo, Eunyoung; Ahn, Ji Eun; Kim, Tae-Jip; Kim, Jeong Hwan; Han, Nam Soo

    2012-10-10

    Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293 is a lactic acid bacterium that converts pyruvate mainly to d-(-)-lactic acid by using d-(-)-lactate dehydrogenase (ldhD). The aim of this study was to identify the gene responsible for d-lactic acid formation in this organism and to characterize the enzyme to facilitate the production of optically pure d-lactic acid. A genomic analysis of L. mesenteroides ATCC 8293 revealed that 7 genes encode lactate-related dehydrogenase. According to transcriptomic, proteomic, and phylogenetic analyses, LEUM_1756 was the major gene responsible for the production of d-lactic acid. The LEUM_1756 gene, of 996bp and encoding 332 amino acids (36.5kDa), was cloned and overexpressed in Escherichia coli BL21(DE3) Star from an inducible pET-21a(+) vector. The enzyme was purified by Ni-NTA column chromatography and showed a specific activity of 4450U/mg, significantly higher than those of other previously reported ldhDs. The gel permeation chromatography analysis showed that the purified enzyme exists as tetramers in solution and this was the first report among lactic acid bacteria. The pH and temperature optima were pH 8.0 and 30°C, respectively, for the pyruvate reduction reaction, and pH 11.0 and 20°C, respectively, for the lactate oxidation reaction. The K(m) kinetic parameters for pyruvate and lactate were 0.58mM and 260mM, respectively. In addition, the k(cat) values for pyruvate and lactate were 2900s(-1) and 2280s(-1), respectively. The enzyme was not inhibited by Ca(2+), Co(2+), Cu(2+), Mg(2+), Mn(2+), Na(+), or urea, but was inhibited by 1mM Zn(2+) and 1mM SDS. PMID:22975125

  6. Alteration in substrate specificity of horse liver alcohol dehydrogenase by an acyclic nicotinamide analog of NAD(+).

    Science.gov (United States)

    Malver, Olaf; Sebastian, Mina J; Oppenheimer, Norman J

    2014-11-01

    A new, acyclic NAD-analog, acycloNAD(+) has been synthesized where the nicotinamide ribosyl moiety has been replaced by the nicotinamide (2-hydroxyethoxy)methyl moiety. The chemical properties of this analog are comparable to those of β-NAD(+) with a redox potential of -324mV and a 341nm λmax for the reduced form. Both yeast alcohol dehydrogenase (YADH) and horse liver alcohol dehydrogenase (HLADH) catalyze the reduction of acycloNAD(+) by primary alcohols. With HLADH 1-butanol has the highest Vmax at 49% that of β-NAD(+). The primary deuterium kinetic isotope effect is greater than 3 indicating a significant contribution to the rate limiting step from cleavage of the carbon-hydrogen bond. The stereochemistry of the hydride transfer in the oxidation of stereospecifically deuterium labeled n-butanol is identical to that for the reaction with β-NAD(+). In contrast to the activity toward primary alcohols there is no detectable reduction of acycloNAD(+) by secondary alcohols with HLADH although these alcohols serve as competitive inhibitors. The net effect is that acycloNAD(+) has converted horse liver ADH from a broad spectrum alcohol dehydrogenase, capable of utilizing either primary or secondary alcohols, into an exclusively primary alcohol dehydrogenase. This is the first example of an NAD analog that alters the substrate specificity of a dehydrogenase and, like site-directed mutagenesis of proteins, establishes that modifications of the coenzyme distance from the active site can be used to alter enzyme function and substrate specificity. These and other results, including the activity with α-NADH, clearly demonstrate the promiscuity of the binding interactions between dehydrogenases and the riboside phosphate of the nicotinamide moiety, thus greatly expanding the possibilities for the design of analogs and inhibitors of specific dehydrogenases.

  7. The conserved Lysine69 residue plays a catalytic role in Mycobacterium tuberculosis shikimate dehydrogenase

    Directory of Open Access Journals (Sweden)

    Rodrigues Valnês

    2009-01-01

    Full Text Available Abstract Background The shikimate pathway is an attractive target for the development of antitubercular agents because it is essential in Mycobacterium tuberculosis, the causative agent of tuberculosis, but absent in humans. M. tuberculosis aroE-encoded shikimate dehydrogenase catalyzes the forth reaction in the shikimate pathway. Structural and functional studies indicate that Lysine69 may be involved in catalysis and/or substrate binding in M. tuberculosis shikimate dehydrogenase. Investigation of the kinetic properties of mutant enzymes can bring important insights about the role of amino acid residues for M. tuberculosis shikimate dehydrogenase. Findings We have performed site-directed mutagenesis, steady-state kinetics, equilibrium binding measurements and molecular modeling for both the wild-type M. tuberculosis shikimate dehydrogenase and the K69A mutant enzymes. The apparent steady-state kinetic parameters for the M. tuberculosis shikimate dehydrogenase were determined; the catalytic constant value for the wild-type enzyme (50 s-1 is 68-fold larger than that for the mutant K69A (0.73 s-1. There was a modest increase in the Michaelis-Menten constant for DHS (K69A = 76 μM; wild-type = 29 μM and NADPH (K69A = 30 μM; wild-type = 11 μM. The equilibrium dissociation constants for wild-type and K69A mutant enzymes are 32 (± 4 μM and 134 (± 21, respectively. Conclusion Our results show that the residue Lysine69 plays a catalytic role and is not involved in substrate binding for the M. tuberculosis shikimate dehydrogenase. These efforts on M. tuberculosis shikimate dehydrogenase catalytic mechanism determination should help the rational design of specific inhibitors, aiming at the development of antitubercular drugs.

  8. Tear Malate Dehydrogenase,Lactate Dehydrogenase and Their Isoenzymes in Normal Chinese Subjects and Patients of Ocular Surface Disorders

    Institute of Scientific and Technical Information of China (English)

    QingGuo; HanchengZhang

    1995-01-01

    Purose:To determine levels of malate dehydrogenase(MDH),lactate dehydroge-nase(LDH)and their isoenzymes in tears of normal Chinese subjects and patients with ocular surface disorders.Methods:The age range of normal subjects was10-88,with136mal and 128fe-male subjects.123patients suffered from ocular surface disorders.Tears were col-lected from lower fornix on Xinghua filter disc(0.1mm thick,5mm in diameter).The values of tearMDHand LDHwere determined by MONARCH-2000Ana-lyzer(U.S.A)Their isoenzymes were separated by acetate cellulose elec-trophoresis and were determined by Model CDS-200light densitometer.Results:The normal values of tear LDH and MDH were 45.51+23.00-81.35+37.84umol·s-1/Land11.00+5.33-19.50+9.17umol·s-1/Lrespectively,dis-regarding sex or eye distriction(P>0.05).The values of tear LDHandMDH in the group aged10-19were significantly lower than in another groups(P<0.05),95%normal ranges of tearMDHaged below19and above20were3.63-19.90umol·s-1/L.THe MDH isoenzymes comprised MDHs and MDHm,the former accounting for80.0-89.1%.The LDH isoenzymes comprised 5varieties.of which the ratioH/Mof subunit H tosubunit Mwas0.196+0.02.Levels of tear LDH,MDHand their isoenzymes in different diseases were various.Conclusions;Tear LDH/MDHratio reflected sensitively the matabolism of corneae and conjunetival epithelium.The changes in LDH isoenzymes were hel-ful to the differential diagnosis of external eye diseases,and the increase of MDHm reflected sensitively the degree of injury to the corneal epithelium.

  9. Mechanism of hyperinsulinism in short-chain 3-hydroxyacyl-CoA dehydrogenase deficiency involves activation of glutamate dehydrogenase.

    Science.gov (United States)

    Li, Changhong; Chen, Pan; Palladino, Andrew; Narayan, Srinivas; Russell, Laurie K; Sayed, Samir; Xiong, Guoxiang; Chen, Jie; Stokes, David; Butt, Yasmeen M; Jones, Patricia M; Collins, Heather W; Cohen, Noam A; Cohen, Akiva S; Nissim, Itzhak; Smith, Thomas J; Strauss, Arnold W; Matschinsky, Franz M; Bennett, Michael J; Stanley, Charles A

    2010-10-01

    The mechanism of insulin dysregulation in children with hyperinsulinism associated with inactivating mutations of short-chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD) was examined in mice with a knock-out of the hadh gene (hadh(-/-)). The hadh(-/-) mice had reduced levels of plasma glucose and elevated plasma insulin levels, similar to children with SCHAD deficiency. hadh(-/-) mice were hypersensitive to oral amino acid with decrease of glucose level and elevation of insulin. Hypersensitivity to oral amino acid in hadh(-/-) mice can be explained by abnormal insulin responses to a physiological mixture of amino acids and increased sensitivity to leucine stimulation in isolated perifused islets. Measurement of cytosolic calcium showed normal basal levels and abnormal responses to amino acids in hadh(-/-) islets. Leucine, glutamine, and alanine are responsible for amino acid hypersensitivity in islets. hadh(-/-) islets have lower intracellular glutamate and aspartate levels, and this decrease can be prevented by high glucose. hadh(-/-) islets also have increased [U-(14)C]glutamine oxidation. In contrast, hadh(-/-) mice have similar glucose tolerance and insulin sensitivity compared with controls. Perifused hadh(-/-) islets showed no differences from controls in response to glucose-stimulated insulin secretion, even with addition of either a medium-chain fatty acid (octanoate) or a long-chain fatty acid (palmitate). Pull-down experiments with SCHAD, anti-SCHAD, or anti-GDH antibodies showed protein-protein interactions between SCHAD and GDH. GDH enzyme kinetics of hadh(-/-) islets showed an increase in GDH affinity for its substrate, α-ketoglutarate. These studies indicate that SCHAD deficiency causes hyperinsulinism by activation of GDH via loss of inhibitory regulation of GDH by SCHAD.

  10. Purification and characterization of ribitol-5-phosphate and xylitol-5-phosphate dehydrogenases from strains of Lactobacillus casei.

    OpenAIRE

    Hausman, S Z; London, J

    1987-01-01

    A simple three-step procedure is described which yields electrophoretically homogeneous preparations of ribitol-5-phosphate dehydrogenase and xylitol-5-phosphate dehydrogenase. The former enzyme is a 115,000-molecular-weight protein composed of two subunits of identical size and is specific for its substrate, ribitol. The xylitol-5-phosphate dehydrogenase exists as a tetrameric protein with a molecular weight of 180,000; this enzyme oxidizes the phosphate esters of both xylitol and D-arabitol...

  11. Cloning of the Arabidopsis and Rice Formaldehyde Dehydrogenase Genes: Implications for the Origin of Plant Adh Enzymes

    OpenAIRE

    Dolferus, R; Osterman, J. C.; Peacock, W. J.; Dennis, E.S.

    1997-01-01

    This article reports the cloning of the genes encoding the Arabidopsis and rice class III ADH enzymes, members of the alcohol dehydrogenase or medium chain reductase/dehydrogenase superfamily of proteins with glutathione-dependent formaldehyde dehydrogenase activity (GSH-FDH). Both genes contain eight introns in exactly the same positions, and these positions are conserved in plant ethanol-active Adh genes (class P). These data provide further evidence that plant class P genes have evolved fr...

  12. Strategy for the isolation of native dehydrogenases with potential for biosensor development from the organism Hyphomicrobium zavarzinii ZV580.

    Science.gov (United States)

    Hilbrig, Frank; Jérôme, Valérie; Salzig, Mark; Freitag, Ruth

    2009-04-17

    Dehydrogenases are interesting candidates for the development of electrochemical biosensors. Most dehydrogenases are characterised by a comparatively broad substrate spectrum, yet highly specific enzymes exist as well. A specific formaldehyde dehydrogenase has, e.g., been described for the organism Hyphomicrobium zavarzinii ZV580. Isolation of enzymes from their natural source instead of a recombinant expression renders the isolation more challenging, as common tools such as affinity tags are no longer available. In this contribution, we develop chromatographic procedures for such isolation tasks. The previously described formaldehyde dehydrogenase was isolated by two procedures, one based on affinity chromatography, the other on hydroxyapatite. Neither procedure yielded an active enzyme. In addition two dehydrogenases, a formaldehyde and a methylamine dehydrogenase, were found in the cell free extract, which had not been described previously. Both enzymes could be isolated to near purity by a sequence of hydroxyapatite and anion exchange chromatography. The new formaldehyde dehydrogenase requires reconstitution with calcium and pyrroloquinoline quinone in order to become active. The enzyme shows no cross-reactivity with methylamine or methanol. The methylamine dehydrogenase catalyses the conversion of methylamine into formaldehyde, hence it could become a technical catalyst for the inverse reaction. This enzyme consists of two types of subunit and may be one of the rare alpha,beta-methylamine dehydrogenases.

  13. Heme binding properties of glyceraldehyde-3-phosphate dehydrogenase.

    Science.gov (United States)

    Hannibal, Luciana; Collins, Daniel; Brassard, Julie; Chakravarti, Ritu; Vempati, Rajesh; Dorlet, Pierre; Santolini, Jérôme; Dawson, John H; Stuehr, Dennis J

    2012-10-30

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a glycolytic enzyme that also functions in transcriptional regulation, oxidative stress, vesicular trafficking, and apoptosis. Because GAPDH is required for the insertion of cellular heme into inducible nitric oxide synthase [Chakravarti, R., et al. (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 18004-18009], we extensively characterized the heme binding properties of GAPDH. Substoichiometric amounts of ferric heme bound to GAPDH (one heme per GAPDH tetramer) to form a low-spin complex with UV-visible maxima at 362, 418, and 537 nm and when reduced to ferrous gave maxima at 424, 527, and 559 nm. Ferric heme association and dissociation rate constants at 10 °C were as follows: k(on) = 17800 M(-1) s(-1), k(off1) = 7.0 × 10(-3) s(-1), and k(off2) = 3.3 × 10(-4) s(-1) (giving approximate affinities of 19-390 nM). Ferrous heme bound more poorly to GAPDH and dissociated with a k(off) of 4.2 × 10(-3) s(-1). Magnetic circular dichroism, resonance Raman, and electron paramagnetic resonance spectroscopic data on the ferric, ferrous, and ferrous-CO complexes of GAPDH showed that the heme is bis-ligated with His as the proximal ligand. The distal ligand in the ferric complex was not displaced by CN(-) or N(3)(-) but in the ferrous complex could be displaced by CO at a rate of 1.75 s(-1) (for >0.2 mM CO). Studies with heme analogues revealed selectivity toward the coordinating metal and porphyrin ring structure. The GAPDH-heme complex was isolated from bacteria induced to express rabbit GAPDH in the presence of δ-aminolevulinic acid. Our finding of heme binding to GAPDH expands the protein's potential roles. The strength, selectivity, reversibility, and redox sensitivity of heme binding to GAPDH are consistent with it performing heme sensing or heme chaperone-like functions in cells.

  14. D- and L-lactate dehydrogenases during invertebrate evolution

    Directory of Open Access Journals (Sweden)

    Stillman Jonathon H

    2008-10-01

    Full Text Available Abstract Background The L-lactate and D-lactate dehydrogenases, which are involved in the reduction of pyruvate to L(--lactate and D(+-lactate, belong to evolutionarily unrelated enzyme families. The genes encoding L-LDH have been used as a model for gene duplication due to the multiple paralogs found in eubacteria, archaebacteria, and eukaryotes. Phylogenetic studies have suggested that several gene duplication events led to the main isozymes of this gene family in chordates, but little is known about the evolution of L-Ldh in invertebrates. While most invertebrates preferentially oxidize L-lactic acid, several species of mollusks, a few arthropods and polychaetes were found to have exclusively D-LDH enzymatic activity. Therefore, it has been suggested that L-LDH and D-LDH are mutually exclusive. However, recent characterization of putative mammalian D-LDH with significant similarity to yeast proteins showing D-LDH activity suggests that at least mammals have the two naturally occurring forms of LDH specific to L- and D-lactate. This study describes the phylogenetic relationships of invertebrate L-LDH and D-LDH with special emphasis on crustaceans, and discusses gene duplication events during the evolution of L-Ldh. Results Our phylogenetic analyses of L-LDH in vertebrates are consistent with the general view that the main isozymes (LDH-A, LDH-B and LDH-C evolved through a series of gene duplications after the vertebrates diverged from tunicates. We report several gene duplication events in the crustacean, Daphnia pulex, and the leech, Helobdella robusta. Several amino acid sequences with strong similarity to putative mammalian D-LDH and to yeast DLD1 with D-LDH activity were found in both vertebrates and invertebrates. Conclusion The presence of both L-Ldh and D-Ldh genes in several chordates and invertebrates suggests that the two enzymatic forms are not necessarily mutually exclusive. Although, the evolution of L-Ldh has been punctuated by

  15. Glucose-6-phosphate dehydrogenase mutations and haplotypes in Mexican Mestizos.

    Science.gov (United States)

    Arámbula, E; Aguilar L, J C; Vaca, G

    2000-08-01

    In a screening for glucose-6-phosphate dehydrogenase (G-6-PD) deficiency in 1985 unrelated male subjects from the general population (Groups A and B) belonging to four states of the Pacific coast, 21 G-6-PD-deficient subjects were detected. Screening for mutations at the G-6-PD gene by PCR-restriction enzyme in these 21 G-6-PD-deficient subjects as well as in 14 G-6-PD-deficient patients with hemolytic anemia belonging to several states of Mexico showed two common G-6-PD variants: G-6-PD A-(202A/376G) (19 cases) and G-6-PD A-(376G/968C) (9 cases). In 7 individuals the mutations responsible for the enzyme deficiency remain to be determined. Furthermore, four silent polymorphic sites at the G-6-PD gene (PvuII, PstI, 1311, and NlaIII) were investigated in the 28 individuals with G-6-PD A- variants and in 137 G-6-PD normal subjects. As expected, only 10 different haplotypes were observed. To date, in our project aiming to determine the molecular basis of G-6-PD deficiency in Mexico, 60 unrelated G-6-PD-deficient Mexican males-25 in previous studies and 35 in the present work-have been studied. More than 75% of these individuals are from states of the Pacific coast (Sinaloa, Nayarit, Jalisco, Michoacán, Guerrero, Oaxaca, and Chiapas). The results show that although G-6-PD deficiency is heterogeneous at the DNA level in Mexico, only three polymorphic variants have been observed: G-6-PD A-(202A/376G) (36 cases), G-6-PD A-(376G/968C) (13 cases), and G-6-PD Seattle(844C) (2 cases). G-6-PD A- variants are relatively distributed homogeneously and both variants explain 82% of the overall prevalence of G-6-PD deficiency. The variant G-6-PD A-(202A/376G) represents 73% of the G-6-PD A- alleles. Our data also show that the variant G-6-PD A-(376G/968C)-which has been observed in Mexico in the context of two different haplotypes-is more common than previously supposed. The three polymorphic variants that we observed in Mexico are on the same haplotypes as found in subjects from

  16. Relationship Between Polymorphism of Methylenetetrahydrofolate Dehydrogenase and Congenital Heart Defect

    Institute of Scientific and Technical Information of China (English)

    JUN CHENG; WEN-LI ZHU; JING-JING DAO; SHU-QING LI; YONG LI

    2005-01-01

    Objective To investigate the relationship between G1958A gene polymorphism of methylenetetrahydrofolate dehydrogenase (MTHFD) and occurrence of congenital heart disease (CHD) in North China. Methods One hundred and ninety-two CHD patients and their parents were included in this study as case group in Liaoning Province by birth defect registration cards, and 124 healthy subjects (age and gender matched) and their parents were simultaneously selected from the same geographic area as control. Their gene polymorphism of MTHFD G1958A locus was examined with PCR-RFLP, and serum folic acid and homocysteine (Hcy) levels were tested with radio-immunoassay and fluorescence polarization immunoassay (FPIA). Results There existed gene polymorphism at MTHFD G1958A locus in healthy subjects living in North China. The percentages of GG, GA, and AA genotype were 57.98%, 35.57%, and 6.45% respectively, and the A allele frequency was 24.23%, which was significantly different from Western population. No difference was observed when comparing genotype distribution and allele frequency between the case and control groups, so was the result from the comparison between genders. The A allele frequency of arterial septal defect patients' mothers (10.87%) was significantly lower than that of controls (28.15%) (P=0.014), with OR=0.31 (95% CI: 0.09-0.84), and no difference in the other subgroups. The percentage of at least one parent carrying A allele in arterial septal defect subgroup (43.48%) was significantly lower than that in controls (69.64%) (P=0.017), with OR=0.34 (95% CI: 0.12-0.92). The analysis of genetic transmission indicated that there was no transmission disequillibrium in CHD nuclear families. Their serum folic acid level was significantly higher than that of controls (P=0.000), and Hcy level of the former was higher than that of the latter with no statistical significance (P>0.05). Serum Hcy and folic acid levels of mothers with gene mutation were lower than those of mothers

  17. Virtual mutagenesis of isocitrate dehydrogenase 1 involved in glioblastoma multiforme

    Institute of Scientific and Technical Information of China (English)

    WANG Ming-dong; SHI Yan-fang; WANG Hong; WANG Jia-liang; MA Wen-bin; WANG Ren-zhi

    2011-01-01

    Background Site A132Arg mutations potentially impair the affinity of isocitrate dehydrogenase 1 (IDH1) for its substrate isocitrate (ICT),consequently reducing the production of α-ketoglutarate and leading to tumor growth through the induction of the hypoxia-inducible factor-1 (HIF-1) pathway.However,given that the roles of other active sites in IDH1 substrate binding remain unclear,we aimed to investigate IDH1 mutation pattern and its influence on enzyme function.Methods Fifteen IDH1 catalytic active site candidates were selected for in silico mutagenesis and protein homology modeling.Binding free energy of the IDH1/ICT complexes with single-site mutations was compared with that of the wild type.The affinity of 10 IDH1 catalytic active sites for the ICT substrate was further calculated.Results The IDH1 active site included seven residues from chain A (A77Thr,A94Ser,A100Arg,A132Arg,A1O9Arg,A275Asp,and A279Asp) and three residues from chain B (B214Thr,B212Lys,and B252Asp) that constituted the substrate ICT-binding site.These residues were located within 0.5 nm of ICT,indicating a potential interaction with the substrate.IDH1 changes of binding free energy (△E) suggested that the A132Arg residue from chain A contributes three hydrogen bonds to the ICT α-carboxyl and β-carboxyl groups,while the other nine residues involved in ICT binding form only one or two hydrogen bonds.Amino acid substitutes at A132Arg,A109Arg,and B212Lys sites,had the greatest effect on enzyme affinity for its substrate.Conclusions Mutations at sites A132Arg,A109Arg,and B212Lys reduced IDH1 affinity for ICT,indicating these active sites may play a central role in substrate binding.Mutations at sites A77Thr,A94Ser,and A275Asp increased the affinity of IDH1 for ICT,which may enhance IDN1 catalytic activity.Mutant IDH1 proteins with higher catalytic activity than the wild-type IDH1 could potentially be used as a novel gene therapy for glioblastoma multiforme.

  18. Glucose-6-phosphate dehydrogenase deficiency in Nigerian children.

    Directory of Open Access Journals (Sweden)

    Olatundun Williams

    Full Text Available 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 (561 males and 561 females aged 1 month to 15 years. The mean age was 7.4 ± 3.2 years. Children of Yoruba ethnicity made up the largest group (77.5% followed by those Igbo descent (10.6% and those of Igede (10.2% and Tiv (1.8% ethnicity. G6PD status was determined using the fluorescent spot method. We found that the overall prevalence of G6PD deficiency was 15.3% (24.1% in males, 6.6% in females. Yoruba children had a higher prevalence (16.9% than Igede (10.5%, Igbo (10.1% and Tiv (5.0% children. The odds of G6PD deficiency were 0.38 times as high in Igbo children compared to Yoruba children (p=0.0500. The odds for Igede and Tiv children were not significantly different from Yoruba children (p=0.7528 and 0.9789 respectively. Mean oxygen saturation, heart rate and hematocrit were not significantly different in G6PD deficient and G6PD sufficient children. The odds of being G6PD deficient were 2.1 times higher in children with scleral icterus than those without (p=0.0351. In conclusion, we determined the prevalence of G6PD deficiency in Nigerian sub-populations. The odds of G6PD deficiency were decreased in Igbo children compared to Yoruba children. There was no association between vital parameters or hematocrit and G6PD deficiency. We found that a history of scleral icterus may increase the odds of G6PD deficiency, but we did not exclude other common causes of icterus such as sickle cell disease or malarial infection.

  19. Glucose-6-phosphate dehydrogenase deficiency in Nigerian children.

    Science.gov (United States)

    Williams, Olatundun; Gbadero, Daniel; Edowhorhu, Grace; Brearley, Ann; Slusher, Tina; 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 (561 males and 561 females) aged 1 month to 15 years. The mean age was 7.4 ± 3.2 years. Children of Yoruba ethnicity made up the largest group (77.5%) followed by those Igbo descent (10.6%) and those of Igede (10.2%) and Tiv (1.8%) ethnicity. G6PD status was determined using the fluorescent spot method. We found that the overall prevalence of G6PD deficiency was 15.3% (24.1% in males, 6.6% in females). Yoruba children had a higher prevalence (16.9%) than Igede (10.5%), Igbo (10.1%) and Tiv (5.0%) children. The odds of G6PD deficiency were 0.38 times as high in Igbo children compared to Yoruba children (p=0.0500). The odds for Igede and Tiv children were not significantly different from Yoruba children (p=0.7528 and 0.9789 respectively). Mean oxygen saturation, heart rate and hematocrit were not significantly different in G6PD deficient and G6PD sufficient children. The odds of being G6PD deficient were 2.1 times higher in children with scleral icterus than those without (p=0.0351). In conclusion, we determined the prevalence of G6PD deficiency in Nigerian sub-populations. The odds of G6PD deficiency were decreased in Igbo children compared to Yoruba children. There was no association between vital parameters or hematocrit and G6PD deficiency. We found that a history of scleral icterus may increase the odds of G6PD deficiency, but we did not exclude other common causes of icterus such as sickle cell disease or malarial infection. PMID:23874768

  20. Catalysis of nitrite generation from nitroglycerin by glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

    Science.gov (United States)

    Seabra, Amedea B; Ouellet, Marc; Antonic, Marija; Chrétien, Michelle N; English, Ann M

    2013-11-30

    Vascular relaxation to nitroglycerin (glyceryl trinitrate; GTN) requires its bioactivation by mechanisms that remain controversial. We report here that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the release of nitrite from GTN. In assays containing dithiothreitol (DTT) and NAD(+), the GTN reductase activity of purified GAPDH produces nitrite and 1,2-GDN as the major products. A vmax of 2.6nmolmin(-)(1)mg(-)(1) was measured for nitrite production by GAPDH from rabbit muscle and a GTN KM of 1.2mM. Reductive denitration of GTN in the absence of DTT results in dose- and time-dependent inhibition of GAPDH dehydrogenase activity. Disulfiram, a thiol-modifying drug, inhibits both the dehydrogenase and GTN reductase activity of GAPDH, while DTT or tris(2-carboxyethyl)phosphine reverse the GTN-induced inhibition. Incubation of intact human erythrocytes or hemolysates with 2mM GTN for 60min results in 50% inhibition of GAPDH's dehydrogenase activity, indicating that GTN is taken up by these cells and that the dehydrogenase is a target of GTN. Thus, erythrocyte GAPDH may contribute to GTN bioactivation.

  1. Fusion of phospholipid vesicles induced by muscle glyceraldehyde-3-phosphate dehydrogenase in the absence of calcium.

    Science.gov (United States)

    Morero, R D; Viñals, A L; Bloj, B; Farías, R N

    1985-04-01

    Ca2+-induced fusion of phospholipid vesicles (phosphatidylcholine/phosphatidic acid, 9:1 mol/mol) prepared by ethanolic injection was followed by five different procedures: resonance energy transfer, light scattering, electron microscopy, intermixing of aqueous content, and gel filtration through Sepharose 4-B. The five methods gave concordant results, showing that vesicles containing only 10% phosphatidic acid can be induced to fuse by millimolar concentrations of Ca2+. When the fusing capability of several soluble proteins was assayed, it was found that concanavalin A, bovine serum albumin, ribonuclease, and protease were inactive. On the other hand, lysozyme, L-lactic dehydrogenase, and muscle and yeast glyceraldehyde-3-phosphate dehydrogenase were capable of inducing vesicle fusion. Glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle, the most extensively studied protein, proved to be very effective: 0.1 microM was enough to induce complete intermixing of bilayer phospholipid vesicles. Under conditions used in this work, fusion was accompanied by leakage of internal contents. The fusing capability of glyceraldehyde-3-phosphate dehydrogenase was not affected by 5 mM ethylenediaminetetraacetic acid. The Ca2+ concentration in the medium, as determined by atomic absorption spectroscopy, was 5 ppm. Heat-denatured enzyme was incapable of inducing fusion. We conclude that glyceraldehyde-3-phosphate dehydrogenase is a soluble protein inherently endowed with the capability of fusing phospholipid vesicles.

  2. Trehalose and sorbitol alter the kinetic pattern of inactivation of glutamate dehydrogenase during drying in levitated microdroplets.

    Science.gov (United States)

    Lorenzen, Elke; Lee, Geoffrey

    2013-12-01

    A single-droplet acoustic levitator was used to determine the drying rate and the kinetics of inactivation of glutamate dehydrogenase in the presence of added trehalose or sorbitol. The solution was also spray dried under the same process condition of drying gas temperature on a bench-top machine. Both trehalose and sorbitol delay the point of onset of enzyme inactivation which lies after the critical point of drying. Both carbohydrates also reduce the apparent rate constant of inactivation calculated during the subsequent inactivation phase. The carbohydrates stabilise, therefore, the enzyme during droplet drying and particle formation mainly during the falling rate drying period. There is no difference between the stabilising effects of the two carbohydrates when examined as levitated single droplets. This suggests the importance of water replacement as a stabilising mechanism in the levitated droplets/particles. On spray drying, the trehalose stabilises the enzyme better than does the sorbitol at a drying gas (outlet) temperature of 60°C. This suggests glass formation with the trehalose but not the sorbitol during the very rapid drying process of small-atomised droplets in the spray dryer.

  3. Comparative 13C metabolic flux analysis of pyruvate dehydrogenase complex-deficient, L-valine-producing Corynebacterium glutamicum.

    Science.gov (United States)

    Bartek, Tobias; Blombach, Bastian; Lang, Siegmund; Eikmanns, Bernhard J; Wiechert, Wolfgang; Oldiges, Marco; Nöh, Katharina; Noack, Stephan

    2011-09-01

    L-Valine can be formed successfully using C. glutamicum strains missing an active pyruvate dehydrogenase enzyme complex (PDHC). Wild-type C. glutamicum and four PDHC-deficient strains were compared by (13)C metabolic flux analysis, especially focusing on the split ratio between glycolysis and the pentose phosphate pathway (PPP). Compared to the wild type, showing a carbon flux of 69% ± 14% through the PPP, a strong increase in the PPP flux was observed in PDHC-deficient strains with a maximum of 113% ± 22%. The shift in the split ratio can be explained by an increased demand of NADPH for l-valine formation. In accordance, the introduction of the Escherichia coli transhydrogenase PntAB, catalyzing the reversible conversion of NADH to NADPH, into an L-valine-producing C. glutamicum strain caused the PPP flux to decrease to 57% ± 6%, which is below the wild-type split ratio. Hence, transhydrogenase activity offers an alternative perspective for sufficient NADPH supply, which is relevant for most amino acid production systems. Moreover, as demonstrated for L-valine, this bypass leads to a significant increase of product yield due to a concurrent reduction in carbon dioxide formation via the PPP.

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

    Science.gov (United States)

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

    2007-01-01

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

  5. Global gene expression analysis of glucose overflow metabolism in Escherichia coli and reduction of aerobic acetate formation.

    Science.gov (United States)

    Veit, Andrea; Polen, Tino; Wendisch, Volker F

    2007-02-01

    During aerobic growth on glucose, Escherichia coli produces acetate in the so-called overflow metabolism. DNA microarray analysis was used to determine the global gene expression patterns of chemostat cultivations of E. coli MG1655 that were characterized by different acetate formation rates during aerobic growth on glucose. A correlation analysis identified that expression of ten genes (sdhCDAB, sucB, sucC, acnB, lpdA, fumC and mdh) encoding the TCA cycle enzymes succinate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinyl-CoA synthetase, aconitase, fumarase and malate dehydrogenase, respectively, and of the acs-yjcH-actP operon for acetate utilization correlated negatively with acetate formation. Relieving transcriptional control of the sdhCDAB-b0725-sucABCD operon by chromosomal promoter exchange mutagenesis yielded a strain with increased specific activities of the TCA cycle enzymes succinate dehydrogenase, alpha-ketoglutarate dehydrogenase and succinyl-CoA synthetase, which are encoded by this operon. The resulting strain produced less acetate and directed more carbon towards carbon dioxide formation than the parent strain MG1655 while maintaining high growth and glucose consumption rates. PMID:17273855

  6. Construction of an integrated enzyme system consisting azoreductase and glucose 1-dehydrogenase for dye removal.

    Science.gov (United States)

    Yang, Yuyi; Wei, Buqing; Zhao, Yuhua; Wang, Jun

    2013-02-01

    Azo dyes are toxic and carcinogenic and are often present in industrial effluents. In this research, azoreductase and glucose 1-dehydrogenase were coupled for both continuous generation of the cofactor NADH and azo dye removal. The results show that 85% maximum relative activity of azoreductase in an integrated enzyme system was obtained at the conditions: 1U azoreductase:10U glucose 1-dehydrogenase, 250mM glucose, 1.0mM NAD(+) and 150μM methyl red. Sensitivity analysis of the factors in the enzyme system affecting dye removal examined by an artificial neural network model shows that the relative importance of enzyme ratio between azoreductase and glucose 1-dehydrogenase was 22%, followed by dye concentration (27%), NAD(+) concentration (23%) and glucose concentration (22%), indicating none of the variables could be ignored in the enzyme system. Batch results show that the enzyme system has application potential for dye removal.

  7. Hydrostatic pressure induces conformational and catalytic changes on two alcohol dehydrogenases but no oligomeric dissociation.

    Science.gov (United States)

    Dallet, S; Legoy, M D

    1996-05-01

    A comparison between the pressure effects on the catalysis of Thermoanaerobium brockii alcohol dehydrogenase (TBADH: a thermostable tetrameric enzyme) and yeast alcohol dehydrogenase (YADH: a mesostable tetrameric enzyme) revealed a different behaviour. YADH activity is continuously inhibited by an increase of pressure, whereas YADH affinity seems less sensitive to pressure. TBADH activity is enhanced by pressure up to 100 MPa. TBADH affinity for alcoholic substrates increases if pressure increases, was TBADH affinity for NADP decreases when pressure increases. Hypothesis has been raised concerning the dissociation of oligomeric enzymes under high hydrostatic pressure ( YADH at all pressures and TBADH for pressures above 100 MPa is not correlated to subunit dissociation. Hence we suggest that enzymes under pressure encounter a molecular rearrangement which can either have a positive or a negative effect on activity. Finally, we have observed that the catalytic behaviour of alcohol dehydrogenases under pressure is connected to their thermostability.

  8. Expression, crystallization and preliminary X-ray crystallographic analysis of alcohol dehydrogenase (ADH) from Kangiella koreensis.

    Science.gov (United States)

    Ngo, Ho-Phuong-Thuy; Hong, Seung-Hye; Hong, Myoung-Ki; Pham, Tan-Viet; Oh, Deok-Kun; Kang, Lin-Woo

    2013-09-01

    Alcohol dehydrogenases (ADHs) are a group of dehydrogenase enzymes that facilitate the interconversion between alcohols and aldehydes or ketones with the reduction of NAD(+) to NADH. In bacteria, some alcohol dehydrogenases catalyze the opposite reaction as part of fermentation to ensure a constant supply of NAD(+). The adh gene from Kangiella koreensis was cloned and the protein (KkADH) was expressed, purified and crystallized. A KkADH crystal diffracted to 2.5 Å resolution and belonged to the monoclinic space group P2(1), with unit-cell parameters a = 94.1, b = 80.9, c = 115.6 Å, β = 111.9°. Four monomers were present in the asymmetric unit, with a corresponding VM of 2.55 Å(3) Da(-1) and a solvent content of 51.8%.

  9. Structural and biochemical insights into 7β-hydroxysteroid dehydrogenase stereoselectivity.

    Science.gov (United States)

    Savino, Simone; Ferrandi, Erica Elisa; Forneris, Federico; Rovida, Stefano; Riva, Sergio; Monti, Daniela; Mattevi, Andrea

    2016-06-01

    Hydroxysteroid dehydrogenases are of great interest as biocatalysts for transformations involving steroid substrates. They feature a high degree of stereo- and regio-selectivity, acting on a defined atom with a specific configuration of the steroid nucleus. The crystal structure of 7β-hydroxysteroid dehydrogenase from Collinsella aerofaciens reveals a loop gating active-site accessibility, the bases of the specificity for NADP(+) , and the general architecture of the steroid binding site. Comparison with 7α-hydroxysteroid dehydrogenase provides a rationale for the opposite stereoselectivity. The presence of a C-terminal extension reshapes the substrate site of the β-selective enzyme, possibly leading to an inverted orientation of the bound substrate. Proteins 2016; 84:859-865. © 2016 Wiley Periodicals, Inc. PMID:27006087

  10. Purification and properties of thiosulfate dehydrogenase from Acidithiobacillus thiooxidans JCM7814.

    Science.gov (United States)

    Nakamura, K; Nakamura, M; Yoshikawa, H; Amano, Y

    2001-01-01

    A key enzyme of the thiosulfate oxidation pathway in Acidithiobacillus thiooxidans JCM7814 was investigated. As a result of assaying the enzymatic activities of thiosulfate dehydrogenase, rhodanese, and thiosulfate reductase at 5.5 of intracellular pH, the activity of thiosulfate dehydrogenase was measured as the key enzyme. The thiosulfate dehydrogenase of A. thiooxidans JCM7814 was purified using three chromatographies. The purified sample was electrophoretically homogeneous. The molecular mass of the enzyme was 27.9 kDa and it was a monomer. This enzyme had cytochrome c. The optimum pH and temperature of this enzyme were 3.5 and 35 degrees C. The enzyme was stable in the pH range from 5 to 7, and it was stable up to 45 degrees C. The isoelectric point of the enzyme was 8.9. This enzyme reacted with thiosulfate as a substrate. The Km was 0.81 mM.

  11. Cloning and expression of glucose 3-dehydrogenase from Halomonas sp. alpha-15 in Escherichia coli.

    Science.gov (United States)

    Kojima, K; Tsugawa, W; Sode, K

    2001-03-23

    The gene encoding glucose 3-dehydrogenase (G3DH) from Halomonas sp. alpha-15 was cloned and expressed in Escherichia coli. An open reading frame of 1686 nucleotides was shown to encode G3DH. The flavine adenine dinucleotide binding motif was found in the N-terminal region of G3DH. The deduced primary structure of G3DH showed about 30% identity to sorbitol dehydrogenase from Gluconobacter oxydans and 2-keto-d-gluconate dehydrogenases from Erwinia herbicola and Pantoea citrea. The folding prediction of G3DH suggested that the 3D structure of G3DH was similar with cholesterol oxidase from Brevibacterium sterolicum or glucose oxidase from Aspergillus niger. PMID:11263965

  12. An X-ray structural study of pyruvate dehydrogenase kinase: A eukaryotic serine kinase with a prokaryotic histidine-kinase fold

    Science.gov (United States)

    Steussy, Calvin Nicklaus, Jr.

    2001-07-01

    Pyruvate Dehydrogenase Kinase is an enzyme that controls the flow of glucose through the eukaryotic cell and contributes to the pathology of diabetes mellitus. Early work on this kinase demonstrated that it has an amino acid sequence much like bacterial histidine kinases, but an activity similar to that of modern serine/threonine kinases. This project utilized the techniques of X-ray crystallography to determine molecular structure of pyruvate dehydrogenase kinase, isozyme 2. The structure was phased using selenium substituted for sulfur in methionine residues, and data at multiple wavelengths was collected at the National Synchrotron Light Source, Brookhaven National Laboratories. PDK 2 was found to fold into a two-domain monomer that forms a dimer through two beta sheets in the C-terminal domain. The N-terminal domain is an alpha-helical bundle while the C-terminal domain is an alpha/beta sandwich. The fold of the C-terminal domain is very similar to that of the prokaryotic histidine kinases, indicating that they share a common ancestor. The catalytic mechanism, however, has evolved to use general base catalysis to activate the serine substrate, rather than the direct nucleophilic attack by the imidazole sidechain used in the prokaryotic kinases. Thus, the structure of the protein echoes its prokaryotic ancestor, while the chemical mechanism has adapted to a serine substrate. The electrostatic surface of PDK2 leads to the suggestion that the lipoyl domain of the pyruvate dehydrogenase kinase, an important associated structure, may bind in the cleft formed between the N- and C-terminal domains. In addition, a network of hydrogen bonds directly connects the nucleotide binding pocket to the dimer interface, suggesting that there may be some interaction between dimer formation and ATP binding or ADP release.

  13. Triiodothyronine (T3)-associated upregulation and downregulation of nuclear T3 binding in the human fibroblast cell (MRC-5)--stimulation of malic enzyme, glucose-6-phosphate-dehydrogenase, and 6-phosphogluconate-dehydrogenase by insulin, but not by T3

    DEFF Research Database (Denmark)

    Matzen, L E; Kristensen, S R; Kvetny, J

    1991-01-01

    The specific nuclear binding of triiodothyronine (T3) (NBT3) and the activity of malic enzyme (ME), glucose-6-phosphate-dehydrogenase (G6PD), and 6-phosphogluconate-dehydrogenase (6PGD) were studied in the human fibroblast cell (MRC-5). The overall apparent binding affinity (Ka) was 2.7 x 10(9) L...

  14. Galaxy Formation

    DEFF Research Database (Denmark)

    Sparre, Martin

    Galaxy formation is an enormously complex discipline due to the many physical processes that play a role in shaping galaxies. The objective of this thesis is to study galaxy formation with two different approaches: First, numerical simulations are used to study the structure of dark matter and how...... galaxies form stars throughout the history of the Universe, and secondly it is shown that observations of gamma-ray bursts (GRBs) can be used to probe galaxies with active star formation in the early Universe. A conclusion from the hydrodynamical simulations is that the galaxies from the stateof-the-art...

  15. Non-redundant functions of two proline dehydrogenase isoforms in Arabidopsis

    OpenAIRE

    Müller Gudrun; Eckard Sonja; Funck Dietmar

    2010-01-01

    Abstract Background Proline (Pro) accumulation is a widespread response of prokaryotic and eukaryotic cells subjected to osmotic stress or dehydration. When the cells are released from stress, Pro is degraded to glutamate by Pro-dehydrogenase (ProDH) and Pyrroline-5-carboxylate dehydrogenase (P5CDH), which are both mitochondrial enzymes in eukaryotes. While P5CDH is a single copy gene in Arabidopsis, two ProDH genes have been identified in the genome. Until now, only ProDH1 (At3g30775) had be...

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

    OpenAIRE

    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...

  17. Isolated tumoral pyruvate dehydrogenase can synthesize acetoin which inhibits pyruvate oxidation as well as other aldehydes.

    Science.gov (United States)

    Baggetto, L G; Lehninger, A L

    1987-05-29

    Oxidation of 1 mM pyruvate by Ehrlich and AS30-D tumor mitochondria is inhibited by acetoin, an unusual and important metabolite of pyruvate utilization by cancer cells, by acetaldehyde, methylglyoxal and excess pyruvate. The respiratory inhibition is reversed by other substrates added to pyruvate and also by 0.5 mM ATP. Kinetic properties of pyruvate dehydrogenase complex isolated from these tumor mitochondria have been studied. This complex appears to be able to synthesize acetoin from acetaldehyde plus pyruvate and is competitively inhibited by acetoin. The role of a new regulatory pattern for tumoral pyruvate dehydrogenase is presented.

  18. Alcohol drinking habits, alcohol dehydrogenase genotypes and risk of acute coronary syndrome

    DEFF Research Database (Denmark)

    Tolstrup, J.S.; Hansen, J.L.; Gronbaek, M.;

    2010-01-01

    Aims: The risk of myocardial infarction is lower among light-to-moderate drinkers compared with abstainers. Results from some previous studies, but not all, suggest that this association is modified by variations in genes coding for alcohol dehydrogenase (ADH). We aimed to test this hypothesis......). Results: Higher alcohol intake (measured as amount or drinking frequency) was associated with lower risk of acute coronary syndrome; however, there was no evidence that these finding were modified by ADH1B or ADH1C genotypes. Conclusions: The importance of functional variation in alcohol dehydrogenase...

  19. 2-methylbutyryl-CoA dehydrogenase deficiency associated with autism and mental retardation

    DEFF Research Database (Denmark)

    Kanavin, Oivind J; Woldseth, Berit; Jellum, Egil;

    2007-01-01

    BACKGROUND: 2-methylbutyryl-CoA dehydrogenase deficiency or short/branched chain acyl-CoA dehydrogenase deficiency (SBCADD) is caused by a defect in the degradation pathway of the amino acid L-isoleucine. METHODS: We report a four-year-old mentally retarded Somali boy with autism and a history...... cases with SBCADD, both originating from Somalia and Eritrea, indicating that it is relatively prevalent in this population. Autism has not previously been described with mutations in this gene, thus expanding the clinical spectrum of SBCADD....

  20. Isolated tumoral pyruvate dehydrogenase can synthesize acetoin which inhibits pyruvate oxidation as well as other aldehydes.

    Science.gov (United States)

    Baggetto, L G; Lehninger, A L

    1987-05-29

    Oxidation of 1 mM pyruvate by Ehrlich and AS30-D tumor mitochondria is inhibited by acetoin, an unusual and important metabolite of pyruvate utilization by cancer cells, by acetaldehyde, methylglyoxal and excess pyruvate. The respiratory inhibition is reversed by other substrates added to pyruvate and also by 0.5 mM ATP. Kinetic properties of pyruvate dehydrogenase complex isolated from these tumor mitochondria have been studied. This complex appears to be able to synthesize acetoin from acetaldehyde plus pyruvate and is competitively inhibited by acetoin. The role of a new regulatory pattern for tumoral pyruvate dehydrogenase is presented. PMID:3593337

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1986-10-01

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

  2. Identification of isobutyryl-CoA dehydrogenase and its deficiency in humans

    DEFF Research Database (Denmark)

    Nguyen, Tien V; Andresen, Brage S; Corydon, Thomas J;

    2002-01-01

    The acyl-CoA dehydrogenases (ACDs) are a family of related enzymes that catalyze the alpha,beta-dehydrogenation of acyl-CoA esters. Two homologues active in branched chain amino acid metabolism have previously been identified. We have used expression in Escherichia coli to produce a previously...... targeted to mitochondria, but inactive when expressed in mammalian cells. These data confirm further the presence of a separated ACD in humans specific to valine catabolism (isobutyryl-CoA dehydrogenase, IBDH), along with the first enzymatic and molecular confirmation of a deficiency of this enzyme...

  3. 2-methylbutyryl-CoA dehydrogenase deficiency associated with autism and mental retardation: a case report

    DEFF Research Database (Denmark)

    Kanavin, Øjvind; Woldseth, Berit; Jellum, Egil;

    2007-01-01

    ABSTRACT: BACKGROUND: 2-methylbutyryl-CoA dehydrogenase deficiency or short/branched chain acyl-CoA dehydrogenase deficiency (SBCADD) is caused by a defect in the degradation pathway of the amino acid L-isoleucine. METHODS: We report a four-year-old mentally retarded Somali boy with autism...... changing the +3 position of intron 3 (c.303+3A > G) in the SBCAD gene. Psychometric testing showed moderate mental retardation and behavioral scores within the autistic spectrum. No beneficial effect was detected after 5 months with a low protein diet. CONCLUSION: This mutation was also found in two...

  4. Preliminary X-ray crystallographic study of glucose dehydrogenase from Thermus thermophilus HB8

    International Nuclear Information System (INIS)

    The glucose dehydrogenase (GDH) protein from T. thermophilus HB8 was cloned, expressed, purified and crystallized. GDH crystals belong to space group P21 and diffract to 1.9 Å resolution. Thermus thermophilus is an aerobic chemoorganotroph that has been found to grow anaerobically in the presence of nitrate. Crystals of glucose dehydrogenase (GDH) from T. thermophilus HB8 belong to space group P21, with unit-cell parameters a = 36.90, b = 132.96, c = 60.78 Å, β = 97.2°. Preliminary studies and molecular-replacement calculations reveal that the asymmetric unit contains two monomers

  5. Function, kinetic properties, crystallization, and regulation of microbial malate dehydrogenase

    Institute of Scientific and Technical Information of China (English)

    Tóshiko TAKAHASHI-ÍÑIGUEZ; Nelly ABURTO-RODRÍGUEZ; Ana Laura VILCHIS-GONZÁLEZ; María Elena FLORES

    2016-01-01

    题目:微生物苹果酸脱氢酶的功能、动力学特征、晶体结构以及调控概苹果酸脱氢酶(MDH)广泛存在于动物、植物以及微生物体内,是生物体进行糖代谢的关键酶之一。在辅酶I(NAD+)或辅酶II(NADP+)的作用下,能够催化草酰乙酸和苹果酸之间相互转化。虽然目前真核微生物中MDH已被广泛研究,但是对原核生物中的这种酶却鲜有报道。因此,有必要对MDH的相关研究信息进行综述,以期更好地了解这种酶的功能。本文综述了细菌相关研究的各种数据信息,进一步挖掘MDH的分子多样性,包括分子量、低聚态、辅因子与底物的结合力,以及酶反应方向的差异等。通过对不同细菌来源的MDH的晶体结构的分析,可鉴别底物与辅因子结合的部位以及形成二聚体的重要残基。对这些结构信息的了解将有利于指导研究人员对酶的结构进行修饰从而提高其催化能力,比如增加酶的活性、辅助因子的结合能力、底物特异性和热稳定性等。另外,本文通过分析比较MDH 系统发生树的重建,将其蛋白超家族分成两个主分支,同时在古生菌、细菌和真核微生物等不同细胞的MDH之间建立联系。%Malate dehydrogenase (MDH) is an enzyme widely distributed among living organisms and is a key protein in the central oxidative pathway. It catalyzes the interconversion between malate and oxaloacetate using NAD+ or NADP+ as a cofactor. Surprisingly, this enzyme has been extensively studied in eukaryotes but there are few reports about this enzyme in prokaryotes. It is necessary to review the relevant information to gain a better understanding of the function of this enzyme. Our review of the data generated from studies in bacteria shows much diversity in their molecular properties, including weight, oligomeric states, cofactor and substrate binding affinities, as wel as differ-ences in the direction

  6. Mutations in the genes encoding 11beta-hydroxysteroid dehydrogenase type 1 and hexose-6-phosphate dehydrogenase interact to cause cortisone reductase deficiency.

    Science.gov (United States)

    Draper, Nicole; Walker, Elizabeth A; Bujalska, Iwona J; Tomlinson, Jeremy W; Chalder, Susan M; Arlt, Wiebke; Lavery, Gareth G; Bedendo, Oliver; Ray, David W; Laing, Ian; Malunowicz, Ewa; White, Perrin C; Hewison, Martin; Mason, Philip J; Connell, John M; Shackleton, Cedric H L; Stewart, Paul M

    2003-08-01

    In cortisone reductase deficiency (CRD), activation of cortisone to cortisol does not occur, resulting in adrenocorticotropin-mediated androgen excess and a phenotype resembling polycystic ovary syndrome (PCOS; refs. 1,2). This suggests a defect in the gene HSD11B1 encoding 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1), a primary regulator of tissue-specific glucocorticoid bioavailability. We identified intronic mutations in HSD11B1 that resulted in reduced gene transcription in three individuals with CRD. In vivo, 11beta-HSD1 catalyzes the reduction of cortisone to cortisol whereas purified enzyme acts as a dehydrogenase converting cortisol to cortisone. Oxo-reductase activity can be regained using a NADPH-regeneration system and the cytosolic enzyme glucose-6-phosphate dehydrogenase. But the catalytic domain of 11beta-HSD1 faces into the lumen of the endoplasmic reticulum (ER; ref. 6). We hypothesized that endolumenal hexose-6-phosphate dehydrogenase (H6PDH) regenerates NADPH in the ER, thereby influencing directionality of 11beta-HSD1 activity. Mutations in exon 5 of H6PD in individuals with CRD attenuated or abolished H6PDH activity. These individuals have mutations in both HSD11B1 and H6PD in a triallelic digenic model of inheritance, resulting in low 11beta-HSD1 expression and ER NADPH generation with loss of 11beta-HSD1 oxo-reductase activity. CRD defines a new ER-specific redox potential and establishes H6PDH as a potential factor in the pathogenesis of PCOS. PMID:12858176

  7. Lactate dehydrogenase from Streptococcus mutans: purification, characterization, and crossed antigenicity with lactate dehydrogenases from Lactobacillus casei, Actinomyces viscosus, and Streptococcus sanguis.

    OpenAIRE

    Sommer, P; Klein, J P; Schöller, M; Van Frank, R M

    1985-01-01

    A cytoplasmic fructose-1,6-diphosphate-dependent lactate dehydrogenase (LDH; EC 1.1.1.27) from Streptococcus mutans OMZ175 was purified to homogeneity as judged by sodium dodecyl sulfate-gel electrophoresis. The purification consisted of ammonium sulfate precipitation of the cytoplasmic fraction, DEAE-Sephacel and Blue-Sepharose CL.6B chromatography, and Sephacryl S200 gel permeation. The catalytic activity of the purified enzyme required the presence of fructose-1,6-diphosphate with a broad ...

  8. Two Zebrafish Alcohol Dehydrogenases Share Common Ancestry with Mammalian Class I, II, IV, and V Alcohol Dehydrogenase Genes but Have Distinct Functional Characteristics*

    OpenAIRE

    Reimers, Mark J.; Hahn, Mark E.; Tanguay, Robert L.

    2004-01-01

    Ethanol is teratogenic to many vertebrates. We are utilizing zebrafish as a model system to determine whether there is an association between ethanol metabolism and ethanol-mediated developmental toxicity. Here we report the isolation and characterization of two cDNAs encoding zebrafish alcohol dehydrogenases (ADHs). Phylogenetic analysis of these zebrafish ADHs indicates that they share a common ancestor with mammalian class I, II, IV, and V ADHs. The genes encoding these zebrafish ADHs have...

  9. Bulge Formation

    CERN Document Server

    Combes, F

    1999-01-01

    The currently discussed theories of bulge formation are reviewed, including the primordial scenario, where bulges form rapidly and then accrete disks, the secular scenario, where bulges are formed by dynamical evolution of disks through bars and galaxy interactions, and some combinations of both, where formation of bulges and disks are more continuous and interleaved. The various scenarios make specific predictions about the relative masses, angular momenta, colours, metallicities of bulges relative to disks, and the bulge-to-disk ratio as a function of time. Dynamical processes relevant to the formation of bulges (bar instabilities, mergers) are described and tested against observed statistics. Current data suggest a dynamical feedback from gravitational instabilities in bulge and disk formation. It is very difficult to discriminate between the various scenarios from surveys at z=0 only, and observations at high redshift are presently the best hope for large progress.

  10. The activity of class I, II, III and IV of alcohol dehydrogenase (ADH) isoenzymes and aldehyde dehydrogenase (ALDH) in brain cancer.

    Science.gov (United States)

    Laniewska-Dunaj, Magdalena; Jelski, Wojciech; Orywal, Karolina; Kochanowicz, Jan; Rutkowski, Robert; Szmitkowski, Maciej

    2013-07-01

    The brain being highly sensitive to the action of alcohol is potentially susceptible to its carcinogenic effects. Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) are the main enzymes involved in ethanol metabolism, which leads to the generation of carcinogenic acetaldehyde. Human brain tissue contains various ADH isoenzymes and possess also ALDH activity. The purpose of this study was to compare the capacity for ethanol metabolism measured by ADH isoenzymes and ALDH activity in cancer tissues and healthy brain cells. The samples were taken from 62 brain cancer patients (36 glioblastoma, 26 meningioma). For the measurement of the activity of class I and II ADH isoenzymes and ALDH activity, the fluorometric methods were used. The total ADH activity and activity of class III and IV isoenzymes were measured by the photometric method. The total activity of ADH, and activity of class I ADH were significantly higher in cancer cells than in healthy tissues. The other tested classes of ADH and ALDH did not show statistically significant differences of activity in cancer and in normal cells. Analysis of the enzymes activity did not show significant differences depending on the location of the tumor. The differences in the activity of total alcohol dehydrogenase, and class I isoenzyme between cancer tissues and healthy brain cells might be a factor for metabolic changes and disturbances in low mature cancer cells and additionally might be a reason for higher level of acetaldehyde which can intensify the carcinogenesis.

  11. Cement Formation

    DEFF Research Database (Denmark)

    Telschow, Samira; Jappe Frandsen, Flemming; Theisen, Kirsten;

    2012-01-01

    Cement production has been subject to several technological changes, each of which requires detailed knowledge about the high multiplicity of processes, especially the high temperature process involved in the rotary kiln. This article gives an introduction to the topic of cement, including......−liquid reactions are discussed, as are the influences of particles sizes on clinker phase formation. Furthermore, a mechanism for clinker phase formation in an industrial rotary kiln reactor is outlined....

  12. Analysis of Quaternary Structure of a [LDH-like] Malate Dehydrogenase of Plasmodium falciparum with Oligomeric Mutants

    Science.gov (United States)

    L-Malate dehydrogenase (PfMDH) from Plasmodium falciparum, the causative agent for the most severe form of malaria, has shown remarkable similarities to L-lactate dehydrogenase (PfLDH). PfMDH is more closely related to [LDH-like] MDHs characterized in archea and other prokaryotes. Initial sequence a...

  13. Metabolic Engineering of Mannitol Production in Lactococcus lactis: Influence of Overexpression of Mannitol 1-Phosphate Dehydrogenase in Different Genetic Backgrounds

    NARCIS (Netherlands)

    Wisselink, H.W.; Mars, A.E.; Meer, van der P.; Eggink, G.; Hugenholtz, J.

    2004-01-01

    To obtain a mannitol-producing Lactococcus lactis strain, the mannitol 1-phosphate dehydrogenase gene (mtlD) from Lactobacillus plantarum was overexpressed in a wild-type strain, a lactate dehydrogenase(LDH)-deficient strain, and a strain with reduced phosphofructokinase activity. High-performance l

  14. Catalytic mechanism of Zn2+-dependent polyol dehydrogenases: kinetic comparison of sheep liver sorbitol dehydrogenase with wild-type and Glu154→Cys forms of yeast xylitol dehydrogenase

    Science.gov (United States)

    Klimacek, Mario; Hellmer, Heidemarie; Nidetzky, Bernd

    2007-01-01

    Co-ordination of catalytic Zn2+ in sorbitol/xylitol dehydrogenases of the medium-chain dehydrogenase/reductase superfamily involves direct or water-mediated interactions from a glutamic acid residue, which substitutes a homologous cysteine ligand in alcohol dehydrogenases of the yeast and liver type. Glu154 of xylitol dehydrogenase from the yeast Galactocandida mastotermitis (termed GmXDH) was mutated to a cysteine residue (E154C) to revert this replacement. In spite of their variable Zn2+ content (0.10–0.40 atom/subunit), purified preparations of E154C exhibited a constant catalytic Zn2+ centre activity (kcat) of 1.19±0.03 s−1 and did not require exogenous Zn2+ for activity or stability. E154C retained 0.019±0.003% and 0.74±0.03% of wild-type catalytic efficiency (kcat/Ksorbitol=7800±700 M−1· s−1) and kcat (=161±4 s−1) for NAD+-dependent oxidation of sorbitol at 25 °C respectively. The pH profile of kcat/Ksorbitol for E154C decreased below an apparent pK of 9.1±0.3, reflecting a shift in pK by about +1.7–1.9 pH units compared with the corresponding pH profiles for GmXDH and sheep liver sorbitol dehydrogenase (termed slSDH). The difference in pK for profiles determined in 1H2O and 2H2O solvent was similar and unusually small for all three enzymes (≈+0.2 log units), suggesting that the observed pK in the binary enzyme–NAD+ complexes could be due to Zn2+-bound water. Under conditions eliminating their different pH-dependences, wild-type and mutant GmXDH displayed similar primary and solvent deuterium kinetic isotope effects of 1.7±0.2 (E154C, 1.7±0.1) and 1.9±0.3 (E154C, 2.4±0.2) on kcat/Ksorbitol respectively. Transient kinetic studies of NAD+ reduction and proton release during sorbitol oxidation by slSDH at pH 8.2 show that two protons are lost with a rate constant of 687±12 s−1 in the pre-steady state, which features a turnover of 0.9±0.1 enzyme equivalents as NADH was produced with a rate constant of 409±3 s−1. The

  15. Streptomyces coelicolor XdhR is a direct target of (p)ppGpp that controls expression of genes encoding xanthine dehydrogenase to promote purine salvage.

    Science.gov (United States)

    Sivapragasam, Smitha; Grove, Anne

    2016-05-01

    The gene encoding Streptomyces coelicolor xanthine dehydrogenase regulator (XdhR) is divergently oriented from xdhABC, which encodes xanthine dehydrogenase (Xdh). Xdh is required for purine salvage pathways. XdhR was previously shown to repress xdhABC expression. We show that XdhR binds the xdhABC-xdhR intergenic region with high affinity (Kd ∼ 0.5 nM). DNaseI footprinting reveals that this complex formation corresponds to XdhR binding the xdhR gene promoter at two adjacent sites; at higher protein concentrations, protection expands to a region that overlaps the transcriptional and translational start sites of xdhABC. While substrates for Xdh have little effect on DNA binding, GTP and ppGpp dissociate the DNA-XdhR complex. Progression of cells to stationary phase, a condition associated with increased (p)ppGpp production, leads to elevated xdhB expression; in contrast, inhibition of Xdh by allopurinol results in xdhB repression. We propose that XdhR is a direct target of (p)ppGpp, and that expression of xdhABC is upregulated during the stringent response to promote purine salvage pathways, maintain GTP homeostasis and ensure continued (p)ppGpp synthesis. During exponential phase growth, basal levels of xdhABC expression may be achieved by GTP serving as a lower-affinity XdhR ligand.

  16. Increasing stability of water-soluble PQQ glucose dehydrogenase by increasing hydrophobic interaction at dimeric interface

    Directory of Open Access Journals (Sweden)

    Ferri Stefano

    2005-02-01

    Full Text Available Abstract Background Water-soluble quinoprotein glucose dehydrogenase (PQQGDH-B from Acinetobacter calcoaceticus has a great potential for application as a glucose sensor constituent. Because this enzyme shows no activity in its monomeric form, correct quaternary structure is essential for the formation of active enzyme. We have previously reported on the increasing of the stability of PQQGDH-B by preventing the subunit dissociation. Previous studies were based on decreasing the entropy of quaternary structure dissociation but not on increasing the interaction between the two subunits. We therefore attempted to introduce a hydrophobic interaction in the dimeric interface to increase the stability of PQQGDH-B. Results Amino acid residues Asn340 and Tyr418 face each other at the dimer interface of PQQGDH-B, however no interaction exists between their side chains. We simultaneously substituted Asn340 to Phe and Tyr418 to Phe or Ile, to create the two mutants Asn340Phe/Tyr418Phe and Asn340Phe/Tyr418Ile. Furthermore, residues Leu280, Val282 and Val342 form a hydrophobic region that faces, on the other subunit, residues Thr416 and Thr417, again without any specific interaction. We simultaneously substituted Thr416 and Thr417 to Val, to create the mutant Thr416Val/Thr417Val. The temperatures resulting in lose of half of the initial activity of the constructed mutants were increased by 3–4°C higher over wild type. All mutants showed 2-fold higher thermal stability at 55°C than the wild-type enzyme, without decreasing their catalytic activities. From the 3D models of all the mutant enzymes, the predicted binding energies were found to be significantly greater that in the wild-type enzyme, consistent with the increases in thermal stabilities. Conclusions We have achieved via site-directed mutagenesis the improvement of the thermal stability of PQQGDH-B by increasing the dimer interface interaction. Through rational design based on the quaternary

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

    DEFF Research Database (Denmark)

    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...

  18. Mutations in the medium chain acyl-CoA dehydrogenase (MCAD) gene

    DEFF Research Database (Denmark)

    Tanaka, K; Yokota, I; Coates, P M;

    1992-01-01

    Medium chain acyl-CoA dehydrogenase (MCAD) catalyzes the first reaction of the beta-oxidation cycle for 4-10-carbon fatty acids. MCAD deficiency is one of the most frequent inborn metabolic disorders in populations of northwestern European origin. In the compilation of data from a worldwide study...

  19. Newborn screening for dihydrolipoamide dehydrogenase deficiency: Citrulline as a useful analyte

    Directory of Open Access Journals (Sweden)

    Shane C. Quinonez

    2014-01-01

    Full Text Available Dihydrolipoamide dehydrogenase deficiency, also known as maple syrup urine disease (MSUD type III, is caused by the deficiency of the E3 subunit of branched chain alpha-ketoacid dehydrogenase (BCKDH, α-ketoglutarate dehydrogenase (αKGDH, and pyruvate dehydrogenase (PDH. DLD deficiency variably presents with either a severe neonatal encephalopathic phenotype or a primarily hepatic phenotype. As a variant form of MSUD, it is considered a core condition recommended for newborn screening. The detection of variant MSUD forms has proven difficult in the past with no asymptomatic DLD deficiency patients identified by current newborn screening strategies. Citrulline has recently been identified as an elevated dried blood spot (DBS metabolite in symptomatic patients affected with DLD deficiency. Here we report the retrospective DBS analysis and second-tier allo-isoleucine testing of 2 DLD deficiency patients. We show that an elevated citrulline and an elevated allo-isoleucine on second-tier testing can be used to successfully detect DLD deficiency. We additionally recommend that DLD deficiency be included in the “citrullinemia/elevated citrulline” ACMG Act Sheet and Algorithm.

  20. Optimization, Application, and Interpretation of Lactate Dehydrogenase Measurements in Microwell Determination of Cell Number and Toxicity

    NARCIS (Netherlands)

    Wolterbeek, H.T.; Van der Meer, A.J.G.M.

    2005-01-01

    The lactate dehydrogenase (LDH) assay was addressed for its sensitivity, disturbances by foaming, and cell number and size. Cells were from a U-251 MG grade IV human glioblastoma brain tumor cell line used in 100-µl well volumes. Cells were counted by microscopy and Coulter counting; assays were LDH

  1. Molecular modeling studies of L-arabinitol 4-dehydrogenase of Hypocrea jecorina

    DEFF Research Database (Denmark)

    Tiwari, Manish; Lee, Jung-Kul

    2010-01-01

    in order to provide better insight into the possible catalytic events in these domains. The 3D structure of NAD(+)-dependent LAD1 was developed based on the crystal structure of human sorbitol dehydrogenase as a template. A series of molecular mechanics and dynamics operations were performed to find...

  2. Furoates and thenoates inhibit pyruvate dehydrogenase kinase 2 allosterically by binding to its pyruvate regulatory site

    NARCIS (Netherlands)

    Masini, Tiziana; Birkaya, Barbara; van Dijk, Simon; Mondal, Milon; Hekelaar, Johan; Jäger, Manuel; Terwisscha van Scheltinga, Anke C; Patel, Mulchand S; Hirsch, Anna K H; Moman, Edelmiro

    2016-01-01

    The last decade has witnessed the reawakening of cancer metabolism as a therapeutic target. In particular, inhibition of pyruvate dehydrogenase kinase (PDK) holds remarkable promise. Dichloroacetic acid (DCA), currently undergoing clinical trials, is a unique PDK inhibitor in which it binds to the a

  3. Unexpected Discovery of Dichloroacetate Derived Adenosine Triphosphate Competitors Targeting Pyruvate Dehydrogenase Kinase To Inhibit Cancer Proliferation.

    Science.gov (United States)

    Zhang, Shao-Lin; Hu, Xiaohui; Zhang, Wen; Tam, Kin Yip

    2016-04-14

    Pyruvate dehydrogenase kinases (PDKs) have recently emerged as an attractive target for cancer therapy. Herein, we prepared a series of compounds derived from dichloroacetate (DCA) which inhibited cancer cells proliferation. For the first time, we have successfully developed DCA derived inhibitors that preferentially bind to the adenosine triphosphate (ATP) pocket of PDK isoform 1 (PDK1).

  4. ISOZYME PROFILES OF LACTIC DEHYDROGENASE AND CREATINE PHOSPHOKINASE IN NEONATAL MOUSE HEARTS

    Science.gov (United States)

    Isozyme profiles of lactic dehydrogenase (LDH) and creatine phosphokinase (CPK) were determined in cardiac tissue of mice during postnatal development. LDH isozymes 1 and 5 showed a definite developmental change, achieving the adult values by 20 days of age, while the other three...

  5. High energy electron beam inactivation of lactate dehydrogenase suspended in different aqueous media

    International Nuclear Information System (INIS)

    The direct and indirect effects of 5 MeV electron beam irradiation at various low temperatures, as well as the influence of the presence or absence of deuterium ions in the suspending medium of the enzyme, on the global enzymatic activity of lactate dehydrogenase have been studied. Frozen lactate dehydrogenase suspensions at 0 degC, -3 degC and -196 degC temperatures have been irradiated with the 5 MeV electron beam of a linear accelerator in the dose range 0-400 Gy. Liquid lactate dehydrogenase suspensions in D2O (99.98 %) and ultrapure water (17 ppm) at 0 degC have been irradiated in the dose range 0 -15 Gy. An exponential decrease was found in the enzymatic activity of irradiated lactate dehydrogenase, at all irradiation temperatures. The drastic decrease in the activity for the enzyme irradiated at 0 degC (total inhibition for a final dose of 100 Gy) indicate that at this temperature the indirect effects of radiation (due to the water radicals induced by radiation in the samples) are predominant. At -3 degC irradiation temperature the indirect effects of radiation are smaller but still present (a total decrease in the enzymatic activity for a dose of 250 Gy), while at -196 degC they are orders of magnitude reduced and the decrease in the enzymatic activity is due almost to the direct interaction of electrons with the macromolecules (70 % for a dose of 400 Gy)

  6. Alcohol dehydrogenase 3 genotype as a risk factor for upper aerodigestive tract cancers

    DEFF Research Database (Denmark)

    Nishimoto, Inês Nobuko; Pinheiro, Nidia A; Rogatto, Silvia R;

    2004-01-01

    OBJECTIVE: To assess alcohol dehydrogenase 3 (ADH3) polymorphism at position Ile349Val as indicator of risk factor for upper aerodigestive tract (UADT) cancer to verify its association with UADT cancer in nonalcoholic or nonsmoking individuals. DESIGN: Cross-sectional study. SETTING: Primary care...

  7. Exercise-induced pyruvate dehydrogenase activation is not affected by 7 days of bed rest

    DEFF Research Database (Denmark)

    Kiilerich, Kristian; Jørgensen, Stine Ringholm; Biensø, Rasmus Sjørup;

    2011-01-01

    To test the hypothesis that physical inactivity impairs the exercise-induced modulation of pyruvate dehydrogenase (PDH), 6 healthy normally physically active male subjects completed 7 days of bed rest. Before and immediately after the bed rest, the subjects completed an OGTT and a one-legged knee...

  8. Cloning and characterization of a thermostable xylitol dehydrogenase from Rhizobium etli CFN42

    DEFF Research Database (Denmark)

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

    2010-01-01

    An NAD(+)-dependent xylitol dehydrogenase from Rhizobium etli CFN42 (ReXDH) was cloned and overexpressed in Escherichia coli. The DNA sequence analysis revealed an open reading frame of 1,044 bp, capable of encoding a polypeptide of 347 amino acid residues with a calculated molecular mass of 35...

  9. Glucose-6-phosphate dehydrogenase-derived NADPH fuels superoxide production in the failing heart

    Science.gov (United States)

    In the failing heart, NADPH oxidase and uncoupled NO synthase utilize cytosolic NADPH to form superoxide. NADPH is supplied principally by the pentose phosphate pathway, whose rate-limiting enzyme is glucose 6-phosphate dehydrogenase (G6PD). Therefore, we hypothesized that cardiac G6PD activation dr...

  10. Myopathy in very-long-chain acyl-CoA dehydrogenase deficiency

    DEFF Research Database (Denmark)

    Scholte, H R; Van Coster, R N; de Jonge, P C;

    1999-01-01

    A 30-year-old man suffered since the age of 13 years from exercise induced episodes of intense generalised muscle pain, weakness and myoglobinuria. Fasting ketogenesis was low, while blood glucose remained normal. Muscle mitochondria failed to oxidise palmitoylcarnitine. Palmitoyl-CoA dehydrogenase...

  11. Separation and Purification of Betaine Aldehyde Dehydrogenase from Wild Suaeda liaotungensis

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    High active betaine aldehyde dehydrogenase (BADH, EC 1.2.1.8) is found in wild Suaeda liaotungensis. The enzyme is purified 206-fold with recovery of 1.5%. It have a specific activity of 2363 nmol/min*mg protein and the molecular mass of each subunit is 64.5 kDa as determined by SDS-PAGE.

  12. Novel approaches for using dehydrogenases and ene-reductases for organic synthesis

    NARCIS (Netherlands)

    Gargiulo, S.

    2015-01-01

    Oxidation of alcohols is a reaction of major interest for organic chemistry. However, the most common chemical routes developed so far involve the use of toxic or hazardous reagents or catalysts that often lack good chemoselectivity. In this respect, alcohol dehydrogenases (ADHs) represent a very va

  13. Growth hormone-induced insulin resistance in human subjects involves reduced pyruvate dehydrogenase activity

    DEFF Research Database (Denmark)

    Nellemann, B.; Vendelbo, M.H.; Nielsen, Thomas Svava;

    2014-01-01

    Insulin resistance induced by growth hormone (GH) is linked to promotion of lipolysis by unknown mechanisms. We hypothesized that suppression of the activity of pyruvate dehydrogenase in the active form (PDHa) underlies GH-induced insulin resistance similar to what is observed during fasting....

  14. Immunolocalization of succinate dehydrogenase in the esophagus epithelium of domesticated mammals

    OpenAIRE

    Meyer, W.; Kacza, J.; I. N. Hornickel; Schoennagel, B.

    2013-01-01

    Using immunohistochemistry and transmission electron microscopy (TEM), the esophagus epithelia of seven domesticated mammals (horse, cattle, goat, pig, dog, laboratory rat, cat) of three nutrition groups (herbivorous, omnivorous, carnivorous) were studied to get first information about energy generation, as demonstrated by succinate dehydrogenase (SDH) activities. Distinct reaction intensities could be observed in all esophageal cell layers of the different species studied reflecting moderate...

  15. Inhibition of dehydrogenase activity in petroleum refinery wastewater bacteria by phenolic compounds

    Directory of Open Access Journals (Sweden)

    Gideon C. Okpokwasili

    2010-04-01

    Full Text Available The toxicity of phenol, 2-nitrophenol, 4-nitrophenol, 2,4-dinitrophenol, 2-chlorophenol, 4-chlorophenol, 4-bromophenol and 3,5-dimethylphenol on Pseudomonas, Bacillus and Escherichia species isolated from petroleum refinery wastewater was assessed via inhibition of dehydrogenase enzyme activity. At low concentrations, 2-nitrophenol, 2-chlorophenol, 4-chlorophenol, 4-bromophenol and 3,5-dimethylphenol stimulated dehydrogenase activity and at sufficient concentrations, phenolic compounds inhibited dehydrogenase activities. Generally, phenol is less toxic than substituted phenols. Estimations of the degree of inhibition/stimulation of dehydrogenase activities showed significant dose-dependent responses that are describable by logistic functions. The toxicity thresholds varied significantly (P < 0.05 among the bacterial strains and phenolic compounds. The median inhibitory concentrations (IC50s ranged from 4.118 ± 0.097 mg.L-1 for 4-nitrophenol against Pseudomonas sp. DAF1 to 1407.997 ± 7.091 mg.L-1 for phenol against Bacillus sp. DISK1. This study suggested that the organisms have moderate sensitivity to phenols and have the potential to be used as indicators for assessment of chemical toxicity. They could also be used as catalysts for degradation of phenols in effluents.

  16. THE CONFORMATIONAL STABILITY OF THE REDOX STATES OF LIPOAMIDE DEHYDROGENASE FROM AZOTOBACTER-VINELANDII

    NARCIS (Netherlands)

    VANBERKEL, WJH; REGELINK, AG; BEINTEMA, JJ; KOK, A

    1991-01-01

    The conformational stability of holo-lipoamide and apo-lipoamide dehydrogenase from Azotobacter vinelandii was studied by thermoinactivation, unfolding and limited proteolysis. The oxidized holoenzyme is thermostable, showing a melting temperature, t(m) = 80-degrees-C. The thermal stability of the h

  17. Often Ignored Facts about the Control of the 2-Oxoglutarate Dehydrogenase Complex

    Science.gov (United States)

    Strumilo, Slawomir

    2005-01-01

    Information about the control of the activity of the 2-oxoglutarate dehydrogenase complex (OGDHC), a key enzyme in the citric acid cycle, is not well covered in the biochemical education literature, especially as it concerns the allosteric regulation of OGDHC by adenine nucleotide and ortophosphate. From experimental work published during the last…

  18. Modification of Rhizopus lactate dehydrogenase for improved resistance to fructose 1,6-bisphosphate

    Science.gov (United States)

    Rhizopus oryzae is frequently used for fermentative production of lactic acid. We determined that one of the key enzymes, lactate dehydrogenase (LDH), involved in synthesis of lactic acid by R. oryzae was significantly inhibited by fructose 1,6-bisphosphate (FBP) at physiological concentrations. Thi...

  19. Angiotensin administration stimulates renal 11 beta-hydroxysteroid dehydrogenase activity in healthy men

    NARCIS (Netherlands)

    Kerstens, MN; van der Kleij, FGH; Boonstra, AH; Sluiter, WJ; van der Molen, JC; Navis, G; Dullaart, RPF

    2004-01-01

    Background. We examined whether acute administration of angiotensin modulates the activity of 11beta-hydroxysteroid dehydrogenase (11betaHSD), the intracellular enzyme catalyzing the interconversion between the hormonally active cortisol and inactive cortisone. Methods. Twenty-one male healthy subje

  20. Prevalence of Long-Chain 3-Hydroxyacyl-CoA Dehydrogenase Deficiency in Estonia

    DEFF Research Database (Denmark)

    Joost, K; Ounap, K; Zordania, R;

    2012-01-01

    The aim of our study was to evaluate the prevalence of long chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD) in the general Estonian population and among patients with symptoms suggestive of fatty acid oxidation (FAO) defects. We collected DNA from a cohort of 1,040 anonymous newborn blo...... prevalence of LCHADD in Estonia would be 1: 91,700....

  1. Medium chain acyl-CoA dehydrogenase deficiency and fatal valproate toxicity

    NARCIS (Netherlands)

    Njolstad, PR; Skjeldal, OH; Agsteribbe, E; Huckriede, A; Wannag, E; Sovik, O; Waaler, PE

    1997-01-01

    A boy with delayed psychomotor development, attention deficit disorder, and therapy-resistant epilepsy was treated with valproate. The patient died of liver failure after 4 months of valproate treatment. Postmortem investigation of cultured fibroblasts suggested medium chain acyl-CoA dehydrogenase d

  2. Catalytic reaction of cytokinin dehydrogenase : preference for quinones as electron acceptors

    NARCIS (Netherlands)

    Frébortová, Jitka; Fraaije, Marco W.; Galuszka, Petr; Šebela, Marek; Peč, Pavel; Hrbáč, Jan; Novák, Ondřej; Bilyeu, Kristin D.; English, James T.; Frébort, Ivo; Sebela, M.; Pec, P.; Hrbac, J.; Frebort, [No Value

    2004-01-01

    The catalytic reaction of cytokinin oxidase/dehydrogenase (EC 1.5.99.12) was studied in detail using the recombinant flavoenzyme from maize. Determination of the redox potential of the covalently linked flavin cofactor revealed a relatively high potential dictating the type of electron acceptor that

  3. Watermelon glyoxysomal malate dehydrogenase is sorted to peroxisomes of the methylotrophic yeast, Hansenula polymorpha

    NARCIS (Netherlands)

    Klei, I.J. van der; Faber, K.N.; Keizer-Gunnink, I.; Gietl, C.; Harder, W.; Veenhuis, M.

    1993-01-01

    We have studied the fate of the watermelon (Citrullus vulgaris Schrad.) glyoxysomal enzyme, malate dehydrogenase (gMDH), after synthesis in the methylotrophic yeast, Hansenula polymorpha. The gene encoding the precursor form of gMDH (pre-gMDH) was cloned in an H. polymorpha expression vector downstr

  4. The intracellular localization of malate dehydrogenase isoenzymes in Pisum arvense roots

    Directory of Open Access Journals (Sweden)

    Genowefa Kubik-Dorosz

    2014-02-01

    Full Text Available Mitochondria and plastids were isolated from Pisum arvense root cells by sucrose density gradient centrifugation. The individual subcellular fractions so obtained were subjected to isoelectric focusing on cellulose acetate strips. Mitochondria and plastids each contained one NAD -malate dehydrogenase, while three isoenzymes were associated with the supernatant.

  5. Communication between L-galactono-¿-lactone dehydrogenase and cytochrome c.

    NARCIS (Netherlands)

    Hervas, M.; Bashir, Q.; Leferink, N.G.H.; Ferreira, P.; Moreno-Beltran, J.B.; Westphal, A.H.; Diaz Moreno, I.; Medina, M.; La Rosa, De M.A.; Ubbink, M.; Navarro, J.A.; Berkel, van W.J.H.

    2013-01-01

    l-galactono-1,4-lactone dehydrogenase (GALDH) catalyzes the terminal step of vitamin C biosynthesis in plant mitochondria. Here we investigated the communication between Arabidopsis thaliana GALDH and its natural electron acceptor cytochrome c (Cc). Using laser-generated radicals we observed the for

  6. The Alcohol Dehydrogenase Kinetics Laboratory: Enhanced Data Analysis and Student-Designed Mini-Projects

    Science.gov (United States)

    Silverstein, Todd P.

    2016-01-01

    A highly instructive, wide-ranging laboratory project in which students study the effects of various parameters on the enzymatic activity of alcohol dehydrogenase has been adapted for the upper-division biochemistry and physical biochemistry laboratory. Our two main goals were to provide enhanced data analysis, featuring nonlinear regression, and…

  7. Star formation

    Energy Technology Data Exchange (ETDEWEB)

    Woodward, P.R.

    1978-09-27

    Theoretical models of star formation are discussed beginning with the earliest stages and ending in the formation of rotating, self-gravitating disks or rings. First a model of the implosion of very diffuse gas clouds is presented which relies upon a shock at the edge of a galactic spiral arm to drive the implosion. Second, models are presented for the formation of a second generation of massive stars in such a cloud once a first generation has formed. These models rely on the ionizing radiation from massive stars or on the supernova shocks produced when these stars explode. Finally, calculations of the gravitational collapse of rotating clouds are discussed with special focus on the question of whether rotating disks or rings are the result of such a collapse. 65 references.

  8. Star formation

    International Nuclear Information System (INIS)

    Theoretical models of star formation are discussed beginning with the earliest stages and ending in the formation of rotating, self-gravitating disks or rings. First a model of the implosion of very diffuse gas clouds is presented which relies upon a shock at the edge of a galactic spiral arm to drive the implosion. Second, models are presented for the formation of a second generation of massive stars in such a cloud once a first generation has formed. These models rely on the ionizing radiation from massive stars or on the supernova shocks produced when these stars explode. Finally, calculations of the gravitational collapse of rotating clouds are discussed with special focus on the question of whether rotating disks or rings are the result of such a collapse. 65 references

  9. Diverging regulation of pyruvate dehydrogenase kinase isoform gene expression in cultured human muscle cells.

    Science.gov (United States)

    Abbot, Emily L; McCormack, James G; Reynet, Christine; Hassall, David G; Buchan, Kevin W; Yeaman, Stephen J

    2005-06-01

    The pyruvate dehydrogenase complex occupies a central and strategic position in muscle intermediary metabolism and is primarily regulated by phosphorylation/dephosphorylation. The identification of multiple isoforms of pyruvate dehydrogenase kinase (PDK1-4) and pyruvate dehydrogenase phosphatase (PDP1-2) has raised intriguing new possibilities for chronic pyruvate dehydrogenase complex control. Experiments to date suggest that PDK4 is the major isoenzyme responsible for changes in pyruvate dehydrogenase complex activity in response to various different metabolic conditions. Using a cultured human skeletal muscle cell model system, we found that expression of both PDK2 and PDK4 mRNA is upregulated in response to glucose deprivation and fatty acid supplementation, the effects of which are reversed by insulin treatment. In addition, insulin directly downregulates PDK2 and PDK4 mRNA transcript abundance via a phosphatidylinositol 3-kinase-dependent pathway, which may involve glycogen synthase kinase-3 but does not utilize the mammalian target of rapamycin or mitogen-activated protein kinase signalling pathways. In order to further elucidate the regulation of PDK, the role of the peroxisome proliferators-activated receptors (PPAR) was investigated using highly potent subtype selective agonists. PPARalpha and PPARdelta agonists were found to specifically upregulate PDK4 mRNA expression, whereas PPARgamma activation selectively decreased PDK2 mRNA transcript abundance. PDP1 mRNA expression was unaffected by all conditions analysed. These results suggest that in human muscle, hormonal and nutritional conditions may control PDK2 and PDK4 mRNA expression via a common signalling mechanism. In addition, PPARs appear to independently regulate specific PDK isoform transcipt levels, which are likely to impart important metabolic mediation of fuel utilization by the muscle. PMID:15955060

  10. Physiological covalent regulation of rat liver branched-chain alpha-ketoacid dehydrogenase

    International Nuclear Information System (INIS)

    A radiochemical assay was developed for measuring branched-chain alpha-ketoacid dehydrogenase activity of Triton X-100 extracts of freeze-clamped rat liver. The proportion of active (dephosphorylated) enzyme was determined by measuring enzyme activities before and after activation of the complex with a broad-specificity phosphoprotein phosphatase. Hepatic branched-chain alpha-ketoacid dehydrogenase activity in normal male Wistar rats was 97% active but decreased to 33% active after 2 days on low-protein (8%) diet and to 13% active after 4 days on the same diet. Restricting protein intake of lean and obese female Zucker rats also caused inactivation of hepatic branched-chain alpha-ketoacid dehydrogenase complex. Essentially all of the enzyme was in the active state in rats maintained for 14 days on either 30 or 50% protein diets. This was also the case for rats maintained on a commercial chow diet (minimum 23% protein). However, maintaining rats on 20, 8, and 0% protein diets decreased the percentage of the active form of the enzyme to 58, 10, and 7% of the total, respectively. Fasting of chow-fed rats for 48 h had no effect on the activity state of hepatic branched-chain alpha-ketoacid dehydrogenase, i.e., 93% of the enzyme remained in the active state compared to 97% for chow-fed rats. However, hepatic enzyme of rats maintained on 8% protein diet was 10% active before starvation and 83% active after 2 days of starvation. Thus, dietary protein deficiency results in inactivation of hepatic branched-chain alpha-ketoacid dehydrogenase complex, presumably as a consequence of low hepatic levels of branched-chain alpha-ketoacids

  11. Pyruvate dehydrogenase kinase 2 and 4 gene deficiency attenuates nociceptive behaviors in a mouse model of acute inflammatory pain.

    Science.gov (United States)

    Jha, Mithilesh Kumar; Rahman, Md Habibur; Park, Dong Ho; Kook, Hyun; Lee, In-Kyu; Lee, Won-Ha; Suk, Kyoungho

    2016-09-01

    Pyruvate dehydrogenase (PDH) kinases (PDKs) 1-4, expressed in peripheral and central tissues, regulate the activity of the PDH complex (PDC). The PDC is an important mitochondrial gatekeeping enzyme that controls cellular metabolism. The role of PDKs in diverse neurological disorders, including neurometabolic aberrations and neurodegeneration, has been described. Implications for a role of PDKs in inflammation and neurometabolic coupling led us to investigate the effect of genetic ablation of PDK2/4 on nociception in a mouse model of acute inflammatory pain. Deficiency in Pdk2 and/or Pdk4 in mice led to attenuation of formalin-induced nociceptive behaviors (flinching, licking, biting, or lifting of the injected paw). Likewise, the pharmacological inhibition of PDKs substantially diminished the nociceptive responses in the second phase of the formalin test. Furthermore, formalin-provoked paw edema formation and mechanical and thermal hypersensitivities were significantly reduced in Pdk2/4-deficient mice. Formalin-driven neutrophil recruitment at the site of inflammation, spinal glial activation, and neuronal sensitization were substantially lessened in the second or late phase of the formalin test in Pdk2/4-deficient animals. Overall, our results suggest that PDK2/4 can be a potential target for the development of pharmacotherapy for the treatment of acute inflammatory pain. © 2016 Wiley Periodicals, Inc. PMID:26931482

  12. Substitution of valine for histidine 265 in carbon monoxide dehydrogenase from Rhodospirillum rubrum affects activity and spectroscopic states.

    Science.gov (United States)

    Spangler, N J; Meyers, M R; Gierke, K L; Kerby, R L; Roberts, G P; Ludden, P W

    1998-02-13

    In carbon monoxide dehydrogenase (CODH) from Rhodospirillum rubrum, histidine 265 was replaced with valine by site-directed mutagenesis of the cooS gene. The altered form of CODH (H265V) had a low nickel content and a dramatically reduced level of catalytic activity. Although treatment with NiCl2 and CoCl2 increased the activity of H265V CODH by severalfold, activity levels remained more than 1000-fold lower than that of wild-type CODH. Histidine 265 was not essential for the formation and stability of the Fe4S4 clusters. The Km and KD for CO as well as the KD for cyanide were relatively unchanged as a result of the amino acid substitution in CODH. The time-dependent reduction of the [Fe4S4]2+ clusters by CO occurred on a time scale of hours, suggesting that, as a consequence of the mutation, a rate-limiting step had been introduced prior to the transfer of electrons from CO to the cubanes in centers B and C. EPR spectra of H265V CODH lacked the gav = 1.86 and gav = 1.87 signals characteristic of reduced forms of the active site (center C) of wild-type CODH. This indicates that the electronic properties of center C have been modified possibly by the disruption or alteration of the ligand-mediated interaction between the nickel site and Fe4S4 chromophore. PMID:9461598

  13. NADP-malate Dehydrogenase Isoforms of Wheat Leaves under Drought: Their Localization, and Some physicochemical and Kinetic Properties

    Directory of Open Access Journals (Sweden)

    H.G. Babayev

    2015-09-01

    Full Text Available Changes in sub-cellular localization, isoenzyme spectrum and kinetic characteristics of NADP-malate dehydrogenase (NADP-MDH, EC 1.1.1.82 in Triticum durum Desf. genotypes with contrasting drought tolerance have been studied. In chloroplast and cytosol fractions of mesophyll cells of wheat flag leaves 70-75% and 25-30% of the total NADP-MDH activity were found to be localized, respectively. Three isoforms of NADP-MDH with molecular weights of 66, 74 and 86 kDa were revealed in the chloroplast fraction, whereas in the cytosolic fraction molecular weights of the isoenzymes were found to be 42, 66 and 74 kDa. Drought caused the formation of a new 90 kDa isoform of the enzyme in the chloroplast fraction in anthesis phase of ontogenesis. In the drought-tolerant genotype the appearance of the new isoform in the chloroplast fraction was accompanied by a more rapid increase in Km and Vmax contrary to the chloroplast fraction of the drought-sensitive genotype manifesting a slight decrease in these parameters, suggesting one of the adaptive traits in forming drought tolerance in C3 plants.

  14. Kinetic and thermodynamic study of the reaction catalyzed by glucose-6-phosphate dehydrogenase with nicotinamide adenine dinucleotide

    Energy Technology Data Exchange (ETDEWEB)

    Martin del Campo, Julia S. [Departamento de Fisica Aplicada, Centro de Investigacion y de Estudios Avanzados - Unidad Merida, Carretera antigua a Progreso Km. 6, A.P. 73 Cordemex, 97310, Merida, Yucatan (Mexico); Patino, Rodrigo, E-mail: rtarkus@mda.cinvestav.mx [Departamento de Fisica Aplicada, Centro de Investigacion y de Estudios Avanzados - Unidad Merida, Carretera antigua a Progreso Km. 6, A.P. 73 Cordemex, 97310, Merida, Yucatan (Mexico)

    2011-04-20

    Research highlights: {yields} The reaction catalyzed by one enzyme of the pentose phosphate pathway was studied. {yields} A spectrophotometric method is proposed for kinetic and thermodynamic analysis. {yields} The pH and the temperature influences are reported on physical chemical properties. {yields} Relative concentrations of substrates are also important in the catalytic process. - Abstract: The enzyme glucose-6-phosphate dehydrogenase (G6PD, EC 1.1.1.49) from Leuconostoc mesenteroides has a dual coenzyme specificity with oxidized nicotinamide adenine dinucleotide (NAD{sub ox}) and oxidized nicotinamide adenine dinucleotide phosphate as electron acceptors. The G6PD coenzyme selection is determined by the metabolic cellular prevailing conditions. In this study a kinetic and thermodynamic analysis is presented for the reaction catalyzed by G6PD from L. mesenteroides with NAD{sub ox} as coenzyme in phosphate buffer. For this work, an in situ spectrophotometric technique was employed based on the detection of one product of the reaction. Substrate and coenzyme concentrations as well as temperature and pH effects were evaluated. The apparent equilibrium constant, the Michaelis constant, and the turnover number were determined as a function of each experimental condition. The standard transformed Gibbs energy of reaction was determined from equilibrium constants at different initial conditions. For the product 6-phospho-D-glucono-1,5-lactone, a value of the standard Gibbs energy of formation is proposed, {Delta}{sub f}G{sup o} = -1784 {+-} 5 kJ mol{sup -1}.

  15. Mitochondrial aldehyde dehydrogenase mediates vasodilator responses of glyceryl trinitrate and sodium nitrite in the pulmonary vascular bed of the rat.

    Science.gov (United States)

    Badejo, Adeleke M; Hodnette, Chris; Dhaliwal, Jasdeep S; Casey, David B; Pankey, Edward; Murthy, Subramanyam N; Nossaman, Bobby D; Hyman, Albert L; Kadowitz, Philip J

    2010-09-01

    It has been reported that mitochondrial aldehyde dehydrogenase (ALDH2) catalyzes the formation of glyceryl dinitrate and inorganic nitrite from glyceryl trinitrate (GTN), leading to an increase in cGMP and vasodilation in the coronary and systemic vascular beds. However, the role of nitric oxide (NO) formed from nitrite in mediating the response to GTN in the pulmonary vascular bed is uncertain. The purpose of the present study was to determine if nitrite plays a role in mediating vasodilator responses to GTN. In this study, intravenous injections of GTN and sodium nitrite decreased pulmonary and systemic arterial pressures and increased cardiac output. The decreases in pulmonary arterial pressure under baseline and elevated tone conditions and decreases in systemic arterial pressure in response to GTN and sodium nitrite were attenuated by cyanamide, an ALDH2 inhibitor, whereas responses to the NO donor, sodium nitroprusside (SNP), were not altered. The decreases in pulmonary and systemic arterial pressure in response to GTN and SNP were not altered by allopurinol, an inhibitor of xanthine oxidoreductase, whereas responses to sodium nitrite were attenuated. GTN was approximately 1,000-fold more potent than sodium nitrite in decreasing pulmonary and systemic arterial pressures. These results suggest that ALDH2 plays an important role in the bioactivation of GTN and nitrite in the pulmonary and systemic vascular beds and that the reduction of nitrite to vasoactive NO does not play an important role in mediating vasodilator responses to GTN in the intact chest rat.

  16. Crystallization and preliminary crystallographic analysis of Gre2p, an NADP+-dependent alcohol dehydrogenase from Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    The alcohol dehydrogenase Gre2p from S. cerevisiae catalyses the stereospecific reduction of a variety of different keto compounds and can therefore be applied as a valuable biocatalyst. The crystallization of the complex of Gre2p with NADP+ and its preliminary X-ray analysis are described. Gre2p [Genes de respuesta a estres (stress-response gene)] from Saccharomyces cerevisiae is a monomeric enzyme of 342 amino acids with a molecular weight of 38.1 kDa. The enzyme catalyses both the stereospecific reduction of keto compounds and the oxidation of various hydroxy compounds and alcohols by the simultaneous consumption of the cofactor NADPH and formation of NADP+. Crystals of a Gre2p complex with NADP+ were grown using PEG 8000 as a precipitant. They belong to the monoclinic space group P21. The current diffraction resolution is 3.2 Å. In spite of the monomeric nature of Gre2p in solution, packing and self-rotation calculations revealed the existence of two Gre2p protomers per asymmetric unit related by a twofold noncrystallographic axis

  17. Expression of protein engineered NADP{sup +}-dependent xylitol dehydrogenase increases ethanol production from xylose in recombinant Saccharomyces cerevisiae

    Energy Technology Data Exchange (ETDEWEB)

    Matsushika, Akinori; Inoue, Hiroyuki; Murakami, Katsuji; Takimura, Osamu; Sawayama, Shigeki [National Institute of Advanced Industrial Science and Technology, Hiroshima (Japan). Biomass Technology Research Center; Watanabe, Seiya; Kodaki, Tsutomu; Makino, Keisuke [Kyoto Univ. (Japan). Inst. of Advanced Energy

    2008-11-15

    A recombinant Saccharomyces cerevisiae strain transformed with xylose reductase (XR) and xylitol dehydrogenase (XDH) genes from Pichia stipitis has the ability to convert xylose to ethanol together with the unfavorable excretion of xylitol, which may be due to cofactor imbalance between NADPH-preferring XR and NAD{sup +}-dependent XDH. To reduce xylitol formation, we have already generated several XDH mutants with a reversal of coenzyme specificity toward NADP{sup +}. In this study, we constructed a set of recombinant S. cerevisiae strains with xylose-fermenting ability, including protein-engineered NADP{sup +}-dependent XDH-expressing strains. The most positive effect on xylose-to-ethanol fermentation was found by using a strain named MA-N5, constructed by chromosomal integration of the gene for NADP{sup +}-dependent XDH along with XR and endogenous xylulokinase genes. The MA-N5 strain had an increase in ethanol production and decrease in xylitol excretion compared with the reference strain expressing wild-type XDH when fermenting not only xylose but also mixed sugars containing glucose and xylose. Furthermore, the MA-N5 strain produced ethanol with a high yield of 0.49 g of ethanol/g of total consumed sugars in the nonsulfuric acid hydrolysate of wood chips. The results demonstrate that glucose and xylose present in the lignocellulosic hydrolysate can be efficiently fermented by this redox-engineered strain. (orig.)

  18. Regulation of enzyme activity of alcohol dehydrogenase through its interactions with pyruvate-ferredoxin oxidoreductase in Thermoanaerobacter tengcongensis.

    Science.gov (United States)

    Wang, Qian; Wang, Quanhui; Tong, Wei; Bai, Xue; Chen, Zhen; Zhao, Jingjing; Zhang, Jiyuan; Liu, Siqi

    2012-01-20

    Alcohol dehydrogenases (ADHs) from thermophilic microorganisms are interesting enzymes that have their potential applications in biotechnology and potentially provide insight into the mechanisms of action of thermo-tolerant proteins. The molecular mechanisms of ADHs under thermal stress in vivo have yet to be explored. Herein, we employed a proteomic strategy to survey the possible interactions of secondary-ADH (2-ADH) with other proteins in Thermoanaerobacter tengcongensis (T. tengcongensis) cultured at 75°C and found that 2-ADH, pyruvate-ferredoxin oxidoreductase (PFOR) and several glycolytic enzymes coexisted in a protein complex. Using anion exchange chromatography, the elution profile indicated that the native 2-ADH was present in two forms, PFOR-bound and PFOR-free. Immuno-precipitation and pull down analysis further validated the interactions between 2-ADH and PFOR. The kinetic behaviours of 2-ADH either in the recombinant or native form were evaluated with different substrates. The enzyme activity of 2-ADH was inhibited in a non-competitive mode by PFOR, implying the interaction of 2-ADH and PFOR negatively regulated alcohol formation. In T. tengcongensis, PFOR is an enzyme complex located at the upstream of 2-ADH in the alcohol generation pathway. These findings, therefore, offered a plausible mechanism for how alcohol metabolism is regulated by hetero-interactions between 2-ADH and PFOR, especially in anaerobic thermophiles. PMID:22222371

  19. Gpd1 and Gpd2 fine-tuning for sustainable reduction of glycerol formation in Saccharomyces cerevisiae

    NARCIS (Netherlands)

    Hubmann, Georg; Guillouet, Stephane; Nevoigt, Elke

    2011-01-01

    Gpd1 and Gpd2 are the two isoforms of glycerol 3-phosphate dehydrogenase (GPDH), which is the rate-controlling enzyme of glycerol formation in Saccharomyces cerevisiae. The two isoenzymes play crucial roles in osmoregulation and redox balancing. Past approaches to increase ethanol yield at the cost

  20. Assessment of freshness and freeze-thawing of sea bream fillets (Sparus aurata) by a cytosolic enzyme: Lactate dehydrogenase.

    Science.gov (United States)

    Diop, Mamadou; Watier, Denis; Masson, Pierre-Yves; Diouf, Amadou; Amara, Rachid; Grard, Thierry; Lencel, Philippe

    2016-11-01

    The evaluation of freshness and freeze-thawing of fish fillets was carried out by assessment of autolysis of cells using a cytosolic enzyme lactate dehydrogenase. Autolysis plays an important role in spoilage of fish and postmortem changes in fish tissue are due to the breakdown of the cellular structures and release of cytoplasmic contents. The outflow of a cytosolic enzyme, lactate dehydrogenase, was studied in sea bream fillets and the Sparus aurata fibroblasts (SAF-1) cell-line during an 8day storage period at +4°C. A significant increase of lactate dehydrogenase release was observed, especially after 5days of storage. The ratio between the free and the total lactate dehydrogenase activity is a promising predictive marker to measure the quality of fresh fish fillets. The effect of freeze-thawing on cytosolic lactate dehydrogenase and lysosomal α-d-glucosidase activities was also tested. Despite the protecting effect of the tissue compared to the cell-line, a loss of lactate dehydrogenase activity, but not of α-d-glucosidase, was observed. In conclusion, lactate dehydrogenase may be used as a marker to both assess freshness of fish and distinguish between fresh and frozen-thawed fish fillets. PMID:27211667

  1. Glucose-6-phosphate dehydrogenase and glutathione reductase activity in methemoglobin reduction by methylene blue and cyst amine: study on glucose-6-phosphate dehydrogenase-deficient individuals, on normal subjects and on riboflavin-treated subjects

    Directory of Open Access Journals (Sweden)

    Benedito Barraviera

    1988-10-01

    Full Text Available The authors have standardized methods for evaluation of the activity of the glucose-6-phosphate dehydrogenase and of glutathione reductase. The general principle of the first method was based on methemoglobin formation by sodium nitrite followed by stimulation of the glucose-6-phosphate dehydrogenase with methylene blue. Forty six adults (23 males and 23 females were studied. Subjects were not G6PD deficient and were aged 20 to 30 years. The results showed that methemoglobin reduction by methylene blue was 154.40 and 139.90 mg/min (p<0.05 for males and females, respectively, in whole blood, and 221.10 and 207.85 mg/min (n.s., respectively, in washed red cells. These data showed that using washed red cells and 0.7g% sodium nitrite concentration produced no differences between sexes and also shortened reading time for the residual amount of methemoglobin to 90 minutes. Glutathione reductase activity was evaluated on the basis of the fact that cystamine (a thiol agent binds to the SH groups of hemoglobin, forming complexes. These complexes are reversed by the action of glutathione reductase, with methemoglobin reduction occurring simultaneously with this reaction. Thirty two adults (16 males and 16 females were studied. Subjects were not G6PD deficient and were aged 20 to 30 years. Methemoglobin reduction by cystamine was 81.27 and 91.13 mg/min (p<0.01 for males and females, respectively. These data showed that using washed red cells and 0.1 M cystamine concentration permits a reading of the residual amount of methemoglobin at 180 minutes of incubation. Glutathione reductase activity was evaluated by methemoglobin reduction by cystamine in 14 females before and after treatment with 10 mg riboflavin per day for 8 days. The results were 73.69 and 94.26 jug/min (p<0.01 before and after treatment, showing that riboflavin treatment increase glutathione reductase activity even in normal individuals. Three Black G6PD-deficient individuals (2 males and 1

  2. Crystallization and preliminary X-ray analysis of the complex of NADH and 3α-hydroxysteroid dehydrogenase from Pseudomonas sp. B-0831

    International Nuclear Information System (INIS)

    The complex of NADH and 3α-HSD from Pseudomonas sp. B-0831 has been crystallized and X-ray diffraction data have been collected to 1.8 Å resolution. The NAD(P)+-dependent enzyme 3α-hydroxysteroid dehydrogenase (3α-HSD) catalyzes the reversible interconversion of hydroxyl and oxo groups at position 3 of the steroid nucleus. The complex of NADH and 3α-HSD from Pseudomonas sp. B-0831 was crystallized by the hanging-drop vapour-diffusion method. Refinement of crystallization conditions with microseeding improved the quality of the X-ray diffraction data to a resolution of 1.8 Å. The crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 62.46, b = 82.25, c = 86.57 Å, and contained two molecules, reflecting dimer formation of 3α-HSD, in the asymmetric unit

  3. 15-hydroxyprostaglandin dehydrogenase activity in vitro in lung and kidney of essential fatty acid-deficient rats

    DEFF Research Database (Denmark)

    Hansen, Harald S.; Toft, B.S.

    1978-01-01

    Weanling rats were fed for 6 months on a diet deficient in essential fatty acids: either fat-free, or with 28% (w/w) partially hydrogenated fish oil. Control rats were fed a diet with 28% (w/w) arachis oil for 6 months. 15-Hydroxyprostaglandin dehydrogenase activity was determined as initial rates...... of the two groups on diets deficient in essential fatty acids as compared to the control group. No difference was observed in dehydrogenase activity in the kidneys. The dehydrogenase may be of importance for the regulation of the level of endogenous prostaglandins and, thus, a decrease in activity could...

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

    Directory of Open Access Journals (Sweden)

    Hahn-Hägerdal Bärbel

    2007-02-01

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

  5. Active site cysteine-null glyceraldehyde-3-phosphate dehydrogenase (GAPDH) rescues nitric oxide-induced cell death.

    Science.gov (United States)

    Kubo, Takeya; Nakajima, Hidemitsu; Nakatsuji, Masatoshi; Itakura, Masanori; Kaneshige, Akihiro; Azuma, Yasu-Taka; Inui, Takashi; Takeuchi, Tadayoshi

    2016-02-29

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a homotetrameric enzyme involved in a key step of glycolysis, also has a role in mediating cell death under nitrosative stress. Our previous reports suggest that nitric oxide-induced intramolecular disulfide-bonding GAPDH aggregation, which occurs through oxidation of the active site cysteine (Cys-152), participates in a mechanism to account for nitric oxide-induced death signaling in some neurodegenerative/neuropsychiatric disorders. Here, we demonstrate a rescue strategy for nitric oxide-induced cell death accompanied by GAPDH aggregation in a mutant with a substitution of Cys-152 to alanine (C152A-GAPDH). Pre-incubation of purified wild-type GAPDH with C152A-GAPDH under exposure to nitric oxide inhibited wild-type GAPDH aggregation in a concentration-dependent manner in vitro. Several lines of structural analysis revealed that C152A-GAPDH extensively interfered with nitric oxide-induced GAPDH-amyloidogenesis. Overexpression of doxycycline-inducible C152A-GAPDH in SH-SY5Y neuroblastoma significantly rescued nitric oxide-induced death, concomitant with the decreased formation of GAPDH aggregates. Further, both co-immunoprecipitation assays and simulation models revealed a heterotetramer composed of one dimer each of wild-type GAPDH and C152A-GAPDH. These results suggest that the C152A-GAPDH mutant acts as a dominant-negative molecule against GAPDH aggregation via the formation of this GAPDH heterotetramer. This study may contribute to a new therapeutic approach utilizing C152A-GAPDH against brain damage in nitrosative stress-related disorders.

  6. Galaxy formation.

    Science.gov (United States)

    Peebles, P J

    1998-01-01

    It is argued that within the standard Big Bang cosmological model the bulk of the mass of the luminous parts of the large galaxies likely had been assembled by redshift z approximately 10. Galaxy assembly this early would be difficult to fit in the widely discussed adiabatic cold dark matter model for structure formation, but it could agree with an isocurvature version in which the cold dark matter is the remnant of a massive scalar field frozen (or squeezed) from quantum fluctuations during inflation. The squeezed field fluctuations would be Gaussian with zero mean, and the distribution of the field mass therefore would be the square of a random Gaussian process. This offers a possibly interesting new direction for the numerical exploration of models for cosmic structure formation.

  7. [Activity of liver mitochondrial NAD+-dependent dehydrogenases of the krebs cycle in rats with acetaminophen-induced hepatitis developed under conditions of alimentary protein deficiency].

    Science.gov (United States)

    Voloshchuk, O N; Kopylchuk, G P

    2016-01-01

    Activity of isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, malate dehydrogenase, and the NAD(+)/NADН ratio were studied in the liver mitochondrial fraction of rats with toxic hepatitis induced by acetaminophen under conditions of alimentary protein deprivation. Acetaminophen-induced hepatitis was characterized by a decrease of isocitrate dehydrogenase, α-ketoglutarate dehydrogenase and malate dehydrogenase activities, while the mitochondrial NAD(+)/NADН ratio remained at the control level. Modeling of acetaminophen-induced hepatitis in rats with alimentary protein caused a more pronounced decrease in the activity of NAD(+)-dependent dehydrogenases studied and a 2.2-fold increase of the mitochondrial NAD(+)/NADН ratio. This suggests that alimentary protein deprivation potentiated drug-induced liver damage.

  8. Lactic dehydrogenase isozyme patterns and alpha-hydroxybutyrate dehydrogenase activities in serum from newborns, patients with ovarian cancer or myocardial infarction.

    Science.gov (United States)

    Kikuchi, Y; Kita, T; Furuya, K; Kato, K

    1988-11-01

    Lactic dehydrogenase (LDH) and alpha-hydroxybutyrate dehydrogenase (HBD) and LDH isozyme patterns were studied in serum from newborns and patients with ovarian cancer or myocardial infarction. LDH and HBD activities from newborns and patients with ovarian cancer or myocardial infarction were significantly increased, compared with those from patients with benign ovarian tumor. These increases were accompanied with a decrease of LDH-H and an increase of LDH-M in serum from newborns and patients with ovarian cancer, while an increase of LDH-H in serum from patients with myocardial infarction was dominant. However, the raised HBD activities in serum from patients with benign ovarian tumor did not affect the LDH isozyme patterns. From analysis of linear regression, a negative correlation between LDH-1 or -2 and HBD activity in serum from patients with ovarian cancer was observed while there was a positive correlation between LDH-4 and HBD activity. Similar patterns in serum from newborns were observed. On the other hand, a positive correlation between LDH-1 and HBD activity and a negative correlation between LDH-4 and HBD activity were found in serum from patients with myocardial infarction.

  9. Uric acid substantially enhances the free radical-induced inactivation of alcohol dehydrogenase.

    Science.gov (United States)

    Kittridge, K J; Willson, R L

    1984-05-01

    Lactate dehydrogenase (LDH) and yeast alcohol dehydrogenase ( YADH ) are inactivated when attacked by hydroxy free radicals (OH). Organic molecules with a high rate constant of reaction with OH such as ascorbate or urate can compete with the enzymes for these strongly oxidising radicals. However, although 10(-3)M ascorbate can substantially protect both LDH and YADH from OH attack, in the presence of 10(-3)M urate only LDH is protected. In the case of YADH an even greater degree of inactivation than with OH occurs. The extent of inactivation is considerably reduced when oxygen is absent, in agreement with a urate peroxy radical perhaps being partly responsible for the increased inactivation of the enzyme.

  10. Regulation of pyruvate dehydrogenase kinase expression by the farnesoid X receptor

    International Nuclear Information System (INIS)

    The pyruvate dehydrogenase complex (PDC) functions as an important junction in intermediary metabolism by influencing the utilization of fat versus carbohydrate as a source of fuel. Activation of PDC is achieved by phosphatases, whereas, inactivation is catalyzed by pyruvate dehydrogenase kinases (PDKs). The expression of PDK4 is highly regulated by the glucocorticoid and peroxisome proliferator-activated receptors. We demonstrate that the farnesoid X receptor (FXR; NR1H4), which regulates a variety of genes involved in lipoprotein metabolism, also regulates the expression of PDK4. Treatment of rat hepatoma cells as well as human primary hepatocytes with FXR agonists stimulates the expression of PDK4 to levels comparable to those obtained with glucocorticoids. In addition, treatment of mice with an FXR agonist significantly increased hepatic PDK4 expression, while concomitantly decreasing plasma triglyceride levels. Thus, activation of FXR may suppress glycolysis and enhance oxidation of fatty acids via inactivation of the PDC by increasing PDK4 expression

  11. Mechanisms underlying regulation of the expression and activities of the mammalian pyruvate dehydrogenase kinases.

    Science.gov (United States)

    Sugden, Mary C; Holness, Mark J

    2006-07-01

    The mechanisms that control mammalian pyruvate dehydrogenase complex (PDC) activity include its phosphorylation (inactivation) by a family of pyruvate dehydrogenase kinases (PDKs 1 - 4). Here we review new developments in the regulation of the activities and expression of the PDKs, in particular PDK2 and PDK4, in relation to glucose and lipid homeostasis. This review describes recent advances relating to the acute and long-term modes of regulation of the PDKs, with particular emphasis on the regulatory roles of nuclear receptors including peroxisome proliferator-activated receptor (PPAR) alpha and Liver X receptor (LXR), PPAR gamma coactivator alpha (PGC-1alpha) and insulin, and the impact of changes in PDK activity and expression in glucose and lipid homeostasis. Since PDK4 may assist in lipid clearance when there is an imbalance between lipid delivery and oxidation, it may represent an attractive target for interventions aimed at rectifying abnormal lipid as well as glucose homeostasis in disease states. PMID:17132539

  12. The effect of heparin and pentosan polysulfate on the thermal stability of yeast alcohol dehydrogenase.

    Science.gov (United States)

    Paulíková, H; Molnárová, M; Podhradský, D

    1998-12-01

    Heparin and pentosan polysulfate as organic polyanions inhibit yeast alcohol dehydrogenase (YADH). The aim of this study was to determine the effect of heparin and pentosan polysulfate on the thermostability of alcohol dehydrogenase. Spectral and kinetic analyses showed that these compounds increase the thermal stability of the enzyme and eliminate entirely thermal aggregation. The thermostabilizing effect of unfractionated heparin and pentosan polysulfate was accelerated in the presence of NAD+. The addition of NAD+ (11 microM) to the incubation medium decreased the inhibition of the YADH activity in the presence of pentosan polysulfate (1.32 microM). Moreover, 38% of the residual activity of YADH was found after a 5-min incubation at 70 degrees C. These findings indicate that heparinoids not only modulate the enzyme activity but also can prevent the protein's thermal denaturation.

  13. Lactate dehydrogenase is not a mitochondrial enzyme in human and mouse vastus lateralis muscle

    DEFF Research Database (Denmark)

    Rasmussen, Hans N; van Hall, Gerrit; Rasmussen, Ulla F

    2002-01-01

    The presence of lactate dehydrogenase in skeletal muscle mitochondria was investigated to clarify whether lactate is a possible substrate for mitochondrial respiration. Mitochondria were prepared from 100 mg samples of human and mouse vastus lateralis muscle. All fractions from the preparation pr......, showed high specific rates of state 3 respiration. This excluded artificial loss from the mitochondria of all activity of a possible LDH. It was concluded that skeletal muscle mitochondria are devoid of LDH and unable to metabolize lactate.......The presence of lactate dehydrogenase in skeletal muscle mitochondria was investigated to clarify whether lactate is a possible substrate for mitochondrial respiration. Mitochondria were prepared from 100 mg samples of human and mouse vastus lateralis muscle. All fractions from the preparation...

  14. Very long chain acyl-coenzyme A dehydrogenase deficiency with adult onset

    DEFF Research Database (Denmark)

    Smelt, A H; Poorthuis, B J; Onkenhout, W;

    1998-01-01

    Very long chain acyl-coenzyme A (acyl-CoA) dehydrogenase (VLCAD) deficiency is a severe disorder of mitochondrial beta-oxidation in infants. We report adult onset of attacks of painful rhabdomyolysis. Gas chromatography identified strongly elevated levels of tetradecenoic acid, 14:1(n-9), tetrade...... be due to residual enzyme activity as a consequence of the two missense mutations. Treatment with L-carnitine and medium chain triglycerides in the diet did not reduce the attacks of rhabdomyolysis.......Very long chain acyl-coenzyme A (acyl-CoA) dehydrogenase (VLCAD) deficiency is a severe disorder of mitochondrial beta-oxidation in infants. We report adult onset of attacks of painful rhabdomyolysis. Gas chromatography identified strongly elevated levels of tetradecenoic acid, 14:1(n-9...

  15. A severe genotype with favourable outcome in very long chain acyl-CoA dehydrogenase deficiency

    DEFF Research Database (Denmark)

    Touma, E H; Rashed, M S; Vianey-Saban, C;

    2001-01-01

    A patient with very long chain acyl-CoA dehydrogenase (VLCAD) deficiency is reported. He had a severe neonatal presentation and cardiomyopathy. He was found to be homozygous for a severe mutation with no residual enzyme activity. Tandem mass spectrometry on dried blood spots revealed increased lo...... chain acylcarnitines. VLCAD enzyme activity was severely decreased to 2% of control levels. Dietary management consisted of skimmed milk supplemented with medium chain triglycerides and L-carnitine. Outcome was good and there was no acute recurrence.......A patient with very long chain acyl-CoA dehydrogenase (VLCAD) deficiency is reported. He had a severe neonatal presentation and cardiomyopathy. He was found to be homozygous for a severe mutation with no residual enzyme activity. Tandem mass spectrometry on dried blood spots revealed increased long...

  16. Crystallization and preliminary X-ray characterization of d-3-hydroxybutyrate dehydrogenase from Pseudomonas fragi

    Energy Technology Data Exchange (ETDEWEB)

    Nakajima, Yoshitaka; Ito, Kiyoshi; Ichihara, Emi; Ogawa, Kyohei; Egawa, Takashi; Xu, Yue; Yoshimoto, Tadashi, E-mail: yosimoto@net.nagasaki-u.ac.jp [Biotechnology Department, Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521 (Japan)

    2005-01-01

    d-3-Hydroxybutyrate dehydrogenase (EC 1.1.1.30) from P. flagi has been crystallized by the hanging-drop method. A recombinant form of d-3-hydroxybutyrate dehydrogenase (EC 1.1.1.30) from Pseudomonas fragi has been crystallized by the hanging-drop method using PEG 3000 as a precipitating agent. The crystals belong to the orthorhombic group P2{sub 1}2{sub 1}2, with unit-cell parameters a = 64.3, b = 99.0, c = 110.2 Å. The crystals are most likely to contain two tetrameric subunits in the asymmetric unit, with a V{sub M} value of 3.29 Å{sup 3} Da{sup −1}. Diffraction data were collected to a 2.0 Å resolution using synchrotron radiation at the BL6A station of the Photon Factory.

  17. Biochemical and molecular characterization of the NAD(+)-dependent isocitrate dehydrogenase from the chemolithotroph Acidithiobacillus thiooxidans.

    Science.gov (United States)

    Inoue, Hiroyuki; Tamura, Takashi; Ehara, Nagisa; Nishito, Akira; Nakayama, Yumi; Maekawa, Makiko; Imada, Katsumi; Tanaka, Hidehiko; Inagaki, Kenji

    2002-08-27

    An isocitrate dehydrogenase (ICDH) with an unique coenzyme specificity from Acidithiobacillus thiooxidans was purified and characterized, and its gene was cloned. The native enzyme was homodimeric with a subunit of M(r) 45000 and showed a 78-fold preference for NAD(+) over NADP(+). The cloned ICDH gene (icd) was expressed in an icd-deficient strain of Escherichia coli EB106; the activity was found in the cell extract. The gene encodes a 429-amino acid polypeptide and is located between open reading frames encoding a putative aconitase gene (upstream of icd) and a putative succinyl-CoA synthase beta-subunit gene (downstream of icd). A. thiooxidans ICDH showed high sequence similarity to bacterial NADP(+)-dependent ICDH rather than eukaryotic NAD(+)-dependent ICDH, but the NAD(+)-preference of the enzyme was suggested due to residues conserved in the coenzyme binding site of the NAD(+)-dependent decarboxylating dehydrogenase.

  18. Pro-haloacetate Nanoparticles for Efficient Cancer Therapy via Pyruvate Dehydrogenase Kinase Modulation

    Science.gov (United States)

    Misra, Santosh K.; Ye, Mao; Ostadhossein, Fatemeh; Pan, Dipanjan

    2016-06-01

    Anticancer agents based on haloacetic acids are developed for inhibition of pyruvate dehydrogenase kinase (PDK), an enzyme responsible for reversing the suppression of mitochondria-dependent apoptosis. Through molecular docking studies mono- and dihaloacetates are identified as potent PDK2 binders and matched their efficiency with dichloroacetic acid. In silico screening directed their conversion to phospholipid prodrugs, which were subsequently self-assembled to pro-haloacetate nanoparticles. Following a thorough physico-chemical characterization, the functional activity of these novel agents was established in wide ranges of human cancer cell lines in vitro and in vivo in rodents. Results indicated that the newly explored PDK modulators can act as efficient agent for cancer regression. A Pyruvate dehydrogenase (PDH) assay mechanistically confirmed that these agents trigger their activity through the mitochondria-dependent apoptosis.

  19. Purification and characterization of methanol dehydrogenase of a serin-producing methylotroph, hyphomicrobium methylovorum

    Energy Technology Data Exchange (ETDEWEB)

    Miyazaki, S.S.; Toki, Shin' ichiro; Izumi, Yoshikazu; Yamada, Hideaki

    1987-08-25

    All methanol-utilizing bacteria except actinomycete contain dyelinked methanol dehydrogenase. Also methol dehydrogenase has been found to participate in the methanol oxidation to formaldehyde necessary for L-serine production by H. methylovorum. This paper describs the purification and characterization of the enzyme from this bacterium. The enzyme is a dimeric protein, both constituting subunits having a molecular mass of about 60,000Da respectively. Optical activity was observed at pH 9.0 and around 35/sup 0/C, and the presence of NH4 was essential for the enzyme activity. The enzyme possesses a broad substrate specificit for primary alcohols, secondary alcohols, substituted alcohols, some aldehyes and others. The enzyme also has such properties as relatively high stability against heat treatment and no inactivation by phenazine methosulfate, etc. (4 figs, 3 tabs, 14 refs)

  20. Covalent immobilization of recombinant Rhizobium etli CFN42 xylitol dehydrogenase onto modified silica nanoparticles

    DEFF Research Database (Denmark)

    Zhang, Ye-Wang; Tiwari, Manish Kumar; Jeya, Marimuthu;

    2011-01-01

    Rare sugars have many applications in food industry, as well as pharmaceutical and nutrition industries. Xylitol dehydrogenase (XDH) can be used to synthesize various rare sugars enzymatically. However, the immobilization of XDH has not been performed to improve the industrial production of rare...... sugars. In this study, silica nanoparticles which have high immobilization efficiency were selected from among several carriers for immobilization of recombinant Rhizobium etli CFN42 xylitol dehydrogenase (ReXDH) and subjected to characterization. Among four different chemical modification methods to......,410 min at 40 °C and from 30 min to 450 min at 50 °C. The K(m) of ReXDH was slightly altered from 17.9 to only 19.2 mM by immobilization. The immobilized ReXDH had significant reusability, as it retained 81% activity after eight cycles of batch conversion of xylitol into L-xylulose. A∼71% conversion and a...

  1. Comparative characterization of two GDP-mannose dehydrogenase genes from Saccharina japonica (Laminariales, Phaeophyceae)

    OpenAIRE

    Zhang, Pengyan; Shao, Zhanru; Jin, Weihua; Duan, Delin

    2016-01-01

    Background Saccharina japonica is an important commercial brown seaweed, its main product is alginate, which is used in food, textile and by the cosmetic and pharmaceutical industries. GDP-mannose dehydrogenase (GMD) is the key enzyme involved in the synthesis of alginate. However, little is known about GMD in S. japonica. Here we report comparative biochemical analysis of two GMD genes in S. japonica. Results Two GMD genes from S. japonica (Sjgmd1, Sjgmd2) were cloned. The open reading frame...

  2. Theoretical Calculations of the Catalytic Triad in Short-Chain Alcohol Dehydrogenases/Reductases

    OpenAIRE

    Gani, Osman A B S M; Adekoya, Olayiwola A; Giurato, Laura; Spyrakis, Francesca; Cozzini, Pietro; Guccione, Salvatore; Winberg, Jan-Olof; Sylte, Ingebrigt

    2007-01-01

    Three highly conserved active site residues (Ser, Tyr, and Lys) of the family of short-chain alcohol dehydrogenases/reductases (SDRs) were demonstrated to be essential for catalytic activity and have been denoted the catalytic triad of SDRs. In this study computational methods were adopted to study the ionization properties of these amino acids in SDRs from Drosophila melanogaster and Drosophila lebanonensis. Three enzyme models, with different ionization scenarios of the catalytic triad that...

  3. Coordination environment of the active-site metal ion of liver alcohol dehydrogenase.

    OpenAIRE

    Makinen, M W; Yim, M B

    1981-01-01

    The coordination environment of the catalytically active metal ion of horse liver alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) has been investigated by electron paramagnetic resonance (EPR) methods with use of the active-site-specific Co2+-reconstituted enzyme. The EPR absorption spectrum of the metal-substituted enzyme is characteristic of a rhombically distorted environment. The spectrum of the enzyme--NAD+ complex shows approximate axial symmetry of the metal ion site, i...

  4. Frequency of glucose-6-phosphate dehydrogenase deficiency in relation to altitude: a malaria hypothesis

    OpenAIRE

    Tzoneva, M.; Bulanov, A. G.; Mavrudieva, M.; Lalchev, S.; Toncheva, D; Tanev, D.

    1980-01-01

    Genetic markers have recently been found to be much more polymorphic than expected. Such extensive human polymorphisms may be partly explained by a number of genetic and environmental factors, including infectious diseases. Malaria, which was very widespread in the past and still poses a problem in many countries today, is a good candidate for research. The association between malaria and glucose-6-phosphate dehydrogenase (G6PD) deficiency is well-known, but more should be done to determine t...

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

    OpenAIRE

    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 ...

  6. Biochemical characterization of prephenate dehydrogenase from the hyperthermophilic bacterium Aquifex aeolicus

    OpenAIRE

    Bonvin, Julie; Aponte, Raphael A.; Marcantonio, Maria; Singh, Sasha; Christendat, Dinesh; Turnbull, Joanne L.

    2006-01-01

    A monofunctional prephenate dehydrogenase (PD) from Aquifex aeolicus was expressed as a His-tagged protein in Escherichia coli and was purified by nickel affinity chromatography allowing the first biochemical and biophysical characterization of a thermostable PD. A. aeolicus PD is susceptible to proteolysis. In this report, the properties of the full-length PD are compared with one of these products, an N-terminally truncated protein variant (Δ19PD) also expressed recombinantly in E. coli. Bo...

  7. Aldehyde Dehydrogenase 1 Is a Tumor Stem Cell-Associated Marker in Lung Cancer

    OpenAIRE

    Jiang, Feng; Qiu, Qi; Khanna, Abha; Todd, Nevins W.; Deepak, Janaki; Xing, Lingxiao; Wang, Huijun; Liu, Zhenqiu; Su, Yun; Stass, Sanford A.; Katz, Ruth L

    2009-01-01

    Tumor contains small population of cancer stem cells (CSC) that are responsible for its maintenance and relapse. Analysis of these CSCs may lead to effective prognostic and therapeutic strategies for the treatment of cancer patients. We report here the identification of CSCs from human lung cancer cells using Aldefluor assay followed by fluorescence-activated cell sorting analysis. Isolated cancer cells with relatively high aldehyde dehydrogenase 1 (ALDH1) activity display in vitro features o...

  8. Androgen-stimulated UDP-glucose dehydrogenase expression limits prostate androgen availability without impacting hyaluronan levels

    OpenAIRE

    Wei, Qin; Galbenus, Robert; Raza, Ashraf; Ronald L. Cerny; Simpson, Melanie A.

    2009-01-01

    UDP-glucose dehydrogenase (UGDH) oxidizes UDP-glucose to UDP-glucuronate, an essential precursor for production of hyaluronan (HA), proteoglycans, and xenobiotic glucuronides. High levels of HA turnover in prostate cancer are correlated with aggressive progression. UGDH expression is high in the normal prostate even though HA accumulation is virtually undetectable. Thus, its normal role in the prostate may be to provide precursors for glucuronosyltransferase enzymes, which inactivate and solu...

  9. 11beta-hydroxysteroid dehydrogenase type 1 regulates glucocorticoid-induced insulin resistance in skeletal muscle.

    LENUS (Irish Health Repository)

    Morgan, Stuart A

    2009-11-01

    Glucocorticoid excess is characterized by increased adiposity, skeletal myopathy, and insulin resistance, but the precise molecular mechanisms are unknown. Within skeletal muscle, 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) converts cortisone (11-dehydrocorticosterone in rodents) to active cortisol (corticosterone in rodents). We aimed to determine the mechanisms underpinning glucocorticoid-induced insulin resistance in skeletal muscle and indentify how 11beta-HSD1 inhibitors improve insulin sensitivity.

  10. Structure of quinoprotein methylamine dehydrogenase at 2.25 A resolution.

    OpenAIRE

    Vellieux, F. M.; Huitema, F; Groendijk, H; Kalk, K H; Jzn, J F; Jongejan, J.A.; Duine, J A; Petratos, K; DRENTH, J.; Hol, W. G.

    1989-01-01

    The three-dimensional structure of quinoprotein methylamine dehydrogenase from Thiobacillus versutus has been determined at 2.25 A resolution by a combination of multiple isomorphous replacement, phase extension by solvent flattening and partial structure phasing using molecular dynamics refinement. In the resulting map, the polypeptide chain for both subunits could be followed and an X-ray sequence was established. The tetrameric enzyme, made up of two heavy (H) and two light (L) subunits, i...

  11. Mitochondrial Pyruvate dehydrogenase E1 of Nosema bombycis: A Marker in Microsporidian evolution

    OpenAIRE

    Tian LI, Xiaoqun DANG, Jinshan XU, Handeng LIU, Guoqing PAN, Zeyang ZHOU

    2009-01-01

    Microsporidia are a group of intracelluar eukaryotic parasites, which can infected almost all animals, including human beings. Till now, no mitochodria but mitosome, a remnant of mitochondria was discovered in this phylum. We present here the mitochondrial pyruvate dehydrogenase E1 (PDH, including PDHα and PDHβ) of the microsporidian Nosema bombycis, the pathogen of silkworm pébrine. Compared with PDH of microsporidian Encephalitozoon cuniculi and Antonospora locustae, both subunits are conse...

  12. Developmental expression of Xenopus short-chain dehydrogenase/reductase 3

    OpenAIRE

    Kam, Richard Kin Ting; Chen, Yonglong; Chan, Sun On; Chan, Wood Yee; Dawid, Igor B.; Hui ZHAO

    2010-01-01

    During early embryonic development, the retinoic acid signaling pathway coordinates with other signaling pathways to regulate body axis patterning and organogenesis. The production of retinoic acid requires two enzymatic reactions, the first of which is the oxidization of vitamin A (all-trans-retinol) to all-trans-retinal, mediated in part by the short-chain dehydrogenase/reductase. Through DNA microarrays, we have identified a gene in Xenopus laevis, which shares a high sequence similarity t...

  13. Discovery of covalent inhibitors of glyceraldehyde-3-phosphate dehydrogenase, a target for the treatment of malaria.

    Science.gov (United States)

    Bruno, Stefano; Pinto, Andrea; Paredi, Gianluca; Tamborini, Lucia; De Micheli, Carlo; La Pietra, Valeria; Marinelli, Luciana; Novellino, Ettore; Conti, Paola; Mozzarelli, Andrea

    2014-09-11

    We developed a new class of covalent inhibitors of Plasmodium falciparum glyceraldehyde-3-phosphate dehydrogenase, a validated target for the treatment of malaria, by screening a small library of 3-bromo-isoxazoline derivatives that inactivate the enzyme through a covalent, selective bond to the catalytic cysteine, as demonstrated by mass spectrometry. Substituents on the isoxazolinic ring modulated the potency up to 20-fold, predominantly due to an electrostatic effect, as assessed by computational analysis. PMID:25137375

  14. The TyrA family of aromatic-pathway dehydrogenases in phylogenetic context

    OpenAIRE

    Wolinsky Murray; Bonner Carol A; Song Jian; Jensen Roy A

    2005-01-01

    Abstract Background The TyrA protein family includes members that catalyze two dehydrogenase reactions in distinct pathways leading to L-tyrosine and a third reaction that is not part of tyrosine biosynthesis. Family members share a catalytic core region of about 30 kDa, where inhibitors operate competitively by acting as substrate mimics. This protein family typifies many that are challenging for bioinformatic analysis because of relatively modest sequence conservation and small size. Result...

  15. Polymorphism of the parasite lactate dehydrogenase gene from Plasmodium vivax Korean isolates

    OpenAIRE

    Shin, Hyun-Il; KIM, Jung-Yeon; Lee, Won-Ja; Sohn, Youngjoo; Lee, Sang-wook; Kang, Yoon-Joong; Lee, Hyeong-Woo

    2013-01-01

    Background Assaying for the parasitic lactate dehydrogenase (pLDH) is widely used as a rapid diagnostic test (RDT), but the efficacy of its serological effectiveness in diagnosis, that is antibody detection ability, is not known. The genetic variation of Korean isolates was analysed, and recombinant protein pLDH was evaluated as a serodiagnostic antigen for the detection of Plasmodium vivax malaria. Methods Genomic DNA was purified, and the pLDH gene of P. vivax was amplified from blood sampl...

  16. Increased riboflavin production by manipulation of inosine 5′-monophosphate dehydrogenase in Ashbya gossypii

    OpenAIRE

    Buey, Ruben M.; Ledesma Amaro, Rodrigo; Balsera, Mónica; Revuelta Doval, José Luis

    2015-01-01

    Guanine nucleotides are the precursors of essential biomolecules including nucleic acids and vitamins such as riboflavin. The enzyme inosine-5′-monophosphate dehydrogenase (IMPDH) catalyzes the ratelimiting step in the guanine nucleotide de novo biosynthetic pathway and plays a key role in controlling the cellular nucleotide pools. Thus, IMPDH is an important metabolic bottleneck in the guanine nucleotide synthesis, susceptible of manipulation by means of metabolic engineering approaches. Her...

  17. Pyranose Dehydrogenase from Agaricus campestris and Agaricus xanthoderma: Characterization and Applications in Carbohydrate Conversions

    OpenAIRE

    Clemens K Peterbauer; Dietmar Haltrich; Petra Staudigl; Iris Krondorfer

    2013-01-01

    Pyranose dehydrogenase (PDH) is a flavin-dependent sugar oxidoreductase that is limited to a rather small group of litter-degrading basidiomycetes. The enzyme is unable to utilize oxygen as an electron acceptor, using substituted benzoquinones and (organo) metal ions instead. PDH displays a broad substrate specificity and intriguing variations in regioselectivity, depending on substrate, enzyme source and reaction conditions. In contrast to the related enzyme pyranose 2-oxidase (POx), PDHs fr...

  18. New studies of the alcohol dehydrogenase cline in D. melanogaster from Mexico.

    Science.gov (United States)

    Pipkin, S B; Franklin-Springer, E; Law, S; Lubega, S

    1976-01-01

    An altitudinal cline of frequencies of alcohol dehydrogenase alleles occurs in D. melanogaster populations of southeastern Mexico. A similar cline of two aldehyde oxidase alleles is present, but frequencies of esterase-6 alleles are not distributed clinically. Collections were made from small dispersed populations. Some gene flow occurred throughout the lowlands according to the distribution of two moderately endemic autosomal inversions and five previously described inversions. The clines are believed dependent on a limited gene flow between temperature races of D. melanogaster.

  19. Targeted Disruption of the Inosine 5′-Monophosphate Dehydrogenase Type I Gene in Mice

    OpenAIRE

    Gu, Jing Jin; Tolin, Amy K.; Jain, Jugnu; Huang, Hai; Santiago, Lalaine; Mitchell, Beverly S.

    2003-01-01

    Inosine 5′-monophosphate dehydrogenase (IMPDH) is the critical, rate-limiting enzyme in the de novo biosynthesis pathway for guanine nucleotides. Two separate isoenzymes, designated IMPDH types I and II, contribute to IMPDH activity. An additional pathway salvages guanine through the activity of hypoxanthine-guanine phosphoribosyltransferase (HPRT) to supply the cell with guanine nucleotides. In order to better understand the relative contributions of IMPDH types I and II and HPRT to normal b...

  20. Functional Distinctions between IMP Dehydrogenase Genes in Providing Mycophenolate Resistance and Guanine Prototrophy to Yeast*

    OpenAIRE

    Hyle, Judith W.; Shaw, Randal J.; Reines, Daniel

    2003-01-01

    IMP dehydrogenase (IMPDH) catalyzes the rate-limiting step in the de novo synthesis of GTP. Yeast with mutations in the transcription elongation machinery are sensitive to inhibitors of this enzyme such as 6-azauracil and mycophenolic acid, at least partly because of their inability to transcriptionally induce IMPDH. To understand the molecular basis of this drug-sensitive phenotype, we have dissected the expression and function of a four-gene family in yeast called IMD1 through IMD4. We show...

  1. The CBS subdomain of inosine 5’-monophosphate dehydrogenase regulates purine nucleotide turnover†

    OpenAIRE

    Pimkin, Maxim; Markham, George D.

    2008-01-01

    Inosine 5’-monophosphate dehydrogenase (IMPDH) catalyzes the rate limiting step in guanine nucleotide biosynthesis. IMPDH has an evolutionary conserved CBS subdomain of unknown function. The subdomain can be deleted without impairing the in vitro IMPDH catalytic activity and is the site for mutations associated with human retinitis pigmentosa. A guanine-prototrophic Escherichia coli strain, MP101, was constructed with the subdomain sequence deleted from the chromosomal gene for IMPDH. The ATP...

  2. Analysis of alcohol dehydrogenase inhibitors from Desmodium styracifolium using centrifugal ultrafiltration coupled with HPLC-MS

    OpenAIRE

    Liu Liangliang; Chen Miao; Chen Xiaoqing

    2015-01-01

    Alcohol dehydrogenase (ADH) inhibitors play an important role in the treatment of human methanol or ethylene glycol poisoning and the suppression of acetaldehyde accumulation in alcoholics. In this study, centrifugal ultrafiltration coupled with high performance liquid chromatography-mass spectrometry (HPLC-MS) was utilized to screen and identify ADH inhibitors from ethyl acetate extract of Desmosium styracifolium (Osb.) Merr. The experiment conditions of c...

  3. Identification of some ectomycorrhizal basidiomycetes by PCR amplification of their gpd (glyceraldehyde-3-phosphate dehydrogenase) genes.

    OpenAIRE

    Kreuzinger, N; Podeu, R; Gruber, F; Göbl, F; Kubicek, C P

    1996-01-01

    Degenerated oligonucleotide primers designed to flank an approximately 1.2-kb fragment of the gene encoding glyceraldehyde-3-phosphate dehydrogenase (gpd) from ascomycetes and basidiomycetes were used to amplify the corresponding gpd fragments from several species of the ectomycorrhizal fungal taxa Boletus, Amanita, and Lactarius. Those from B. edulis, A. muscaria, and L. deterrimus were cloned and sequenced. The respective nucleotide sequences of these gene fragments showed a moderate degree...

  4. Regulation of pyruvate dehydrogenase kinase activity by protein thiol-disulfide exchange.

    OpenAIRE

    Pettit, F H; Humphreys, J; Reed, L J

    1982-01-01

    Endogenous kinase activity of highly purified pyruvate dehydrogenase complex from bovine kidney is markedly inhibited by N-ethylmaleimide and by certain disulfides. Inhibition by disulfides is highly specific and is reversed by thiols. 5,5'-Dithiobis(2-nitrobenzoate) is the most potent inhibitor, showing significant inhibition at a concentration as low as 1 microM. Cystamine, oxidized glutathione, pantethine, lipoic acid, lipoamide, ergothionine, insulin, oxytocin, and vasopressin were ineffe...

  5. The effect of pH and ADP on ammonia affinity for human glutamate dehydrogenases

    DEFF Research Database (Denmark)

    Zaganas, Ioannis; Pajecka, Kamilla; Nielsen, Camilla Wendel;

    2013-01-01

    Glutamate dehydrogenase (GDH) uses ammonia to reversibly convert α-ketoglutarate to glutamate using NADP(H) and NAD(H) as cofactors. While GDH in most mammals is encoded by a single GLUD1 gene, humans and other primates have acquired a GLUD2 gene with distinct tissue expression profile. The two h...... of the kidney during systemic acidosis. The reverse could apply for conditions of local or systemic hyperammonemia or alkalosis....

  6. Characterization of the Two Neurospora crassa Cellobiose Dehydrogenases and Their Connection to Oxidative Cellulose Degradation

    OpenAIRE

    Sygmund, Christoph; Kracher, Daniel; Scheiblbrandner, Stefan; Zahma, Kawah; Felice, Alfons K. G.; Harreither, Wolfgang; Kittl, Roman; Ludwig, Roland

    2012-01-01

    The genome of Neurospora crassa encodes two different cellobiose dehydrogenases (CDHs) with a sequence identity of only 53%. So far, only CDH IIA, which is induced during growth on cellulose and features a C-terminal carbohydrate binding module (CBM), was detected in the secretome of N. crassa and preliminarily characterized. CDH IIB is not significantly upregulated during growth on cellulosic material and lacks a CBM. Since CDH IIB could not be identified in the secretome, both CDHs were rec...

  7. Simple and efficient expression of Agaricus meleagris pyranose dehydrogenase in Pichia pastoris

    OpenAIRE

    Sygmund, Christoph; Gutmann, Alexander; Krondorfer, Iris; Kujawa, Magdalena; Glieder, Anton; Pscheidt, Beate; Haltrich, Dietmar; Peterbauer, Clemens; Kittl, Roman

    2011-01-01

    Pyranose dehydrogenase (PDH) is a fungal flavin-dependent sugar oxidoreductase that is highly interesting for applications in organic synthesis or electrochemistry. The low expression levels of the filamentous fungus Agaricus meleagris as well as the demand for engineered PDH make heterologous expression necessary. Recently, Aspergillus species were described to efficiently secrete recombinant PDH. Here, we evaluate recombinant protein production with expression hosts more suitable for geneti...

  8. Structure-based drug design of 11β-hydroxysteroid dehydrogenase type 1 inhibitors

    OpenAIRE

    Adie, Jillian E.

    2010-01-01

    The enzyme 11β-Hydroxysteroid Dehydrogenase 1 (11β-HSD1) catalyses the intracellular biosynthesis of the active glucocorticoid cortisol. Tissue specific dysregulation of the enzyme has been implicated in the development of metabolic syndrome and other associated diseases. Experiments with transgenic mice and prototype inhibitors show that inhibition of 11β-HSD1 in visceral adipose tissue and liver leads to a resistance of diet-induced hyperglycemia and a favourable lipid and lipoprotein profi...

  9. Differential pulse voltammetric studies on the effects of Al(Ⅲ) on the lactate dehydrogenase activity

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    In this paper, differential pulse voltammetry (DPV) was applied to study the effects of aluminum Al(Ⅲ) on the lactate dehydrogenase (LDH) activity. Michaelis-Menten constant (KNADHm) and maximum velocity (vmax) in the enzyme promoting catalytic reaction of "pyruvate(Pyr) + NADH + H+ LDH(=) lactate + NAD+" under different conditions by monitoring DPV reduction current of NAD+ were reported.(C) 2007 Shu Ping Bi. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.

  10. Changes in short-chain acyl-coA dehydrogenase during rat cardiac development and stress

    OpenAIRE

    Huang, Jinxian; Xu, Lipeng; Huang, Qiuju; Luo, Jiani; Liu, Peiqing; Chen, Shaorui; Yuan, Xi; Lu, Yao; Wang, Ping; Zhou, Sigui

    2015-01-01

    This study was designed to investigate the expression of short-chain acyl-CoA dehydrogenase (SCAD), a key enzyme of fatty acid β-oxidation, during rat heart development and the difference of SCAD between pathological and physiological cardiac hypertrophy. The expression of SCAD was lowest in the foetal and neonatal heart, which had time-dependent increase during normal heart development. In contrast, a significant decrease in SCAD expression was observed in different ages of spontaneously hyp...

  11. Bilateral recurrent auricular pseudocyst: Importance of fine-needle aspiration cytology and lactate dehydrogenase estimation

    Directory of Open Access Journals (Sweden)

    Kalyan Khan

    2013-01-01

    Full Text Available Auricular pseudocyst or Idiopathic cystic chondromalacia is a rare, benign condition characterized by a focal noninflammatory cystic swelling on the pinna, occurring usually in young male patients. Bilaterality and recurrence have been reported rarely. We report a case of bilateral, recurrent auricular pseudocyst in a young male patient, where fine needle aspiration cytology coupled with fluid lactate dehydrogenase level estimation was diagnostic. Repeated surgery was avoided and conservative treatment was proved to be effective.

  12. XoxF-Type Methanol Dehydrogenase from the Anaerobic Methanotroph “Candidatus Methylomirabilis oxyfera”

    OpenAIRE

    Wu, Ming L.; Wessels, Hans J. C. T.; Pol, Arjan; Op den Camp, Huub J. M.; Mike S.M. Jetten; van Niftrik, Laura; Keltjens, Jan T.

    2014-01-01

    “Candidatus Methylomirabilis oxyfera” is a newly discovered anaerobic methanotroph that, surprisingly, oxidizes methane through an aerobic methane oxidation pathway. The second step in this aerobic pathway is the oxidation of methanol. In Gram-negative bacteria, the reaction is catalyzed by pyrroloquinoline quinone (PQQ)-dependent methanol dehydrogenase (MDH). The genome of “Ca. Methylomirabilis oxyfera” putatively encodes three different MDHs that are localized in one large gene cluster: one...

  13. Structure and Protein–Protein Interactions of Methanol Dehydrogenase from Methylococcus capsulatus (Bath)

    OpenAIRE

    Culpepper, Megen A.; Rosenzweig, Amy C.

    2014-01-01

    In the initial steps of their metabolic pathway, methanotrophic bacteria oxidize methane to methanol with methane monooxygenases (MMOs) and methanol to formaldehyde with methanol dehydrogenases (MDHs). Several lines of evidence suggest that the membrane-bound or particulate MMO (pMMO) and MDH interact to form a metabolic supercomplex. To further investigate the possible existence of such a supercomplex, native MDH from Methylococcus capsulatus (Bath) has been purified and characterized by siz...

  14. Prolonged QTc Interval in Association With Medium-Chain Acyl-Coenzyme A Dehydrogenase Deficiency

    OpenAIRE

    Wiles, Jason R.; Leslie, Nancy; Knilans, Timothy K.; Akinbi, Henry

    2014-01-01

    Medium-chain acyl-coenzyme A dehydrogenase (MCAD) deficiency is the most common disorder of mitochondrial fatty acid oxidation. We report a term male infant who presented at 3 days of age with hypoglycemia, compensated metabolic acidosis, hypocalcemia, and prolonged QTc interval. Pregnancy was complicated by maternal premature atrial contractions and premature ventricular contractions. Prolongation of the QTc interval resolved after correction of metabolic derangements. The newborn screen was...

  15. Duplication of Locus Coding of Malate Dehydrogenase in Populus tomentosa Carr.

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    Horizontal starch-gel electrophoresis was used to study crude enzyme extraction from young leaves of 234 clones of Populus tomentosa Carr. selected from nine provenances in North China. Ten enzyme systems were resolved. One hundred and fifty-six clones showing unusual allozyme band patterns at locus Mdh-1 were found. Three allozyme bands at locus Mdh-1 were 9:6:1 in concentration. Further studies on the electrophoretic patterns of ground mixed pollen extraction of 30 male clones selected at random from the 156 clones were conducted and it was found that allozyme bands at locus Mdh-1 were composed of two dark-stained bands and a weak band. Only one group of the malate dehydrogenase (MDH) zymogram composed of two bands was obtained from the electrophoretic segregation of pollen leachate of the same clones. A comparison of the electrophoretic patterns one another suggested that the locus Mdh-1 coding malate dehydrogenase in diploid species of P. tomentosa was duplicated. The duplicate gene locus possessed three same alleles and was located in mitochondria. The locus duplication of alleles coding malate dehydrogenase in P. tomentosa was discovered and reported for the first time.

  16. Structure-guided development of specific pyruvate dehydrogenase kinase inhibitors targeting the ATP-binding pocket.

    Science.gov (United States)

    Tso, Shih-Chia; Qi, Xiangbing; Gui, Wen-Jun; Wu, Cheng-Yang; Chuang, Jacinta L; Wernstedt-Asterholm, Ingrid; Morlock, Lorraine K; Owens, Kyle R; Scherer, Philipp E; Williams, Noelle S; Tambar, Uttam K; Wynn, R Max; Chuang, David T

    2014-02-14

    Pyruvate dehydrogenase kinase isoforms (PDKs 1-4) negatively regulate activity of the mitochondrial pyruvate dehydrogenase complex by reversible phosphorylation. PDK isoforms are up-regulated in obesity, diabetes, heart failure, and cancer and are potential therapeutic targets for these important human diseases. Here, we employed a structure-guided design to convert a known Hsp90 inhibitor to a series of highly specific PDK inhibitors, based on structural conservation in the ATP-binding pocket. The key step involved the substitution of a carbonyl group in the parent compound with a sulfonyl in the PDK inhibitors. The final compound of this series, 2-[(2,4-dihydroxyphenyl)sulfonyl]isoindoline-4,6-diol, designated PS10, inhibits all four PDK isoforms with IC50 = 0.8 μM for PDK2. The administration of PS10 (70 mg/kg) to diet-induced obese mice significantly augments pyruvate dehydrogenase complex activity with reduced phosphorylation in different tissues. Prolonged PS10 treatments result in improved glucose tolerance and notably lessened hepatic steatosis in the mouse model. The results support the pharmacological approach of targeting PDK to control both glucose and fat levels in obesity and type 2 diabetes. PMID:24356970

  17. Pyruvate dehydrogenase complex activity controls metabolic and malignant phenotype in cancer cells.

    Science.gov (United States)

    McFate, Thomas; Mohyeldin, Ahmed; Lu, Huasheng; Thakar, Jay; Henriques, Jeremy; Halim, Nader D; Wu, Hong; Schell, Michael J; Tsang, Tsz Mon; Teahan, Orla; Zhou, Shaoyu; Califano, Joseph A; Jeoung, Nam Ho; Harris, Robert A; Verma, Ajay

    2008-08-15

    High lactate generation and low glucose oxidation, despite normal oxygen conditions, are commonly seen in cancer cells and tumors. Historically known as the Warburg effect, this altered metabolic phenotype has long been correlated with malignant progression and poor clinical outcome. However, the mechanistic relationship between altered glucose metabolism and malignancy remains poorly understood. Here we show that inhibition of pyruvate dehydrogenase complex (PDC) activity contributes to the Warburg metabolic and malignant phenotype in human head and neck squamous cell carcinoma. PDC inhibition occurs via enhanced expression of pyruvate dehydrogenase kinase-1 (PDK-1), which results in inhibitory phosphorylation of the pyruvate dehydrogenase alpha (PDHalpha) subunit. We also demonstrate that PDC inhibition in cancer cells is associated with normoxic stabilization of the malignancy-promoting transcription factor hypoxia-inducible factor-1alpha (HIF-1alpha) by glycolytic metabolites. Knockdown of PDK-1 via short hairpin RNA lowers PDHalpha phosphorylation, restores PDC activity, reverts the Warburg metabolic phenotype, decreases normoxic HIF-1alpha expression, lowers hypoxic cell survival, decreases invasiveness, and inhibits tumor growth. PDK-1 is an HIF-1-regulated gene, and these data suggest that the buildup of glycolytic metabolites, resulting from high PDK-1 expression, may in turn promote HIF-1 activation, thus sustaining a feed-forward loop for malignant progression. In addition to providing anabolic support for cancer cells, altered fuel metabolism thus supports a malignant phenotype. Correction of metabolic abnormalities offers unique opportunities for cancer treatment and may potentially synergize with other cancer therapies. PMID:18541534

  18. Detection of enzymes dehydrogenases and proteases inBrugia malayi filarial parasites.

    Science.gov (United States)

    Bhandary, Y P; Krithika, K N; Kulkarni, Sandeep; Reddy, M V R; Harinath, B C

    2006-03-01

    Lymphatic filariasis caused mainly by infection fromW. bancrofti andB. malayi remains a major cause of clinical morbidity in tropical and subtropical countries. Analysis ofB. malayi mf, infective larval and adult worm lysates for the activity of enzymes led to the demonstration of activities of three key enzymes of carbohydrate metabolism viz., Malate dehydrogenase (MDH), Malic enzyme (ME) and Glucose-6-phosphate dehydrogenase (G6PDH) in all the three stages of the parasite. The specific activity of all the three dehydrogenases was significantly high in mf lysate compared to their activity in lysates of the other two stages (PFlouride (PMSF). In sodium do-decyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), using gelatin copolymerized gel, the microfilarial lysate showed 3 protease molecules of 40 kDa, 180 kDa and 200 kDa and the L(3) larval lysate had 6 protease molecules of 18, 25, 37, 49, 70 and 200 kDa size.

  19. Surviving environmental stress: the role of betaine aldehyde dehydrogenase in marine crustaceans

    Directory of Open Access Journals (Sweden)

    NA Stephens-Camacho

    2015-02-01

    Full Text Available Betaine aldehyde dehydrogenase (BADH belongs to the aldehyde dehydrogenases (ALDH family, an ancestral group of enzymes responsible for aldehyde detoxification in several organisms. The BADH enzyme catalyzes the irreversible oxidation of betaine aldehyde to glycine betaine (GB an important osmoptrotector and osmoregulator accumulated in response to cellular osmotic stress. The BADH enzymes have been extensively described in terrestrial organisms, but information in marine crustaceans remains scarce. Research on crustacean stress-adaptive capacity to environmental stressors relates GB accumulation in response to salinity variations. Although GB de novo synthesis is confirmed on crustaceans, its metabolic pathways and regulation mechanism are unexplored. In this work, the state of the knowledge of betaine aldehyde dehydrogenase enzymes in marine crustaceans is summarized, as a mechanism to overcome the deleterious effects of changes in temperature, salinity and dissolved oxygen concentration in seawater. The purpose of this review is to provide a more comprehensive overview to set the basis for exploring novel functions and properties of BADHs on the response of crustaceans to environmental stress.

  20. Cellular and subcellular localization of hexokinase, glutamate dehydrogenase, and alanine aminotransferase in the honeybee drone retina.

    Science.gov (United States)

    Veuthey, A L; Tsacopoulos, M; Millan de Ruiz, L; Perrottet, P

    1994-05-01

    Subcellular localization of hexokinase in the honeybee drone retina was examined following fractionation of cell homogenate using differential centrifugation. Nearly all hexokinase activity was found in the cytosolic fraction, following a similar distribution as the cytosolic enzymatic marker, phosphoglycerate kinase. The distribution of enzymatic markers of mitochondria (succinate dehydrogenase, rotenone-insensitive cytochrome c reductase, and adenylate kinase) indicated that the outer mitochondrial membrane was partly damaged, but their distributions were different from that of hexokinase. The activity of hexokinase in purified suspensions of cells was fivefold higher in glial cells than in photoreceptors. This result is consistent with the hypothesis based on quantitative 2-deoxy[3H]glucose autoradiography that only glial cells phosphorylate significant amounts of glucose to glucose-6-phosphate. The activities of alanine aminotransferase and to a lesser extent of glutamate dehydrogenase were higher in the cytosolic than in the mitochondrial fraction. This important cytosolic activity of glutamate dehydrogenase was consistent with the higher activity found in mitochondria-poor glial cells. In conclusion, this distribution of enzymes is consistent with the model of metabolic interactions between glial and photoreceptor cells in the intact bee retina. PMID:8158142

  1. [Possible ways of regulating detoxifying processes in the alcohol dehydrogenase reaction with pantothenic acid derivatives].

    Science.gov (United States)

    Chernikevich, I P; Dorofeev, B F; Moĭseenok, A G

    1993-01-01

    Oxidation of derivatives and precursors of pantothenic acid was studied in alcohol dehydrogenase reactions. Despite the presence of free hydroxymethyl groups in a number of pantothenic acid derivatives only panthenol with Km = 8 x 10(-3) M was shown to serve as a substrate for alcohol dehydrogenase from horse liver tissue (EC 1.1.1.1) Pantethine, sodium phosphopantothenate, CoA and acetyl-CoA decreased the rate of ethanol oxidation, where pantethine and sodium phosphopantothenate were competitive inhibitors, while CoA and acetyl-CoA inhibited the enzyme noncompetitively Ki = 1.2 x 10(-2) M, 2.1 x 10(-2) M, 4.4 x 10(-4) M and 5.1 x 10(-4) M, respectively. Metabolic precursors, which were different from pantothenic acid in their structure, were not involved in the alcohol dehydrogenase reaction. Possible regulation of alcohol intoxication using derivatives and precursors of vitamin B3 is discussed. PMID:8511887

  2. Human liver alcohol dehydrogenase. 1. The primary structure of the beta 1 beta 1 isoenzyme.

    Science.gov (United States)

    Hempel, J; Bühler, R; Kaiser, R; Holmquist, B; de Zalenski, C; von Wartburg, J P; Vallee, B; Jörnvall, H

    1984-12-17

    Determination of the amino acid sequence of the beta 1 subunit from the class I (pyrazole-sensitive) human liver alcohol dehydrogenase isoenzyme beta 1 beta 1 revealed a 373-residue structure differing at 48 positions (including a gap) from that of the subunit of the well studied horse liver alcohol dehydrogenase EE isoenzyme. The structure deduced is compatible with known differences in composition, ultraviolet absorbance, electrophoretic mobility and catalytic properties between the horse and human enzymes. All zinc-liganding residues of the horse E subunit are strictly conserved in the human beta 1 subunit, despite an earlier report of a mutation involving Cys-46. This residue therefore remains conserved in all known alcohol dehydrogenase structures. However, the total cysteine content of the beta 1 structure is raised from 14 in the subunit of the horse enzyme to 15 by a Tyr----Cys exchange. Most exchanges are on the surface of the molecule and of a well conserved nature. Substitutions close to the catalytic centre are of interest to explain the altered substrate specificity and different catalytic activity of the beta 1 homodimer. Functionally, a Ser----Thr exchange at position 48 appears to be of special importance, since Thr-48 in beta 1 instead of Ser-48 in the horse enzyme can restrict available space. Four other substitutions also line the active-site pocket, and appear to constitute partly compensated exchanges. PMID:6391920

  3. The suitability of saliva for detection of glucose-6-phosphate dehydrogenase deficiency.

    Science.gov (United States)

    Beamont, A H; Miguel, A; Goos, C M; Vermeesch-Markslag, A M; Hermans, A; Vermorken, A J

    1988-01-01

    Saliva was investigated for its suitability as a biopsy tissue for the determination of glucose-6-phosphate dehydrogenase deficiency. It appears that there is a significant difference between the activity of the enzyme in patients and controls. However, some controls have very low values making discrimination between patients and controls using a qualitative method impossible. Glucose-6-phosphate dehydrogenase deficiency is a relevant clinical problem in many rural areas in developing countries. Existing methods for determination of the deficiency in blood and hair follicles do not meet the criteria necessary for their large scale introduction in the areas of the world that are concerned by the problem. The present study shows that saliva is not a suitable alternative. Between the three biopsy tissues compared: blood, hair follicles and saliva, hair follicles remain most attractive since their isolation hardly involves the risk of infection. A simplified method for the detection of glucose-6-phosphate dehydrogenase activity in hair follicles that would allow health service workers in the field to determine the carrier status of pregnant women might form the basis for a future kernicterus prevention programme. PMID:3221843

  4. Novel Diketopiperazine Dihydroorotate Dehydrogenase Inhibitors Purified from Traditional Tibetan Animal Medicine Osteon Myospalacem Baileyi.

    Science.gov (United States)

    Jiang, Lei; Wen, Huaixiu; Shao, Yun; Yu, Ruitao; Liu, Zenggen; Wang, Shuo; Wang, Qilan; Zhao, Xiaohui; Zhang, Peng; Tao, Yanduo; Mei, Lijuan

    2015-10-01

    Traditional Tibetan medicine provides an abundant source of knowledge on human ailments and their treatment. As such, it is necessary to explore their active single compounds used to treat these ailments to discover lead compounds with good pharmacologic properties. In this present work, animal medicine, Osteon Myospalacem Baileyi extracts have been separated using a two-dimensional preparative chromatographic method to obtain single compounds with high purity as part of the following pharmacological research. Five high-purity cyclic dipeptides from chromatography work were studied for their dihydroorotate dehydrogenase inhibitory activity on recombinant human dihydroorotate dehydrogenase enzyme and compound Fr. 1-4 was found to contain satisfying inhibition activity. The molecular modeling study suggests that the active compound Fr. 1-4 may have a teriflunomide-like binding mode. Then, the energy decomposition study suggests that the hydrogen bond between Fr. 1-4 and Arg136 can improve the binding mode to indirectly increase the van der Waals binding energy. All the results above together come to the conclusion that the 2, 5-diketopiperazine structure group can interact with the polar residues well in the active pocket using electrostatic power. If some proper hydrophobic groups can be added to the sides of the 2, 5-diketopiperazine group, it is believed that better 2, 5-diketopiperazine dihydroorotate dehydrogenase inhibitors will be found in the future.

  5. Purification, characterization, and cDNA cloning of opine dehydrogenases from the polychaete rockworm Marphysa sanguinea.

    Science.gov (United States)

    Endo, Noriyuki; Kan-no, Nobuhiro; Nagahisa, Eizoh

    2007-06-01

    Alanopine dehydrogenase (AlDH) and three isoforms of strombine/alanopine dehydrogenase (St/AlDH) were purified from muscle tissue of the polychaete rockworm Marphysa sanguinea. The four enzymes, which can be distinguished by the isoelectric point, are monomeric 42 kDa proteins, possess similar pH-activity profiles, and display specificity for pyruvate and NAD(H). The three isoforms of St/AlDH show equivalent Km and Vmax for glycine and L-alanine and for D-strombine and meso-alanopine. Free amino acid levels in the muscle and D-strombine accumulation in vivo during muscle activity suggest that St/AlDHs function physiologically as StDH. AlDH shows specificity for L-alanine and meso-alanopine, but not for glycine or D-strombine. The amino acid sequences of AlDH and one of the St/AlDH isoforms were determined by a combination of amino acid sequence analysis and cDNA cloning. St/AlDH cDNA consisted of 1586 bp nucleotides that encode a 399-residue protein (43,346.70 Da), and AlDH cDNA consisted of 1587 bp nucleotides that encode a 399-residue protein (43,886.68 Da). The two amino acid sequences deduced from the cDNA displayed 67% amino acid identity, with greatest similarity to that of tauropine dehydrogenase from the polychaete Arabella iricolor. PMID:17350870

  6. Enzyme inhibition assay for pyruvate dehydrogenase complex: Clinical utility for the diagnosis of primary biliary cirrhosis

    Institute of Scientific and Technical Information of China (English)

    Katsuhisa Omagri; Hiroaki Hazama; Shigeru Kohno

    2005-01-01

    Primary biliary cirrhosis (PBC) is usually diagnosed by the presence of characteristic histopathological features of the liver and/or antimitochondrial antibodies (AMA) in the serum traditionally detected by immunofluorescence.Recently, new and more accurate serological assays for the detection of AMA, such as enzyme-linked immunosorbent assay (ELISA), immunoblotting, and enzyme inhibition assay, have been developed. Of these,the enzyme inhibition assay for the detection of antipyruvate dehydrogenase complex (PDC) antibodies offers certain advantages such as objectivity, rapidity,simplicity, and low cost. Since this assay has almost 100% specificity, it may have particular applicability in screening the at-risk segment of the population in developing countries. Moreover, this assay could be also used for monitoring the disease course in PBC. Almost all sera of PBC-suspected patients can be confirmed for PBC or non-PBC by the combination results of immunoblotting and enzyme inhibition assay without histopathological examination. For the development of a "complete" or "gold standard" diagnostic assay for PBC, similar assays of the enzyme inhibition for anti2-oxoglutarate dehydrogenase complex (OGDC) and anti-branched chain oxo-acid dehydrogenase complex (BCOADC) antibodies will be needed in future.

  7. [Use of immobilization in the study of glyceraldehyde 3-phosphate dehydrogenase. Immobilized monomers].

    Science.gov (United States)

    Muronets, V I; Ashmarina, L I; Asriiants, R A; Nagradova, N K

    1982-06-01

    Active immobilized monomers of glyceraldehyde 3-phosphate dehydrogenase were prepared by means of dissociation of the tetrameric enzyme molecule covalently bound to Sepharose via a single subunit. The conditions were elaborated to achieve the inactivation and solubilization of the non-covalently bound subunits leaving the monomer coupled to the matrix intact. This procedure differs from the previously developed method of matrix-bound oligomeric enzymes dissociation in a detail which was found to be essentially important. The widely used method includes complete denaturation of all subunits during treatment with urea followed by reactivation of the immobilized one, whereas only the non-covalently bound subunits suffer denaturation under the conditions developed in the present work. The immobilized monomers of glyceraldehyde 3-phosphate dehydrogenase exhibit Vmax and Km (for NAD and substrate) values similar to those found for the immobilized tetramer. Reassociation of the immobilized monomers with soluble enzyme subunits obtained in the presence of urea produces matrix-bound tetrameric species. Immobilized trimers ae formed upon incubation of matrix-bound monomers in a diluted apoenzyme solution. The immobilized monomeric, trimeric and tetrameric enzyme species were used to study the role of subunit interactions in cooperative phenomena exhibited by the dehydrogenase. PMID:7115810

  8. Acute overexpression of lactate dehydrogenase-A perturbs beta-cell mitochondrial metabolism and insulin secretion.

    Science.gov (United States)

    Ainscow, E K; Zhao, C; Rutter, G A

    2000-07-01

    Islet beta-cells express low levels of lactate dehydrogenase and have high glycerol phosphate dehydrogenase activity. To determine whether this configuration favors oxidative glucose metabolism via mitochondria in the beta-cell and is important for beta-cell metabolic signal transduction, we have determined the effects on glucose metabolism and insulin secretion of acute overexpression of the skeletal muscle isoform of lactate dehydrogenase (LDH)-A. Monitored in single MIN6 beta-cells, LDH hyperexpression (achieved by intranuclear cDNA microinjection or adenoviral infection) diminished the response to glucose of both phases of increases in mitochondrial NAD(P)H, as well as increases in mitochondrial membrane potential, cytosolic free ATP, and cystolic free Ca2+. These effects were observed at all glucose concentrations, but were most pronounced at submaximal glucose levels. Correspondingly, adenoviral vector-mediated LDH-A overexpression reduced insulin secretion stimulated by 11 mmol/l glucose and the subsequent response to stimulation with 30 mmol/l glucose, but it was without significant effect when the concentration of glucose was raised acutely from 3 to 30 mmol/l. Thus, overexpression of LDH activity interferes with normal glucose metabolism and insulin secretion in the islet beta-cell type, and it may therefore be directly responsible for insulin secretory defects in some forms of type 2 diabetes. The results also reinforce the view that glucose-derived pyruvate metabolism in the mitochondrion is critical for glucose-stimulated insulin secretion in the beta-cell.

  9. Steroid Biomarkers and Genetic Studies Reveal Inactivating Mutations in Hexose-6-Phosphate Dehydrogenase in Patients with Cortisone Reductase Deficiency

    OpenAIRE

    Lavery, Gareth G.; Walker, Elizabeth A.; Tiganescu, Ana; Ride, Jon P.; Shackleton, Cedric H. L.; Tomlinson, Jeremy W.; Connell, John M C; Ray, David W; Biason-Lauber, Anna; Malunowicz, Ewa M.; Arlt, Wiebke; Stewart, Paul M.

    2008-01-01

    Context: Cortisone reductase deficiency (CRD) is characterized by a failure to regenerate cortisol from cortisone via 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), resulting in increased cortisol clearance, activation of the hypothalamic-pituitary-axis (HPA) and ACTH-mediated adrenal androgen excess. 11β-HSD1 oxoreductase activity requires the reduced nicotinamide adenine dinucleotide phosphate-generating enzyme hexose-6-phosphate dehydrogenase (H6PDH) within the endoplasmic reticulum. ...

  10. The methanol dehydrogenase structural gene mxaF and its use as a functional gene probe for methanotrophs and methylotrophs.

    OpenAIRE

    McDonald, I R; Murrell, J.C.

    1997-01-01

    The methanol dehydrogenase gene mxaF, encoding the large subunit of the enzyme, was amplified from the DNA of a number of representative methanotrophs, methyletrophs, and environmental samples by PCR using primers designed from regions of conserved amino acid sequence identified by comparison of three known sequences of the large subunit of methanol dehydrogenase. The resulting 550-bp PCR products were cloned and sequenced. Analysis of the predicted amino acid sequences corresponding to these...

  11. Pivotal Role of the C-terminal DW-motif in Mediating Inhibition of Pyruvate Dehydrogenase Kinase 2 by Dichloroacetate*

    OpenAIRE

    Li, Jun; Kato, Masato; Chuang, David T.

    2009-01-01

    The mitochondrial pyruvate dehydrogenase complex (PDC) is down-regulated by phosphorylation catalyzed by pyruvate dehydrogenase kinase (PDK) isoforms 1–4. Overexpression of PDK isoforms and therefore reduced PDC activity prevails in cancer and diabetes. In the present study, we investigated the role of the invariant C-terminal DW-motif in inhibition of human PDK2 by dichloroacetate (DCA). Substitutions were made in the DW-motif (Asp-382 and Trp-383) and its interacting residues (Tyr-145 and A...

  12. Pentitol phosphate dehydrogenases: Discovery, characterization and use in D-arabitol and xylitol production by metabolically engineered Bacillus subtilis

    OpenAIRE

    Povelainen, Mira

    2008-01-01

    The ultimate goal of this study has been to construct metabolically engineered microbial strains capable of fermenting glucose into pentitols D-arabitol and, especially, xylitol. The path that was chosen to achieve this goal required discovery, isolation and sequencing of at least two pentitol phosphate dehydrogenases of different specificity, followed by cloning and expression of their genes and characterization of recombinant arabitol and xylitol phosphate dehydrogenases. An enzyme of ...

  13. Ethanol utilization regulatory protein: profile alignments give no evidence of origin through aldehyde and alcohol dehydrogenase gene fusion.

    OpenAIRE

    Nicholas, H B; Persson, B; Jörnvall, H; Hempel, J.

    1995-01-01

    The suggestion that the ethanol regulatory protein from Aspergillus has its evolutionary origin in a gene fusion between aldehyde and alcohol dehydrogenase genes (Hawkins AR, Lamb HK, Radford A, Moore JD, 1994, Gene 146:145-158) has been tested by profile analysis with aldehyde and alcohol dehydrogenase family profiles. We show that the degree and kind of similarity observed between these profiles and the ethanol regulatory protein sequence is that expected from random sequences of the same c...

  14. Retinoic acid response element in the human alcohol dehydrogenase gene ADH3: implications for regulation of retinoic acid synthesis.

    OpenAIRE

    Duester, G; Shean, M L; McBride, M S; Stewart, M J

    1991-01-01

    Retinoic acid regulation of one member of the human class I alcohol dehydrogenase (ADH) gene family was demonstrated, suggesting that the retinol dehydrogenase function of ADH may play a regulatory role in the biosynthetic pathway for retinoic acid. Promoter activity of human ADH3, but not ADH1 or ADH2, was shown to be activated by retinoic acid in transient transfection assays of Hep3B human hepatoma cells. Deletion mapping experiments identified a region in the ADH3 promoter located between...

  15. Physiological regulation of isocitrate dehydrogenase and the role of 2-oxoglutarate in Prochlorococcus sp. strain PCC 9511.

    Directory of Open Access Journals (Sweden)

    María Agustina Domínguez-Martín

    Full Text Available The enzyme isocitrate dehydrogenase (ICDH; EC 1.1.1.42 catalyzes the oxidative decarboxylation of isocitrate, to produce 2-oxoglutarate. The incompleteness of the tricarboxylic acids cycle in marine cyanobacteria confers a special importance to isocitrate dehydrogenase in the C/N balance, since 2-oxoglutarate can only be metabolized through the glutamine synthetase/glutamate synthase pathway. The physiological regulation of isocitrate dehydrogenase was studied in cultures of Prochlorococcus sp. strain PCC 9511, by measuring enzyme activity and concentration using the NADPH production assay and Western blotting, respectively. The enzyme activity showed little changes under nitrogen or phosphorus starvation, or upon addition of the inhibitors DCMU, DBMIB and MSX. Azaserine, an inhibitor of glutamate synthase, induced clear increases in the isocitrate dehydrogenase activity and icd gene expression after 24 h, and also in the 2-oxoglutarate concentration. Iron starvation had the most significant effect, inducing a complete loss of isocitrate dehydrogenase activity, possibly mediated by a process of oxidative inactivation, while its concentration was unaffected. Our results suggest that isocitrate dehydrogenase responds to changes in the intracellular concentration of 2-oxoglutarate and to the redox status of the cells in Prochlorococcus.

  16. Further studies on the bioaffinity chromatography of NAD(+)-dependent dehydrogenases using the locking-on effect.

    Science.gov (United States)

    O'Carra, P; Griffin, T; O'Flaherty, M; Kelly, N; Mulcahy, P

    1996-10-17

    Previous studies have capitalized on ordered kinetic mechanisms in the design of biospecific affinity chromatographic methods for highly efficient purifications and mechanistic studies of enzymes. The most direct tactic has been the use of immobilised analogues of the following, usually enzyme-specific substrates, e.g., lactate/pyruvate in the case of lactate dehydrogenase for which NAD+ is the leading substrate. Such immobilised specific substrates are, however, often difficult or impossible to synthesise. The locking-on strategy reverses the tactic by using the more accessible immobilised leading substrate, immobilised NAD+, as adsorbent with soluble analogues of the enzyme-specific ligands (e.g., lactate in the case of lactate dehydrogenase) providing a substantial reinforcement of biospecific adsorption sufficient to effect adsorptive selection of an enzyme from a group of enzymes such as the NAD(+)-specific enzymes. The value of this approach is demonstrated using model studies with lactate dehydrogenase (LDH, EC 1.1.1.27), alcohol dehydrogenase (ADH, EC 1.1.1.1), glutamate dehydrogenase (GDH, EC 1.4.1.3) and malate dehydrogenase (MDH, EC 1.1.1.37). Purification of bovine liver GDH in high yield from crude extracts is described using the tactic. PMID:8917627

  17. Differing features of proteins in membranes may result in antioxidant or prooxidant action: opposite effects on lipid peroxidation of alcohol dehydrogenase and albumin in liposomal systems.

    Science.gov (United States)

    Riedl, A; Shamsi, Z; Anderton, M; Goldfarb, P; Wiseman, A

    1996-02-01

    The influence of 3 thiol-containing compounds, bovine serum albumin (fatty acid free: BSA), glutathione (GSH) and yeast alcohol dehydrogenase (YADH) on lipid peroxidation in multilamellar liposomes, prepared from ox-brain phospholipid, was investigated. Thiol-compounds were added either before liposome formation, or after liposome formation; and their effects compared to a positive control. Bovine serum albumin (BSA), an acidic hydrophilic protein, displays a small, concentration dependent, antioxidant effect when added to preformed liposomes. A much larger antioxidant effect was observed when the BSA was entrapped inside the liposome, by adding BSA just prior to liposome preparation. In contrast, a Zn(2+) containing redox enzyme, YADH, a basic hydrophobic membrane-associating protein, displays a large pro-oxidant effect at much lower concentrations especially when entrapped inside the liposome. This was observed also with GSH; but per mole of -SH, YADH was about 18 times as powerful a pro-oxidant perhaps because of structural changes to the membrane. Oxidized glutathione and N-acetylcysteine were also pro-oxidant (cysteine and cystine showed little effect). Formation of thiyl radicals may occur in the presence of iron ions with these pro-oxidant sulphur-containing compounds. Partial protection against lipid peroxidation was observed with EDTA, desferrioxamine and protoporphyrin (IX), potent iron-chelating agents.

  18. Determination of Dehydrogenase Activities Involved in D-Glucose Oxidation in Gluconobacter and Acetobacter Strains.

    Science.gov (United States)

    Sainz, Florencia; Jesús Torija, María; Matsutani, Minenosuke; Kataoka, Naoya; Yakushi, Toshiharu; Matsushita, Kazunobu; Mas, Albert

    2016-01-01

    Acetic acid bacteria (AAB) are known for rapid and incomplete oxidation of an extensively variety of alcohols and carbohydrates, resulting in the accumulation of organic acids as the final products. These oxidative fermentations in AAB are catalyzed by PQQ- or FAD- dependent membrane-bound dehydrogenases. In the present study, the enzyme activity of the membrane-bound dehydrogenases [membrane-bound PQQ-glucose dehydrogenase (mGDH), D-gluconate dehydrogenase (GADH) and membrane-bound glycerol dehydrogenase (GLDH)] involved in the oxidation of D-glucose and D-gluconic acid (GA) was determined in six strains of three different species of AAB (three natural and three type strains). Moreover, the effect of these activities on the production of related metabolites [GA, 2-keto-D-gluconic acid (2KGA) and 5-keto-D-gluconic acid (5KGA)] was analyzed. The natural strains belonging to Gluconobacter showed a high mGDH activity and low activity in GADH and GLDH, whereas the Acetobacter malorum strain presented low activity in the three enzymes. Nevertheless, no correlation was observed between the activity of these enzymes and the concentration of the corresponding metabolites. In fact, all the tested strains were able to oxidize D-glucose to GA, being maximal at the late exponential phase of the AAB growth (24 h), which coincided with D-glucose exhaustion and the maximum mGDH activity. Instead, only some of the tested strains were capable of producing 2KGA and/or 5KGA. In the case of Gluconobacter oxydans strains, no 2KGA production was detected which is related to the absence of GADH activity after 24 h, while in the remaining strains, detection of GADH activity after 24 h resulted in a high accumulation of 2KGA. Therefore, it is possible to choose the best strain depending on the desired product composition. Moreover, the sequences of these genes were used to construct phylogenetic trees. According to the sequence of gcd, gene coding for mGDH, Acetobacter and Komagataeibacter

  19. Determination of dehydrogenase activities involved in D-glucose oxidation in Gluconobacter and Acetobacter strains

    Directory of Open Access Journals (Sweden)

    Florencia Sainz

    2016-08-01

    Full Text Available Acetic acid bacteria (AAB are known for rapid and incomplete oxidation of an extensively variety of alcohols and carbohydrates, resulting in the accumulation of organic acids as the final products. These oxidative fermentations in AAB are catalyzed by PQQ- or FAD- dependent membrane bound dehydrogenases. In the present study, the enzyme activity of the membrane bound dehydrogenases (membrane-bound PQQ-glucose dehydrogenase (mGDH, D-gluconate dehydrogenase (GADH and membrane-bound glycerol dehydrogenase (GLDH involved in the oxidation of D-glucose and D-gluconic acid (GA was determined in six strains of three different species of AAB (three natural and three type strains. Moreover, the effect of these activities on the production of related metabolites (GA, 2-keto-D-gluconic acid (2KGA and 5-keto-D-gluconic acid (5KGA was analyzed. The natural strains belonging to Gluconobacter showed a high mGDH activity and low activity in GADH and GLDH, whereas the A. malorum strain presented low activity in the three enzymes. Nevertheless, no correlation was observed between the activity of these enzymes and the concentration of the corresponding metabolites. In fact, all the tested strains were able to oxidize D-glucose to GA, being maximal at the late exponential phase of the AAB growth (24 h, which coincided with glucose exhaustion and the maximum mGDH activity. Instead, only some of the tested strains were capable of producing 2KGA and/or 5KGA. In the case of G. oxydans strains, no 2KGA production was detected which is related to the absence of GADH activity after 24 h, while in the remaining strains, detection of GADH activity after 24h resulted in a high accumulation of 2KGA. Therefore, it is possible to choose the best strain depending on the desired product composition.Moreover, the sequences of these genes were used to construct phylogenetic trees. According to the sequence of gcd, gene coding for mGDH, Acetobacter and Komagataeibacter were

  20. Determination of Dehydrogenase Activities Involved in D-Glucose Oxidation in Gluconobacter and Acetobacter Strains

    Science.gov (United States)

    Sainz, Florencia; Jesús Torija, María; Matsutani, Minenosuke; Kataoka, Naoya; Yakushi, Toshiharu; Matsushita, Kazunobu; Mas, Albert

    2016-01-01

    Acetic acid bacteria (AAB) are known for rapid and incomplete oxidation of an extensively variety of alcohols and carbohydrates, resulting in the accumulation of organic acids as the final products. These oxidative fermentations in AAB are catalyzed by PQQ- or FAD- dependent membrane-bound dehydrogenases. In the present study, the enzyme activity of the membrane-bound dehydrogenases [membrane-bound PQQ-glucose dehydrogenase (mGDH), D-gluconate dehydrogenase (GADH) and membrane-bound glycerol dehydrogenase (GLDH)] involved in the oxidation of D-glucose and D-gluconic acid (GA) was determined in six strains of three different species of AAB (three natural and three type strains). Moreover, the effect of these activities on the production of related metabolites [GA, 2-keto-D-gluconic acid (2KGA) and 5-keto-D-gluconic acid (5KGA)] was analyzed. The natural strains belonging to Gluconobacter showed a high mGDH activity and low activity in GADH and GLDH, whereas the Acetobacter malorum strain presented low activity in the three enzymes. Nevertheless, no correlation was observed between the activity of these enzymes and the concentration of the corresponding metabolites. In fact, all the tested strains were able to oxidize D-glucose to GA, being maximal at the late exponential phase of the AAB growth (24 h), which coincided with D-glucose exhaustion and the maximum mGDH activity. Instead, only some of the tested strains were capable of producing 2KGA and/or 5KGA. In the case of Gluconobacter oxydans strains, no 2KGA production was detected which is related to the absence of GADH activity after 24 h, while in the remaining strains, detection of GADH activity after 24 h resulted in a high accumulation of 2KGA. Therefore, it is possible to choose the best strain depending on the desired product composition. Moreover, the sequences of these genes were used to construct phylogenetic trees. According to the sequence of gcd, gene coding for mGDH, Acetobacter and Komagataeibacter

  1. Cyclic AMP and AKAP-mediated targeting of protein kinase A regulates lactate dehydrogenase subunit A mRNA stability.

    Science.gov (United States)

    Jungmann, Richard A; Kiryukhina, Olga

    2005-07-01

    Expression of the lactate dehydrogenase A subunit (ldh-A) gene is controlled through transcriptional as well as post-transcriptional mechanisms. Both mechanisms involve activation of protein kinase A (PKA) into its subunits and subsequent phosphorylation and activation of several key regulatory factors. In rat C6 glioma cells, post-transcriptional gene regulation occurs through PKA-mediated stabilization of LDH-A mRNA and subsequent increase of intracellular LDH-A mRNA levels. Previous studies have demonstrated a cAMP-stabilizing region (CSR) located in the LDH-A 3'-untranslated region which, in combination with several phosphorylated CSR-binding proteins (CSR-BP), regulates the PKA-mediated stabilization of LDH-A mRNA. However, the mechanistic details of interaction of CSR with proteins as they pertain to mRNA stabilization by PKA are so far largely unknown. In this study we tested the hypothesis that ribosomal protein extracts (RSW) from glioma cells contain PKA regulatory (RII) and catalytic (C) subunits that, in combination with a protein kinase A anchoring protein (AKAP 95) and CSR-BPs participate in forming CSR-protein complexes that are responsible for mRNA stability regulation. To demonstrate the importance of CSR-protein complex formation, the PKA subunits and AKAP 95 were removed from the RSW by immunoprecipitation, and the antigen-deleted RSW were subjected to CSR binding analysis using gel mobility shift and UV cross-linking. It was shown that AKAP 95 as well as RII formed a direct linkage with CSR during CSR-protein complex formation. In contrast, the catalytic subunit formed part of the CSR-protein complex but did not bind to CSR directly in a covalent linkage. To determine whether formation of CSR complexes that included C, RII, and AKAP 95 constituted a functional event and was necessary for mRNA stabilization, cell-free decay reactions were carried out with RSW extracts, and the kinetics of decay of LDH-A mRNA was determined. Depletion of PKA

  2. Coupled reactions on bioparticles: Stereoselective reduction with cofactor regeneration on PhaC inclusion bodies.

    Science.gov (United States)

    Spieler, Valerie; Valldorf, Bernhard; Maaß, Franziska; Kleinschek, Alexander; Hüttenhain, Stefan H; Kolmar, Harald

    2016-07-01

    Chiral alcohols are important building blocks for specialty chemicals and pharmaceuticals. The production of chiral alcohols from ketones can be carried out stereo selectively with alcohol dehydrogenases (ADHs). To establish a process for cost-effective enzyme immobilization on solid phase for application in ketone reduction, we used an established enzyme pair consisting of ADH from Rhodococcus erythropolis and formate dehydrogenase (FDH) from Candida boidinii for NADH cofactor regeneration and co-immobilized them on modified poly-p-hydroxybutyrate synthase (PhaC)-inclusion bodies that were recombinantly produced in Escherichia coli cells. After separate production of genetically engineered and recombinantly produced enzymes and particles, cell lysates were combined and enzymes endowed with a Kcoil were captured on the surface of the Ecoil presenting particles due to coiled-coil interaction. Enzyme-loaded particles could be easily purified by centrifugation. Total conversion of 4'-chloroacetophenone to (S)-4-chloro-α-methylbenzyl alcohol could be accomplished using enzyme-loaded particles, catalytic amounts of NAD(+) and formate as substrates for FDH. Chiral GC-MS analysis revealed that immobilized ADH retained enantioselectivity with 99 % enantiomeric excess. In conclusion, this strategy may become a cost-effective alternative to coupled reactions using purified enzymes.

  3. Simultaneous involvement of a tungsten-containing aldehyde:ferredoxin oxidoreductase and a phenylacetaldehyde dehydrogenase in anaerobic phenylalanine metabolism.

    Science.gov (United States)

    Debnar-Daumler, Carlotta; Seubert, Andreas; Schmitt, Georg; Heider, Johann

    2014-01-01

    Anaerobic phenylalanine metabolism in the denitrifying betaproteobacterium Aromatoleum aromaticum is initiated by conversion of phenylalanine to phenylacetate, which is further metabolized via benzoyl-coenzyme A (CoA). The formation of phenylacetate is catalyzed by phenylalanine transaminase, phenylpyruvate decarboxylase, and a phenylacetaldehyde-oxidizing enzyme. The presence of these enzymes was detected in extracts of cells grown with phenylalanine and nitrate. We found that two distinct enzymes are involved in the oxidation of phenylacetaldehyde to phenylacetate, an aldehyde:ferredoxin oxidoreductase (AOR) and a phenylacetaldehyde dehydrogenase (PDH). Based on sequence comparison, growth studies with various tungstate concentrations, and metal analysis of the enriched enzyme, AOR was shown to be a tungsten-containing enzyme, necessitating specific cofactor biosynthetic pathways for molybdenum- and tungsten-dependent enzymes simultaneously. We predict from the genome sequence that most enzymes of molybdopterin biosynthesis are shared, while the molybdate/tungstate uptake systems are duplicated and specialized paralogs of the sulfur-inserting MoaD and the metal-inserting MoeA proteins seem to be involved in dedicating biosynthesis toward molybdenum or tungsten cofactors. We also characterized PDH biochemically and identified both NAD(+) and NADP(+) as electron acceptors. We identified the gene coding for the enzyme and purified a recombinant Strep-tagged PDH variant. The homotetrameric enzyme is highly specific for phenylacetaldehyde, has cooperative kinetics toward the substrate, and shows considerable substrate inhibition. Our data suggest that A. aromaticum utilizes PDH as the primary enzyme during anaerobic phenylalanine degradation, whereas AOR is not essential for the metabolic pathway. We hypothesize a function as a detoxifying enzyme if high aldehyde concentrations accumulate in the cytoplasm, which would lead to substrate inhibition of PDH.

  4. Characterization of mannitol-2-dehydrogenase in Saccharina japonica: evidence for a new polyol-specific long-chain dehydrogenases/reductase.

    Directory of Open Access Journals (Sweden)

    Zhanru Shao

    Full Text Available Mannitol plays a crucial role in brown algae, acting as carbon storage, organic osmolytes and antioxidant. Transcriptomic analysis of Saccharina japonica revealed that the relative genes involved in the mannitol cycle are existent. Full-length sequence of mannitol-2-dehydrogenase (M2DH gene was obtained, with one open reading frame of 2,007 bp which encodes 668 amino acids. Cis-regulatory elements for response to methyl jasmonic acid, light and drought existed in the 5'-upstream region. Phylogenetic analysis indicated that SjM2DH has an ancient prokaryotic origin, and is probably acquired by horizontal gene transfer event. Multiple alignment and spatial structure prediction displayed a series of conserved functional residues, motifs and domains, which favored that SjM2DH belongs to the polyol-specific long-chain dehydrogenases/reductase (PSLDR family. Expressional profiles of SjM2DH in the juvenile sporophytes showed that it was influenced by saline, oxidative and desiccative factors. SjM2DH was over-expressed in Escherichia coli, and the cell-free extracts with recombinant SjM2DH displayed high activity on D-fructose reduction reaction. The analysis on SjM2DH gene structure and biochemical parameters reached a consensus that activity of SjM2DH is NADH-dependent and metal ion-independent. The characterization of SjM2DH showed that M2DH is a new member of PSLDR family and play an important role in mannitol metabolism in S. japonica.

  5. Structure of NADP+-dependent glutamate dehydrogenase from Escherichia coli - reflections on the basis of coenzyme specificity in the family of glutamate dehydrogenases

    Energy Technology Data Exchange (ETDEWEB)

    Sharkey, Michael A.; Oliveira, Tânia F.; Engel, Paul C.; Khan, Amir R. [Trinity; (FCT/UNL); (UC-Dublin)

    2013-09-05

    Glutamate dehydrogenases catalyse the oxidative deamination of L-glutamate to α-ketoglutarate, using NAD+ and/or NADP+ as a cofactor. Subunits of homo-hexameric bacterial enzymes comprise a substrate-binding domain I followed by a nucleotide-binding domain II. The reaction occurs in a catalytic cleft between the two domains. Although conserved residues in the nucleotide-binding domains of various dehydrogenases have been linked to cofactor preferences, the structural basis for specificity in the GDH family remains poorly understood. Here, the refined crystal structure of Escherichia coli GDH in the absence of reactants is described at 2.5-Å resolution. Modelling of NADP+ in domain II reveals the potential contribution of positively charged residues from a neighbouring α-helical hairpin to phosphate recognition. In addition, a serine that follows the P7 aspartate is presumed to form a hydrogen bond with the 2'-phosphate. Mutagenesis and kinetic analysis confirms the importance of these residues in NADP+ recognition. Surprisingly, one of the positively charged residues is conserved in all sequences of NAD+-dependent enzymes, but the conformations adopted by the corresponding regions in proteins whose structure has been solved preclude their contribution to the coordination of the 2'-ribose phosphate of NADP+. These studies clarify the sequence–structure relationships in bacterial GDHs, revealing that identical residues may specify different coenzyme preferences, depending on the structural context. Primary sequence alone is therefore not a reliable guide for predicting coenzyme specificity. We also consider how it is possible for a single sequence to accommodate both coenzymes in the dual-specificity GDHs of animals.

  6. Comparative genomics of aldehyde dehydrogenase 5a1 (succinate semialdehyde dehydrogenase and accumulation of gamma-hydroxybutyrate associated with its deficiency

    Directory of Open Access Journals (Sweden)

    Malaspina Patrizia

    2009-01-01

    Full Text Available Abstract Succinic semialdehyde dehydrogenase (SSADH; aldehyde dehydrogenase 5A1 [ALDH5A1]; locus 6p22 occupies a central position in central nervous system (CNS neurotransmitter metabolism as one of two enzymes necessary for γ-aminobutyric acid (GABA recycling from the synaptic cleft. Its importance is highlighted by the neurometabolic disease associated with its inherited deficiency in humans, as well as the severe epileptic phenotype observed in Aldh5a1-/- knockout mice. Expanding evidence now suggests, however, that even subtle decreases in human SSADH activity, associated with rare and common single nucleotide polymorphisms, may produce subclinical pathological effects. SSADH, in conjunction with aldo-keto reductase 7A2 (AKR7A2, represent two neural enzymes responsible for further catabolism of succinic semialdehyde, producing either succinate (SSADH or γ-hydroxybutyrate (GHB; AKR7A2. A GABA analogue, GHB is a short-chain fatty alcohol with unusual properties in the CNS and a long pharmacological history. Moreover, SSADH occupies a further role in the CNS as the enzyme responsible for further metabolism of the lipid peroxidation aldehyde 4-hydroxy-2-nonenal (4-HNE, an intermediate known to induce oxidant stress. Accordingly, subtle decreases in SSADH activity may have the capacity to lead to regional accumulation of neurotoxic intermediates (GHB, 4-HNE. Polymorphisms in SSADH gene structure may also associate with quantitative traits, including intelligence quotient and life expectancy. Further population-based studies of human SSADH activity promise to reveal additional properties of its function and additional roles in CNS tissue.

  7. [Possible participation of mitochondria in formation of peroxisomes in yeasts].

    Science.gov (United States)

    Kozlova, T M; Meĭsel', M N

    1976-01-01

    The origin of peroxisomes in yeast organisms is still unknown. These organelles are believed to be formed, similar to animal cells, from the endoplasmatic reticulum. However, this has not been confirmed directly. Peroxisomes are often found to be in contact with channels of the endoplasmatic reticulum and, in our experiments, with mitochondria of yeast organisms, especially those which utilize oleic acid, n-alkanes and methanol as a sole source of carbon. In Rhodotorula, peroxisomes are characterized by the same "bean" configuration and paired arrangement imitating "copulation" as mitocondria. In Kloeckera boidinii, a mitochondrion was transformed into a peroxisome and cristae were lost. A part of the peroxisome still possessed a double membrane typical of mitochondria while another part had a single membrane characteristic of peroxisomes. Further studies are being carried out in order to find if this is a general relationship or one of possibilities. PMID:1034867

  8. Pattern Formation

    Science.gov (United States)

    Hoyle, Rebecca

    2006-03-01

    From the stripes of a zebra and the spots on a leopard's back to the ripples on a sandy beach or desert dune, regular patterns arise everywhere in nature. The appearance and evolution of these phenomena has been a focus of recent research activity across several disciplines. This book provides an introduction to the range of mathematical theory and methods used to analyse and explain these often intricate and beautiful patterns. Bringing together several different approaches, from group theoretic methods to envelope equations and theory of patterns in large-aspect ratio-systems, the book also provides insight behind the selection of one pattern over another. Suitable as an upper-undergraduate textbook for mathematics students or as a fascinating, engaging, and fully illustrated resource for readers in physics and biology, Rebecca Hoyle's book, using a non-partisan approach, unifies a range of techniques used by active researchers in this growing field. Accessible description of the mathematical theory behind fascinating pattern formation in areas such as biology, physics and materials science Collects recent research for the first time in an upper level textbook Features a number of exercises - with solutions online - and worked examples

  9. Interdependence of coenzyme-induced conformational work and binding potential in yeast alcohol and porcine heart lactate dehydrogenases: a hydrogen-deuterium exchange study

    Energy Technology Data Exchange (ETDEWEB)

    De Weck, Z.; Pande, J.; Kaegi, J.H.R.

    1987-07-28

    Binding of NAD coenzymes to yeast alcohol dehydrogenase (YADH) and porcine heart lactate dehydrogenase (PHLDH) was studied by hydrogen-deuterium exchange with the infrared technique. Conformational changes in the enzymes specific to the coenzymes and their fragments were observed, and the pH dependence of the exchange reaction shows that it conforms to the EX-2 scheme. In both YADH and PHLDH the magnitude of the conformational change as measured by exchange retardation is considerably larger for the NAD/sup +/ than for NADH. Studies with coenzyme fragments like ADP-ribose, ADP, and AMP also highlight the lack of rigorous correlation between structural features such as charge and size and their influence on exchange behavior. Ternary complexes such as YADH-NAD/sup +/-pyrazole, PHLDH-NAD/sup +/-oxalate, and PHLDH-NADH-oxamate, which mimic the transition state, have a significantly more pronounced effect on exchange rates than the corresponding binary complexes. The outstanding feature of this study is the demonstration that in the binary enzyme-coenzyme complexes the more loosely bound NAD/sup +/ is more effective in retarding exchange than the more firmly bound NADH. These differences are attributed to the unequal structural constraints exerted by the two coenzymes upon the enzymes, which translate to unequal expenditure of transconformational work in the formation of the two complexes. The opposing variation in the free energy of binding and the transconformational work expended can be viewed as an unequal partitioning of the net free energy gain resulting from the protein-ligand interaction into a binding term and that required for conformational change.

  10. Interdependence of coenzyme-induced conformational work and binding potential in yeast alcohol and porcine heart lactate dehydrogenases: a hydrogen-deuterium exchange study.

    Science.gov (United States)

    De Weck, Z; Pande, J; Kägi, J H

    1987-07-28

    Binding of NAD coenzymes to yeast alcohol dehydrogenase (YADH) and porcine heart lactate dehydrogenase (PHLDH) was studied by hydrogen-deuterium exchange with the infrared technique. Conformational changes in the enzymes specific to the coenzymes and their fragments were observed, and the pH dependence of the exchange reaction shows that it conforms to the EX-2 scheme. In both YADH and PHLDH the magnitude of the conformational change of measured by exchange retardation is considerably larger for NAD+ than for NADH. Studies with coenzyme fragments like ADP-ribose, ADP, and AMP also highlight the lack of rigorous correlation between structural features such as charge and size and their influence on exchange behavior. Ternary complexes such as YADH-NAD+-pyrazole, PHLDH-NAD+-oxalate, and PHLDH-NADH-oxamate, which mimic the transition state, have a significantly more pronounced effect on exchange rates than the corresponding binary complexes. The outstanding feature of this study is the demonstration that in the binary enzyme-coenzyme complexes the more loosely bound NAD+ is more effective in retarding exchange than the more firmly bound NADH. These differences are attributed to the unequal structural constraints exerted by the two coenzymes upon the enzymes, which translate to unequal expenditure of transconformational work in the formation of the two complexes. The opposing variation in the free energy of binding and the transconformational work expended can be viewed as an unequal partitioning of the net free energy gain resulting from the protein-ligand interaction into a binding term and that required for conformational change.

  11. The PduQ enzyme is an alcohol dehydrogenase used to recycle NAD+ internally within the Pdu microcompartment of Salmonella enterica.

    Directory of Open Access Journals (Sweden)

    Shouqiang Cheng

    Full Text Available Salmonella enterica uses a bacterial microcompartment (MCP for coenzyme B(12-dependent 1,2-propanediol (1,2-PD utilization (Pdu. The Pdu MCP consists of a protein shell that encapsulates enzymes and cofactors required for metabolizing 1,2-PD as a carbon and energy source. Here we show that the PduQ protein of S. enterica is an iron-dependent alcohol dehydrogenase used for 1,2-PD catabolism. PduQ is also demonstrated to be a new component of the Pdu MCP. In addition, a series of in vivo and in vitro studies show that a primary function of PduQ is to recycle NADH to NAD(+ internally within the Pdu MCP in order to supply propionaldehyde dehydrogenase (PduP with its required cofactor (NAD(+. Genetic tests determined that a pduQ deletion mutant grew slower than wild-type Salmonella on 1,2-PD and that this phenotype was not complemented by a non-MCP associated Adh2 from Zymomonas that catalyzes the same reaction. This suggests that PduQ has a MCP-specific function. We also found that a pduQ deletion mutant had no growth defect in a genetic background having a second mutation that prevents MCP formation which further supports a MCP-specific role for PduQ. Moreover, studies with purified Pdu MCPs demonstrated that the PduQ enzyme can convert NADH to NAD(+ to supply the PduP reaction in vitro. Cumulatively, these studies show that the PduQ enzyme is used to recycle NADH to NAD(+ internally within the Pdu MCP. To our knowledge, this is the first report of internal recycling as a mechanism for cofactor homeostasis within a bacterial MCP.

  12. The human L-threonine 3-dehydrogenase gene is an expressed pseudogene

    Directory of Open Access Journals (Sweden)

    Edgar Alasdair J

    2002-10-01

    Full Text Available Abstract Background L-threonine is an indispensable amino acid. One of the major L-threonine degradation pathways is the conversion of L-threonine via 2-amino-3-ketobutyrate to glycine. L-threonine dehydrogenase (EC 1.1.1.103 is the first enzyme in the pathway and catalyses the reaction: L-threonine + NAD+ = 2-amino-3-ketobutyrate + NADH. The murine and porcine L-threonine dehydrogenase genes (TDH have been identified previously, but the human gene has not been identified. Results The human TDH gene is located at 8p23-22 and has 8 exons spanning 10 kb that would have been expected to encode a 369 residue ORF. However, 2 cDNA TDH transcripts encode truncated proteins of 157 and 230 residues. These truncated proteins are the result of 3 mutations within the gene. There is a SNP, A to G, present in the genomic DNA sequence of some individuals which results in the loss of the acceptor splice site preceding exon 4. The acceptor splice site preceding exon 6 was lost in all 23 individuals genotyped and there is an in-frame stop codon in exon 6 (CGA to TGA resulting in arginine-214 being replaced by a stop codon. These truncated proteins would be non-functional since they have lost part of the NAD+ binding motif and the COOH terminal domain that is thought to be involved in binding L-threonine. TDH mRNA was present in all tissues examined. Conclusions The human L-threonine 3-dehydrogenase gene is an expressed pseudogene having lost the splice acceptor site preceding exon 6 and codon arginine-214 (CGA is mutated to a stop codon (TGA.

  13. An artificial intelligence approach to motif discovery in protein sequences: application to steriod dehydrogenases.

    Science.gov (United States)

    Bailey, T L; Baker, M E; Elkan, C P

    1997-05-01

    MEME (Multiple Expectation-maximization for Motif Elicitation) is a unique new software tool that uses artificial intelligence techniques to discover motifs shared by a set of protein sequences in a fully automated manner. This paper is the first detailed study of the use of MEME to analyse a large, biologically relevant set of sequences, and to evaluate the sensitivity and accuracy of MEME in identifying structurally important motifs. For this purpose, we chose the short-chain alcohol dehydrogenase superfamily because it is large and phylogenetically diverse, providing a test of how well MEME can work on sequences with low amino acid similarity. Moreover, this dataset contains enzymes of biological importance, and because several enzymes have known X-ray crystallographic structures, we can test the usefulness of MEME for structural analysis. The first six motifs from MEME map onto structurally important alpha-helices and beta-strands on Streptomyces hydrogenans 20beta-hydroxysteroid dehydrogenase. We also describe MAST (Motif Alignment Search Tool), which conveniently uses output from MEME for searching databases such as SWISS-PROT and Genpept. MAST provides statistical measures that permit a rigorous evaluation of the significance of database searches with individual motifs or groups of motifs. A database search of Genpept90 by MAST with the log-odds matrix of the first six motifs obtained from MEME yields a bimodal output, demonstrating the selectivity of MAST. We show for the first time, using primary sequence analysis, that bacterial sugar epimerases are homologs of short-chain dehydrogenases. MEME and MAST will be increasingly useful as genome sequencing provides large datasets of phylogenetically divergent sequences of biomedical interest. PMID:9366496

  14. NADP-dependent mannitol dehydrogenase, a major allergen of Cladosporium herbarum.

    Science.gov (United States)

    Simon-Nobbe, Birgit; Denk, Ursula; Schneider, Peter Bernhard; Radauer, Christian; Teige, Markus; Crameri, Reto; Hawranek, Thomas; Lang, Roland; Richter, Klaus; Schmid-Grendelmeier, Peter; Nobbe, Stephan; Hartl, Arnulf; Breitenbach, Michael

    2006-06-16

    Cladosporium herbarum is an important allergenic fungal species that has been reported to cause allergic diseases in nearly all climatic zones. 5-30% of the allergic population displays IgE antibodies against molds. Sensitization to Cladosporium has often been associated with severe asthma and less frequently with chronic urticaria and atopic eczema. However, no dominant major allergen of this species has been found so far. We present cloning, production, and characterization of NADP-dependent mannitol dehydrogenase of C. herbarum (Cla h 8) and show that this protein is a major allergen that is recognized by IgE antibodies of approximately 57% of all Cladosporium allergic patients. This is the highest percentage of patients reacting with any Cladosporium allergen characterized so far. Cla h 8 was purified to homogeneity by standard chromatographic methods, and both N-terminal and internal amino acid sequences of protein fragments were determined. Enzymatic analysis of the purified natural protein revealed that this allergen represents a NADP-dependent mannitol dehydrogenase that interconverts mannitol and d-fructose. It is a soluble, non-glycosylated cytoplasmic protein. Two-dimensional protein analysis indicated that mannitol dehydrogenase is present as a single isoform. The cDNA encoding Cla h 8 was cloned from a lambda-ZAP library constructed from hyphae and spores. The recombinant non-fusion protein was expressed in Escherichia coli and purified to homogeneity. Its immunological and biochemical identity with the natural protein was shown by enzyme activity tests, CD spectroscopy, IgE immunoblots with sera of patients, and by skin prick testing of Cladosporium allergic patients. This protein therefore is a new major allergen of C. herbarum.

  15. Retinol dehydrogenase, RDH1l, is essential for the heart development and cardiac performance in zebrafish

    Institute of Scientific and Technical Information of China (English)

    WANG Wei; ZHANG Li-feng; GUI Yong-hao; SONG Hou-yan

    2013-01-01

    Background Retinoic acid (RA) is a potent signaling molecule that plays pleiotropic roles in patterning,morphogenesis,and organogenesis during embryonic development.The synthesis from retinol (vitamin A) to retinoic acid requires two sequential oxidative steps.The first step involves the oxidation of retinol to retinal through the action of retinol dehydrogenases.Retinol dehydrogenases1l (RDH1l) is a novel zebrafish retinol dehydrogenase.Herein we investigated the role of zebrafish RDH1l in heart development and cardiac performance in detail.Methods RDH1l specific morpholino was used to reduce the function of RDH1l in zebrafish.The gene expressions were observed by using whole mount in situ hybridization.Heart rates were observed and recorded under the microscope from 24 to 72 hours post fertilization (hpf).The cardiac performance was analyzed by measuring ventricular shortening fraction (VSF).Results The knock-down of RDH1l led to abnormal neural crest cells migration and reduced numbers of neural crest cells in RDH1l morphant embryos.The reduced numbers of cardiac neural crest cells also can be seen in RDH1l morphant embryos.Furthermore,the morpholino-mediated knock-down of RDH1l resulted in the abnormal heart loop.The left-right determining genes expression pattern was altered in RDH1l morphant embryos.The impaired cardiac performance was observed in RDH1l morphant embryos.Taken together,these data demonstrate that RDH1l is essential for the heart development and cardiac performance in zebrafish.Conclusions RDH1l plays a important role in the neural crest cells development,and then ultimately affects the heart loop and cardiac performance.These results show for the first time that an enzyme involved in the retinol to retinaldehyde conversion participate in the heart development and cardiac performance in zebrafish.

  16. Human liver alcohol dehydrogenase. 2. The primary structure of the gamma 1 protein chain.

    Science.gov (United States)

    Bühler, R; Hempel, J; Kaiser, R; de Zalenski, C; von Wartburg, J P; Jörnvall, H

    1984-12-17

    The primary structure of the gamma 1 subunit of human liver alcohol dehydrogenase isoenzyme gamma 1 gamma 1 was deduced by characterization of 36 tryptic and 2 CNBr peptides. The polypeptide chain is composed of 373 amino acid residues. gamma 1 differs from the beta 1 subunit of human liver alcohol dehydrogenase at 21 positions, and from the E subunit of horse liver alcohol dehydrogenase at 43 positions including a gap at position 128 as in the beta 1 subunit. All zinc-liganding residues from the E subunit of the horse protein and the beta 1 subunit of the human enzyme are conserved, but like beta 1, gamma 1 also has an additional cysteine residue at position 286 (in the positional numbering system of the horse enzyme) due to a Tyr----Cys exchange. Most amino acid exchanges preserve the properties of the residues affected and are largely located on the surface of the molecules, away from the active site and the coenzyme binding region. However, eight positions with charge differences in relation to the E subunit of the horse enzyme are noticed. These result in a net positive charge increase of one in gamma 1 versus E, explaining the electrophoretic mobilities on starch gels. Of functional significance is the conservation of Ser-48 in gamma 1 relative to E. The residue is close to the active site but different (Thr-48) in the beta 1 subunit of the human enzyme. Thus, the closer structural relationship between human gamma 1 and horse E enzyme subunit than between beta 1 and E is also reflected in functionally important residues, explaining a greater similarity between gamma 1 gamma 1 and EE than between beta 1 beta 1 and EE. PMID:6391921

  17. Soil dehydrogenase in a land degradation-rehabilitation gradient: observations from a savanna site with a wet/dry seasonal cycle.

    Science.gov (United States)

    Doi, Ryoichi; Ranamukhaarachchi, Senaratne Leelananda

    2009-01-01

    Soil dehydrogenase activity is a good indicator of overall microbial activity in soil, and it can serve as a good indicator of soil condition. However, seasonal changes in soil moisture content may have an effect on soil dehydrogenase activity, making an accurate assessment of soil condition difficult. In this study, we attempted to determine the significance of soil dehydrogenase activity for assessing soil condition, and we attempted to find a way to account for the influence of soil moisture content on soil dehydrogenase activity.' Soils were sampled in dry evergreen forest (original vegetation), bare ground (severely degraded) and Acacia plantation plots established on bare ground in 1986 and 1987 in Sakaerat, Thailand. Soil physico-chemical characteristics and dehydrogenase activity in the Acacia plantation soil had few differences from those in the evergreen forest soil. Soil dehydrogenase activity varied significantly between the bare ground and the forests regardless of the season (wet or dry), while the season did not produce a significant variation in soil dehydrogenase activity, as determined by repeated measures analysis of variance (p=0.077). The physico-chemical data provided the first principal component as a good measure of soil fertility. Values of soil dehydrogenase activity significantly correlated to scores of the soil samples of the first principal component (R=0.787, p<0.001). We found that soil dehydrogenase activity is a useful indicator of the extent of soil degradation and the rehabilitative effects of reforestation in this part of Thailand. PMID:19637703

  18. Purification of a branched-chain keto acid dehydrogenase from Pseudomonas putida.

    OpenAIRE

    Sokatch, J R; McCully, V; Roberts, C M

    1981-01-01

    We purified branched-chain keto acid dehydrogenase to a specific activity of 10 mumol/min per mg of protein from Pseudomonas putida grown on valine. The purified enzyme was active with 2-ketoisovalerate, 2-ketoisocaproate, and 2-keto-3-methylvalerate in a ratio of 1.0:0.8:0.7 but showed no activity with either pyruvate or 2-ketoglutarate. There were four polypeptides in the purified enzyme (molecular weights, 49,000, 46,000, 39,000, and 37,000). The purified enzyme was deficient in the specif...

  19. l-Valine Production with Pyruvate Dehydrogenase Complex-Deficient Corynebacterium glutamicum▿

    OpenAIRE

    Blombach, Bastian; Schreiner, Mark E.; Holátko, Jiří; Bartek, Tobias; Oldiges, Marco; Eikmanns, Bernhard J.

    2007-01-01

    Corynebacterium glutamicum was engineered for the production of l-valine from glucose by deletion of the aceE gene encoding the E1p enzyme of the pyruvate dehydrogenase complex and additional overexpression of the ilvBNCE genes encoding the l-valine biosynthetic enzymes acetohydroxyacid synthase, isomeroreductase, and transaminase B. In the absence of cellular growth, C. glutamicum ΔaceE showed a relatively high intracellular concentration of pyruvate (25.9 mM) and produced significant amount...

  20. Screen-Printed Carbon Electrodes Modified by Rhodium Dioxide and Glucose Dehydrogenase

    OpenAIRE

    Vojtěch Polan; Jan Soukup; Karel Vytřas

    2011-01-01

    The described glucose biosensor is based on a screen-printed carbon electrode (SPCE) modified by rhodium dioxide, which functions as a mediator. The electrode is further modified by the enzyme glucose dehydrogenase, which is immobilized on the electrode's surface through electropolymerization with m-phenylenediamine. The enzyme biosensor was optimized and tested in model glucose samples. The biosensor showed a linear range of 500–5000 mg L−1 of glucose with a detection limit of 210 mg L−1 (es...

  1. Neutral metal-bound water is the base catalyst in liver alcohol dehydrogenase.

    OpenAIRE

    Makinen, M W; Maret, W.; Yim, M B

    1983-01-01

    The catalytic role of the active site metal-water complex in horse liver alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) is investigated on the basis of a comparative analysis of the pH dependence of steady-state kinetic parameters of the native and active-site-specific Co2+-reconstituted enzyme and on the basis of assignment of the coordination environment of the Co2+ by electron paramagnetic resonance methods. The pH dependence of the kinetic parameters for the oxidation of ...

  2. Evidence of redox imbalance in a patient with succinic semialdehyde dehydrogenase deficiency

    Directory of Open Access Journals (Sweden)

    Anna-Kaisa Niemi

    2014-01-01

    Full Text Available The pathophysiology of succinic semialdehyde dehydrogenase (SSADH deficiency is not completely understood. Oxidative stress, mitochondrial pathology, and low reduced glutathione levels have been demonstrated in mice, but no studies have been reported in humans. We report on a patient with SSADH deficiency in whom we found low levels of blood reduced glutathione (GSH, and elevations of dicarboxylic acids in urine, suggestive of possible redox imbalance and/or mitochondrial dysfunction. Thus, targeting the oxidative stress axis may be a potential therapeutic approach if our findings are confirmed in other patients.

  3. Mechanism of glucose-6-phosphate dehydrogenase-mediated regulation of coronary artery contractility

    OpenAIRE

    Ata, Hirotaka; Rawat, Dhwajbhadur K.; Lincoln, Thomas; Gupte, Sachin A.

    2011-01-01

    We previously identified glucose-6-phosphate dehydrogenase (G6PD) as a regulator of vascular smooth muscle contraction. In this study, we tested our hypothesis that G6PD activated by KCl via a phosphatase and tensin homologue deleted on chromosome 10 (PTEN)-protein kinase C (PKC) pathway increases vascular smooth muscle contraction and that inhibition of G6PD relaxes smooth muscle by decreasing intracellular Ca2+ ([Ca2+]i) and Ca2+ sensitivity to the myofilament. Here we show that G6PD is act...

  4. The protective effects of osmolytes on yeast alcohol dehydrogenase conformational stability and aggregation.

    Science.gov (United States)

    Han, Hong-Yan; Yao, Zhi-Gang; Gong, Cheng-Liang; Xu, Wei-An

    2010-08-01

    The protective effects of four osmolytes (trehalose, dimethysulfoxide, glycine and proline) on the conformational stability and aggregation of guanidine-denatured yeast alcohol dehydrogenase (YADH) have been investigated in this paper. The results show that the four osmolytes protect YADH against unfolding and inactivation by reducing ki (inactivation rate constants), increasing DeltaDeltaGi (transition free energy changes at 25 degrees C), increasing Cm (value for the midpoint of denaturation) and decreasing its ANS-binding fluorescence intensity. Furthermore, these osmolytes can prevent YADH aggregation in a concentration-dependent manner during YADH refolding.

  5. Glucose replaces glutamate as energy substrate to fuel glutamate uptake in glutamate dehydrogenase-deficient astrocytes

    DEFF Research Database (Denmark)

    Pajęcka, Kamilla; Nissen, Jakob D; Stridh, Malin H;

    2015-01-01

    Cultured astrocytes treated with siRNA to knock down glutamate dehydrogenase (GDH) were used to investigate whether this enzyme is important for the utilization of glutamate as an energy substrate. By incubation of these cells in media containing different concentrations of glutamate (range 100......-500 µM) in the presence or in the absence of glucose, the metabolism of these substrates was studied by using tritiated glutamate or 2-deoxyglucose as tracers. In addition, the cellular contents of glutamate and ATP were determined. The astrocytes were able to maintain physiological levels of ATP...

  6. Very long-chain acyl CoA dehydrogenase deficiency which was accepted as infanticide.

    Science.gov (United States)

    Eminoglu, Tuba F; Tumer, Leyla; Okur, Ilyas; Ezgu, Fatih S; Biberoglu, Gursel; Hasanoglu, Alev

    2011-07-15

    Very-long-chain acyl-coenzyme A (CoA) dehydrogenase deficiency (VLCADD) (OMIM #201475) is an autosomal recessive disorder of fatty acid oxidation. Major phenotypic expressions are hypoketotic hypoglycemia, hepatomegaly, cardiomyopathy, myopathy, rhabdomyolysis, elevated creatinine kinase, and lipid infiltration of liver and muscle. At the same time, it is a rare cause of Sudden Infant Death Syndrome (SIDS) or unexplained death in the neonatal period [1-4]. We report a patient with VLCADD whose parents were investigated for infanticide because her three previous siblings had suddenly died after normal deliveries.

  7. Nox5 forms a functional oligomer mediated by self-association of its dehydrogenase domain†

    OpenAIRE

    Kawahara, Tsukasa; Jackson, Heather M.; Smith, Susan M. E.; Simpson, Paul D.; Lambeth, J. David

    2011-01-01

    Nox5 belongs to the calcium-regulated subfamily of NADPH oxidases (Nox). Like other calcium-regulated Noxes, Nox5 has an EF hand-containing calcium-binding domain at its N-terminus, a transmembrane heme-containing region and a C-terminal dehydrogenase (DH) domain that binds FAD and NADPH. While Nox1-4 require regulatory subunits including p22phox, Nox5 activity does not depend on any subunits. We found that inactive point mutants and truncated forms of Nox5 (including the naturally expressed ...

  8. Metabolic control of cell division in α-proteobacteria by a NAD-dependent glutamate dehydrogenase.

    Science.gov (United States)

    Beaufay, François; De Bolle, Xavier; Hallez, Régis

    2016-01-01

    Prior to initiate energy-consuming processes, such as DNA replication or cell division, cells need to evaluate their metabolic status. We have recently identified and characterized a new connection between metabolism and cell division in the α-proteobacterium Caulobacter crescentus. We showed that an NAD-dependent glutamate dehydrogenase (GdhZ) coordinates growth with cell division according to its enzymatic activity. Here we report the conserved role of GdhZ in controlling cell division in another α-proteobacterium, the facultative intracellular pathogen Brucella abortus. We also discuss the importance of amino acids as a main carbon source for α-proteobacteria.

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

    OpenAIRE

    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...

  10. Isolated 2-methylbutyrylglycinuria caused by short/branched-chain acyl-CoA dehydrogenase deficiency

    DEFF Research Database (Denmark)

    Andresen, B S; Christensen, E; Corydon, T J;

    2000-01-01

    of homozygosity for a 1228G-->A mutation in the patient. This mutation was not present in 118 control chromosomes. In vitro transcription/translation experiments and overexpression in COS cells confirmed the disease-causing nature of the mutant SBCAD protein and showed that ACAD-8 is an isobutyryl......-CoA dehydrogenase and that both wild-type proteins are imported into mitochondria and form tetramers. In conclusion, we report the first mutation in the SBCAD gene, show that it results in an isolated defect in isoleucine catabolism, and indicate that ACAD-8 is a mitochondrial enzyme that functions in valine...

  11. Dehydrogenase activity of liver parenchyma in mice exposed to arsenic in drinking water

    Energy Technology Data Exchange (ETDEWEB)

    Bencko, V.; Rossner, P.; Mokry, M.

    1975-01-01

    The objective was to detect changes in dehydrogenase activity (DHA) of liver parenchyma in mice exposed to arsenic in drinking water (for 2, 4, 8, 16, 32 and 64 days). The concentrations used guaranteed threshold, above-threshold and toxic doses of arsenic. A concentration of 5 mg/l produced no significant difference (in comparison with control), a concentration of 250 mg/l caused a deep decline in DHA activity. A concentration of 50 mg/l reduced DHA solely in the first exposure interval. It was found that DHA activity was substantially less altered by arsenic than the metabolic consumption of oxygen under identical conditions.

  12. Dehydrogenase activity of technogenic soils of former sulphur mines (Yavoriv and Nemyriv, Ukraine)

    OpenAIRE

    T. Włodarczyk; M. Brzezińska; Maryskevych, O.; V. Levyk

    2007-01-01

    The dehydrogenase activity (an index of the total soil biological activity), sulphur content, pH and Corg were determined in technogenic soils of the former (1954-1994) sulphur mines in Ukraine (open pit and underground sulphur melting, Yavoriv and Nemyriv, respectively). The soils were neither managed nor reclaimed, and underwent natural self-restoration processes. Soils of former open pit sulphur mine showed Corg of 0.07-1.29%, pH of 7-7.9 and a high SO4-S content (1.7-14.7 g kg-1). Dehydro...

  13. Glucose-6-Phosphate Dehydrogenase Deficiency and Adrenal Hemorrhage in a Filipino Neonate with Hyperbilirubinemia

    Directory of Open Access Journals (Sweden)

    Akira Ohishi

    2013-05-01

    Full Text Available We report on a Filipino neonate with early onset and prolonged hyperbilirubinemia who was delivered by a vacuum extraction due to a prolonged labor. Subsequent studies revealed adrenal hemorrhage and glucose-6-phosphate dehydrogenase (G6PD deficiency. It is likely that asphyxia and resultant hypoxia underlie the occurrence of adrenal hemorrhage and the clinical manifestation of G6PD deficiency and that the presence of the two events explains the early onset and prolonged hyperbilirubinemia of this neonate. Our results represent the importance of examining possible underlying factors for the development of severe, early onset, or prolonged hyperbilirubinemia.

  14. The Structural Basis of Cryptosporidium-Specific IMP Dehydrogenase Inhibitor Selectivity

    Energy Technology Data Exchange (ETDEWEB)

    MacPherson, Iain S.; Kirubakaran, Sivapriya; Gorla, Suresh Kumar; Riera, Thomas V.; D’Aquino, J. Alejandro; Zhang, Minjia; Cuny, Gregory D.; Hedstrom, Lizbeth (BWH); (Brandeis)

    2010-03-29

    Cryptosporidium parvum is a potential biowarfare agent, an important AIDS pathogen, and a major cause of diarrhea and malnutrition. No vaccines or effective drug treatment exist to combat Cryptosporidium infection. This parasite relies on inosine 5{prime}-monophosphate dehydrogenase (IMPDH) to obtain guanine nucleotides, and inhibition of this enzyme blocks parasite proliferation. Here, we report the first crystal structures of CpIMPDH. These structures reveal the structural basis of inhibitor selectivity and suggest a strategy for further optimization. Using this information, we have synthesized low-nanomolar inhibitors that display 10{sup 3} selectivity for the parasite enzyme over human IMPDH2.

  15. Isolation of an Aldehyde Dehydrogenase Involved in the Oxidation of Fluoroacetaldehyde to Fluoroacetate in Streptomyces cattleya

    Science.gov (United States)

    Murphy, Cormac D.; Moss, Steven J.; O'Hagan, David

    2001-01-01

    Streptomyces cattleya is unusual in that it produces fluoroacetate and 4-fluorothreonine as secondary metabolites. We now report the isolation of an NAD+-dependent fluoroacetaldehyde dehydrogenase from S. cattleya that mediates the oxidation of fluoroacetaldehyde to fluoroacetate. This is the first enzyme to be identified that is directly involved in fluorometabolite biosynthesis. Production of the enzyme begins in late exponential growth and continues into the stationary phase. Measurement of kinetic parameters shows that the enzyme has a high affinity for fluoroacetaldehyde and glycoaldehyde, but not acetaldehyde. PMID:11571203

  16. Gd(-) Muret and gd(-) Colomiers, two new variants of glucose-6-phosphate dehydrogenase associated with favism.

    Science.gov (United States)

    Vergnes, H; Ribet, A; Bommelaer, G; Amadieu, J; Brun, H

    1981-01-01

    Two males subjects are described with hitherto undescribed glucose-6-phosphate dehydrogenase (G6PD) variants. The first is of French ancestry, the second of Sicilian extraction. Each subject suffered from acute hemolytic anemia following ingestion of broad beans (Vicia fava). In both cases the hemolytic crisis occurred in a late period of life (29 and 58 years). No previous hemolytic crisis was recorded. The electrophoretic and kinetic properties of the mutant enzymes examined after purification from the red cells allowed each to be distinguished from other G6PD variants reported until now. The first variant was named Gd(-) Muret, the other Gd(-) Colomiers. PMID:7250973

  17. Enzymatic Kinetic Properties of the Lactate Dehydrogenase Isoenzyme C4 of the Plateau Pika (Ochotona curzoniae

    Directory of Open Access Journals (Sweden)

    Yang Wang

    2016-01-01

    Full Text Available Testis-specific lactate dehydrogenase (LDH-C4 is one of the lactate dehydrogenase (LDH isozymes that catalyze the terminal reaction of pyruvate to lactate in the glycolytic pathway. LDH-C4 in mammals was previously thought to be expressed only in spermatozoa and testis and not in other tissues. Plateau pika (Ochotona curzoniae belongs to the genus Ochotona of the Ochotonidea family. It is a hypoxia-tolerant species living in remote mountain areas at altitudes of 3000–5000 m above sea level on the Qinghai-Tibet Plateau. Surprisingly, Ldh-c is expressed not only in its testis and sperm, but also in somatic tissues of plateau pika. To shed light on the function of LDH-C4 in somatic cells, Ldh-a, Ldh-b, and Ldh-c of plateau pika were subcloned into bacterial expression vectors. The pure enzymes of Lactate Dehydrogenase A4 (LDH-A4, Lactate Dehydrogenase B4 (LDH-B4, and LDH-C4 were prepared by a series of expression and purification processes, and the three enzymes were identified by the method of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE and native polyacrylamide gel electrophoresis (PAGE. The enzymatic kinetics properties of these enzymes were studied by Lineweaver-Burk double-reciprocal plots. The results showed the Michaelis constant (Km of LDH-C4 for pyruvate and lactate was 0.052 and 4.934 mmol/L, respectively, with an approximate 90 times higher affinity of LDH-C4 for pyruvate than for lactate. At relatively high concentrations of lactate, the inhibition constant (Ki of the LDH isoenzymes varied: LDH-A4 (Ki = 26.900 mmol/L, LDH-B4 (Ki = 23.800 mmol/L, and LDH-C4 (Ki = 65.500 mmol/L. These data suggest that inhibition of lactate by LDH-A4 and LDH-B4 were stronger than LDH-C4. In light of the enzymatic kinetics properties, we suggest that the plateau pika can reduce reliance on oxygen supply and enhance its adaptation to the hypoxic environments due to increased anaerobic glycolysis by LDH-C4.

  18. Cerium Regulates Expression of Alternative Methanol Dehydrogenases in Methylosinus trichosporium OB3b

    OpenAIRE

    Farhan Ul Haque, Muhammad; Kalidass, Bhagyalakshmi; Bandow, Nathan; Turpin, Erick A.; DiSpirito, Alan A.; Semrau, Jeremy D.

    2015-01-01

    Methanotrophs have multiple methane monooxygenases that are well known to be regulated by copper, i.e., a “copper switch.” At low copper/biomass ratios the soluble methane monooxygenase (sMMO) is expressed while expression and activity of the particulate methane monooxygenase (pMMO) increases with increasing availability of copper. In many methanotrophs there are also multiple methanol dehydrogenases (MeDHs), one based on Mxa and another based on Xox. Mxa-MeDH is known to have calcium in its ...

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

    Directory of Open Access Journals (Sweden)

    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.

  20. Role of NAD+-Dependent Malate Dehydrogenase in the Metabolism of Methylomicrobium alcaliphilum 20Z and Methylosinus trichosporium OB3b

    Directory of Open Access Journals (Sweden)

    Olga N. Rozova

    2015-02-01

    Full Text Available We have expressed the l-malate dehydrogenase (MDH genes from aerobic methanotrophs Methylomicrobium alcaliphilum 20Z and Methylosinus trichosporium OB3b as his-tagged proteins in Escherichia coli. The substrate specificities, enzymatic kinetics and oligomeric states of the MDHs have been characterized. Both MDHs were NAD+-specific and thermostable enzymes not affected by metal ions or various organic metabolites. The MDH from M. alcaliphilum 20Z was a homodimeric (2 × 35 kDa enzyme displaying nearly equal reductive (malate formation and oxidative (oxaloacetate formation activities and higher affinity to malate (Km = 0.11 mM than to oxaloacetate (Km = 0.34 mM. The MDH from M. trichosporium OB3b was homotetrameric (4 × 35 kDa, two-fold more active in the reaction of oxaloacetate reduction compared to malate oxidation and exhibiting higher affinity to oxaloacetate (Km = 0.059 mM than to malate (Km = 1.28 mM. The kcat/Km ratios indicated that the enzyme from M. alcaliphilum 20Z had a remarkably high catalytic efficiency for malate oxidation, while the MDH of M. trichosporium OB3b was preferable for oxaloacetate reduction. The metabolic roles of the enzymes in the specific metabolism of the two methanotrophs are discussed.