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Sample records for boidinii formate dehydrogenase

  1. 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.

  2. Expression of NAD+-dependent formate dehydrogenase in Enterobacter aerogenes and its involvement in anaerobic metabolism and H2 production.

    Science.gov (United States)

    Lu, Yuan; Zhao, Hongxin; Zhang, Chong; Lai, Qiheng; Wu, Xi; Xing, Xin-Hui

    2009-10-01

    An expression system for NAD(+)-dependent formate dehydrogenase gene (fdh1), from Candida boidinii, was constructed and cloned into Enterobacter aerogenes IAM1183. With the fdh1 expression, the total H(2) yield was attributed to a decrease in activity of the lactate pathway and an increase of the formate pathway flux due to the NADH regeneration. Analysis of the redox state balance and ethanol-to-acetate ratio in the fdhl-expressed strain showed that increased reducing power arose from the reconstruction of NADH regeneration pathway from formate thereby contributing to the improved H(2) production.

  3. Alteration of hydrogen metabolism of ldh-deleted Enterobacter aerogenes by overexpression of NAD+-dependent formate dehydrogenase.

    Science.gov (United States)

    Lu, Yuan; Zhao, Hongxin; Zhang, Chong; Lai, Qiheng; Wu, Xi; Xing, Xin-Hui

    2010-03-01

    The NAD+-dependent formate dehydrogenase FDH1 gene (fdh1), cloned from Candida boidinii, was expressed in the ldh-deleted mutant of Enterobacter aerogenes IAM1183 strain. The plasmid of pCom10 driven by the PalkB promoter was used to construct the fdh1 expression system and thus introduce a new dihydronicotinamide adenine dinucleotide (NADH) regeneration pathway from formate in the ldh-deleted mutant. The knockout of NADH-consuming lactate pathway affected the whole cellular metabolism, and the hydrogen yield increased by 11.4% compared with the wild strain. Expression of fdh1 in the ldh-deleted mutant caused lower final cell concentration and final pH after 16 h cultivation, and finally resulted in 86.8% of increase in hydrogen yield per mole consumed glucose. The analysis of cellular metabolites and estimated redox state balance in the fdhl-expressed strain showed that more excess of reducing power was formed by the rewired NADH regeneration pathway, changing the metabolic distribution and promoting the hydrogen production.

  4. 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

  5. Site Saturation Mutagenesis Applications on Candida methylica Formate Dehydrogenase

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    Gülşah P. Özgün

    2016-01-01

    Full Text Available In NADH regeneration, Candida methylica formate dehydrogenase (cmFDH is a highly significant enzyme in pharmaceutical industry. In this work, site saturation mutagenesis (SSM which is a combination of both rational design and directed evolution approaches is applied to alter the coenzyme specificity of NAD+-dependent cmFDH from NAD+ to NADP+ and increase its thermostability. For this aim, two separate libraries are constructed for screening a change in coenzyme specificity and an increase in thermostability. To alter the coenzyme specificity, in the coenzyme binding domain, positions at 195, 196, and 197 are subjected to two rounds of SSM and screening which enabled the identification of two double mutants D195S/Q197T and D195S/Y196L. These mutants increase the overall catalytic efficiency of NAD+ to 5.6×104-fold and 5×104-fold value, respectively. To increase the thermostability of cmFDH, the conserved residue at position 1 in the catalytic domain of cmFDH is subjected to SSM. The thermodynamic and kinetic results suggest that 8 mutations on the first residue can be tolerated. Among all mutants, M1L has the best residual activity after incubation at 60°C with 17%. These studies emphasize that SSM is an efficient method for creating “smarter libraries” for improving the properties of cmFDH.

  6. Genetic analysis of methylotrophic yeast Candida boidinii PLD1.

    Science.gov (United States)

    Lahtchev, K; Penkova, R; Ivanova, V; Tuneva, D

    1992-04-01

    This paper reports the initial experiments for genetic analysis of the haploid methylotrophic yeast Candida boidinii PLD1. The collection of multiply marked auxotrophic mutants was obtained after treatment with UV-light or X-rays. Protoplasts from several mutants were fused by the PEG-CA2+ technique and five prototrophic hybrids were isolated. The genetic structure of the hybrids was studied by means of spontaneous and induced mitotic segregation. Our data suggest that hybrids are diploids, heterozygous by parental auxotrophic markers. We obtained genetic linkage between mutations lys2-8-met-3 from one hand and ade-17-arg-24 from the other. The genetic maps constructed showed similar characteristics concerning both the order of the markers and their map distances.

  7. Molecular characterization of hap complex components responsible for methanol-inducible gene expression in the methylotrophic yeast Candida boidinii.

    Science.gov (United States)

    Oda, Saori; Yurimoto, Hiroya; Nitta, Nobuhisa; Sasano, Yu; Sakai, Yasuyoshi

    2015-03-01

    We identified genes encoding components of the Hap complex, CbHAP2, CbHAP3, and CbHAP5, as transcription factors regulating methanol-inducible gene expression in the methylotrophic yeast Candida boidinii. We found that the Cbhap2Δ, Cbhap3Δ, and Cbhap5Δ gene-disrupted strains showed severe growth defects on methanol but not on glucose and nonfermentable carbon sources such as ethanol and glycerol. In these disruptants, the transcriptional activities of methanol-inducible promoters were significantly decreased compared to those of the wild-type strain, indicating that CbHap2p, CbHap3p, and CbHap5p play indispensable roles in methanol-inducible gene expression. Further molecular and biochemical analyses demonstrated that CbHap2p, CbHap3p, and CbHap5p localized to the nucleus and bound to the promoter regions of methanol-inducible genes regardless of the carbon source, and heterotrimer formation was suggested to be necessary for binding to DNA. Unexpectedly, distinct from Saccharomyces cerevisiae, the Hap complex functioned in methanol-specific induction rather than glucose derepression in C. boidinii. Our results shed light on a novel function of the Hap complex in methanol-inducible gene expression in methylotrophic yeasts.

  8. 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.

  9. Phosphorylation of formate dehydrogenase in potato tuber mitochondria

    DEFF Research Database (Denmark)

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

    2003-01-01

    of phosphorylation of both FDH and PDH was strongly decreased by NAD+, formate, and pyruvate, indicating that reversible phosphorylation of FDH and PDHs was regulated in a similar fashion. At low oxygen concentrations inside the intact potato tubers, FDH activity was strongly increased relative to cytochrome c...... oxidase activity pointing to a possible involvement of FDH in hypoxic metabolism. Computational sequence analysis indicated that a conserved local sequence motif of pyruvate formate-lyase is found in the Arabidopsis thaliana genome, and this enzyme might be the source of formate for FDH in plants....

  10. Complex formation between malate dehydrogenase and isocitrate dehydrogenase from Bacillus subtilis is regulated by tricarboxylic acid cycle metabolites.

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    Bartholomae, Maike; Meyer, Frederik M; Commichau, Fabian M; Burkovski, Andreas; Hillen, Wolfgang; Seidel, Gerald

    2014-02-01

    In Bacillus subtilis, recent in vivo studies revealed that particular enzymes of the tricarboxylic acid cycle form complexes that allow an efficient transfer of metabolites. Remarkably, a complex of the malate dehydrogenase (Mdh) (EC 1.1.1.37) with isocitrate dehydrogenase (Icd) (EC 1.1.1.42) was identified, although both enzymes do not catalyze subsequent reactions. In the present study, the interactions between these enzymes were characterized in vitro by surface plasmon resonance in the absence and presence of their substrates and cofactors. These analyses revealed a weak but specific interaction between Mdh and Icd, which was specifically stimulated by a mixture of substrates and cofactors of Icd: isocitrate, NADP(+) and Mg(2+). Wild-type Icd converted these substrates too fast, preventing any valid quantitative analysis of the interaction with Mdh. Therefore, binding of the IcdS104P mutant to Mdh was quantified because the mutation reduced the enzymatic activity by 174-fold but did not affect the stimulatory effect of substrates and cofactors on Icd-Mdh complex formation. The analysis of the unstimulated Mdh-IcdS104P interaction revealed kinetic constants of k(a) = 2.0 ± 0.2 × 10(2) m(-1) ·s(-1) and k(d) = 1.0 ± 0.1 × 10(-3) ·s(-1) and a K(D) value of 5.0 ± 0.1 μm. Addition of isocitrate, NADP(+) and Mg(2+) stimulated the affinity of IcdS104P to Mdh by 33-fold (K(D) = 0.15 ± 0.01 μm, k(a) = 1.7 ± 0.7 × 10(3) m(-1) ·s(-1), k(d) = 2.6 ± 0.6 × 10(-4) ·s(-1)). Analyses of the enzymatic activities of wild-type Icd and Mdh showed that Icd activity doubles in the presence of Mdh, whereas Mdh activity was slightly reduced by Icd. In summary, these data indicate substrate control of complex formation in the tricarboxylic acid cycle metabolon assembly and maintenance of the α-ketoglutarate supply for amino acid anabolism in vivo.

  11. 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

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

    OpenAIRE

    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...

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

    Science.gov (United States)

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

    1993-06-01

    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 in monomeric forms in C. boidinii cells. The C. boidinii URA3 gene was overexpressed in C. boidinii on these CARS vectors. CARS1 and CARS2 were found to function as an autonomous replicating element in Saccharomyces cerevisiae as well. Different portions of the CARS1 sequence were needed for autonomous replicating activity in C. boidinii and S. cerevisiae. C. boidinii could also be transformed with vectors harboring a CARS fragment and the S. cerevisiae URA3 gene.

  14. [Mechanism of malate dehydrogenase isoform formation in Sphaerotilus natans D-507 under different cultivation conditions].

    Science.gov (United States)

    Eprintsev, A T; Falaleeva, M I; Arabtseva, M A; Lavrinenko, I A; Parfenova, I V; Grechkina, M V; Abud, F S

    2011-01-01

    Electrophoretically homogenous preparations of malate dehydrogenase (MDH) isoforms of the bacteria Sphaerotilus natans D-507 with specific activity 7.46 U/mg and 5.74 U/mg with respect to protein concentration have been obtained. The dimeric isoform of the enzyme was shown to function under organotrophic growth conditions, whereas the tetrameric isoform was induced under mixotrophic cultivation conditions. PCR-analysis revealed a single gene encoding the malate dehydrogenase molecule. The topography of the MDH isoform surface was studied by atomic-force microscopy, and a 3D-structure of the enzyme was obtained. Spectraphotometric analysis data allowed us to suggest that stabilization of the tetrameric form of MDH is due to additional bounds implicated in the quaternary structure formation.

  15. Identification of disulfide bond formation between MitoNEET and glutamate dehydrogenase 1.

    Science.gov (United States)

    Roberts, Morgan E; Crail, Jacquelyn P; Laffoon, Megan M; Fernandez, William G; Menze, Michael A; Konkle, Mary E

    2013-12-17

    MitoNEET is a protein that was identified as a drug target for diabetes, but its cellular function as well as its role in diabetes remains elusive. Protein pull-down experiments identified glutamate dehydrogenase 1 (GDH1) as a potential binding partner. GDH1 is a key metabolic enzyme with emerging roles in insulin regulation. MitoNEET forms a covalent complex with GDH1 through disulfide bond formation and acts as an activator. Proteomic analysis identified the specific cysteine residues that participate in the disulfide bond. This is the first report that effectively links mitoNEET to activation of the insulin regulator GDH1.

  16. CO2 Photoreduction by Formate Dehydrogenase and a Ru-Complex in a Nanoporous Glass Reactor.

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    Noji, Tomoyasu; Jin, Tetsuro; Nango, Mamoru; Kamiya, Nobuo; Amao, Yutaka

    2017-02-01

    In this study, we demonstrated the conversion of CO2 to formic acid under ambient conditions in a photoreduction nanoporous reactor using a photosensitizer, methyl viologen (MV(2+)), and formate dehydrogenase (FDH). The overall efficiency of this reactor was 14 times higher than that of the equivalent solution. The accumulation rate of formic acid in the nanopores of 50 nm is 83 times faster than that in the equivalent solution. Thus, this CO2 photoreduction nanoporous glass reactor will be useful as an artificial photosynthesis system that converts CO2 to fuel.

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

    DEFF Research Database (Denmark)

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

    2001-01-01

    a homolactic pattern of fermentation. Only after lactate dehydrogenase activity was reduced ninefold compared to the wild-type was the growth rate significantly affected, and the ldh mutants started to produce mixed-acid products (formate, acetate, and ethanol in addition to lactate). Flux control coefficients...... were determined and it was found that lactate dehydrogenase exerted virtually no control on the glycolytic flux at the wild-type enzyme level and also not on the flux catalyzed by the enzyme itself, i.e. on the lactate production. As expected, the flux towards the mixed-acid products was strongly...... 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...

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

    Science.gov (United States)

    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.

  19. 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.

  20. Phosphorylation controls the functioning of Staphylococcus aureus isocitrate dehydrogenase--favours biofilm formation.

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    Prasad, U Venkateswara; Vasu, D; Yeswanth, S; Swarupa, V; Sunitha, M M; Choudhary, A; Sarma, P V G K

    2015-01-01

    Isocitrate dehydrogenase (IDH) gene from Staphylococcus aureus ATCC12600 was cloned, sequenced and characterized (HM067707). PknB site was observed in the active site of IDH; thus, it was predicted as IDH may be regulated by phosphorylation. Therefore, in this study, PknB, alkaline phosphatase III (SAOV 2675) and IDH genes (JN695616, JN645811 and HM067707) of S. aureus ATCC12600 were over expressed from clones PV 1, UVPALP-3 and UVIDH 1. On passing the cytosloic fractions through nickel metal chelate column, pure enzymes were obtained. Phosphorylation of pure IDH by PknB resulted in the complete loss of activity and was restored upon dephosphorylation with SAOV 2675 which indicated that phosphorylation and dephosphorylation regulate IDH activity in S. aureus. Further, when S. aureus ATCC12600 was grown in BHI broth, decreased IDH activity and increased biofilm units were observed; therefore, this regulation of IDH alters redox status in this pathogen favouring biofilm formation.

  1. 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.

  2. 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.

  3. 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.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  6. Highly stable and reusable immobilized formate dehydrogenases: Promising biocatalysts for in situ regeneration of NADH

    Directory of Open Access Journals (Sweden)

    Barış Binay

    2016-02-01

    Full Text Available This study aimed to prepare robust immobilized formate dehydrogenase (FDH preparations which can be used as effective biocatalysts along with functional oxidoreductases, in which in situ regeneration of NADH is required. For this purpose, Candida methylica FDH was covalently immobilized onto Immobead 150 support (FDHI150, Immobead 150 support modified with ethylenediamine and then activated with glutaraldehyde (FDHIGLU, and Immobead 150 support functionalized with aldehyde groups (FDHIALD. The highest immobilization yield and activity yield were obtained as 90% and 132%, respectively when Immobead 150 functionalized with aldehyde groups was used as support. The half-life times (t1/2 of free FDH, FDHI150, FDHIGLU and FDHIALD were calculated as 10.6, 28.9, 22.4 and 38.5 h, respectively at 35 °C. FDHI150, FDHIGLU and FDHIALD retained 69, 38 and 51% of their initial activities, respectively after 10 reuses. The results show that the FDHI150, FDHIGLU and FDHIALD offer feasible potentials for in situ regeneration of NADH.

  7. Do dynamic effects play a significant role in enzymatic catalysis? A theoretical analysis of formate dehydrogenase.

    Science.gov (United States)

    Roca, Maite; Oliva, Mónica; Castillo, Raquel; Moliner, Vicente; Tuñón, Iñaki

    2010-10-04

    A theoretical study of the protein dynamic effects on the hydride transfer between the formate anion and nicotinamide adenine dinucleotide (NAD(+)), catalyzed by formate dehydrogenase (FDH), is presented in this paper. The analysis of free downhill molecular dynamic trajectories, performed in the enzyme and compared with the reaction in aqueous solution, has allowed the study of the dynamic coupling between the reacting fragments and the protein or the solvent water molecules, as well as an estimation of the dynamic effect contribution to the catalytic effect from calculation of the transmission coefficient in the enzyme and in solution. The obtained transmission coefficients for the enzyme and in solution were 0.46±0.04 and 0.20±0.03, respectively. These values represent a contribution to catalysis of 0.5 kcal mol(-1), which, although small, is not negligible keeping in mind the low efficiency of FDH. The analysis of the reactive trajectories also reveals how the relative movements of some amino acids, mainly His332 and Arg284, precede and promote the chemical reaction. In spite of these movements, the time-dependent evolution of the electric field created by the enzyme on the key atoms of the reaction reveals a permanent field, which reduces the work required to reach the transition state, with a concomitant polarization of the cofactor. Finally, application of Grote-Hynes theory has allowed the identification of the modes responsible for the substrate-environment coupling, showing how some protein motions take place simultaneously with the reaction. Thus, the equilibrium approach would provide, in this case, an overestimation of the catalyzed rate constant.

  8. 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

  9. 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.

  10. Formation of Nitric Oxide by Aldehyde Dehydrogenase-2 Is Necessary and Sufficient for Vascular Bioactivation of Nitroglycerin*

    Science.gov (United States)

    Opelt, Marissa; Eroglu, Emrah; Waldeck-Weiermair, Markus; Russwurm, Michael; Koesling, Doris; Malli, Roland; Graier, Wolfgang F.; Fassett, John T.; Schrammel, Astrid; Mayer, Bernd

    2016-01-01

    Aldehyde dehydrogenase-2 (ALDH2) catalyzes vascular bioactivation of the antianginal drug nitroglycerin (GTN), resulting in activation of soluble guanylate cyclase (sGC) and cGMP-mediated vasodilation. We have previously shown that a minor reaction of ALDH2-catalyzed GTN bioconversion, accounting for about 5% of the main clearance-based turnover yielding inorganic nitrite, results in direct NO formation and concluded that this minor pathway could provide the link between vascular GTN metabolism and activation of sGC. However, lack of detectable NO at therapeutically relevant GTN concentrations (≤1 μm) in vascular tissue called into question the biological significance of NO formation by purified ALDH2. We addressed this issue and used a novel, highly sensitive genetically encoded fluorescent NO probe (geNOp) to visualize intracellular NO formation at low GTN concentrations (≤1 μm) in cultured vascular smooth muscle cells (VSMC) expressing an ALDH2 mutant that reduces GTN to NO but lacks clearance-based GTN denitration activity. NO formation was compared with GTN-induced activation of sGC. The addition of 1 μm GTN to VSMC expressing either wild-type or C301S/C303S ALDH2 resulted in pronounced intracellular NO elevation, with maximal concentrations of 7 and 17 nm, respectively. Formation of GTN-derived NO correlated well with activation of purified sGC in VSMC lysates and cGMP accumulation in intact porcine aortic endothelial cells infected with wild-type or mutant ALDH2. Formation of NO and cGMP accumulation were inhibited by ALDH inhibitors chloral hydrate and daidzin. The present study demonstrates that ALDH2-catalyzed NO formation is necessary and sufficient for GTN bioactivation in VSMC. PMID:27679490

  11. 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 .

  12. 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.

  13. 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.

  14. Control of oxygen free radical formation from mitochondrial complex I: roles for protein kinase A and pyruvate dehydrogenase kinase.

    Science.gov (United States)

    Raha, Sandeep; Myint, A Tomoko; Johnstone, Leslie; Robinson, Brian H

    2002-03-01

    Human NADH CoQ oxidoreductase is composed of a total of 43 subunits and has been demonstrated to be a major site for the production of superoxide by mitochondria. Incubation of rat heart mitochondria with ATP resulted in the phosphorylation of two mitochondrial membrane proteins, one with a M(r) of 6 kDa consistent with the NDUFA1 (MWFE), and one at 18kDa consistent with either NDUFS4 (AQDQ) or NDUFB7 (B18). Phosphorylation of both subunits was enhanced by cAMP derivatives and protein kinase A (PKA) and was inhibited by PKA inhibitors (PKAi). When mitochondrial membranes were incubated with pyruvate dehydrogenase kinase, phosphorylation of an 18kDa protein but not a 6kDa protein was observed. NADH cytochrome c reductase activity was decreased and superoxide production rates with NADH as substrate were increased. On the other hand, with protein kinase A-driven phosphorylation, NADH cytochrome c reductase was increased and superoxide production decreased. Overall there was a 4-fold variation in electron transport rates observable at the extremes of these phosphorylation events. This suggests that electron flow through complex I and the production of oxygen free radicals can be regulated by phosphorylation events. In light of these observations we discuss a potential model for the dual regulation of complex I and the production of oxygen free radicals by both PKA and PDH kinase.

  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. 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

    and regenerates active cortisol (corticosterone) from circulating inert 11-keto forms. The aim of the present study was to investigate the role of this intracrine activation of GCs on normal bone physiology in vivo using mice deficient in 11betaHSD1 (HSD1(-/-)). The HSD1(-/-) mice exhibited no significant changes...... in cortical or trabecular bone mass compared with wild-type (Wt) mice. Aged HSD1(-/-) mice showed age-related bone loss similar to that observed in Wt mice. Histomorphometric analysis showed similar bone formation and bone resorption parameters in HSD1(-/-) and Wt mice. However, examination of bone marrow...

  17. 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

  18. Glucose-6-phosphate dehydrogenase

    Science.gov (United States)

    ... 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 protein that helps red ...

  19. Efficient reduction of the formation of by-products and improvement of production yield of 2,3-butanediol by a combined deletion of alcohol dehydrogenase, acetate kinase-phosphotransacetylase, and lactate dehydrogenase genes in metabolically engineered Klebsiella oxytoca in mineral salts medium.

    Science.gov (United States)

    Jantama, Kaemwich; Polyiam, Pattharasedthi; Khunnonkwao, Panwana; Chan, Sitha; Sangproo, Maytawadee; Khor, Kirin; Jantama, Sirima Suvarnakuta; Kanchanatawee, Sunthorn

    2015-07-01

    Klebsiella oxytoca KMS005 (∆adhE∆ackA-pta∆ldhA) was metabolically engineered to improve 2,3-butanediol (BDO) yield. Elimination of alcohol dehydrogenase E (adhE), acetate kinase A-phosphotransacetylase (ackA-pta), and lactate dehydrogenase A (ldhA) enzymes allowed BDO production as a primary pathway for NADH re-oxidation, and significantly reduced by-products. KMS005 was screened for the efficient glucose utilization by metabolic evolution. KMS005-73T improved BDO production at a concentration of 23.5±0.5 g/L with yield of 0.46±0.02 g/g in mineral salts medium containing 50 g/L glucose in a shake flask. KMS005-73T also exhibited BDO yields of about 0.40-0.42 g/g from sugarcane molasses, cassava starch, and maltodextrin. During fed-batch fermentation, KMS005-73T produced BDO at a concentration, yield, and overall and specific productivities of 117.4±4.5 g/L, 0.49±0.02 g/g, 1.20±0.05 g/Lh, and 27.2±1.1 g/gCDW, respectively. No acetoin, lactate, and formate were detected, and only trace amounts of acetate and ethanol were formed. The strain also produced the least by-products and the highest BDO yield among other Klebsiella strains previously developed.

  20. 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.

  1. Glucose-6-phosphate dehydrogenase deficiency

    Science.gov (United States)

    ... 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 in which ...

  2. Studies on lipoamide dehydrogenase.

    NARCIS (Netherlands)

    Benen, J.A.E.

    1992-01-01

    At the onset of the investigations described in this thesis progress was being made on the elucidation of the crystal structure of the Azotobactervinelandii lipoamide dehydrogenase. Also the gene encoding this enzyme was cloned in our laboratory. By this, a firm basis was laid to start site directed

  3. 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.

  4. 15 Hypoxyprostaglandin dehydrogenase. A review

    DEFF Research Database (Denmark)

    Hansen, Harald S.

    1976-01-01

    A review is given on the enzyme 15 hydroxyprostaglandin dehydrogenase. The determination, activity, distribution, purification, properties and physiological aspects are discussed. 128 references.......A review is given on the enzyme 15 hydroxyprostaglandin dehydrogenase. The determination, activity, distribution, purification, properties and physiological aspects are discussed. 128 references....

  5. 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.

  6. Properties and subunit structure of pig heart pyruvate dehydrogenase.

    Science.gov (United States)

    Hamada, M; Hiraoka, T; Koike, K; Ogasahara, K; Kanzaki, T

    1976-06-01

    Pyruvate dehydrogenase [EC 1.2.4.1] was separated from the pyruvate dehydrogenase complex and its molecular weight was estimated to be about 150,000 by sedimentation equilibrium methods. The enzyme was dissociated into two subunits (alpha and beta), with estimated molecular weights of 41,000 (alpha) and 36,000 (beta), respectively, by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The subunits were separated by phosphocellulose column chromatography and their chemical properties were examined. The subunit structure of the pyruvate dehydrogenase was assigned as alpha2beta2. The content of right-handed alpha-helix in the enzyme molecule was estimated to be about 29 and 28% by optical rotatory dispersion and by circular dichroism, respectively. The enzyme contained no thiamine-PP, and its dehydrogenase activity was completely dependent on added thiamine-PP and partially dependent on added Mg2+ and Ca2+. The Km value of pyruvate dehydrogenase for thiamine diphosphate was estimated to be 6.5 X 10(-5) M in the presence of Mg2+ or Ca2+. The enzyme showed highly specific activity for thiamine-PP dependent oxidation of both pyruvate and alpha-ketobutyrate, but it also showed some activity with alpha-ketovalerate, alpha-ketoisocaproate, and alpha-ketoisovalerate. The pyruvate dehydrogenase activity was strongly inhibited by bivalent heavy metal ions and by sulfhydryl inhibitors; and the enzyme molecule contained 27 moles of 5,5'-dithiobis(2-nitrobenzoic acid)-reactive sulfhydryl groups and a total of 36 moles of sulfhydryl groups. The inhibitory effect of p-chloromercuribenzoate was prevented by preincubating the enzyme with thiamine-PP plus pyruvate. The structure of pyruvate dehydrogenase necessary for formation of the complex is also reported.

  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. Formats

    Directory of Open Access Journals (Sweden)

    Gehmann, Ulrich

    2012-03-01

    Full Text Available In the following, a new conceptual framework for investigating nowadays’ “technical” phenomena shall be introduced, that of formats. The thesis is that processes of formatting account for our recent conditions of life, and will do so in the very next future. It are processes whose foundations have been laid in modernity and which will further unfold for the time being. These processes are embedded in the format of the value chain, a circumstance making them resilient to change. In addition, they are resilient in themselves since forming interconnected systems of reciprocal causal circuits.Which leads to an overall situation that our entire “Lebenswelt” became formatted to an extent we don’t fully realize, even influencing our very percep-tion of it.

  9. Michael hydratase alcohol dehydrogenase or just alcohol dehydrogenase?

    NARCIS (Netherlands)

    Resch, V.A.; Jin, J.; Chen, B.S.; Hanefeld, U.

    2014-01-01

    The Michael hydratase – alcohol dehydrogenase (MhyADH) from Alicycliphilus denitrificans was previously identified as a bi-functional enzyme performing a hydration of α,β-unsaturated ketones and subsequent oxidation of the formed alcohols. The investigations of the bi-functionality were based on a s

  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. 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

  12. Genetics Home Reference: dihydropyrimidine dehydrogenase deficiency

    Science.gov (United States)

    ... of the skin on the palms and soles (hand-foot syndrome); shortness of breath; and hair loss may also ... dehydrogenase deficiency , with its early-onset neurological symptoms, is a rare disorder. Its prevalence is ...

  13. Isocitrate dehydrogenase mutations in gliomas.

    Science.gov (United States)

    Waitkus, Matthew S; Diplas, Bill H; Yan, Hai

    2016-01-01

    Over the last decade, extraordinary progress has been made in elucidating the underlying genetic causes of gliomas. In 2008, our understanding of glioma genetics was revolutionized when mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) were identified in the vast majority of progressive gliomas and secondary glioblastomas (GBMs). IDH enzymes normally catalyze the decarboxylation of isocitrate to generate α-ketoglutarate (αKG), but recurrent mutations at Arg(132) of IDH1 and Arg(172) of IDH2 confer a neomorphic enzyme activity that catalyzes reduction of αKG into the putative oncometabolite D-2-hydroxyglutate (D2HG). D2HG inhibits αKG-dependent dioxygenases and is thought to create a cellular state permissive to malignant transformation by altering cellular epigenetics and blocking normal differentiation processes. Herein, we discuss the relevant literature on mechanistic studies of IDH1/2 mutations in gliomas, and we review the potential impact of IDH1/2 mutations on molecular classification and glioma therapy.

  14. 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.

  15. 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

  16. 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.

  17. Aluminum decreases the glutathione regeneration by the inhibition of NADP-isocitrate dehydrogenase in mitochondria.

    Science.gov (United States)

    Murakami, Keiko; Yoshino, Masataka

    2004-12-15

    Effect of aluminum on the NADPH supply and glutathione regeneration in mitochondria was analyzed. Reduced glutathione acted as a principal scavenger of reactive oxygen species in mitochondria. Aluminum inhibited the regeneration of glutathione from the oxidized form, and the effect was due to the inhibition of NADP-isocitrate dehydrogenase the only enzyme supplying NADPH in mitochondria. In cytosol, aluminum inhibited the glutathione regeneration dependent on NADPH supply by malic enzyme and NADP-isocitrate dehydrogenase, but did not affect the glucose 6-phosphate dehydrogenase dependent glutathione formation. Aluminum can cause oxidative damage on cellular biological processes by inhibiting glutathione regeneration through the inhibition of NADPH supply in mitochondria, but only a little inhibitory effect on the glutathione generation in cytosol.

  18. Serum lactic dehydrogenase isoenzymes and serum hydroxy butyric dehydrogenase in myocardial infarction

    Directory of Open Access Journals (Sweden)

    Kanekar D

    1979-01-01

    Full Text Available Total serum lactate dehydrogenase activity in cases of myocar-dial infarct is difficult to interpret as abnormal values can occur in diseases of liver, kidney and skeletal muscle. The estimation of its isoenzymes is of better diagnostic help because of its tissue specificity. Serum LDH isoenzymes were studied in patients o f myocardial infarction and results are quantitated by densitometry. As LDH 1 represents serum hydroxybutyric dehydrogenase when 2-oxylbutyrate is used as substrate, serum hydroxybutyric dehydro-genase was also estimated in above patients. Greater specificity in diagnosis is achieved with SHBDH because of its myocardial nature and lower incidence of false positive results.

  19. 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.

  20. Alcohol dehydrogenase – physiological and diagnostic Importance

    Directory of Open Access Journals (Sweden)

    Magdalena Łaniewska-Dunaj

    2013-08-01

    Full Text Available Alcohol dehydrogenase (ADH is a polymorphic enzyme, existing in multiple isoenzymes divided into several classes and localized in different organs. ADH plays a significant role in the metabolism of many biologically important substances, catalyzing the oxidation or reduction of a wide spectrum of specific substrates. The best characterized function of ADH is protection against excess of ethanol and some other exogenous xenobiotics and products of lipid peroxidation. The isoenzymes of alcohol dehydrogenase also participate in the metabolism of retinol and serotonin. The total alcohol dehydrogenase activity is significantly higher in cancer tissues than in healthy organs (e.g. liver, stomach, colorectum. The changes in activity of particular ADH isoenzymes in the sera of patients with different cancers (especially of the digestive system seem to be caused by release of these isoenzymes from cancer cells, and may play a potential role as markers of this cancer. The particular isoenzymes of ADH present in the serum may indicate the cancer localization. Alcohol dehydrogenase may also be useful for diagnostics of non-cancerous liver diseases (e.g. viral hepatitis, non-alcoholic cirrhosis.

  1. 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

  2. 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.

  3. 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).

  4. Glutamate oxidation in astrocytes: Roles of glutamate dehydrogenase and aminotransferases

    DEFF Research Database (Denmark)

    McKenna, Mary C; Stridh, Malin H; McNair, Laura Frendrup;

    2016-01-01

    The cellular distribution of transporters and enzymes related to glutamate metabolism led to the concept of the glutamate–glutamine cycle. Glutamate is released as a neurotransmitter and taken up primarily by astrocytes ensheathing the synapses. The glutamate carbon skeleton is transferred back...... oxidative degradation; thus, quantitative formation of glutamine from the glutamate taken up is not possible. Oxidation of glutamate is initiated by transamination catalyzed by an aminotransferase, or oxidative deamination catalyzed by glutamate dehydrogenase (GDH). We discuss methods available to elucidate...... the enzymes that mediate this conversion. Methods include pharmacological tools such as the transaminase inhibitor aminooxyacetic acid, studies using GDH knockout mice, and siRNA-mediated knockdown of GDH in astrocytes. Studies in brain slices incubated with [15N]glutamate demonstrated activity of GDH...

  5. Hybridizability of gamma-irradiated lactic dehydrogenase

    Energy Technology Data Exchange (ETDEWEB)

    Saito, M.

    1976-03-01

    The hybridizabilities of the gamma-irradiated chicken heart and pig muscle lactic dehydrogenases were estimated by hybridizing the irradiated enzymes with the unirradiated pig heart lactic dehydrogenase. The disc gel electrophoretic patterns of the inter- and intraspecific hybrids showed that the LDH activity of the pig heart isozyme band increased as a function of dose. This observation was analyzed upon the binomial redistribution pattern of the recombined subunits. The result shows that the hybridizabilities of both the chicken heart and pig muscle isozymes decreased along with the loss of catalytic activity and the release from substrate inhibition. The titration of free SH groups of the irradiated chicken isozyme suggested that the unfolding of the peptide chain destroyed the specific tertiary structure needed for the binding of subunits. (auth)

  6. Purification of arogenate dehydrogenase from Phenylobacterium immobile.

    Science.gov (United States)

    Mayer, E; Waldner-Sander, S; Keller, B; Keller, E; Lingens, F

    1985-01-07

    Phenylobacterium immobile, a bacterium which is able to degrade the herbicide chloridazon, utilizes for L-tyrosine synthesis arogenate as an obligatory intermediate which is converted in the final biosynthetic step by a dehydrogenase to tyrosine. This enzyme, the arogenate dehydrogenase, has been purified for the first time in a 5-step procedure to homogeneity as confirmed by electrophoresis. The Mr of the enzyme that consists of two identical subunits amounts to 69000 as established by gel electrophoresis after cross-linking the enzyme with dimethylsuberimidate. The Km values were 0.09 mM for arogenate and 0.02 mM for NAD+. The enzyme has a high specificity with respect to its substrate arogenate.

  7. Oxidative stress and bovine liver diseases: Role of glutathione peroxidase and glucose6‐phosphate dehydrogenase

    OpenAIRE

    Abd Ellah, Mahmoud Rushdi; OKADA, Keiji; Yasuda, Jun

    2007-01-01

    This article summarizes the different types of free radicals, antioxidants and the effect of oxidative stress on the activities of glutathione peroxidase and glucose6‐phosphate dehydrogenase in bovine liver diseases. A growing body of evidence suggests that the formation of reactive oxygen species is a common occurrence associated with most if not all disease processes. The overall importance of reactive oxygen species to the progression and severity of various disease state...

  8. Isocitrate dehydrogenase 1 and 2 mutations in cholangiocarcinoma.

    Science.gov (United States)

    Kipp, Benjamin R; Voss, Jesse S; Kerr, Sarah E; Barr Fritcher, Emily G; Graham, Rondell P; Zhang, Lizhi; Highsmith, W Edward; Zhang, Jun; Roberts, Lewis R; Gores, Gregory J; Halling, Kevin C

    2012-10-01

    Somatic mutations in isocitrate dehydrogenase 1 and 2 genes are common in gliomas and help stratify patients with brain cancer into histologic and molecular subtypes. However, these mutations are considered rare in other solid tumors. The aims of this study were to determine the frequency of isocitrate dehydrogenase 1 and 2 mutations in cholangiocarcinoma and to assess histopathologic differences between specimens with and without an isocitrate dehydrogenase mutation. We sequenced 94 formalin-fixed, paraffin-embedded cholangiocarcinoma (67 intrahepatic and 27 extrahepatic) assessing for isocitrate dehydrogenase 1 (codon 132) and isocitrate dehydrogenase 2 (codons 140 and 172) mutations. Multiple histopathologic characteristics were also evaluated and compared with isocitrate dehydrogenase 1/2 mutation status. Of the 94 evaluated specimens, 21 (22%) had a mutation including 14 isocitrate dehydrogenase 1 and 7 isocitrate dehydrogenase 2 mutations. Isocitrate dehydrogenase mutations were more frequently observed in intrahepatic cholangiocarcinoma than in extrahepatic cholangiocarcinoma (28% versus 7%, respectively; P = .030). The 14 isocitrate dehydrogenase 1 mutations were R132C (n = 9), R132S (n = 2), R132G (n = 2), and R132L (n = 1). The 7 isocitrate dehydrogenase 2 mutations were R172K (n = 5), R172M (n = 1), and R172G (n = 1). Isocitrate dehydrogenase mutations were more frequently observed in tumors with clear cell change (P < .001) and poorly differentiated histology (P = .012). The results of this study show for the first time that isocitrate dehydrogenase 1 and 2 genes are mutated in cholangiocarcinoma. The results of this study are encouraging because it identifies a new potential target for genotype-directed therapeutic trials and may represent a potential biomarker for earlier detection of cholangiocarcinoma in a subset of cases.

  9. 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.

  10. A rare disease-associated mutation in the medium-chain acyl-CoA dehydrogenase (MCAD) gene changes a conserved arginine, previously shown to be functionally essential in short-chain acyl-CoA dehydrogenase (SCAD)

    DEFF Research Database (Denmark)

    Andresen, B S; Bross, P; Jensen, T G

    1993-01-01

    157 mutation was verified in genomic DNA from the patient and her mother by a PCR-based assay. The mutation changes conserved arginine at position 28 (R28C) of the mature MCAD protein. The effect of the T157 mutation on MCAD protein was investigated by expression of mutant MCAD cDNA in COS-7 cells......-chain acyl-CoA dehydrogenase (SCAD) gene of a patient with SCAD deficiency, suggesting that the conserved arginine is crucial for formation of active enzyme in the straight-chain acyl-CoA dehydrogenases....

  11. 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

  12. Structure of a bacterial enzyme regulated by phosphorylation, isocitrate dehydrogenase.

    OpenAIRE

    1989-01-01

    The structure of isocitrate dehydrogenase [threo-DS-isocitrate: NADP+ oxidoreductase (decarboxylating), EC 1.1.1.42] from Escherichia coli has been solved and refined at 2.5 A resolution and is topologically different from that of any other dehydrogenase. This enzyme, a dimer of identical 416-residue subunits, is inactivated by phosphorylation at Ser-113, which lies at the edge of an interdomain pocket that also contains many residues conserved between isocitrate dehydrogenase and isopropylma...

  13. Malate dehydrogenase: a model for structure, evolution, and catalysis.

    OpenAIRE

    1994-01-01

    Malate dehydrogenases are widely distributed and alignment of the amino acid sequences show that the enzyme has diverged into 2 main phylogenetic groups. Multiple amino acid sequence alignments of malate dehydrogenases also show that there is a low degree of primary structural similarity, apart from in several positions crucial for nucleotide binding, catalysis, and the subunit interface. The 3-dimensional structures of several malate dehydrogenases are similar, despite their low amino acid s...

  14. 21 CFR 862.1500 - Malic dehydrogenase test system.

    Science.gov (United States)

    2010-04-01

    ... plasma. Malic dehydrogenase measurements are used in the diagnosis and treatment of muscle and liver diseases, myocardial infarctions, cancer, and blood disorders such as myelogenous (produced in the...

  15. Placental glucose dehydrogenase polymorphism in Koreans.

    Science.gov (United States)

    Kim, Y J; Paik, S G; Park, H Y

    1994-12-01

    The genetic polymorphism of placental glucose dehydrogenase (GDH) was investigated in 300 Korean placentae using horizontal starch gel electrophoresis. The allele frequencies for GDH1, GDH2 and GDH3 were 0.537, 0.440 and 0.005, respectively, which were similar to those in Japanese. We also observed an anodal allele which was similar to the GDH4 originally reported in Chinese populations at a low frequency of 0.015. An additional new cathodal allele (named GDH6) was observed in the present study with a very low frequency of 0.003.

  16. 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...

  17. Peculiarities of the inhibition of the pyruvate dehydrogenase complex by thiamine thiazolone diphosphate in vitro and in intact mitochondria

    Energy Technology Data Exchange (ETDEWEB)

    Yakovleva, G.M.; Strumilo, S.A.; Gorenshtein, B.I.; Ostrovskii, Yu.M.

    1986-07-10

    Thiamine thiazolone diphosphate (TTPP) possesses the ability to penetrate through the mitochondrial membrane and inhibit the pyruvate dehydrogenase complex in intact mitochondria, TTPP inhibits the activity of the complex of animal origin according to a mixed type (K/sub i/ 5 x 10/sup -8/ M) and yeast pyruvate decarboxylase according to a competitive type (K/sub i/ 5 x 10/sup -6/ M) with respect to thiamine diphosphate (TPP). Decarboxylation of pyruvate in intact and lysed rat liver and brain mitochondria is inhibited in the presence of TTPP significantly more weakly than the total activity of the pyruvate dehydrogenase complex, determined according to the formation of acetyl-CoA. It is suggested that TTPP, as an analog of the transition state, acts only in dehydrogenase reactions but not at the stage of simple decarboxylation of pyruvate.

  18. 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.

  19. 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

  20. 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%.

  1. 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.

  2. 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

    2016-01-01

    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. PMID:26729717

  3. 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.

  4. 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).

  5. 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.

  6. 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.

  7. 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.

  8. Asparaginyl deamidation in two glutamate dehydrogenase isoenzymes from Saccharomyces cerevisiae.

    Science.gov (United States)

    DeLuna, Alexander; Quezada, Héctor; Gómez-Puyou, Armando; González, Alicia

    2005-03-25

    The non-enzymatic deamidation of asparaginyl residues is a major source of spontaneous damage of several proteins under physiological conditions. In many cases, deamidation and isoaspartyl formation alters the biological activity or stability of the native polypeptide. Rates of deamidation of particular residues depend on many factors including protein structure and solvent exposure. Here, we investigated the spontaneous deamidation of the two NADP-glutamate dehydrogenase isoenzymes from Saccharomyces cerevisiae, which have different kinetic properties and are differentially expressed in this yeast. Our results show that Asn54, present in Gdh3p but missing in the GDH1-encoded homologue, is readily deamidated in vitro under alkaline conditions. Relative to the native enzyme, deamidated Gdh3p shows reduced protein stability. The different deamidation rates of the two isoenzymes could explain to some extent, the relative in vivo instability of the allosteric Gdh3p enzyme, compared to that of Gdh1p. It is thus possible that spontaneous asparaginyl modification could play a role in the metabolic regulation of ammonium assimilation and glutamate biosynthesis.

  9. Leucaena sp. recombinant cinnamyl alcohol dehydrogenase: purification and physicochemical characterization.

    Science.gov (United States)

    Patel, Parth; Gupta, Neha; Gaikwad, Sushama; Agrawal, Dinesh C; Khan, Bashir M

    2014-02-01

    Cinnamyl alcohol dehydrogenase is a broad substrate specificity enzyme catalyzing the final step in monolignol biosynthesis, leading to lignin formation in plants. Here, we report characterization of a recombinant CAD homologue (LlCAD2) isolated from Leucaena leucocephala. LlCAD2 is 80 kDa homo-dimer associated with non-covalent interactions, having substrate preference toward sinapaldehyde with Kcat/Km of 11.6×10(6) (M(-1) s(-1)), and a possible involvement of histidine at the active site. The enzyme remains stable up to 40 °C, with the deactivation rate constant (Kd(*)) and half-life (t1/2) of 0.002 and 5h, respectively. LlCAD2 showed optimal activity at pH 6.5 and 9 for reduction and oxidation reactions, respectively, and was stable between pH 7 and 9, with the deactivation rate constant (Kd(*)) and half-life (t1/2) of 7.5×10(-4) and 15 h, respectively. It is a Zn-metalloenzyme with 4 Zn(2+) per dimer, however, was inhibited in presence of externally supplemented Zn(2+) ions. The enzyme was resistant to osmolytes, reducing agents and non-ionic detergents.

  10. Metabolism of the novel IMP dehydrogenase inhibitor benzamide riboside.

    Science.gov (United States)

    Jäger, Walter; Salamon, Alexandra; Szekeres, Thomas

    2002-04-01

    Benzamide riboside (BR) is a novel anticancer agent exhibiting pronounced activity against several human tumor cell lines via the inhibition of inosine 5'-monophosphate dehydrogenase (IMPDH) that catalyzes the formation of xanthine 5'-monophosphate from inosine 5'-monophosphate and nicotinamide adenine dinucleotide, thereby restricting the biosynthesis of guanylates. Phosphorylation of BR to its 5'-monophosphate derivative appears to be ubiquitous in most cells catalyzed by the enzymes, adenosine kinase, nicotinamide nucleoside kinase and 5' nucleotidase. BR 5'-monophosphate is then converted to the active metabolite benzamide adenine dinucleotide (BAD) by NMN adenylyltransferase, the rate-limiting enzyme in the biosynthesis of NAD. As BAD is more potent in the inhibition of IMPDH than BR and BR 5'-monophosphate, cytotoxicity of BR is closely connected with intercellular metabolism to BAD. However, intracellular BAD level is also affected by BADase activity, a phosphodiesterase which hydrolyzes BAD to BR-5'-monophosphate and AMP. A recent study demonstrates enzymatic deamination of BR to non-cytotoxic benzene carboxylic acid (BR-COOH) as the main hepatic BR biotransformation product in rat liver. As the IMPDH inhibitors tiazofurin and ribavirin exhibit predominant accumulation and biotransformation in liver, hepatic metabolism may be an important factor also for BR activation and inactivation and should be considered in human liver during cancer therapy when BR is used as a single drug or in combination with other anticancer agents.

  11. 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.

  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. 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.

  14. External NAD(P)H dehydrogenases in Acanthamoeba castellanii mitochondria.

    Science.gov (United States)

    Antos-Krzeminska, Nina; Jarmuszkiewicz, Wieslawa

    2014-09-01

    The mitochondrial respiratory chain of plants and some fungi contains multiple rotenone-insensitive NAD(P)H dehydrogenases, of which at least two are located on the outer surface of the inner membrane (i.e., external NADH and external NADPH dehydrogenases). Annotated sequences of the putative alternative NAD(P)H dehydrogenases of the protozoan Acanthamoeba castellanii demonstrated similarity to plant and fungal sequences. We also studied activity of these dehydrogenases in isolated A. castellanii mitochondria. External NADPH oxidation was observed for the first time in protist mitochondria. The coupling parameters were similar for external NADH oxidation and external NADPH oxidation, indicating similar efficiencies of ATP synthesis. Both external NADH oxidation and external NADPH oxidation had an optimal pH of 6.8 independent of relevant ubiquinol-oxidizing pathways, the cytochrome pathway or a GMP-stimulated alternative oxidase. The maximal oxidizing activity with external NADH was almost double that with external NADPH. However, a lower Michaelis constant (K(M)) value for external NADPH oxidation was observed compared to that for external NADH oxidation. Stimulation by Ca(2+) was approximately 10 times higher for external NADPH oxidation, while NADH dehydrogenase(s) appeared to be slightly dependent on Ca(2+). Our results indicate that external NAD(P)H dehydrogenases similar to those in plant and fungal mitochondria function in mitochondria of A. castellanii.

  15. Cell wall-associated malate dehydrogenase activity from maize roots.

    Science.gov (United States)

    Hadži-Tašković Šukalović, Vesna; Vuletić, Mirjana; Marković, Ksenija; Vučinić, Zeljko

    2011-10-01

    Isolated cell walls from maize (Zea mays L.) roots exhibited ionically and covalently bound NAD-specific malate dehydrogenase activity. The enzyme catalyses a rapid reduction of oxaloacetate and much slower oxidation of malate. The kinetic and regulatory properties of the cell wall enzyme solubilized with 1M NaCl were different from those published for soluble, mitochondrial or plasma membrane malate dehydrogenase with respect to their ATP, Pi, and pH dependence. Isoelectric focusing of ionically-bound proteins and specific staining for malate dehydrogenase revealed characteristic isoforms present in cell wall isolate, different from those present in plasma membranes and crude homogenate. Much greater activity of cell wall-associated malate dehydrogenase was detected in the intensively growing lateral roots compared to primary root with decreased growth rates. Presence of Zn(2+) and Cu(2+) in the assay medium inhibited the activity of the wall-associated malate dehydrogenase. Exposure of maize plants to excess concentrations of Zn(2+) and Cu(2+) in the hydroponic solution inhibited lateral root growth, decreased malate dehydrogenase activity and changed isoform profiles. The results presented show that cell wall malate dehydrogenase is truly a wall-bound enzyme, and not an artefact of cytoplasmic contamination, involved in the developmental processes, and detoxification of heavy metals.

  16. Priapism and glucose-6-phosphate dehydrogenase deficiency: An underestimated correlation?

    Directory of Open Access Journals (Sweden)

    Aldo Franco De Rose

    2016-10-01

    Full Text Available Priapism is a rare clinical condition characterized by a persistent erection unrelated to sexual excitement. Often the etiology is idiopathic. Three cases of priapism in glucose-6-phosphate dehydrogenase (G6PD deficiency patients have been described in literature. We present the case of a 39-year-old man with glucose- 6-phosphate dehydrogenase deficiency, who reached out to our department for the arising of a non-ischemic priapism without arteriolacunar fistula. We suggest that the glucose-6-phosphate dehydrogenase deficiency could be an underestimated risk factor for priapism.

  17. 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

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

    OpenAIRE

    1984-01-01

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

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

    OpenAIRE

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

    1982-01-01

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

  20. 21 CFR 862.1380 - Hydroxybutyric dehydrogenase test system.

    Science.gov (United States)

    2010-04-01

    ... dehydrogenase (HBD) in plasma or serum. HBD measurements are used in the diagnosis and treatment of myocardial infarction, renal damage (such as rejection of transplants), certain hematological diseases (such as...

  1. Genetics Home Reference: glucose-6-phosphate dehydrogenase deficiency

    Science.gov (United States)

    ... enzyme is involved in the normal processing of carbohydrates. It also protects red blood cells from the ... of glucose-6-phosphate dehydrogenase or alter its structure, this enzyme can no longer play its protective ...

  2. 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.

  3. Formaldehyde degradation in Corynebacterium glutamicum involves acetaldehyde dehydrogenase and mycothiol-dependent formaldehyde dehydrogenase.

    Science.gov (United States)

    Lessmeier, Lennart; Hoefener, Michael; Wendisch, Volker F

    2013-12-01

    Corynebacterium glutamicum, a Gram-positive soil bacterium belonging to the actinomycetes, is able to degrade formaldehyde but the enzyme(s) involved in this detoxification process were not known. Acetaldehyde dehydrogenase Ald, which is essential for ethanol utilization, and FadH, characterized here as NAD-linked mycothiol-dependent formaldehyde dehydrogenase, were shown to be responsible for formaldehyde oxidation since a mutant lacking ald and fadH could not oxidize formaldehyde resulting in the inability to grow when formaldehyde was added to the medium. Moreover, C. glutamicum ΔaldΔfadH did not grow with vanillate, a carbon source giving rise to intracellular formaldehyde. FadH from C. glutamicum was purified from recombinant Escherichia coli and shown to be active as a homotetramer. Mycothiol-dependent formaldehyde oxidation revealed Km values of 0.6 mM for mycothiol and 4.3 mM for formaldehyde and a Vmax of 7.7 U mg(-1). FadH from C. glutamicum also possesses zinc-dependent, but mycothiol-independent alcohol dehydrogenase activity with a preference for short chain primary alcohols such as ethanol (Km = 330 mM, Vmax = 9.6 U mg(-1)), 1-propanol (Km = 150 mM, Vmax = 5 U mg(-1)) and 1-butanol (Km = 50 mM, Vmax = 0.8 U mg(-1)). Formaldehyde detoxification system by Ald and mycothiol-dependent FadH is essential for tolerance of C. glutamicum to external stress by free formaldehyde in its habitat and for growth with natural substrates like vanillate, which are metabolized with concomitant release of formaldehyde.

  4. 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.

  5. Resurrecting ancestral alcohol dehydrogenases from yeast.

    Science.gov (United States)

    Thomson, J Michael; Gaucher, Eric A; Burgan, Michelle F; De Kee, Danny W; Li, Tang; Aris, John P; Benner, Steven A

    2005-06-01

    Modern yeast living in fleshy fruits rapidly convert sugars into bulk ethanol through pyruvate. Pyruvate loses carbon dioxide to produce acetaldehyde, which is reduced by alcohol dehydrogenase 1 (Adh1) to ethanol, which accumulates. Yeast later consumes the accumulated ethanol, exploiting Adh2, an Adh1 homolog differing by 24 (of 348) amino acids. As many microorganisms cannot grow in ethanol, accumulated ethanol may help yeast defend resources in the fruit. We report here the resurrection of the last common ancestor of Adh1 and Adh2, called Adh(A). The kinetic behavior of Adh(A) suggests that the ancestor was optimized to make (not consume) ethanol. This is consistent with the hypothesis that before the Adh1-Adh2 duplication, yeast did not accumulate ethanol for later consumption but rather used Adh(A) to recycle NADH generated in the glycolytic pathway. Silent nucleotide dating suggests that the Adh1-Adh2 duplication occurred near the time of duplication of several other proteins involved in the accumulation of ethanol, possibly in the Cretaceous age when fleshy fruits arose. These results help to connect the chemical behavior of these enzymes through systems analysis to a time of global ecosystem change, a small but useful step towards a planetary systems biology.

  6. Liver alcohol dehydrogenase immobilized on polyvinylidene difluoride.

    Science.gov (United States)

    Roig, M G; Bello, J F; Moreno de Vega, M A; Cachaza, J M; Kennedy, J F

    1990-01-01

    A physical method for immobilization of liver alcohol dehydrogenase (ADH) by hydrophobic adsorption onto a supporting membrane of polyvinylidene difluoride (PVDF) was performed. Simultaneously, a physicochemical characterization of the immobilized enzyme regarding its kinetic behaviour was performed. The activity/pH profile observed points to an effect of pH on activity that is completely different from the case of ADH in solution. The disturbance in the typical bell-shaped profile owing to the fact that the enzyme was immobilized is explained on the basis of a potent limitation to the diffusion of the protons in the support. The findings of the present work also reveal the existence of an effect that limits free external diffusion of the substrate towards and/or the product from the support; this effect seems to be the determinant of the overall rate of the enzymatic reaction and is thus of great importance in the effective kinetic behaviour (v([S])) of immobilized ADH, whose kinetic behaviour is complex (non-Michaelian), as may be seen from the lack of linearity observed in the corresponding double reciprocal and Eadie-Hofstee plots. By non-linear regression numerical analysis of the v([S]) data and application of the F-test for model discrimination, the minimum rate equation necessary to describe the intrinsic kinetic behaviour of PVDF-immobilized ADH proved to be one of the polynomial quotient type of degree 2:2 (in substrate concentration).

  7. 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...

  8. Daidzin: a potent, selective inhibitor of human mitochondrial aldehyde dehydrogenase.

    OpenAIRE

    Keung, W M; Vallee, B L

    1993-01-01

    Human mitochondrial aldehyde dehydrogenase (ALDH-I) is potently, reversibly, and selectively inhibited by an isoflavone isolated from Radix puerariae and identified as daidzin, the 7-glucoside of 4',7-dihydroxyisoflavone. Kinetic analysis with formaldehyde as substrate reveals that daidzin inhibits ALDH-I competitively with respect to formaldehyde with a Ki of 40 nM, and uncompetitively with respect to the coenzyme NAD+. The human cytosolic aldehyde dehydrogenase isozyme (ALDH-II) is nearly 3...

  9. Effects of deletion of glycerol-3-phosphate dehydrogenase and glutamate dehydrogenase genes on glycerol and ethanol metabolism in recombinant Saccharomyces cerevisiae.

    Science.gov (United States)

    Kim, Jin-Woo; Chin, Young-Wook; Park, Yong-Cheol; Seo, Jin-Ho

    2012-01-01

    Bioethanol is currently used as an alternative fuel for gasoline worldwide. For economic production of bioethanol by Saccharomyces cerevisiae, formation of a main by-product, glycerol, should be prevented or minimized in order to reduce a separation cost of ethanol from fermentation broth. In this study, S. cerevisiae was engineered to investigate the effects of the sole and double disruption of NADH-dependent glycerol-3-phosphate dehydrogenase 1 (GPD1) and NADPH-requiring glutamate dehydrogenase 1 (GDH1) on the production of glycerol and ethanol from glucose. Even though sole deletion of GPD1 or GDH1 reduced glycerol production, double deletion of GPD1 and GDH1 resulted in the lowest glycerol concentration of 2.31 g/L, which was 46.4% lower than the wild-type strain. Interestingly, the recombinant S. cerevisiae ∆GPD1∆GDH1 strain showed a slight improvement in ethanol yield (0.414 g/g) compared with the wild-type strain (0.406 g/g). Genetic engineering of the glycerol and glutamate metabolic pathways modified NAD(P)H-requiring metabolic pathways and exerted a positive effect on glycerol reduction without affecting ethanol production.

  10. Plants Possess a Cyclic Mitochondrial Metabolic Pathway similar to the Mammalian Metabolic Repair Mechanism Involving Malate Dehydrogenase and l-2-Hydroxyglutarate Dehydrogenase.

    Science.gov (United States)

    Hüdig, Meike; Maier, Alexander; Scherrers, Isabell; Seidel, Laura; Jansen, Erwin E W; Mettler-Altmann, Tabea; Engqvist, Martin K M; Maurino, Veronica G

    2015-09-01

    Enzymatic side reactions can give rise to the formation of wasteful and toxic products that are removed by metabolite repair pathways. In this work, we identify and characterize a mitochondrial metabolic repair mechanism in Arabidopsis thaliana involving malate dehydrogenase (mMDH) and l-2-hydroxyglutarate dehydrogenase (l-2HGDH). We analyze the kinetic properties of both A. thaliana mMDH isoforms, and show that they produce l-2-hydroxyglutarate (l-2HG) from 2-ketoglutarate (2-KG) at low rates in side reactions. We identify A. thaliana l-2HGDH as a mitochondrial FAD-containing oxidase that converts l-2HG back to 2-KG. Using loss-of-function mutants, we show that the electrons produced in the l-2HGDH reaction are transferred to the mitochondrial electron transport chain through the electron transfer protein (ETF). Thus, plants possess the biochemical components of an l-2HG metabolic repair system identical to that found in mammals. While deficiencies in the metabolism of l-2HG result in fatal disorders in mammals, accumulation of l-2HG in plants does not adversely affect their development under a range of tested conditions. However, orthologs of l-2HGDH are found in all examined genomes of viridiplantae, indicating that the repair reaction we identified makes an essential contribution to plant fitness in as yet unidentified conditions in the wild.

  11. 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.

  12. Inhibition of 3(17)beta-hydroxysteroid dehydrogenase from Pseudomonas testosteroni by steroidal A ring fused pyrazoles.

    Science.gov (United States)

    Levy, M A; Holt, D A; Brandt, M; Metcalf, B W

    1987-04-21

    Several 2,3- and 3,4-steroidal fused pyrazoles have been investigated as potential inhibitors of NAD(P)H-dependent steroid oxidoreductases. These compounds are proven to be potent, specific inhibitors for 3(17) beta-hydroxysteroid dehydrogenase from Pseudomonas testosteroni with Ki values of 6-100 nM. In contrast, the activities of 3 alpha,20 beta-hydroxysteroid dehydrogenase from Streptomyces hydrogenans, steroid 5 alpha-reductase from rat prostate, and 3 alpha-hydroxysteroid dehydrogenase from rat liver were unaffected by micromolar concentrations of these compounds. Product and dead-end inhibition studies indicate an ordered association to the beta-dehydrogenase with the cofactor binding prior to substrate or inhibitor. From the results of double inhibition experiments, it is proposed that inhibition occurs through formation of an enzyme-NAD+-inhibitor ternate. On the basis of pH profiles of Vm/Km, Vm, and 1/Ki and of absorbance difference spectra, a hypothetical mechanism of inhibition by the steroidal pyrazoles, drawn by analogy from the inhibition of liver alcohol dehydrogenase by alkylpyrazoles [Theorell, H., & Yonetani, T. (1963) Biochem. Z. 338, 537-553; Andersson, P., Kvassman, J. K., Lindström, A., Oldén, B., & Pettersson, G. (1981) Eur. J. Biochem. 113, 549-554], is reconsidered. The pH studies and enzyme modification experiments by diethyl pyrocarbonate suggest the involvement of histidine in binding of the inhibitor. A modified proposal for the structure of the enzyme-NAD+-steroidal pyrazole complex is proposed.(ABSTRACT TRUNCATED AT 250 WORDS)

  13. 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).

  14. 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.

  15. Characterization of interactions of dihydrolipoamide dehydrogenase with its binding protein in the human pyruvate dehydrogenase complex

    Energy Technology Data Exchange (ETDEWEB)

    Park, Yun-Hee [Department of Biochemistry, School of Medicine and Biomedical Sciences, University at Buffalo, State University of New York, Buffalo, NY 14214 (United States); Patel, Mulchand S., E-mail: mspatel@buffalo.edu [Department of Biochemistry, School of Medicine and Biomedical Sciences, University at Buffalo, State University of New York, Buffalo, NY 14214 (United States)

    2010-05-07

    Unlike pyruvate dehydrogenase complexes (PDCs) from prokaryotes, PDCs from higher eukaryotes have an additional structural component, E3-binding protein (BP), for binding of dihydrolipoamide dehydrogenase (E3) in the complex. Based on the 3D structure of the subcomplex of human (h) E3 with the di-domain (L3S1) of hBP, the amino acid residues (H348, D413, Y438, and R447) of hE3 for binding to hBP were substituted singly by alanine or other residues. These substitutions did not have large effects on hE3 activity when measured in its free form. However, when these hE3 mutants were reconstituted in the complex, the PDC activity was significantly reduced to 9% for Y438A, 20% for Y438H, and 18% for D413A. The binding of hE3 mutants with L3S1 determined by isothermal titration calorimetry revealed that the binding affinities of the Y438A, Y438H, and D413A mutants to L3S1 were severely reduced (1019-, 607-, and 402-fold, respectively). Unlike wild-type hE3 the binding of the Y438A mutant to L3S1 was accompanied by an unfavorable enthalpy change and a large positive entropy change. These results indicate that hE3-Y438 and hE3-D413 play important roles in binding of hE3 to hBP.

  16. Aqueous soluble tetrazolium/formazan MTS as an indicator of NADH- and NADPH-dependent dehydrogenase activity.

    Science.gov (United States)

    Dunigan, D D; Waters, S B; Owen, T C

    1995-10-01

    Recently a new tetrazolium was described for the use of monitoring cell viability in culture. This tetrazolium, commonly referred to as MTS [3-(4,5-dimethylthiazol-2-yl)- 5-(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt], has the unusual property that it can be reduced to a water-soluble formazan. beta-Nicotinamide adenine dinucleotide/reduced (NADH) and beta-nicotinamide adenine dinucleotide phosphate/reduced (NADPH) are examples of physiologically important reducing agents. In cell-free studies, MTS was reduce to the soluble formazan in the presence of NADH and NADPH, and reaction were compared to those with dithiothreitol (DTT) or 2-mercaptoethanol (2-ME). The efficiency of these reactions was enhanced 1000-fold by the presence of phenazine methosulfate. Selectivity in the electron transfer from NADPH was slightly greater than NADH, and NADPH or NADH was much greater than the thiols DTT or 2-ME. Generation of either NADH or NADPH in solution by malate dehydrogenase or isocitrate dehydrogenase, respectively, was monitored by the MTS reduction reaction. The rate of formazan formation was comparable to the formation of NADH or NADPH. This system represents a useful tool for evaluating reaction kinetics in solutions of NAD- or NADP-dependent dehydrogenase enzymes, and these reactions can be performed in typical biological buffers containing reducing agents without significant interference to the MTS/formazan system.

  17. 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.

  18. The Role of Pyruvate Dehydrogenase Kinase in Diabetes and Obesity

    Directory of Open Access Journals (Sweden)

    In-Kyu Lee

    2014-06-01

    Full Text Available The pyruvate dehydrogenase complex (PDC is an emerging target for the treatment of metabolic syndrome. To maintain a steady-state concentration of adenosine triphosphate during the feed-fast cycle, cells require efficient utilization of fatty acid and glucose, which is controlled by the PDC. The PDC converts pyruvate, coenzyme A (CoA, and oxidized nicotinamide adenine dinucleotide (NAD+ into acetyl-CoA, reduced form of nicotinamide adenine dinucleotide (NADH, and carbon dioxide. The activity of the PDC is up- and down-regulated by pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase, respectively. In addition, pyruvate is a key intermediate of glucose oxidation and an important precursor for the synthesis of glucose, glycerol, fatty acids, and nonessential amino acids.

  19. 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.

  20. 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

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. [Effect of overexpression of malate dehydrogenase on succinic acid production in Escherichia coli NZN111].

    Science.gov (United States)

    Liang, Liya; Ma, Jiangfeng; Liu, Rongming; Wang, Guangming; Xu, Bing; Zhang, Min; Jiang, Min

    2011-07-01

    Escherichia coli NZN111 is a double mutant with lactate dehydrogenase (ldhA) and pyruvate formate-lyase (pflB) inactivated. Under anaerobic conditions, disequilibrium of coenzyme NADH and NAD+ causes Escherichia coli NZN111 losing the glucose utilizing capability. In this study, we constructed a recombinant strain E. coli NZN111/pTrc99a-mdh and overexpressed the mdh gene with 0.3 mmol/L of IPTG under anaerobic fermentation condition in sealed bottles. The specific malate dehydrogenase (MDH) activity in the recombinant strain was 14.8-fold higher than that in E. coli NZN111. The NADH/ NAD+ ratio decreased from 0.64 to 0.26 and the concentration of NAD+ and NADH increased 1.5-fold and 0.2-fold respectively. Under anaerobic conditions, the recombinant strain possessed the capability of growth and glucose absorption. We took dual-phase fermentation for succinate production. After the dry cell weight (DCW) reached 6.4 g/L under aerobic conditions, the cell culture was changed to anaerobic conditions. After 15 h, 14.75 g/L glucose was consumed and succinic acid reached 15.18 g/L. The yield of succinic acid was 1.03 g/g Glu and the productivity of succinic acid was 1.012 g/(L x h).

  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. Malate dehydrogenase in phototrophic purple bacteria: purification, molecular weight, and quaternary structure.

    OpenAIRE

    1987-01-01

    The citric acid cycle enzyme malate dehydrogenase was purified to homogeneity from the nonsulfur purple bacteria Rhodobacter capsulatus, Rhodospirillum rubrum, Rhodomicrobium vannielii, and Rhodocyclus purpureus. Malate dehydrogenase was purified from each species by either a single- or a two-step protocol: triazine dye affinity chromatography was the key step in purification of malate dehydrogenase in all cases. Purification of malate dehydrogenase resulted in a 130- to 240-fold increase in ...

  8. Mutations associated with succinate dehydrogenase D-related malignant paragangliomas.

    NARCIS (Netherlands)

    Timmers, H.J.L.M.; Pacak, K.; Bertherat, J.; Lenders, J.W.M.; Duet, M.; Eisenhofer, G.; Stratakis, C.A.; Niccoli-Sire, P.; Tran, B.H.; Burnichon, N.; Gimenez-Roqueplo, A.P.

    2008-01-01

    OBJECTIVE: Hereditary paraganglioma (PGL) syndromes result from germline mutations in genes encoding subunits B, C and D of the mitochondrial enzyme succinate dehydrogenase (SDHB, SDHC and SDHD). SDHB-related PGLs are known in particular for their high malignant potential. Recently, however, maligna

  9. [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...

  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. Medium-chain acyl-CoA dehydrogenase deficiency

    DEFF Research Database (Denmark)

    Waddell, Leigh; Wiley, Veronica; Carpenter, Kevin

    2006-01-01

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

  12. 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

  13. Succinate dehydrogenase is the regulator of respiration in Mycobacterium tuberculosis.

    Directory of Open Access Journals (Sweden)

    Travis Hartman

    2014-11-01

    Full Text Available In chronic infection, Mycobacterium tuberculosis bacilli are thought to enter a metabolic program that provides sufficient energy for maintenance of the protonmotive force, but is insufficient to meet the demands of cellular growth. We sought to understand this metabolic downshift genetically by targeting succinate dehydrogenase, the enzyme which couples the growth processes controlled by the TCA cycle with the energy production resulting from the electron transport chain. M. tuberculosis contains two operons which are predicted to encode succinate dehydrogenase enzymes (sdh-1 and sdh-2; we found that deletion of Sdh1 contributes to an inability to survive long term stationary phase. Stable isotope labeling and mass spectrometry revealed that Sdh1 functions as a succinate dehydrogenase during aerobic growth, and that Sdh2 is dispensable for this catalysis, but partially overlapping activities ensure that the loss of one enzyme can incompletely compensate for loss of the other. Deletion of Sdh1 disturbs the rate of respiration via the mycobacterial electron transport chain, resulting in an increased proportion of reduced electron carrier (menaquinol which leads to increased oxygen consumption. The loss of respiratory control leads to an inability to recover from stationary phase. We propose a model in which succinate dehydrogenase is a governor of cellular respiration in the adaptation to low oxygen environments.

  14. 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.

  15. 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...

  16. Nondecarboxylating and decarboxylating isocitrate dehydrogenases: oxalosuccinate reductase as an ancestral form of isocitrate dehydrogenase.

    Science.gov (United States)

    Aoshima, Miho; Igarashi, Yasuo

    2008-03-01

    Isocitrate dehydrogenase (ICDH) from Hydrogenobacter thermophilus catalyzes the reduction of oxalosuccinate, which corresponds to the second step of the reductive carboxylation of 2-oxoglutarate in the reductive tricarboxylic acid cycle. In this study, the oxidation reaction catalyzed by H. thermophilus ICDH was kinetically analyzed. As a result, a rapid equilibrium random-order mechanism was suggested. The affinities of both substrates (isocitrate and NAD+) toward the enzyme were extremely low compared to other known ICDHs. The binding activities of isocitrate and NAD+ were not independent; rather, the binding of one substrate considerably promoted the binding of the other. A product inhibition assay demonstrated that NADH is a potent inhibitor, although 2-oxoglutarate did not exhibit an inhibitory effect. Further chromatographic analysis demonstrated that oxalosuccinate, rather than 2-oxoglutarate, is the reaction product. Thus, it was shown that H. thermophilus ICDH is a nondecarboxylating ICDH that catalyzes the conversion between isocitrate and oxalosuccinate by oxidation and reduction. This nondecarboxylating ICDH is distinct from well-known decarboxylating ICDHs and should be categorized as a new enzyme. Oxalosuccinate-reducing enzyme may be the ancestral form of ICDH, which evolved to the extant isocitrate oxidative decarboxylating enzyme by acquiring higher substrate affinities.

  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. Inhibition effects of some metal ions on the rat liver 6-phosphogluconate dehydrogenase

    Science.gov (United States)

    Adem, Şevki; Kayhan, Naciye

    2016-04-01

    6-phosphogluconate dehydrogenase is an enzyme in the pentose phosphate path. The main functions of the pathway are the manufacture of the reduced coenzyme NADPH and the formation of ribose 5-phosphate for nucleic acid synthesis and nucleotide. Both NADPH and ribose 5-phosphate involve a critical biochemical process. Metals have been recognized as important toxic agents for living for a long time. It has been considered that they lead to in the emergence of many diseases. To evaluate whether metals is effect towards rat liver 6PGD, we apply various concentrations of metals and enzyme inhibition was analyzed using enzyme activity assays. The IC50 values of Pb+2, Cr+3, Co+2, Ni+2, Cd+2, and Va+2, metals on rat liver 6PGD were calculated as 138,138, 169, 214, 280, and 350 µM, respectively.

  1. Equilibrium concentrations for pyruvate dehydrogenase and the citric acid cycle at specified concentrations of certain coenzymes.

    Science.gov (United States)

    Alberty, Robert A

    2004-04-01

    It is of interest to calculate equilibrium compositions of systems of biochemical reactions at specified concentrations of coenzymes because these reactants tend to be in steady states. Thermodynamic calculations under these conditions require the definition of a further transformed Gibbs energy G" by use of a Legendre transform. These calculations are applied to the pyruvate dehydrogenase reaction plus the citric acid cycle, but steady-state concentrations of CoA, acetyl-CoA and succinyl-CoA cannot be specified because they are involved in the conservation of carbon atoms. These calculations require the use of linear algebra to obtain further transformed Gibbs energies of formation of reactants and computer programs to calculate equilibrium compositions. At specified temperature, pH, ionic strength and specified concentrations of several coenzymes, the equilibrium composition depends on the specified concentrations of the coenzymes and the initial amounts of reactants.

  2. Effect of AMP on mRNA binding by yeast NAD+-specific isocitrate dehydrogenase.

    Science.gov (United States)

    Anderson, Sondra L; Schirf, Virgil; McAlister-Henn, L

    2002-06-04

    Yeast mitochondrial NAD+-specific isocitrate dehydrogenase (IDH) has previously been shown to bind specifically to 5'-untranslated regions of yeast mitochondrial mRNAs, and transcripts containing these regions have been found to allosterically inhibit activity of the enzyme. This inhibition is relieved by AMP, an allosteric activator of this regulatory enzyme of the tricarboxylic acid cycle. We further investigated these enzyme/ligand interactions to determine if binding of RNA and AMP by IDH is competitive or independent. Gel mobility shift experiments indicated no effect of AMP on formation of an IDH/RNA complex. Similarly, sedimentation velocity ultracentrifugation experiments used to analyze interactions in solution indicated that AMP alone had little effect on the formation or stability of an RNA/IDH complex. However, when these sedimentation experiments were conducted in the presence of isocitrate, which has been shown to be essential for binding of AMP by IDH, the proportion of RNA sedimenting in a complex with IDH was significantly reduced by AMP. These results suggest that AMP can affect the binding of RNA by IDH but that this effect is apparent only in the presence of substrate. They also suggest that the catalytic activity of IDH in vivo may be subject to complex allosteric control determined by relative mitochondrial concentrations of mRNA, isocitrate, and AMP. We also found evidence for binding of 5'-untranslated regions of mitochondrial mRNAs by yeast mitochondrial NADP+-specific isocitrate dehydrogenase (IDP1) but not by the corresponding cytosolic isozyme (IDP2). However, this appears to be a nonspecific interaction since no evidence was obtained for any effect on the catalytic activity of IDP1.

  3. Expression of lactate dehydrogenase C correlates with poor prognosis in renal cell carcinoma.

    Science.gov (United States)

    Hua, Yibo; Liang, Chao; Zhu, Jundong; Miao, Chenkui; Yu, Yajie; Xu, Aimin; Zhang, Jianzhong; Li, Pu; Li, Shuang; Bao, Meiling; Yang, Jie; Qin, Chao; Wang, Zengjun

    2017-03-01

    Lactate dehydrogenase C is an isoenzyme of lactate dehydrogenase and a member of the cancer-testis antigens family. In this study, we aimed to investigate the expression and functional role of lactate dehydrogenase C and its basic mechanisms in renal cell carcinoma. First, a total of 133 cases of renal cell carcinoma samples were analysed in a tissue microarray, and Kaplan-Meier survival curve analyses were performed to investigate the correlation between lactate dehydrogenase C expression and renal cell carcinoma progression. Lactate dehydrogenase C protein levels and messenger RNA levels were significantly upregulated in renal cell carcinoma tissues, and the patients with positive lactate dehydrogenase C expression had a shorter progression-free survival, indicating the oncogenic role of lactate dehydrogenase C in renal cell carcinoma. In addition, further cytological experiments demonstrated that lactate dehydrogenase C could prompt renal cell carcinoma cells to produce lactate, and increase metastatic and invasive potential of renal cell carcinoma cells. Furthermore, lactate dehydrogenase C could induce the epithelial-mesenchymal transition process and matrix metalloproteinase-9 expression. In summary, these findings showed lactate dehydrogenase C was associated with poor prognosis in renal cell carcinoma and played a pivotal role in the migration and invasion of renal cell carcinoma cells. Lactate dehydrogenase C may act as a novel biomarker for renal cell carcinoma progression and a potential therapeutic target for the treatment of renal cell carcinoma.

  4. 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

  5. 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

  6. SERUM LACTATE DEHYDROGENASE AS A PROGNOSTIC MARKER IN BREAST CANCER

    Directory of Open Access Journals (Sweden)

    Hardik

    2015-11-01

    Full Text Available : BACKGROUND: Breast cancer a multifactorial disease and one of the most dreaded of human diseases that claims the lives of thousands of women all over the globe every year. This may probably due to the fact that it remains undiagnosed at an early stage perhaps due to lack of awareness amongst the females and the fact that most cancers do not produce any symptoms until the tumour are too large to be removed surgically. Hence there is need to detect cancer at an early stage. AIM: Estimation of diagnostic importance and prognostication of serum Lactate dehydrogenase in cases on breast cancer. SETTINGS AND DESIGN: An observational study was conducted in Acharya Vinoba Bhave Rural Hospital, Sawangi (Meghe, Wardha which included 44 confirmed cases of carcinoma breast and 44 normal healthy females admitted in AVBRH in a span of 2 years. METHODS AND MATERIAL: Determination of serum LDH was done using TC matrix analyser. The values of LDH were obtained on presentation, 21 days after intervention, 2 months after intervention and 6 months after intervention. The values of LDH on presentation in both the groups were compared. The decline in the values of LDH were observed with the due course of treatment. Chisquare test and Student’s Unpaired and paired t test were used for statistical analysis. RESULT: The mean Lactate dehydrogenase on presentation was in study group and control group was 564.38±219.41 IU/L and 404.18±101.32 IU/L respectively (p<0.05. The levels of Lactate dehydrogenase decreased with due course of treatment. The levels of LDH were proportionate to the stage of disease. CONCLUSION: The results of the study concludes cost effective usefulness of serum Lactate dehydrogenase in early detection of breast cancer and to assess its prognostic importance which can be done in smaller laboratories. The traditional model of DS-

  7. Characterization of Flavin-Containing Opine Dehydrogenase from Bacteria.

    Directory of Open Access Journals (Sweden)

    Seiya Watanabe

    Full Text Available Opines, in particular nopaline and octopine, are specific compounds found in crown gall tumor tissues induced by infections with Agrobacterium species, and are synthesized by well-studied NAD(PH-dependent dehydrogenases (synthases, which catalyze the reductive condensation of α-ketoglutarate or pyruvate with L-arginine. The corresponding genes are transferred into plant cells via a tumor-inducing (Ti plasmid. In addition to the reverse oxidative reaction(s, the genes noxB-noxA and ooxB-ooxA are considered to be involved in opine catabolism as (membrane-associated oxidases; however, their properties have not yet been elucidated in detail due to the difficulties associated with purification (and preservation. We herein successfully expressed Nox/Oox-like genes from Pseudomonas putida in P. putida cells. The purified protein consisted of different α-, β-, and γ-subunits encoded by the OdhA, OdhB, and OdhC genes, which were arranged in tandem on the chromosome (OdhB-C-A, and exhibited dehydrogenase (but not oxidase activity toward nopaline in the presence of artificial electron acceptors such as 2,6-dichloroindophenol. The enzyme contained FAD, FMN, and [2Fe-2S]-iron sulfur as prosthetic groups. On the other hand, the gene cluster from Bradyrhizobium japonicum consisted of OdhB1-C-A-B2, from which two proteins, OdhAB1C and OdhAB2C, appeared through the assembly of each β-subunit together with common α- and γ-subunits. A poor phylogenetic relationship was detected between OdhB1 and OdhB2 in spite of them both functioning as octopine dehydrogenases, which provided clear evidence for the acquisition of novel functions by "subunit-exchange". To the best of our knowledge, this is the first study to have examined flavin-containing opine dehydrogenase.

  8. GLUTAMATE DEHYDROGENASE 1 AND SIRT4 REGULATE GLIAL DEVELOPMENT

    OpenAIRE

    Komlos, Daniel; Mann, Kara D.; Zhuo, Yue; Ricupero, Christopher L.; Hart, Ronald P.; Liu, Alice Y.-C.; Firestein, Bonnie L.

    2012-01-01

    Congenital hyperinsulinism/hyperammonemia (HI/HA) syndrome is caused by an activation mutation of glutamate dehydrogenase 1 (GDH1), a mitochondrial enzyme responsible for the reversible interconversion between glutamate and α-ketoglutarate. The syndrome presents clinically with hyperammonemia, significant episodic hypoglycemia, seizures, and a frequent incidences of developmental and learning defects. Clinical research has implicated that although some of the developmental and neurological de...

  9. 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.)

  10. 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...

  11. Plasma Lactate Dehydrogenase Levels Predict Mortality in Acute Aortic Syndromes

    OpenAIRE

    Morello, Fulvio; Ravetti, Anna; Nazerian, Peiman; Liedl, Giovanni; Veglio, Maria Grazia; Battista, Stefania; Vanni, Simone; Pivetta, Emanuele; Montrucchio, Giuseppe; Mengozzi, Giulio; Rinaldi, Mauro; Moiraghi, Corrado; Lupia, Enrico

    2016-01-01

    Abstract In acute aortic syndromes (AAS), organ malperfusion represents a key event impacting both on diagnosis and outcome. Increased levels of plasma lactate dehydrogenase (LDH), a biomarker of malperfusion, have been reported in AAS, but the performance of LDH for the diagnosis of AAS and the relation of LDH with outcome in AAS have not been evaluated so far. This was a bi-centric prospective diagnostic accuracy study and a cohort outcome study. From 2008 to 2014, patients from 2 Emergency...

  12. 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.

  13. Recent advances in biotechnological applications of alcohol dehydrogenases.

    Science.gov (United States)

    Zheng, Yu-Guo; Yin, Huan-Huan; Yu, Dao-Fu; Chen, Xiang; Tang, Xiao-Ling; Zhang, Xiao-Jian; Xue, Ya-Ping; Wang, Ya-Jun; Liu, Zhi-Qiang

    2017-02-01

    Alcohol dehydrogenases (ADHs), which belong to the oxidoreductase superfamily, catalyze the interconversion between alcohols and aldehydes or ketones with high stereoselectivity under mild conditions. ADHs are widely employed as biocatalysts for the dynamic kinetic resolution of racemic substrates and for the preparation of enantiomerically pure chemicals. This review provides an overview of biotechnological applications for ADHs in the production of chiral pharmaceuticals and fine chemicals.

  14. Characterization of two β-decarboxylating dehydrogenases from Sulfolobus acidocaldarius.

    Science.gov (United States)

    Takahashi, Kento; Nakanishi, Fumika; Tomita, Takeo; Akiyama, Nagisa; Lassak, Kerstin; Albers, Sonja-Verena; Kuzuyama, Tomohisa; Nishiyama, Makoto

    2016-11-01

    Sulfolobus acidocaldarius, a hyperthermoacidophilic archaeon, possesses two β-decarboxylating dehydrogenase genes, saci_0600 and saci_2375, in its genome, which suggests that it uses these enzymes for three similar reactions in lysine biosynthesis through 2-aminoadipate, leucine biosynthesis, and the tricarboxylic acid cycle. To elucidate their roles, these two genes were expressed in Escherichia coli in the present study and their gene products were characterized. Saci_0600 recognized 3-isopropylmalate as a substrate, but exhibited slight and no activity for homoisocitrate and isocitrate, respectively. Saci_2375 exhibited distinct and similar activities for isocitrate and homoisocitrate, but no detectable activity for 3-isopropylmalate. These results suggest that Saci_0600 is a 3-isopropylmalate dehydrogenase for leucine biosynthesis and Saci_2375 is a dual function enzyme serving as isocitrate-homoisocitrate dehydrogenase. The crystal structure of Saci_0600 was determined as a closed-form complex that binds 3-isopropylmalate and Mg(2+), thereby revealing the structural basis for the extreme thermostability and novel-type recognition of the 3-isopropyl moiety of the substrate.

  15. 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.

  16. Daidzin: a potent, selective inhibitor of human mitochondrial aldehyde dehydrogenase.

    Science.gov (United States)

    Keung, W M; Vallee, B L

    1993-02-15

    Human mitochondrial aldehyde dehydrogenase (ALDH-I) is potently, reversibly, and selectively inhibited by an isoflavone isolated from Radix puerariae and identified as daidzin, the 7-glucoside of 4',7-dihydroxyisoflavone. Kinetic analysis with formaldehyde as substrate reveals that daidzin inhibits ALDH-I competitively with respect to formaldehyde with a Ki of 40 nM, and uncompetitively with respect to the coenzyme NAD+. The human cytosolic aldehyde dehydrogenase isozyme (ALDH-II) is nearly 3 orders of magnitude less sensitive to daidzin inhibition. Daidzin does not inhibit human class I, II, or III alcohol dehydrogenases, nor does it have any significant effect on biological systems that are known to be affected by other isoflavones. Among more than 40 structurally related compounds surveyed, 12 inhibit ALDH-I, but only prunetin and 5-hydroxydaidzin (genistin) combine high selectivity and potency, although they are 7- to 15-fold less potent than daidzin. Structure-function relationships have established a basis for the design and synthesis of additional ALDH inhibitors that could both be yet more potent and specific.

  17. Purification, crystallization and preliminary X-ray analysis of bifunctional isocitrate dehydrogenase kinase/phosphatase in complex with its substrate, isocitrate dehydrogenase, from Escherichia coli

    OpenAIRE

    2009-01-01

    The protein complex of bifunctional isocitrate dehydrogenase kinase/phosphatase with its substrate, isocitrate dehydrogenase, has been crystallized for structural analysis. A complete data set was collected from the complex crystal and processed to 2.9 Å resolution.

  18. Isocitrate dehydrogenase 1 Gene Mutation Is Associated with Prognosis in Clinical Low-Grade Gliomas.

    Directory of Open Access Journals (Sweden)

    Ming-Yang Li

    Full Text Available Isocitrate dehydrogenase 1 gene mutations are found in most World Health Organization grade II and III gliomas and secondary glioblastomas. Isocitrate dehydrogenase 1 mutations are known to have prognostic value in high-grade gliomas. However, their prognostic significance in low-grade gliomas remains controversial. We determined the predictive and prognostic value of isocitrate dehydrogenase 1 status in low-grade gliomas. The association of isocitrate dehydrogenase 1 status with clinicopathological and genetic factors was also evaluated. Clinical information and genetic data including isocitrate dehydrogenase 1 mutation, O 6-methylguanine DNA methyltransferase promoter methylation, 1p/19q chromosome loss, and TP53 mutation of 417 low-grade gliomas were collected from the Chinese Glioma Genome Atlas database. Kaplan-Meier and Cox proportional hazards regression analyses were performed to evaluate the prognostic effect of clinical characteristics and molecular biomarkers. Isocitrate dehydrogenase 1 mutation was identified as an independent prognostic factor for overall, but not progression-free, survival. Notably, isocitrate dehydrogenase 1 mutation was found to be a significant prognostic factor in patients with oligodendrogliomas, but not in patients with astrocytomas. Furthermore, O 6-methylguanine DNA methyltransferase promoter methylation (p = 0.017 and TP53 mutation (p < 0.001, but not 1p/19q loss (p = 0.834, occurred at a higher frequency in isocitrate dehydrogenase 1-mutated tumors than in isocitrate dehydrogenase 1 wild-type tumors. Younger patient age (p = 0.041 and frontal lobe location (p = 0.010 were significantly correlated with isocitrate dehydrogenase 1 mutation. Chemotherapy did not provide a survival benefit in patients with isocitrate dehydrogenase 1-mutated tumors. Isocitrate dehydrogenase 1 mutation was an independent prognostic factor in low-grade gliomas, whereas it showed no predictive value for chemotherapy response

  19. Isocitrate dehydrogenase 1 Gene Mutation Is Associated with Prognosis in Clinical Low-Grade Gliomas.

    Science.gov (United States)

    Li, Ming-Yang; Wang, Yin-Yan; Cai, Jin-Quan; Zhang, Chuan-Bao; Wang, Kuan-Yu; Cheng, Wen; Liu, Yan-Wei; Zhang, Wei; Jiang, Tao

    2015-01-01

    Isocitrate dehydrogenase 1 gene mutations are found in most World Health Organization grade II and III gliomas and secondary glioblastomas. Isocitrate dehydrogenase 1 mutations are known to have prognostic value in high-grade gliomas. However, their prognostic significance in low-grade gliomas remains controversial. We determined the predictive and prognostic value of isocitrate dehydrogenase 1 status in low-grade gliomas. The association of isocitrate dehydrogenase 1 status with clinicopathological and genetic factors was also evaluated. Clinical information and genetic data including isocitrate dehydrogenase 1 mutation, O 6-methylguanine DNA methyltransferase promoter methylation, 1p/19q chromosome loss, and TP53 mutation of 417 low-grade gliomas were collected from the Chinese Glioma Genome Atlas database. Kaplan-Meier and Cox proportional hazards regression analyses were performed to evaluate the prognostic effect of clinical characteristics and molecular biomarkers. Isocitrate dehydrogenase 1 mutation was identified as an independent prognostic factor for overall, but not progression-free, survival. Notably, isocitrate dehydrogenase 1 mutation was found to be a significant prognostic factor in patients with oligodendrogliomas, but not in patients with astrocytomas. Furthermore, O 6-methylguanine DNA methyltransferase promoter methylation (p = 0.017) and TP53 mutation (p isocitrate dehydrogenase 1-mutated tumors than in isocitrate dehydrogenase 1 wild-type tumors. Younger patient age (p = 0.041) and frontal lobe location (p = 0.010) were significantly correlated with isocitrate dehydrogenase 1 mutation. Chemotherapy did not provide a survival benefit in patients with isocitrate dehydrogenase 1-mutated tumors. Isocitrate dehydrogenase 1 mutation was an independent prognostic factor in low-grade gliomas, whereas it showed no predictive value for chemotherapy response. Isocitrate dehydrogenase 1 mutation was highly associated with O 6-methylguanine DNA

  20. High substrate specificity of ipsdienol dehydrogenase (IDOLDH), a short-chain dehydrogenase from Ips pini bark beetles.

    Science.gov (United States)

    Figueroa-Teran, Rubi; Pak, Heidi; Blomquist, Gary J; Tittiger, Claus

    2016-09-01

    Ips spp. bark beetles use ipsdienol, ipsenol, ipsdienone and ipsenone as aggregation pheromone components and pheromone precursors. For Ips pini, the short-chain oxidoreductase ipsdienol dehydrogenase (IDOLDH) converts (-)-ipsdienol to ipsdienone, and thus likely plays a role in determining pheromone composition. In order to further understand the role of IDOLDH in pheromone biosynthesis, we compared IDOLDH to its nearest functionally characterized ortholog with a solved structure: human L-3-hydroxyacyl-CoA dehydrogenase type II/ amyloid-β binding alcohol dehydrogenase (hHADH II/ABAD), and conducted functional assays of recombinant IDOLDH to determine substrate and product ranges and structural characteristics. Although IDOLDH and hHADH II/ABAD had only 35% sequence identity, their predicted tertiary structures had high identity. We found IDOLDH is a functional homo-tetramer. In addition to oxidizing (-)-ipsdienol, IDOLDH readily converted racemic ipsenol to ipsenone, and stereo-specifically reduced both ketones to their corresponding (-)-alcohols. The (+)-enantiomers were never observed as products. Assays with various substrate analogs showed IDOLDH had high substrate specificity for (-)-ipsdienol, ipsenol, ipsenone and ipsdienone, supporting that IDOLDH functions as a pheromone-biosynthetic enzyme. These results suggest that different IDOLDH orthologs and or activity levels contribute to differences in Ips spp. pheromone composition.

  1. Interaction between glutamate dehydrogenase (GDH) and L-leucine catabolic enzymes: intersecting metabolic pathways.

    Science.gov (United States)

    Hutson, Susan M; Islam, Mohammad Mainul; Zaganas, Ioannis

    2011-09-01

    Branched-chain amino acids (BCAAs) catabolism follows sequential reactions and their metabolites intersect with other metabolic pathways. The initial enzymes in BCAA metabolism, the mitochondrial branched-chain aminotransferase (BCATm), which deaminates the BCAAs to branched-chain α-keto acids (BCKAs); and the branched-chain α-keto acid dehydrogenase enzyme complex (BCKDC), which oxidatively decarboxylates the BCKAs, are organized in a supramolecular complex termed metabolon. Glutamate dehydrogenase (GDH1) is found in the metabolon in rat tissues. Bovine GDH1 binds to the pyridoxamine 5'-phosphate (PMP)-form of human BCATm (PMP-BCATm) but not to pyridoxal 5'-phosphate (PLP)-BCATm in vitro. This protein interaction facilitates reamination of the α-ketoglutarate (αKG) product of the GDH1 oxidative deamination reaction. Human GDH1 appears to act like bovine GDH1 but human GDH2 does not show the same enhancement of BCKDC enzyme activities. Another metabolic enzyme is also found in the metabolon is pyruvate carboxylase (PC). Kinetic results suggest that PC binds to the E1 decarboxylase of BCKDC but does not effect BCAA catabolism. The protein interaction of BCATm and GDH1 promotes regeneration of PLP-BCATm which then binds to BCKDC resulting in channeling of the BCKA products from BCATm first half reaction to E1 and promoting BCAA oxidation and net nitrogen transfer from BCAAs. The cycling of nitrogen through glutamate via the actions of BCATm and GDH1 releases free ammonia. Formation of ammonia may be important for astrocyte glutamine synthesis in the central nervous system. In peripheral tissue association of BCATm and GDH1 would promote BCAA oxidation at physiologically relevant BCAA concentrations.

  2. 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.

  3. FAD binding properties of a cytosolic version of Escherichia coli NADH dehydrogenase-2.

    Science.gov (United States)

    Villegas, Josefina M; Valle, Lorena; Morán Vieyra, Faustino E; Rintoul, María R; Borsarelli, Claudio D; Rapisarda, Viviana A

    2014-03-01

    Respiratory NADH dehydrogenase-2 (NDH-2) of Escherichia coli is a peripheral membrane-bound flavoprotein. By eliminating its C-terminal region, a water soluble truncated version was obtained in our laboratory. Overall conformation of the mutant version resembles the wild-type protein. Considering these data and the fact that the mutant was obtained as an apo-protein, the truncated version is an ideal model to study the interaction between the enzyme and its cofactor. Here, the FAD binding properties of this version were characterized using far-UV circular dichroism (CD), differential scanning calorimetry (DSC), limited proteolysis, and steady-state and dynamic fluorescence spectroscopy. CD spectra, thermal unfolding and DSC profiles did not reveal any major difference in secondary structure between apo- and holo-protein. In addition, digestion site accessibility and tertiary conformation were similar for both proteins, as seen by comparable chymotryptic cleavage patterns. FAD binding to the apo-protein produced a parallel increment of both FAD fluorescence quantum yield and steady-state emission anisotropy. On the other hand, addition of FAD quenched the intrinsic fluorescence emission of the truncated protein, indicating that the flavin cofactor should be closely located to the protein Trp residues. Analysis of the steady-state and dynamic fluorescence data confirms the formation of the holo-protein with a 1:1 binding stoichiometry and an association constant KA=7.0(±0.8)×10(4)M(-1). Taken together, the FAD-protein interaction is energetically favorable and the addition of FAD is not necessary to induce the enzyme folded state. For the first time, a detailed characterization of the flavin:protein interaction was performed among alternative NADH dehydrogenases.

  4. Buformin suppresses the expression of glyceraldehyde 3-phosphate dehydrogenase.

    Science.gov (United States)

    Yano, Akiko; Kubota, Masafumi; Iguchi, Kazuhiro; Usui, Shigeyuki; Hirano, Kazuyuki

    2006-05-01

    The biguanides metformin and buformin, which are clinically used for diabetes mellitus, are known to improve resistance to insulin in patients. Biguanides were reported to cause lactic acidosis as a side effect. Since the mechanism of the side effect still remains obscure, we have examined genes whose expression changes by treating HepG2 cells with buformin in order to elucidate the mechanisms of the side effect. A subtraction cDNA library was constructed by the method of suppressive subtractive hybridization and the screening of the library was performed with cDNA probes prepared from HepG2 cells treated with or without buformin for 12 h. The expression of the gene and the protein obtained by the screening was monitored by real-time RT-PCR with specific primers and Western blotting with specific antibody. The amounts of ATP and NAD+ were determined with luciferase and alcohol dehydrogenase, respectively. We found that expression of the glyceraldehyde 3-phosphate dehydrogenase (GAPD) gene was suppressed by treating HepG2 cells with 0.25 mM buformin for 12 h as a result of the library screening. The decrease in the expression depended on the treatment period. The amount of GAPD protein also decreased simultaneously with the suppression of the gene expression by the treatment with buformin. The amount of ATP and NAD+ in the HepG2 cells treated with buformin decreased to 10 and 20% of the control, respectively. These observations imply that the biguanide causes deactivation of the glycolytic pathway and subsequently the accumulation of pyruvate and NADH and a decrease in NAD+. Therefore, the reaction equilibrium catalyzed by lactate dehydrogenase leans towards lactate production and this may result in lactic acidosis.

  5. [Dihydropirymidine dehydrogenase (DPD)--a toxicity marker for 5-fluorouracil?].

    Science.gov (United States)

    Jedrzychowska, Adriana; Dołegowska, Barbara

    2013-01-01

    In proceedings relating to patients suffering from cancer, an important step is predicting response and toxicity to treatment. Depending on the type of cancer, physicians use the generally accepted schema of treatment, for example pharmacotherapy. 5-fluorouracil (5-FU) is the most widely used anticancer drug in chemotherapy for colon, breast, and head and neck cancer. Patients with dihydropyrimidine dehydrogenase (DPD) deficiency, which is responsible for the metabolism of 5-FU, may experience severe side effects during treatment, and even death. In many publications the need for determining the activity of DPD is discussed, which would protect the patient from the numerous side effects of treatment. However, in practice these assays are not done routinely, despite the high demand. In most cases, a genetic test is used to detect changes in the gene encoding DPD (such as in the USA), but because of the large number of mutations the genetic test cannot be used as a screening test. Dihydropyrimidine dehydrogenase activity has been shown to have high variability among the general population, with an estimated proportion of at least 3-5% of individuals showing low or deficient DPD activity. In this publication we presents data about average dihydropirymidine dehydrogenase activity in various populations of the world (e.g. Japan, Ghana, Great Britain) including gender differences and collected information about the possibility of determination of DPD activity in different countries. Detection of reduced DPD activity in patients with planned chemotherapy will allow a lower dosage of 5-FU or alternative treatment without exposing them to adverse reactions.

  6. 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.

  7. Arteriovenous malformation within an isocitrate dehydrogenase 1 mutated anaplastic oligodendroglioma

    Directory of Open Access Journals (Sweden)

    Grace Lai

    2015-01-01

    Full Text Available Background: The co-occurrence of intracranial arteriovenous malformations (AVMs and cerebral neoplasms is exceedingly rare but may harbor implications pertaining to the molecular medicine of brain cancer pathogenesis. Case Description: Here, we present a case of de novo AVM within an isocitrate dehydrogenase 1 mutated anaplastic oligodendroglioma (WHO Grade III and review the potential contribution of this mutation to aberrant angiogenesis as an interesting case study in molecular medicine. Conclusion: The co-occurrence of an IDH1 mutated neoplasm and AVM supports the hypothesis that IDH1 mutations may contribute to aberrant angiogenesis and vascular malformation.

  8. Deracemization of Secondary Alcohols by using a Single Alcohol Dehydrogenase

    KAUST Repository

    Karume, Ibrahim

    2016-03-01

    © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. We developed a single-enzyme-mediated two-step approach for deracemization of secondary alcohols. A single mutant of Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase enables the nonstereoselective oxidation of racemic alcohols to ketones, followed by a stereoselective reduction process. Varying the amounts of acetone and 2-propanol cosubstrates controls the stereoselectivities of the consecutive oxidation and reduction reactions, respectively. We used one enzyme to accomplish the deracemization of secondary alcohols with up to >99% ee and >99.5% recovery in one pot and without the need to isolate the prochiral ketone intermediate.

  9. 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...

  10. Structures of citrate synthase and malate dehydrogenase of Mycobacterium tuberculosis.

    Science.gov (United States)

    Ferraris, Davide M; Spallek, Ralf; Oehlmann, Wulf; Singh, Mahavir; Rizzi, Menico

    2015-02-01

    The tricarboxylic acid (TCA) cycle is a central metabolic pathway of all aerobic organisms and is responsible for the synthesis of many important precursors and molecules. TCA cycle plays a key role in the metabolism of Mycobacterium tuberculosis and is involved in the adaptation process of the bacteria to the host immune response. We present here the first crystal structures of M. tuberculosis malate dehydrogenase and citrate synthase, two consecutive enzymes of the TCA, at 2.6 Å and 1.5 Å resolution, respectively. General analogies and local differences with the previously reported homologous protein structures are described.

  11. 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...

  12. 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.

  13. Selective inhibition of 6-phosphogluconate dehydrogenase from Trypanosoma brucei

    Science.gov (United States)

    Bertelli, Massimo; El-Bastawissy, Eman; Knaggs, Michael H.; Barrett, Michael P.; Hanau, Stefania; Gilbert, Ian H.

    2001-05-01

    A number of triphenylmethane derivatives have been screened against 6-phosphogluconate dehydrogenase from Trypanosoma brucei and sheep liver. Some of these compounds show good inhibition of the enzymes and also selectivity towards the parasite enzyme. Modelling was undertaken to dock the compounds into the active sites of both enzymes. Using a combination of DOCK 3.5 and FLEXIDOCK a correlation was obtained between docking score and both activity for the enzymes and selectivity. Visualisation of the docked structures of the inhibitors in the active sites of the enzymes yielded a possible explanation of the selectivity for the parasite enzyme.

  14. 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...

  15. 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.

  16. 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.

  17. Characterization of molybdenum and tungsten formate dehydrogenases from sulfate reducing bacteria

    OpenAIRE

    Mota, Cristiano de Sousa

    2011-01-01

    Dissertação apresentada para a obtenção do Grau de Doutor em Bioquímica, especialidade Bioquímica-Física pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia Fundação para a Ciência e Tecnologia - funding (Grant SFRH/BD/32478/2006).

  18. 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

  19. 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

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. In Silico Analysis of Arabidopsis thaliana Peroxisomal 6-Phosphogluconate Dehydrogenase

    Directory of Open Access Journals (Sweden)

    Álvaro D. Fernández-Fernández

    2016-01-01

    Full Text Available NADPH, whose regeneration is critical for reductive biosynthesis and detoxification pathways, is an essential component in cell redox homeostasis. Peroxisomes are subcellular organelles with a complex biochemical machinery involved in signaling and stress processes by molecules such as hydrogen peroxide (H2O2 and nitric oxide (NO. NADPH is required by several peroxisomal enzymes involved in β-oxidation, NO, and glutathione (GSH generation. Plants have various NADPH-generating dehydrogenases, one of which is 6-phosphogluconate dehydrogenase (6PGDH. Arabidopsis contains three 6PGDH genes that probably are encoded for cytosolic, chloroplastic/mitochondrial, and peroxisomal isozymes, although their specific functions remain largely unknown. This study focuses on the in silico analysis of the biochemical characteristics and gene expression of peroxisomal 6PGDH (p6PGDH with the aim of understanding its potential function in the peroxisomal NADPH-recycling system. The data show that a group of plant 6PGDHs contains an archetypal type 1 peroxisomal targeting signal (PTS, while in silico gene expression analysis using affymetrix microarray data suggests that Arabidopsis p6PGDH appears to be mainly involved in xenobiotic response, growth, and developmental processes.

  5. 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.

  6. Structural analysis of fungus-derived FAD glucose dehydrogenase.

    Science.gov (United States)

    Yoshida, Hiromi; Sakai, Genki; Mori, Kazushige; Kojima, Katsuhiro; Kamitori, Shigehiro; Sode, Koji

    2015-08-27

    We report the first three-dimensional structure of fungus-derived glucose dehydrogenase using flavin adenine dinucleotide (FAD) as the cofactor. This is currently the most advanced and popular enzyme used in glucose sensor strips manufactured for glycemic control by diabetic patients. We prepared recombinant nonglycosylated FAD-dependent glucose dehydrogenase (FADGDH) derived from Aspergillus flavus (AfGDH) and obtained the X-ray structures of the binary complex of enzyme and reduced FAD at a resolution of 1.78 Å and the ternary complex with reduced FAD and D-glucono-1,5-lactone (LGC) at a resolution of 1.57 Å. The overall structure is similar to that of fungal glucose oxidases (GOxs) reported till date. The ternary complex with reduced FAD and LGC revealed the residues recognizing the substrate. His505 and His548 were subjected for site-directed mutagenesis studies, and these two residues were revealed to form the catalytic pair, as those conserved in GOxs. The absence of residues that recognize the sixth hydroxyl group of the glucose of AfGDH, and the presence of significant cavity around the active site may account for this enzyme activity toward xylose. The structural information will contribute to the further engineering of FADGDH for use in more reliable and economical biosensing technology for diabetes management.

  7. 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.

  8. 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.

  9. Selective methanol or formate production during continuous CO₂ fermentation by the acetogen biocatalysts engineered via integration of synthetic pathways using Tn7-tool.

    Science.gov (United States)

    Tyurin, Michael; Kiriukhin, Michael

    2013-09-01

    Methanol-resistant mutant acetogen Clostridium sp. MT1424 originally producing only 365 mM acetate from CO₂/CO was engineered to eliminate acetate production and spore formation using Cre-lox66/lox71-system to power subsequent methanol production via expressing synthetic methanol dehydrogenase, formaldehyde dehydrogenase and formate dehydrogenase, three copies of each, assembled in cluster and integrated to chromosome using Tn7-based approach. Production of 2.2 M methanol was steady (p IGCC power plant. Hydrogen may be produced in situ via powered by solar panels electrolysis.

  10. 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...... populations.SUBJECTS/METHODS: A nested case-control study (1269 cases matched to 2107controls by sex, age, study centre and date of blood collection) was conducted within the European Prospective Investigation into Cancer and Nutrition (EPIC) to evaluate the impact of rs1229984 (ADH1B), rs1573496 (ADH7...

  11. 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.

  12. 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

  13. Role of phosphoenolpyruvate in the NADP-isocitrate dehydrogenase and isocitrate lyase reaction in Escherichia coli.

    Science.gov (United States)

    Ogawa, Tadashi; Murakami, Keiko; Mori, Hirotada; Ishii, Nobuyoshi; Tomita, Masaru; Yoshin, Masataka

    2007-02-01

    Phosphoenolpyruvate inhibited Escherichia coli NADP-isocitrate dehydrogenase allosterically (Ki of 0.31 mM) and isocitrate lyase uncompetitively (Ki' of 0.893 mM). Phosphoenolpyruvate enhances the uncompetitive inhibition of isocitrate lyase by increasing isocitrate, which protects isocitrate dehydrogenase from the inhibition, and contributes to the control through the tricarboxylic acid cycle and glyoxylate shunt.

  14. Role of Phosphoenolpyruvate in the NADP-Isocitrate Dehydrogenase and Isocitrate Lyase Reaction in Escherichia coli▿

    OpenAIRE

    2006-01-01

    Phosphoenolpyruvate inhibited Escherichia coli NADP-isocitrate dehydrogenase allosterically (Ki of 0.31 mM) and isocitrate lyase uncompetitively (Ki′ of 0.893 mM). Phosphoenolpyruvate enhances the uncompetitive inhibition of isocitrate lyase by increasing isocitrate, which protects isocitrate dehydrogenase from the inhibition, and contributes to the control through the tricarboxylic acid cycle and glyoxylate shunt.

  15. P450BM3 fused to phosphite dehydrogenase allows phosphite-driven selective oxidations

    NARCIS (Netherlands)

    Beyer, Nina; Kulig, Justyna K; Bartsch, Anette; Hayes, Martin A; Janssen, Dick B; Fraaije, Marco W

    2016-01-01

    To facilitate the wider application of the NADPH-dependent P450BM3, we fused the monooxygenase with a phosphite dehydrogenase (PTDH). The resulting monooxygenase-dehydrogenase fusion enzyme acts as a self-sufficient bifunctional catalyst, accepting phosphite as a cheap electron donor for the regener

  16. 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.

  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. Increased IMP dehydrogenase gene expression in solid tumor tissues and tumor cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Collart, F.R.; Chubb, C.B.; Mirkin, B.L.; Huberman, E.

    1992-07-10

    IMP dehydrogenase, a regulatory enzyme of guanine nucleotide biosynthesis, may play a role in cell proliferation and malignancy. To assess this possibility, we examined IMP dehydrogenase expression in a series of human solid tumor tissues and tumor cell lines in comparison with their normal counterparts. Increased IMP dehydrogenase gene expression was observed in brain tumors relative to normal brain tissue and in sarcoma cells relative to normal fibroblasts. Similarly, in several B- and T-lymphoid leukemia cell lines, elevated levels of IMP dehydrogenase mRNA and cellular enzyme were observed in comparison with the levels in peripheral blood lymphocytes. These results are consistent with an association between increased IMP dehydrogenase expression and either enhanced cell proliferation or malignant transformation.

  19. 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

  20. 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...

  1. Branched-chain amino acid metabolon: interaction of glutamate dehydrogenase with the mitochondrial branched-chain aminotransferase (BCATm).

    Science.gov (United States)

    Islam, Mohammad Mainul; Nautiyal, Manisha; Wynn, R Max; Mobley, James A; Chuang, David T; Hutson, Susan M

    2010-01-01

    The catabolic pathway for branched-chain amino acids includes deamination followed by oxidative decarboxylation of the deaminated product branched-chain alpha-keto acids, catalyzed by the mitochondrial branched-chain aminotransferase (BCATm) and branched-chain alpha-keto acid dehydrogenase enzyme complex (BCKDC). We found that BCATm binds to the E1 decarboxylase of BCKDC, forming a metabolon that allows channeling of branched-chain alpha-keto acids from BCATm to E1. The protein complex also contains glutamate dehydrogenase (GDH1), 4-nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1, pyruvate carboxylase, and BCKDC kinase. GDH1 binds to the pyridoxamine 5'-phosphate (PMP) form of BCATm (PMP-BCATm) but not to the pyridoxal 5'-phosphate-BCATm and other metabolon proteins. Leucine activates GDH1, and oxidative deamination of glutamate is increased further by addition of PMP-BCATm. Isoleucine and valine are not allosteric activators of GDH1, but in the presence of 5'-phosphate-BCATm, they convert BCATm to PMP-BCATm, stimulating GDH1 activity. Sensitivity to ADP activation of GDH1 was unaffected by PMP-BCATm; however, addition of a 3 or higher molar ratio of PMP-BCATm to GDH1 protected GDH1 from GTP inhibition by 50%. Kinetic results suggest that GDH1 facilitates regeneration of the form of BCATm that binds to E1 decarboxylase of the BCKDC, promotes metabolon formation, branched-chain amino acid oxidation, and cycling of nitrogen through glutamate.

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

    Directory of Open Access Journals (Sweden)

    Tao Gao

    Full Text Available 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.

  3. 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.

  4. 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.

  5. Pyruvate dehydrogenase complex in cerebral ischemia-reperfusion injury

    Directory of Open Access Journals (Sweden)

    Alexa Thibodeau

    2016-01-01

    Full Text Available Pyruvate dehydrogenase (PDH complex is a mitochondrial matrix enzyme that serves a critical role in the conversion of anaerobic to aerobic cerebral energy. The regulatory complexity of PDH, coupled with its significant influence in brain metabolism, underscores its susceptibility to, and significance in, ischemia-reperfusion injury. Here, we evaluate proposed mechanisms of PDH-mediated neurodysfunction in stroke, including oxidative stress, altered regulatory enzymatic control, and loss of PDH activity. We also describe the neuroprotective influence of antioxidants, dichloroacetate, acetyl-L-carnitine, and combined therapy with ethanol and normobaric oxygen, explained in relation to PDH modulation. Our review highlights the significance of PDH impairment in stroke injury through an understanding of the mechanisms by which it is modulated, as well as an exploration of neuroprotective strategies available to limit its impairment.

  6. Fabricating polystyrene fiber-dehydrogenase assemble as a functional biocatalyst.

    Science.gov (United States)

    An, Hongjie; Jin, Bo; Dai, Sheng

    2015-01-01

    Immobilization of the enzymes on nano-structured materials is a promising approach to enhance enzyme stabilization, activation and reusability. This study aimed to develop polystyrene fiber-enzyme assembles to catalyze model formaldehyde to methanol dehydrogenation reaction, which is an essential step for bioconversion of CO2 to a renewable bioenergy. We fabricated and modified electrospun polystyrene fibers, which showed high capability to immobilize dehydrogenase for the fiber-enzyme assembles. Results from evaluation of biochemical activities of the fiber-enzyme assemble showed that nitriation with the nitric/sulfuric acid ratio (v/v, 10:1) and silanization treatment delivered desirable enzyme activity and long-term storage stability, showing great promising toward future large-scale applications.

  7. Glucose-6 phosphate dehydrogenase deficiency and psychotic illness

    Directory of Open Access Journals (Sweden)

    Vijender Singh

    2012-01-01

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

  8. 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.

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

    Science.gov (United States)

    Casida, L. E., Jr.

    1977-01-01

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

  10. Encapsulation of Alcohol Dehydrogenase in Mannitol by Spray Drying

    Directory of Open Access Journals (Sweden)

    Hirokazu Shiga

    2014-03-01

    Full Text Available 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 due to the quick crystallization of mannitol during spray drying that resulted in the impairment of enzyme protection ability in comparison to its amorphous form. Maltodextin (dextrose equivalent = 11 was used to reduce the crystallization of mannitol. The addition of maltodextrin increased ADH activity and drastically changed the powder X-ray diffractogram of the spray-dried powders.

  11. Encapsulation of alcohol dehydrogenase in mannitol by spray drying.

    Science.gov (United States)

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

    2014-03-24

    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 due to the quick crystallization of mannitol during spray drying that resulted in the impairment of enzyme protection ability in comparison to its amorphous form. Maltodextin (dextrose equivalent = 11) was used to reduce the crystallization of mannitol. The addition of maltodextrin increased ADH activity and drastically changed the powder X-ray diffractogram of the spray-dried powders.

  12. Kinetics of myoglobin redox form stabilization by malate dehydrogenase.

    Science.gov (United States)

    Mohan, Anand; Muthukrishnan, S; Hunt, Melvin C; Barstow, Thomas J; Houser, Terry A

    2010-06-09

    This study reports the reduction of metmyoglobin (MMb) via oxidation of malate to oxaloacetate and the regeneration of reduced nicotinamide adenine dinucleotide (NADH) via malate dehydrogenase (MDH). Two experiments were conducted to evaluate a malate-MDH-NADH system as a possible mechanism for MMb reduction. In experiment 1, kinetics of MDH and MMb reduction were determined, and the results showed that increasing concentrations of oxidized nicotinamide adenine dinucleotide (NAD(+)) and l-malate also increased (p malate and NAD(+) added. Reduction of MMb in the muscle extracts via MDH was NAD(+), malate, and extract concentration dependent (p malate can replenish NADH via MDH activity in post-mortem muscle, ultimately resulting in a more functional meat color.

  13. In vitro interaction between psychotropic drugs and alcohol dehydrogenase activity.

    Science.gov (United States)

    Roig, M G; Bello, F; Burguillo, F J; Cachaza, J M; Kennedy, J F

    1991-03-01

    A series of CNS-stimulating and -depressant drugs have been studied for their in vitro interaction with horse liver alcohol dehydrogenase (ADH) activity. The depressant drugs studied included barbital, phenobarbital, thiopental, nitrazepam, chlorpromazine, sulpiride, clomethiazole, Li2CO3, diazepam, phenytoin, ethosuximide, morphine, and codeine. The stimulant drugs were theophylline, caffeine, amphetamine, imipramine, chlorimipramine, amitriptyline, and tranylcypromine. The results were as follows. First, ADH activity was inhibited by the action of chlorpromazine, tranylcypromine, imipramine, chlorimipramine, amitriptyline, sulpiride, amphetamine, codeine, ethosuximide, morphine, clomethiazole, nitrazepam, Li2CO3, theophylline, and phenobarbital, in descending order of inhibitory effect. Second, inhibition followed by activation of ADH activity was observed for imipramine and chlorimipramine. Third, activation of ADH activity was observed for phenytoin. Finally, the following drugs were not seen to exert any effect on ADH activity: barbital, thiopental, diazepam, and caffeine.

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

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    Keiki Nagaharu

    2016-01-01

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

  15. Mechanistic enzymology of CO dehydrogenase from Clostridium thermoaceticum

    Energy Technology Data Exchange (ETDEWEB)

    Ragsdale, S.W.

    1992-01-01

    The final steps in acetyl-CoA biosynthesis by anaerobic bacteria are performed by carbon monoxide dehydrogenase (CODH), a nickel/iron-sulfur protein. An important achievement was to establish conditions under which acetyl-CoA synthesis by purified enzymes equals the in vivo rate of acetate synthesis. Under these optimized conditions we established that the rate limiting step in the synthesis of acetyl-CoA from methyl-H[sub 4]folate, CO and CoA is likely to be the methylation of CODH by the methylated corrinoid/iron-sulfur protein. We then focused on stopped flow studies of this rate limiting transmethylation reaction and established its mechanism. We have studied the carbonylation of CODH by infrared and resonance Raman spectroscopy and determined that the [Ni-Fe[sup 3-4]S[sub 4

  16. Crystallographic analysis of FAD-dependent glucose dehydrogenase.

    Science.gov (United States)

    Komori, Hirofumi; Inaka, Koji; Furubayashi, Naoki; Honda, Michinari; Higuchi, Yoshiki

    2015-08-01

    An FAD-dependent glucose dehydrogenase (GDH) from Aspergillus terreus was purified and crystallized at 293 K using the sitting-drop vapour-diffusion method. A data set was collected to a resolution of 1.6 Å from a single crystal at 100 K using a rotating-anode X-ray source. The crystal belonged to space group P21, with unit-cell parameters a = 56.56, b = 135.74, c = 74.13 Å, β = 90.37°. The asymmetric unit contained two molecules of GDH. The Matthews coefficient was calculated to be 2.2 Å(3) Da(-1) and the solvent content was estimated to be 44%.

  17. Conjugated bilirubin in neonates with glucose-6-phosphate dehydrogenase deficiency.

    Science.gov (United States)

    Kaplan, M; Rubaltelli, F F; Hammerman, C; Vilei, M T; Leiter, C; Abramov, A; Muraca, M

    1996-05-01

    We used a system capable of measuring conjugated bilirubin and its monoconjugated and diconjugated fractions in serum to assess bilirubin conjugation in 29 glucose-6-phosphate dehydrogenase (G6PD)-deficient, term, male newborn infants and 35 control subjects; all had serum bilirubin levels > or = 256 mumol/L (15 mg/dI). The median value for diconjugated bilirubin was lower in the G6PD-deficient neonates than in control subjects (0.06 (range 0.00 to 1.84) vs 0.21 (range 0.00 to 1.02) mumol/L, p = 0.006). Diglucuronide was undetectable in 11 (38.9%) of the G6PD-deficient infants versus 3 (8.6%) of the control subjects (p = 0.015). These findings imply a partial defect of bilirubin conjugation not previously demonstrated in G6PD-deficient newborn infants.

  18. Lactate dehydrogenase (LDH isoenzymes patterns in ocular tumours

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    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.

  19. 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.

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

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    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.

  1. Biochemical and structural characterization of Plasmodium falciparum glutamate dehydrogenase 2.

    Science.gov (United States)

    Zocher, Kathleen; Fritz-Wolf, Karin; Kehr, Sebastian; Fischer, Marina; Rahlfs, Stefan; Becker, Katja

    2012-05-01

    Glutamate dehydrogenases (GDHs) play key roles in cellular redox, amino acid, and energy metabolism, thus representing potential targets for pharmacological interventions. Here we studied the functional network provided by the three known glutamate dehydrogenases of the malaria parasite Plasmodium falciparum. The recombinant production of the previously described PfGDH1 as hexahistidyl-tagged proteins was optimized. Additionally, PfGDH2 was cloned, recombinantly produced, and characterized. Like PfGDH1, PfGDH2 is an NADP(H)-dependent enzyme with a specific activity comparable to PfGDH1 but with slightly higher K(m) values for its substrates. The three-dimensional structure of hexameric PfGDH2 was solved to 3.1 Å resolution. The overall structure shows high similarity with PfGDH1 but with significant differences occurring at the subunit interface. As in mammalian GDH1, in PfGDH2 the subunit-subunit interactions are mainly assisted by hydrogen bonds and hydrophobic interactions, whereas in PfGDH1 these contacts are mediated by networks of salt bridges and hydrogen bonds. In accordance with this, the known bovine GDH inhibitors hexachlorophene, GW5074, and bithionol were more effective on PfGDH2 than on PfGDH1. Subcellular localization was determined for all three plasmodial GDHs by fusion with the green fluorescent protein. Based on our data, PfGDH1 and PfGDH3 are cytosolic proteins whereas PfGDH2 clearly localizes to the apicoplast, a plastid-like organelle specific for apicomplexan parasites. This study provides new insights into the structure and function of GDH isoenzymes of P. falciparum, which represent potential targets for the development of novel antimalarial drugs.

  2. Redesigning the substrate specificity of an enzyme: isocitrate dehydrogenase.

    Science.gov (United States)

    Doyle, S A; Fung, S Y; Koshland, D E

    2000-11-21

    Despite the structural similarities between isocitrate and isopropylmalate, isocitrate dehydrogenase (IDH) exhibits a strong preference for its natural substrate. Using a combination of rational and random mutagenesis, we have engineered IDH to use isopropylmalate as a substrate. Rationally designed mutations were based on comparison of IDH to a similar enzyme, isopropylmalate dehydrogenase (IPMDH). A chimeric enzyme that replaced an active site loop-helix motif with IPMDH sequences exhibited no activity toward isopropylmalate, and site-directed mutants that replaced IDH residues with their IPMDH equivalents only showed small improvements in k(cat). Random mutants targeted the IDH active site at positions 113 (substituted with glutamate), 115, and 116 (both randomized) and were screened for activity toward isopropylmalate. Six mutants were identified that exhibited up to an 8-fold improvement in k(cat) and increased the apparent binding affinity by as much as a factor of 80. In addition to the S113E mutation, five other mutants contained substitutions at positions 115 and/or 116. Most small hydrophobic substitutions at position 116 improved activity, possibly by generating space to accommodate the isopropyl group of isopropylmalate; however, substitution with serine yielded the most improvement in k(cat). Only two substitutions were identified at position 115, which suggests a more specific role for the wild-type asparagine residue in the utilization of isopropylmalate. Since interactions between neighboring residues in this region greatly influenced the effects of each other in unexpected ways, structural solutions were best identified in combinations, as allowed by random mutagenesis.

  3. 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.

  4. Aminolevulinate dehydrogenase polymorphisms did not modified lead serum and memory relationship

    Directory of Open Access Journals (Sweden)

    Lantip Rujito

    2015-12-01

    Full Text Available BACKGROUND Lead accumulation in the blood widely known affecting the formation of heme and oxygen transport processes in vital organs, Leading to organ failure including the brain synapses. Lead affinity has been recognized influenced by constitutional genotype of aminolevulinate dehydrogenase (ALAD, which encodes for heme synthesis. This research aimed to determine the relationship between plumbum (Pb and short term memory on each ALAD gene genotyping (ALAD 1-1, ALAD 1-2 or ALAD 2-2 in gas station workers. METHODS Seventy six probands from gas station workers were recruited to participate in this research. Each probands was carried out ALAD genotyping using polymerase chain reaction-restriction fragment length polymorphism (PCRRFLP method, lead serum level using atomic absorbent spectrophotometer (AAS, and short term memory was measurement by intelligence structure test (IST. RESULTS Proportion of δ ALAD 1-1, 1-2, and 2-2 were 91.8%, 8.2% and 0% respectively. Lead serum showed 15.84 ppb in homozygous 1-1, and 20.79 ppb in heterozygous. Short term memory in the probands varied from 85 until 117, with average in 99.71. There was significant negative relationship between lead serum and short term memory (r=-0.24; p=0.038. However, we could not find any significant correlation in each δ ALAD genotypes. CONCLUSION The δ ALAD genotypes did not modified the relationship between serum lead level and short term memory in gas station workers.

  5. Alternative splicing of testis-specific lactate dehydrogenase C gene in mammals and pigeon.

    Science.gov (United States)

    Huang, Lin; Lin, Yaqiu; Jin, Suyu; Liu, Wei; Xu, Yaou; Zheng, Yucai

    2012-04-01

    The objective of the present study was to confirm the widespread existence of alternative splicing of lactate dehydrogenase c (ldhc) gene in mammals. RT-PCR was employed to amplify cDNAs of ldhc from testes of mammals including pig, dog, rabbit, cat, rat, and mouse, as well as pigeon. Two to six kinds of splice variants of ldhc were observed in the seven species as a result of deletion of one or more exons or insertion of partial sequence of an intron in the mature mRNA. The deleted exons occur mostly in exons 5, 4, 6, and 3. The insertion of a partial sequence of introns, which resulted in an abnormal stop codon in the inserted intron sequence, was observed only in dog and rat. The deletion of exons also resulted in a reading frame shift and formation of a stop codon in some variants. No alternative splicing was observed for ldha and ldhb genes in testis of yak. Native polyacrylamide gel electrophoresis and Western blot analysis revealed no obvious LDH-C4 activity derived from expressed ldhc variants. Our results demonstrated the widespread and unique existence of alternative splicing of ldhc genes in mammals.

  6. A high-throughput fluorescence-based assay for Plasmodium dihydroorotate dehydrogenase inhibitor screening.

    Science.gov (United States)

    Caballero, Iván; Lafuente, María José; Gamo, Francisco-Javier; Cid, Concepción

    2016-08-01

    Plasmodium dihydroorotate dehydrogenase (DHODH) is a mitochondrial membrane-associated flavoenzyme that catalyzes the rate-limiting step of de novo pyrimidine biosynthesis. DHODH is a validated target for malaria, and DSM265, a potent inhibitor, is currently in clinical trials. The enzyme catalyzes the oxidation of dihydroorotate to orotate using flavin mononucleotide (FMN) as cofactor in the first half of the reaction. Reoxidation of FMN to regenerate the active enzyme is mediated by ubiquinone (CoQD), which is the physiological final electron acceptor and second substrate of the reaction. We have developed a fluorescence-based high-throughput enzymatic assay to find DHODH inhibitors. In this assay, the CoQD has been replaced by a redox-sensitive fluorogenic dye, resazurin, which changes to a fluorescent state on reduction to resorufin. Remarkably, the assay sensitivity to find competitive inhibitors of the second substrate is higher than that reported for the standard colorimetric assay. It is amenable to 1536-well plates with Z' values close to 0.8. The fact that the human enzyme can also be assayed in the same format opens additional applications of this assay to the discovery of inhibitors to treat cancer, transplant rejection, autoimmune diseases, and other diseases mediated by rapid cellular growth.

  7. 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.

  8. 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.

  9. Nicotine promotes Streptococcus mutans extracellular polysaccharide synthesis, cell aggregation and overall lactate dehydrogenase activity.

    Science.gov (United States)

    Huang, R; Li, M; Gregory, R L

    2015-08-01

    Several epidemiology studies have reported a positive relationship between smoking and dental caries. Nicotine, an alkaloid component of tobacco, has been demonstrated to stimulate biofilm formation and metabolic activity of Streptococcus mutans, one of the most important pathogens of dental caries. The first aim of the present study was to explore the possible mechanisms leading to increased biofilm by nicotine treatment from three aspects, extracellular polysaccharides (EPS) synthesis, glucosyltransferase (Gtf) synthesis and glucan-binding protein (Gbp) synthesis at the mRNA and protein levels. The second aim was to investigate how nicotine affects S. mutans virulence, particular in lactate dehydrogenase (LDH) activity. Confocal laser scanning microscopy results demonstrated that both biofilm bacterial cell numbers and EPS were increased by nicotine. Gtf and GbpA protein expression of S. mutans planktonic cells were upregulated while GbpB protein expression of biofilm cells were downregulated by nicotine. The mRNA expression trends of those genes were mostly consistent with results on protein level but not statistically significant, and gtfD and gbpD of biofilm cells were inhibited. Nicotine was not directly involved in S. mutans LDH activity. However, since it increases the total number of bacterial cells in biofilm, the overall LDH activity of S. mutans biofilm is increased. In conclusion, nicotine stimulates S. mutans planktonic cell Gtf and Gbp expression. This leads to more planktonic cells attaching to the dental biofilm. Increased cell numbers within biofilm results in higher overall LDH activity. This contributes to caries development in smokers.

  10. 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.

  11. A new paper-based analytical device for detection of Glucose-6-phosphate dehydrogenase deficiency.

    Science.gov (United States)

    Kaewarsa, Phuritat; Laiwattanapaisal, Wanida; Palasuwan, Attakorn; Palasuwan, Duangdao

    2017-03-01

    Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a genetic haemolytic disorder. Most persons with G6PD deficiency are asymptomatic, but exposure to oxidant drugs, such as the anti-malarial drug primaquine, may induce haemolysis, which is commonly found in Asian countries. A reliable test is necessary for diagnosing the deficiency to prevent an acute haemolytic crisis. This study proposes a novel quantitative method to detect G6PD deficiency using paper-based analytical devices (G6PDD-PAD). Wax printing was utilized for fabricating circular reaction zone patterns in paper. The colorimetric assay is based on the formation of formazan via a reduction of tetra-nitro blue tetrazolium (TNBT) by the G6PD enzyme on G6PDD-PAD. Detection was achieved by capturing the colour using a desktop scanner and the colour intensity was analysed with Adobe Photoshop C56. The results showed that the G6PD activity analysed by G6PDD-PAD was highly correlated with the standard biochemical assay (SBA) (r(2)=0.87, pPAD and the SBA (mean bias 1.4 IU/gHb). The detection limit was 0 IU/gHb of G6PD activity. This study demonstrates the feasibility of using G6PDD-PAD. This simple, low-cost test ($0.1/test) should be useful for diagnosing G6PD deficiency in resource-limited settings.

  12. 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.

  13. 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.

  14. Formate hydrogenlyase in the hyperthermophilic archaeon, Thermococcus litoralis

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    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.

  15. Polymorphisms of alcohol dehydrogenase-2 and aldehyde dehydrogenase-2 and esophageal cancer risk in Southeast Chinese males

    Institute of Scientific and Technical Information of China (English)

    Jian-Hua Ding; Su-Ping Li; Hai-Xia Cao; Jian-Zhong Wu; Chang-Ming Gao; Ping Su; Yan-Ting Liu; Jian-Nong Zhou; Jun Chang; Gen-Hong Yao

    2009-01-01

    AIM: To evaluate the impact of alcohol dehydrogenase-2 (ADH2) and aldehyde dehydrogenase-2 (ALDH2) polymorphisms on esophageal cancer susceptibility in Southeast Chinese males. METHODS: Two hundred and twenty-one esophageal cancer patients and 191 healthy controls from Taixing city in Jiangsu Province were enrolled in this study. ADH2 and ALDH2 genotypes were examined by polymerase chain reaction and denaturing highperformance liquid chromatography. Unconditional logistic regression was used to calculate the odds ratios (OR) and 95% confidence interval (CI). RESULTS: The ADH G allele carriers were more susceptible to esophageal cancer, but no association was found between ADH2 genotypes and risk of esophageal cancer when disregarding alcohol drinking status. Regardless of ADH2 genotype, ALDH2G/A or A/A carriers had significantly increased risk of developing esophageal cancer, with homozygous individuals showing higher esophageal cancer risk than those who were heterozygous. A significant interaction between ALDH2 and drinking was detected regarding esophageal cancer risk; the OR was 3.05 (95% CI: 1.49-6.25). Compared with non-drinkers carrying both ALDH2 G/G and ADH2 A/A, drinkers carrying both ALDH2 A allele and ADH2 G allele showed a significantly higher risk of developing esophageal cancer (OR = 8.36, 95% CI: 2.98-23.46).CONCLUSION: Both ADH2 G allele and ALDH2 A allele significantly increase the risk of esophageal cancer development in Southeast Chinese males. ALDH2 A allele significantly increases the risk of esophageal cancer development especially in alcohol drinkers. Alcohol drinkers carrying both ADH2 G allele and ALDH2 A allele have a higher risk of developing esophageal cancer.

  16. 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.

  17. Improved production of propionic acid in Propionibacterium jensenii via combinational overexpression of glycerol dehydrogenase and malate dehydrogenase from Klebsiella pneumoniae.

    Science.gov (United States)

    Liu, Long; Zhuge, Xin; Shin, Hyun-Dong; Chen, Rachel R; Li, Jianghua; Du, Guocheng; Chen, Jian

    2015-04-01

    Microbial production of propionic acid (PA), an important chemical building block used as a preservative and chemical intermediate, has gained increasing attention for its environmental friendliness over traditional petrochemical processes. In previous studies, we constructed a shuttle vector as a useful tool for engineering Propionibacterium jensenii, a potential candidate for efficient PA synthesis. In this study, we identified the key metabolites for PA synthesis in P. jensenii by examining the influence of metabolic intermediate addition on PA synthesis with glycerol as a carbon source under anaerobic conditions. We also further improved PA production via the overexpression of the identified corresponding enzymes, namely, glycerol dehydrogenase (GDH), malate dehydrogenase (MDH), and fumarate hydratase (FUM). Compared to those in wild-type P. jensenii, the activities of these enzymes in the engineered strains were 2.91- ± 0.17- to 8.12- ± 0.37-fold higher. The transcription levels of the corresponding enzymes in the engineered strains were 2.85- ± 0.19- to 8.07- ± 0.63-fold higher than those in the wild type. The coexpression of GDH and MDH increased the PA titer from 26.95 ± 1.21 g/liter in wild-type P. jensenii to 39.43 ± 1.90 g/liter in the engineered strains. This study identified the key metabolic nodes limiting PA overproduction in P. jensenii and further improved PA titers via the coexpression of GDH and MDH, making the engineered P. jensenii strain a potential industrial producer of PA.

  18. 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.

  19. Overexpression and nuclear accumulation of glyceraldehyde-3-phosphate dehydrogenase in a transgenic mouse model of Huntington's disease.

    Science.gov (United States)

    Senatorov, Vladimir V; Charles, Vinod; Reddy, P H; Tagle, Dan A; Chuang, De-Maw

    2003-03-01

    Huntington's disease is due to an expansion of CAG repeats in the huntingtin gene. Huntingtin interacts with several proteins including glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We performed immunohistochemical analysis of GAPDH expression in the brains of transgenic mice carrying the huntingtin gene with 89 CAG repeats. In all wild-type animals examined, GAPDH was evenly distributed among the different cell types throughout the brain. In contrast, the majority of transgenic mice showed GAPDH overexpression, with the most prominent GAPDH changes observed in the caudate putamen, globus pallidus, neocortex, and hippocampal formation. Double staining for NeuN and GFAP revealed that GAPDH overexpression occurred exclusively in neurons. Nissl staining analysis of the neocortex and caudate putamen indicated 24 and 27% of cell loss in transgenic mice, respectively. Subcellular fluorescence analysis revealed a predominant increase in GAPDH immunostaining in the nucleus. Thus, we conclude that mutation of huntingtin is associated with GAPDH overexpression and nuclear translocation in discrete populations of brain neurons.

  20. 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.

  1. 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

  2. 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.

  3. Glyceraldehyde-3-phosphate Dehydrogenase Aggregates Accelerate Amyloid-β Amyloidogenesis in Alzheimer Disease*

    Science.gov (United States)

    Itakura, Masanori; Nakajima, Hidemitsu; Kubo, Takeya; Semi, Yuko; Kume, Satoshi; Higashida, Shusaku; Kaneshige, Akihiro; Kuwamura, Mitsuru; Harada, Naoki; Kita, Akinori; Azuma, Yasu-Taka; Yamaji, Ryoichi; Inui, Takashi; Takeuchi, Tadayoshi

    2015-01-01

    Alzheimer disease (AD) is a progressive neurodegenerative disorder characterized by loss of neurons and formation of pathological extracellular deposits induced by amyloid-β peptide (Aβ). Numerous studies have established Aβ amyloidogenesis as a hallmark of AD pathogenesis, particularly with respect to mitochondrial dysfunction. We have previously shown that glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forms amyloid-like aggregates upon exposure to oxidative stress and that these aggregates contribute to neuronal cell death. Here, we report that GAPDH aggregates accelerate Aβ amyloidogenesis and subsequent neuronal cell death both in vitro and in vivo. Co-incubation of Aβ40 with small amounts of GAPDH aggregates significantly enhanced Aβ40 amyloidogenesis, as assessed by in vitro thioflavin-T assays. Similarly, structural analyses using Congo red staining, circular dichroism, and atomic force microscopy revealed that GAPDH aggregates induced Aβ40 amyloidogenesis. In PC12 cells, GAPDH aggregates augmented Aβ40-induced cell death, concomitant with disruption of mitochondrial membrane potential. Furthermore, mice injected intracerebroventricularly with Aβ40 co-incubated with GAPDH aggregates exhibited Aβ40-induced pyramidal cell death and gliosis in the hippocampal CA3 region. These observations were accompanied by nuclear translocation of apoptosis-inducing factor and cytosolic release of cytochrome c from mitochondria. Finally, in the 3×Tg-AD mouse model of AD, GAPDH/Aβ co-aggregation and mitochondrial dysfunction were consistently detected in an age-dependent manner, and Aβ aggregate formation was attenuated by GAPDH siRNA treatment. Thus, this study suggests that GAPDH aggregates accelerate Aβ amyloidogenesis, subsequently leading to mitochondrial dysfunction and neuronal cell death in the pathogenesis of AD. PMID:26359500

  4. 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...... and internal rotenone-insensitive NADH dehydrogenase activity are also light dependent. These findings suggest that NDA has a role in photorespiration and might be identical to the previously unidentified internal rotenone-insensitive NADH dehydrogenase....

  5. 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.

  6. 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

  7. 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

  8. 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

  9. 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.

  10. Heterozygosity of the sheep: Polymorphism of 'malic enzyme', isocitrate dehydrogenase (NADP+), catalase and esterase.

    Science.gov (United States)

    Baker, C M; Manwell, C

    1977-04-01

    In contrast to other reports, it is found that the sheep has approximately as much enzyme variation as man. Most of the genetically interpretable enzyme variation in heart, liver, kidney and muscle from 52 sheep (Merinos or Merino crosses) is in the NADP-dependent dehydrogenases [two 'malic enzymes' and the supernatant isocitrate dehydrogenase (NADP+)] and in the esterases. Ten different loci for NAD-dependent dehydrogenases are electrophoretically monomorphic, as are five different NADH diaphorases from heart muscle and 15 different major proteins from skeletal muscle. It is highly statistically significant that NADP-dependent dehydrogenases and esterases are polymorphic but representatives of several other major classes of enzymes are not. The physiological significance of this polymorphism may be related to the role of these enzymes in growth and detoxication, sheep having been selected by man for faster growth, of wool or of carcass, and for grazing a wide variety of plants.

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

    Directory of Open Access Journals (Sweden)

    Alparslan Merdin

    2014-03-01

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

  12. 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...

  13. 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...

  14. Undetected Toxicity Risk in Pharmacogenetic Testing for Dihydropyrimidine Dehydrogenase

    Directory of Open Access Journals (Sweden)

    Felicia Stefania Falvella

    2015-04-01

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

  15. The role of glutamate dehydrogenase in mammalian ammonia metabolism.

    Science.gov (United States)

    Spanaki, Cleanthe; Plaitakis, Andreas

    2012-01-01

    Glutamate dehydrogenase (GDH) catalyzes the reversible inter-conversion of glutamate to α-ketoglutarate and ammonia. High levels of GDH activity is found in mammalian liver, kidney, brain, and pancreas. In the liver, GDH reaction appears to be close-to-equilibrium, providing the appropriate ratio of ammonia and amino acids for urea synthesis in periportal hepatocytes. In addition, GDH produces glutamate for glutamine synthesis in a small rim of pericentral hepatocytes. Hence, hepatic GDH can be either a source for ammonia or an ammonia scavenger. In the kidney, GDH function produces ammonia from glutamate to control acidosis. In the human, the presence of two differentially regulated isoforms (hGDH1 and hGDH2) suggests a complex role for GDH in ammonia homeostasis. Whereas hGDH1 is sensitive to GTP inhibition, hGDH2 has dissociated its function from GTP control. Furthermore, hGDH2 shows a lower optimal pH than hGDH1. The hGDH2 enzyme is selectively expressed in human astrocytes and Sertoli cells, probably facilitating metabolic recycling processes essential for their supportive role. Here, we report that hGDH2 is also expressed in the epithelial cells lining the convoluted tubules of the renal cortex. As hGDH2 functions more efficiently under acidotic conditions without the operation of the GTP energy switch, its presence in the kidney may increase the efficacy of the organ to maintain acid base equilibrium.

  16. 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.

  17. Regulation of L-threonine dehydrogenase in somatic cell reprogramming.

    Science.gov (United States)

    Han, Chuanchun; Gu, Hao; Wang, Jiaxu; Lu, Weiguang; Mei, Yide; Wu, Mian

    2013-05-01

    Increasing evidence suggests that metabolic remodeling plays an important role in the regulation of somatic cell reprogramming. Threonine catabolism mediated by L-threonine dehydrogenase (TDH) has been recognized as a specific metabolic trait of mouse embryonic stem cells. However, it remains unknown whether TDH-mediated threonine catabolism could regulate reprogramming. Here, we report TDH as a novel regulator of somatic cell reprogramming. Knockdown of TDH inhibits, whereas induction of TDH enhances reprogramming efficiency. Moreover, microRNA-9 post-transcriptionally regulates the expression of TDH and thereby inhibits reprogramming efficiency. Furthermore, protein arginine methyltransferase (PRMT5) interacts with TDH and mediates its post-translational arginine methylation. PRMT5 appears to regulate TDH enzyme activity through both methyltransferase-dependent and -independent mechanisms. Functionally, TDH-facilitated reprogramming efficiency is further enhanced by PRMT5. These results suggest that TDH-mediated threonine catabolism controls somatic cell reprogramming and indicate the importance of post-transcriptional and post-translational regulation of TDH.

  18. Glutamate dehydrogenase 1 and SIRT4 regulate glial development.

    Science.gov (United States)

    Komlos, Daniel; Mann, Kara D; Zhuo, Yue; Ricupero, Christopher L; Hart, Ronald P; Liu, Alice Y-C; Firestein, Bonnie L

    2013-03-01

    Congenital hyperinsulinism/hyperammonemia (HI/HA) syndrome is caused by an activation mutation of glutamate dehydrogenase 1 (GDH1), a mitochondrial enzyme responsible for the reversible interconversion between glutamate and α-ketoglutarate. The syndrome presents clinically with hyperammonemia, significant episodic hypoglycemia, seizures, and frequent incidences of developmental and learning defects. Clinical research has implicated that although some of the developmental and neurological defects may be attributed to hypoglycemia, some characteristics cannot be ascribed to low glucose and as hyperammonemia is generally mild and asymptomatic, there exists the possibility that altered GDH1 activity within the brain leads to some clinical changes. GDH1 is allosterically regulated by many factors, and has been shown to be inhibited by the ADP-ribosyltransferase sirtuin 4 (SIRT4), a mitochondrially localized sirtuin. Here we show that SIRT4 is localized to mitochondria within the brain. SIRT4 is highly expressed in glial cells, specifically astrocytes, in the postnatal brain and in radial glia during embryogenesis. Furthermore, SIRT4 protein decreases in expression during development. We show that factors known to allosterically regulate GDH1 alter gliogenesis in CTX8 cells, a novel radial glial cell line. We find that SIRT4 and GDH1 overexpression play antagonistic roles in regulating gliogenesis and that a mutant variant of GDH1 found in HI/HA patients accelerates the development of glia from cultured radial glia cells.

  19. Regulation by ammonium of glutamate dehydrogenase (NADP+) from Saccharomyces cerevisiae.

    Science.gov (United States)

    Bogonez, E; Satrústegui, J; Machado, A

    1985-06-01

    The activity of glutamate dehydrogenase (NADP+) (EC 1.4.1.4; NADP-GDH) of Saccharomyces cerevisiae is decreased under conditions in which intracellular ammonia concentrations increases. A high internal ammonia concentration can be obtained (a) by increasing the ammonium sulphate concentration in the culture medium, and (b) by growing the yeast either in acetate + ammonia media, where the pH of the medium rises during growth, or in heavily buffered glucose + ammonia media at pH 7.5. Under these conditions cellular oxoglutarate concentrations do not vary and changes in NADP-GDH activity appear to provide a constant rate of oxoglutarate utilization. The following results suggest that the decrease in NADP-GDH activity in ammonia-accumulating yeast cells is brought about by repression of synthesis: (i) after a shift to high ammonium sulphate concentrations, the number of units of activity per cell decreased as the inverse of cell doubling; and (ii) the rate of degradation of labelled NADP-GDH was essentially the same in ammonia-accumulating yeast cells and in controls, whereas the synthesis constant was much lower in the ammonia-accumulating cells than in the controls.

  20. 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

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

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    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.

  2. 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.

  3. 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

  4. Isocitrate dehydrogenase 1 and 2 mutations in gliomas.

    Science.gov (United States)

    Megova, Magdalena; Drabek, Jiri; Koudelakova, Vladimira; Trojanec, Radek; Kalita, Ondrej; Hajduch, Marian

    2014-12-01

    Over the past few years, new biomarkers have allowed a deeper insight into gliomagenesis and facilitated the identification of possible targets for glioma therapy. Isocitrate dehydrogenase (IDH) 1 and IDH2 mutations have been shown to be promising biomarkers for monitoring disease prognosis and predicting the response to treatment. This review summarizes recent findings in this field. Web of Science, Science Direct, and PubMed online databases were used to search for publications investigating the role of IDH in glioma. References were identified by searching for the keywords "IDH1 or IDH2 and glioma and diagnostic or predictive or prognostic" in papers published from January, 2008, to April, 2014. Only papers in English were reviewed. Publications available only as an abstract were not included. IDH1/2 mutations are tightly associated with grade II and III gliomas and secondary glioblastomas, with better prognosis and production of a recently described oncometabolite, 2-hydroxyglutarate (2HG). Although the contradictory positive effect of IDH mutation on prognosis and negative role of 2HG in tumor transformation remain unresolved, the future direction of personalized treatment strategies targeted to glioma development is likely to focus on IDH1/2 mutations.

  5. Alternative splicing regulates targeting of malate dehydrogenase in Yarrowia lipolytica.

    Science.gov (United States)

    Kabran, Philomène; Rossignol, Tristan; Gaillardin, Claude; Nicaud, Jean-Marc; Neuvéglise, Cécile

    2012-06-01

    Alternative pre-mRNA splicing is a major mechanism contributing to the proteome complexity of most eukaryotes, especially mammals. In less complex organisms, such as yeasts, the numbers of genes that contain introns are low and cases of alternative splicing (AS) with functional implications are rare. We report the first case of AS with functional consequences in the yeast Yarrowia lipolytica. The splicing pattern was found to govern the cellular localization of malate dehydrogenase, an enzyme of the central carbon metabolism. This ubiquitous enzyme is involved in the tricarboxylic acid cycle in mitochondria and in the glyoxylate cycle, which takes place in peroxisomes and the cytosol. In Saccharomyces cerevisiae, three genes encode three compartment-specific enzymes. In contrast, only two genes exist in Y. lipolytica. One gene (YlMDH1, YALI0D16753g) encodes a predicted mitochondrial protein, whereas the second gene (YlMDH2, YALI0E14190g) generates the cytosolic and peroxisomal forms through the alternative use of two 3'-splice sites in the second intron. Both splicing variants were detected in cDNA libraries obtained from cells grown under different conditions. Mutants expressing the individual YlMdh2p isoforms tagged with fluorescent proteins confirmed that they localized to either the cytosolic or the peroxisomal compartment.

  6. Multiple soluble malate dehydrogenase of Geophagus brasiliensis (Cichlidae, Perciformes

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    Aquino-Silva Maria Regina de

    1998-01-01

    Full Text Available A recent locus duplication hypothesis for sMDH-B* was proposed to explain the complex electrophoretic pattern of six bands detected for the soluble form of malate dehydrogenase (MDH, EC 1.1.1.37 in 84% of the Geophagus brasiliensis (Cichlidae, Perciformes analyzed (AB1B2 individuals. Klebe's serial dilutions were carried out in skeletal muscle extracts. B1 and B2 subunits had the same visual end-points, reflecting a nondivergent pattern for these B-duplicated genes. Since there is no evidence of polyploidy in the Cichlidae family, MDH-B* loci must have evolved from regional gene duplication. Tissue specificities, thermostability and kinetic tests resulted in similar responses from both B-isoforms, in both sMDH phenotypes, suggesting that these more recently duplicated loci underwent the same regulatory gene action. Similar results obtained with the two sMDH phenotypes did not show any indication of a six-banded specimen adaptive advantage in subtropical regions.

  7. Characterization of malate dehydrogenase from the hyperthermophilic archaeon Pyrobaculum islandicum.

    Science.gov (United States)

    Yennaco, Lynda J; Hu, Yajing; Holden, James F

    2007-09-01

    Native and recombinant malate dehydrogenase (MDH) was characterized from the hyperthermophilic, facultatively autotrophic archaeon Pyrobaculum islandicum. The enzyme is a homotetramer with a subunit mass of 33 kDa. The activity kinetics of the native and recombinant proteins are the same. The apparent K ( m ) values of the recombinant protein for oxaloacetate (OAA) and NADH (at 80 degrees C and pH 8.0) were 15 and 86 microM, respectively, with specific activity as high as 470 U mg(-1). Activity decreased more than 90% when NADPH was used. The catalytic efficiency of OAA reduction by P. islandicum MDH using NADH was significantly higher than that reported for any other archaeal MDH. Unlike other archaeal MDHs, specific activity of the P. islandicum MDH back-reaction also decreased more than 90% when malate and NAD(+) were used as substrates and was not detected with NADP(+). A phylogenetic tree of 31 archaeal MDHs shows that they fall into 5 distinct groups separated largely along taxonomic lines suggesting minimal lateral mdh transfer between Archaea.

  8. Cytosolic malate dehydrogenase regulates senescence in human fibroblasts.

    Science.gov (United States)

    Lee, Seung-Min; Dho, So Hee; Ju, Sung-Kyu; Maeng, Jin-Soo; Kim, Jeong-Yoon; Kwon, Ki-Sun

    2012-10-01

    Carbohydrate metabolism changes during cellular senescence. Cytosolic malate dehydrogenase (MDH1) catalyzes the reversible reduction of oxaloacetate to malate at the expense of reduced nicotinamide adenine dinucleotide (NADH). Here, we show that MDH1 plays a critical role in the cellular senescence of human fibroblasts. We observed that the activity of MDH1 was reduced in old human dermal fibroblasts (HDFs) [population doublings (PD) 56], suggesting a link between decreased MDH1 protein levels and aging. Knockdown of MDH1 in young HDFs (PD 20) and the IMR90 human fibroblast cell line resulted in the appearance of significant cellular senescence features, including senescence-associated β-galactosidase staining, flattened and enlarged morphology, increased population doubling time, and elevated p16(INK4A) and p21(CIP1) protein levels. Cytosolic NAD/NADH ratios were decreased in old HDFs to the same extent as in MDH1 knockdown HDFs, suggesting that cytosolic NAD depletion is related to cellular senescence. We found that AMP-activated protein kinase, a sensor of cellular energy, was activated in MDH1 knockdown cells. We also found that sirtuin 1 (SIRT1) deacetylase, a controller of cellular senescence, was decreased in MDH1 knockdown cells. These results indicate that the decrease in MDH1 and subsequent reduction in NAD/NADH ratio, which causes SIRT1 inhibition, is a likely carbohydrate metabolism-controlled cellular senescence mechanism.

  9. 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.

  10. Human choline dehydrogenase: medical promises and biochemical challenges.

    Science.gov (United States)

    Salvi, Francesca; Gadda, Giovanni

    2013-09-15

    Human choline dehydrogenase (CHD) is located in the inner membrane of mitochondria primarily in liver and kidney and catalyzes the oxidation of choline to glycine betaine. Its physiological role is to regulate the concentrations of choline and glycine betaine in the blood and cells. Choline is important for regulation of gene expression, the biosynthesis of lipoproteins and membrane phospholipids and for the biosynthesis of the neurotransmitter acetylcholine; glycine betaine plays important roles as a primary intracellular osmoprotectant and as methyl donor for the biosynthesis of methionine from homocysteine, a required step for the synthesis of the ubiquitous methyl donor S-adenosyl methionine. Recently, CHD has generated considerable medical attention due to its association with various human pathologies, including male infertility, homocysteinuria, breast cancer and metabolic syndrome. Despite the renewed interest, the biochemical characterization of the enzyme has lagged behind due to difficulties in the obtainment of purified, active and stable enzyme. This review article summarizes the medical relevance and the physiological roles of human CHD, highlights the biochemical knowledge on the enzyme, and provides an analysis based on the comparison of the protein sequence with that of bacterial choline oxidase, for which structural and biochemical information is available.

  11. The PQQ-alcohol dehydrogenase of Gluconacetobacter diazotrophicus.

    Science.gov (United States)

    Gómez-Manzo, Saúl; Contreras-Zentella, Martha; González-Valdez, Alejandra; Sosa-Torres, Martha; Arreguín-Espinoza, Roberto; Escamilla-Marván, Edgardo

    2008-06-30

    The oxidation of ethanol to acetic acid is the most characteristic process in acetic acid bacteria. Gluconacetobacter diazotrophicus is rather unique among the acetic acid bacteria as it carries out nitrogen fixation and is a true endophyte, originally isolated from sugar cane. Aside its peculiar life style, Ga. diazotrophicus, possesses a constitutive membrane-bound oxidase system for ethanol. The Alcohol dehydrogenase complex (ADH) of Ga. diazotrophicus was purified to homogeneity from the membrane fraction. It-exhibited two subunits with molecular masses of 71.4 kDa and 43.5 kDa. A positive peroxidase reaction confirmed the presence of cytochrome c in both subunits. Pyrroloquinoline quinone (PQQ) of ADH was identified by UV-visible light and fluorescence spectroscopy. The enzyme was purified in its full reduced state; potassium ferricyanide induced its oxidation. Ethanol or acetaldehyde restored the full reduced state. The enzyme showed an isoelectric point (pI) of 6.1 and its optimal pH was 6.0. Both ethanol and acetaldehyde were oxidized at almost the same rate, thus suggesting that the ADH complex of Ga. diazotrophicus could be kinetically competent to catalyze, at least in vitro, the double oxidation of ethanol to acetic acid.

  12. Serum alcohol dehydrogenase levels in patients with mental disorders.

    Science.gov (United States)

    Kravos, Matej; Malesic, Ivan; Levanic, Suzana

    2005-11-01

    Alcohol dehydrogenase (ADH) was assessed in 81 patients admitted to hospital for treatment for alcohol dependence with or without liver cirrhosis, 20 patients with bipolar disorder treated with lithium carbonate and 41 patients with various mental disorders treated with psychopharmacologic agents. Testing the hypothesis of the arithmetic mean showed that in alcohol dependents the arithmetic mean of ADH activity (12.19 nkat/l+/-5.61) differs significantly from that in healthy subjects (4.45 nkat/l+/-2.31) and in the group with ethanol poisoning (6.24 nkat/l+/-3.65) there is none. In the group with bipolar disorder, treated with lithium (7.39 nkat/l+/-3.11) and, in the group of patients treated with psychiatric drugs because of various mental disorders (7.79 nkat/l+/-8.51), the differences are statistically significant. In our opinion, assessing ADH activity in the sera of alcohol dependents could be an additional marker advantageous to the diagnostics, course and monitoring of therapy in such patients. In the groups of patients with mental disorders treated with psychotropic drugs, the increased ADH activity was found to be a more sensitive marker for the detection of drug hepatotoxicity.

  13. 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.

  14. 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.

  15. [Palmitoyl-CoA-dehydrogenase from rabbit adrenals, liver and myocardium].

    Science.gov (United States)

    Doroshkevich, N A; Mandrik, K A; Vinogradov, V V

    1988-01-01

    Partially purified preparations of palmitoyl-CoA dehydrogenase from rabbit adrenal glands, liver and heart tissues exhibited similar kinetic parameters. Km value constituted 6.58, 5.26 and 6.67 microM for the enzyme from adrenal glands, liver and heart tissues, respectively. At the same time, palmitoyl-CoA dehydrogenase possessed lower catalytic capacity in adrenal glands due to the decreased amount of the enzyme as compared with that of liver or heart tissues.

  16. Requirement of succinate dehydrogenase activity for symbiotic bacteroid differentiation of Rhizobium meliloti in alfalfa nodules.

    OpenAIRE

    Gardiol, A E; Truchet, G L; Dazzo, F. B.

    1987-01-01

    Transmission electron microscopy was used to study the cellular morphologies of a wild-type Rhizobium meliloti strain (L5-30), a nitrogen fixation-ineffective (Fix-) succinate dehydrogenase mutant (Sdh-) strain, and a Fix+ Sdh+ revertant strain within alfalfa nodules and after free-living growth in a minimal medium containing 27 mM mannitol plus 20 mM succinate. The results showed a requirement of succinate dehydrogenase activity for symbiotic differentiation and maintenance of R. meliloti ba...

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. Elementary steps in the reaction of the pyruvate dehydrogenase complex from pig heart. Kinetics of thiamine diphosphate binding to the complex.

    Science.gov (United States)

    Sümegi, B; Alkonyi, I

    1983-11-02

    In the progress curve of the reaction of the pyruvate dehydrogenase complex, a lag phase was observed when the concentration of thiamin diphosphate was lower than usual (about 0.2-1 mM) in the enzyme assay. The length of the lag phase was dependent on thiamin diphosphate concentration, ranging from 0.2 min to 2 min as the thiamin diphosphate concentration varied from 800 nM to 22 nM. The lag phase was also observed in the elementary steps catalyzed by the pyruvate dehydrogenase component. A Km value of 107 nM was found for thiamin diphosphate with respect to the steady-state reaction rate following the lag phase. The pre-steady-state kinetic data indicate that the resulting lag phase was the consequence of a slow holoenzyme formation from apoenzyme and thiamin diphosphate. The thiamin diphosphate can bind to the pyruvate dehydrogenase complex in the absence of pyruvate, but the presence of 2 mM pyruvate increases the rate constant of binding from 1.4 X 10(4) M-1 S-1 to 1.3 X 10(5) M-1 S-1 and decreases the rate constant of dissociation from 2.3 X 10(-2) S-1 to 4.1 X 10(-3) S-1. On the other hand, the effect of pyruvate on the thiamin diphosphate binding revealed the existence of a thiamin-diphosphate-independent pyruvate-binding site in the pyruvate dehydrogenase complex. Direct evidence was also obtained with fluorescence techniques for the existence of this binding site and the dissociation constant of pyruvate was found to be 0.38 mM. On the basis of these data we have proposed a random mechanism for the binding of pyruvate and thiamin diphosphate to the complex. Binding of substrates to the enzyme complex caused an increase in the fluorescence of the dansylaziridine-labelled pyruvate dehydrogenase complex, showing that binding of substrates to the complex is accompanied by structural changes.

  2. Evidence for horizontal gene transfer of anaerobic carbon monoxide dehydrogenases

    Directory of Open Access Journals (Sweden)

    Stephen eTechtmann

    2012-04-01

    Full Text Available Carbon monoxide (CO is commonly known as a toxic gas, yet it is used by both aerobic and anaerobic bacteria and many archaea. In this study, we determined the prevalence of anaerobic carbon monoxide dehydrogenases (anaerobic CODHs, or [Ni,Fe]-CODHs in currently available genomic sequence databases. More than 6% (185 genomes out of 2887 bacterial and archaeal genome sequences in the IMG database possess at least one gene encoding [Ni,Fe]-CODH, the key enzyme for anaerobic CO utilization. The phylogenetic study of this extended protein family revealed nine distinct clades of [Ni,Fe]-CODHs. These clades consisted of [Ni,Fe]-CODHs that, while apparently monophyletic within the clades, were encoded by microorganisms of disparate phylogeny, based on 16S rRNA sequences, and widely ranging physiology. Following this discovery, it was therefore of interest to examine the extent and possible routes of horizontal gene transfer (HGT affecting [Ni,Fe]-CODH genes and gene clusters that include [Ni,Fe]-CODHs.The genome sequence of the extreme thermophile Thermosinus carboxydivorans was used as a case study for HGT. The [Ni,Fe]-CODH operon of T. carboxydivorans differs from its whole genome in its G+C content by 8.2 mol%. Here, we apply statistical methods to establish acquisition by T. carboxydivorans of the gene cluster including [Ni,Fe]-CODH via HGT. Analysis of tetranucleotide frequency and codon usage with application of the Kullback-Leibler divergence metric showed that the [Ni,Fe]-CODH-1 operon of T. carboxidyvorans is quite dissimilar to the whole genome. Using the same metrics, the T. carboxydivorans [Ni,Fe]-CODH-1 operon is highly similar to the genome of the phylogenetically distant anaerobic carboxydotroph Carboxydothermus hydrogenoformans. These results allow to assume recent HTG of the gene cluster from a relative of C. hydrogenoformans to T. carboxydivorans or a more ancient transfer from a C. hydrogenoformans ancestor to a T. carboxydivorans

  3. Cloning, characterization, and engineering of fungal L-arabinitol dehydrogenases.

    Science.gov (United States)

    Kim, Byoungjin; Sullivan, Ryan P; Zhao, Huimin

    2010-07-01

    L-Arabinitol 4-dehydrogenase (LAD) catalyzes the conversion of L-arabinitol to L-xylulose with concomitant NAD(+) reduction in fungal L-arabinose catabolism. It is an important enzyme in the development of recombinant organisms that convert L: -arabinose to fuels and chemicals. Here, we report the cloning, characterization, and engineering of four fungal LADs from Penicillium chrysogenum, Pichia guilliermondii, Aspergillus niger, and Trichoderma longibrachiatum, respectively. The LAD from P. guilliermondii was inactive, while the other three LADs were NAD(+)-dependent and showed high catalytic activities, with P. chrysogenum LAD being the most active. T. longibrachiatum LAD was the most thermally stable and showed the maximum activity in the temperature range of 55-65 degrees C with the other LADs showed the maximum activity in the temperature range of 40-50 degrees C. These LADs were active from pH 7 to 11 with an optimal pH of 9.4. Site-directed mutagenesis was used to alter the cofactor specificity of these LADs. In a T. longibrachiatum LAD mutant, the cofactor preference toward NADP(+) was increased by 2.5 x 10(4)-fold, whereas the cofactor preference toward NADP(+) of the P. chrysogenum and A. niger LAD mutants was also drastically improved, albeit at the expense of significantly reduced catalytic efficiencies. The wild-type LADs and their mutants with altered cofactor specificity could be used to investigate the functionality of the fungal L-arabinose pathways in the development of recombinant organisms for efficient microbial L-arabinose utilization.

  4. Identification of Hedgehog pathway responsive glioblastomas by isocitrate dehydrogenase mutation.

    Science.gov (United States)

    Gerardo Valadez, J; Grover, Vandana K; Carter, Melissa D; Calcutt, M Wade; Abiria, Sunday A; Lundberg, Christopher J; Williams, Thomas V; Cooper, Michael K

    2013-01-28

    The Hedgehog (Hh) pathway regulates the growth of a subset of adult gliomas and better definition of Hh-responsive subtypes could enhance the clinical utility of monitoring and targeting this pathway in patients. Somatic mutations of the isocitrate dehydrogenase (IDH) genes occur frequently in WHO grades II and III gliomas and WHO grade IV secondary glioblastomas. Hh pathway activation in WHO grades II and III gliomas suggests that it might also be operational in glioblastomas that developed from lower-grade lesions. To evaluate this possibility and to better define the molecular and histopathological glioma subtypes that are Hh-responsive, IDH genes were sequenced in adult glioma specimens assayed for an operant Hh pathway. The proportions of grades II-IV specimens with IDH mutations correlated with the proportions that expressed elevated levels of the Hh gene target PTCH1. Indices of an operational Hh pathway were measured in all primary cultures and xenografts derived from IDH-mutant glioma specimens, including IDH-mutant glioblastomas. In contrast, the Hh pathway was not operational in glioblastomas that lacked IDH mutation or history of antecedent lower-grade disease. IDH mutation is not required for an operant pathway however, as significant Hh pathway modulation was also measured in grade III gliomas with wild-type IDH sequences. These results indicate that the Hh pathway is operational in grades II and III gliomas and glioblastomas with molecular or histopathological evidence for evolvement from lower-grade gliomas. Lastly, these findings suggest that gliomas sharing this molecularly defined route of progression arise in Hh-responsive cell types.

  5. 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.

  6. 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.

  7. The expression of succinate dehydrogenase in breast phyllodes tumor.

    Science.gov (United States)

    Choi, Junjeong; Kim, Do Hee; Jung, WooHee; Koo, Ja Seung

    2014-10-01

    The purpose of this study is to investigate the expression of succinate dehydrogenase (SDH)A, SDHB, and HIF-1α in phyllodes tumors and the association with clinic-pathologic factors. Using tissue microarray (TMA) for 206 phyllodes tumor cases, we performed immunohistochemical stains for SDHA, SDHB, and HIF-1α and analyzed their expression in regard to clinicopathologic parameters of each case. The cases were comprised of 156 benign, 34 borderline, and 16 malignant phyllodes tumors. The expression of stromal SDHA and epithelial- and stromal- SDHB increased as the tumor progressed from benign to malignant (P⟨0.001). There were five stromal SDHA-negative cases and 31 stromal SDHB-negative cases. SDHB negativity was associated with a lower histologic grade (P=0.054) and lower stromal atypia (P=0.048). Univariate analysis revealed that a shorter disease free survival (DFS) was associated with stromal SDHB high-positivity (P=0.013) and a shorter overall survival (OS) was associated with high-positivity of stromal SDHA and SDHB (P⟨0.001 and P⟨0.001, respectively). The multivariate Cox analysis with the variables stromal cellularity, stromal atypia, stromal mitosis, stromal overgrowth, tumor margin, stromal SDHA expression, and stromal SDHB expression revealed that stromal overgrowth was associated with a shorter DFS (hazard ratio: 24.78, 95% CI: 3.126-196.5, P=0.002) and a shorter OS (hazard ratio: 176.7, 95% CI: 8.466-3691, P=0.001). In conclusion, Tumor grade is positively correlated with SDHA and SDHB expression in the tumor stroma in phyllodes tumors of the breast. This result may be attributed to the increased metabolic demand in high grade tumors.

  8. 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

  9. 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

  10. Aldehyde Dehydrogenase 1A1: Friend or Foe to Female Metabolism?

    Directory of Open Access Journals (Sweden)

    Jennifer M. Petrosino

    2014-03-01

    Full Text Available In this review, we summarize recent advances in understanding vitamin A-dependent regulation of sex-specific differences in metabolic diseases, inflammation, and certain cancers. We focus on the characterization of the aldehyde dehydrogenase-1 family of enzymes (ALDH1A1, ALDH1A2, ALDH1A3 that catalyze conversion of retinaldehyde to retinoic acid. Additionally, we propose a “horizontal transfer of signaling” from estrogen to retinoids through the action of ALDH1A1. Although estrogen does not directly influence expression of Aldh1a1, it has the ability to suppress Aldh1a2 and Aldh1a3, thereby establishing a female-specific mechanism for retinoic acid generation in target tissues. ALDH1A1 regulates adipogenesis, abdominal fat formation, glucose tolerance, and suppression of thermogenesis in adipocytes; in B cells, ALDH1A1 plays a protective role by inducing oncogene suppressors Rara and Pparg. Considering the conflicting responses of Aldh1a1 in a multitude of physiological processes, only tissue-specific regulation of Aldh1a1 can result in therapeutic effects. We have shown through successful implantation of tissue-specific Aldh1a1−/− preadipocytes that thermogenesis can be induced in wild-type adipose tissues to resolve diet-induced visceral obesity in females. We will briefly discuss the emerging role of ALDH1A1 in multiple myeloma, the regulation of reproduction, and immune responses, and conclude by discussing the role of ALDH1A1 in future therapeutic applications.

  11. Binding Mode and Selectivity of Steroids towards Glucose-6-phosphate Dehydrogenase from the Pathogen Trypanosoma cruzi.

    Science.gov (United States)

    Ortiz, Cecilia; Moraca, Francesca; Medeiros, Andrea; Botta, Maurizio; Hamilton, Niall; Comini, Marcelo A

    2016-03-17

    Glucose-6-phosphate dehydrogenase (G6PDH) plays a housekeeping role in cell metabolism by generating reducing power (NADPH) and fueling the production of nucleotide precursors (ribose-5-phosphate). Based on its indispensability for pathogenic parasites from the genus Trypanosoma, G6PDH is considered a drug target candidate. Several steroid-like scaffolds were previously reported to target the activity of G6PDH. Epiandrosterone (EA) is an uncompetitive inhibitor of trypanosomal G6PDH for which its binding site to the enzyme remains unknown. Molecular simulation studies with the structure of Trypanosoma cruzi G6PDH revealed that EA binds in a pocket close to the G6P binding-site and protrudes into the active site blocking the interaction between substrates and hence catalysis. Site directed mutagenesis revealed the important steroid-stabilizing effect of residues (L80, K83 and K84) located on helix α-1 of T. cruzi G6PDH. The higher affinity and potency of 16α-Br EA by T. cruzi G6PDH is explained by the formation of a halogen bond with the hydrogen from the terminal amide of the NADP+-nicotinamide. At variance with the human enzyme, the inclusion of a 21-hydroxypregnane-20-one moiety to a 3β-substituted steroid is detrimental for T. cruzi G6PDH inhibition. The species-specificity of certain steroid derivatives towards the parasite G6PDH and the corresponding biochemically validated binding models disclosed in this work may prove valuable for the development of selective inhibitors against the pathogen's enzyme.

  12. 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.

  13. Biochemical, cellular, and biophysical characterization of a potent inhibitor of mutant isocitrate dehydrogenase IDH1.

    Science.gov (United States)

    Davis, Mindy I; Gross, Stefan; Shen, Min; Straley, Kimberly S; Pragani, Rajan; Lea, Wendy A; Popovici-Muller, Janeta; DeLaBarre, Byron; Artin, Erin; Thorne, Natasha; Auld, Douglas S; Li, Zhuyin; Dang, Lenny; Boxer, Matthew B; Simeonov, Anton

    2014-05-16

    Two mutant forms (R132H and R132C) of isocitrate dehydrogenase 1 (IDH1) have been associated with a number of cancers including glioblastoma and acute myeloid leukemia. These mutations confer a neomorphic activity of 2-hydroxyglutarate (2-HG) production, and 2-HG has previously been implicated as an oncometabolite. Inhibitors of mutant IDH1 can potentially be used to treat these diseases. In this study, we investigated the mechanism of action of a newly discovered inhibitor, ML309, using biochemical, cellular, and biophysical approaches. Substrate binding and product inhibition studies helped to further elucidate the IDH1 R132H catalytic cycle. This rapidly equilibrating inhibitor is active in both biochemical and cellular assays. The (+) isomer is active (IC50 = 68 nm), whereas the (-) isomer is over 400-fold less active (IC50 = 29 μm) for IDH1 R132H inhibition. IDH1 R132C was similarly inhibited by (+)-ML309. WT IDH1 was largely unaffected by (+)-ML309 (IC50 >36 μm). Kinetic analyses combined with microscale thermophoresis and surface plasmon resonance indicate that this reversible inhibitor binds to IDH1 R132H competitively with respect to α-ketoglutarate and uncompetitively with respect to NADPH. A reaction scheme for IDH1 R132H inhibition by ML309 is proposed in which ML309 binds to IDH1 R132H after formation of the IDH1 R132H NADPH complex. ML309 was also able to inhibit 2-HG production in a glioblastoma cell line (IC50 = 250 nm) and had minimal cytotoxicity. In the presence of racemic ML309, 2-HG levels drop rapidly. This drop was sustained until 48 h, at which point the compound was washed out and 2-HG levels recovered.

  14. Expanded Hematopoietic Progenitor Cells Reselected for High Aldehyde Dehydrogenase Activity Demonstrate Islet Regenerative Functions.

    Science.gov (United States)

    Seneviratne, Ayesh K; Bell, Gillian I; Sherman, Stephen E; Cooper, Tyler T; Putman, David M; Hess, David A

    2016-04-01

    Human umbilical cord blood (UCB) hematopoietic progenitor cells (HPC) purified for high aldehyde dehydrogenase activity (ALDH(hi) ) stimulate islet regeneration after transplantation into mice with streptozotocin-induced β cell deletion. However, ALDH(hi) cells represent a rare progenitor subset and widespread use of UCB ALDH(hi) cells to stimulate islet regeneration will require progenitor cell expansion without loss of islet regenerative functions. Here we demonstrate that prospectively purified UCB ALDH(hi) cells expand efficiently under serum-free, xeno-free conditions with minimal growth factor supplementation. Consistent with the concept that ALDH-activity is decreased as progenitor cells differentiate, kinetic analyses over 9 days revealed the frequency of ALDH(hi) cells diminished as culture time progressed such that total ALDH(hi) cell number was maximal (increased 3-fold) at day 6. Subsequently, day 6 expanded cells (bulk cells) were sorted after culture to reselect differentiated progeny with low ALDH-activity (ALDH(lo) subset) from less differentiated progeny with high ALDH-activity (ALDH(hi) subset). The ALDH(hi) subset retained primitive cell surface marker coexpression (32.0% ± 7.0% CD34(+) /CD38(-) cells, 37.0% ± 6.9% CD34(+) /CD133(+) cells), and demonstrated increased hematopoietic colony forming cell function compared with the ALDH(lo) subset. Notably, bulk cells or ALDH(lo) cells did not possess the functional capacity to lower hyperglycemia after transplantation into streptozotocin-treated NOD/SCID mice. However, transplantation of the repurified ALDH(hi) subset significantly reduced hyperglycemia, improved glucose tolerance, and increased islet-associated cell proliferation and capillary formation. Thus, expansion and delivery of reselected UCB cells that retain high ALDH-activity after short-term culture represents an improved strategy for the development of cellular therapies to enhance islet regeneration in situ.

  15. [Catabolism of carnitine: products of carnitine decarboxylase and carnitine dehydrogenase in vivo].

    Science.gov (United States)

    Seim, H; Löster, H; Strack, E

    1980-09-01

    1) Rats and mice were given large oral or subcutaneous doses of (-)-L-, (+)-D- and DL-carnitine (5 mg/g body weight). The carnitine metabolites, beta-methylcholine and acetonyltrimethylammonium, were isolated from the urine by special methods, and determined as their characteristic derivatives (2,4-dinitrophenylhydrazone and butyric ester) by thin-layer chromatography or photometry. 2) beta-Methylcholine, the product of carnitine decarboxylase, was not excreted, even when animals were heavily dosed with both carnitine isomers, with or without starvation. 3) After the administration of (+)-D- and DL-carnitine, both species excreted acetonyltrimethylammonium, which is already known as the spontaneous decarboxylation product of dehydrocarnitine (product of carnitine dehydrogenase) in bacteria. Injection of 0.71 mmol (+)-D-carnitine resulted in the excretion of 5.0 mumol (average) acetonyltrimethylammonium per mouse during the 48 h post injection. Under the same conditions, rats produced up to 40 mumol acetonyltrimethylammonium. The ratio of excreted acetonyltrimethylammonium to injected (+)-D-carnitine depended on the method of administration and the dose. 4) Production of the pharmacologically active (+)-acetyl-L-beta-methylcholine is not to be expected, following high exogenous doses of (-)-L-carnitine or (-)-acetyl-L-carnitine. The chief metabolites are trimethylamine, trimethylamine oxide and gamma-butyrobetaine (this journal 361, 1059), and both the (-)-L-carnitine pool and exogenous (-)-L-carnitine are dehydrogenated or decarboxylated only to a very small extent, if at all. When DL-carnitine is used therapeutically, the formation of acetonyltrimethylammonium must be taken into account.

  16. Puriifcation and characterization of a thermostable glutamate dehydrogenase from a thermophilic microorganism from Deception Island, Antarctica

    Institute of Scientific and Technical Information of China (English)

    Patricio A Flores M; Freddy Boehmwald; Jenny M Blamey A

    2016-01-01

    Glutamate dehydrogenase (GDH) catalyzes the oxidative deamination of glutamate toα-ketoglutarate and ammonium ions. Currently the determination of ammonium and glutamate is carried out using a bovine GDH enzyme, which lacks optimal thermostability for long term storage at room temperature. From samples of Deception Island, Antarctica, we obtained the thermophilic bacteria PID 15 belonging to theBacillus genera with high GDH speciifc activity. This new enzyme exhibited NAD+dependent activity and no activity was observed when NADP+was used. This enzyme shows a speciifc activity of 4.7 U∙mg-1 for the oxidative deamination reaction and 15.4 U∙mg-1 for the reduction ofα-ketoglutarate. This enzyme has an optimum temperature of 65°C and pH of 8.5 for the oxidative deamination. For the reduction ofα-ketoglutarate, the optimum temperature is 60°C, with a pH of 8.0. One of the most important characteristics of this enzyme is its ability to retain more than 60% of its activity when it is incubated for 8 h at 65°C. The enzyme is also able to retain full activity when it is incubated for 48 d at 4°C and over 80% of its activity when it is incubated at 25°C. Characterization of its kinetics suggests that it primarily catalyzes the formation of α-ketoglutarate. This enzyme has an important biological role in the catabolism of glutamate and may have some interesting biotechnological applications based on its thermostable properties.

  17. Pyruvate formate lyase acts as a formate supplier for metabolic processes during anaerobiosis in Staphylococcus aureus.

    Science.gov (United States)

    Leibig, Martina; Liebeke, Manuel; Mader, Diana; Lalk, Michael; Peschel, Andreas; Götz, Friedrich

    2011-02-01

    Previous studies demonstrated an upregulation of pyruvate formate lyase (Pfl) and NAD-dependent formate dehydrogenase (Fdh) in Staphylococcus aureus biofilms. To investigate their physiological role, we constructed fdh and pfl deletion mutants (Δfdh and Δpfl). Although formate dehydrogenase activity in the fdh mutant was lost, it showed little phenotypic alterations under oxygen-limited conditions. In contrast, the pfl mutant displayed pleiotropic effects and revealed the importance of formate production for anabolic metabolism. In the pfl mutant, no formate was produced, glucose consumption was delayed, and ethanol production was decreased, whereas acetate and lactate production were unaffected. All metabolic alterations could be restored by addition of formate or complementation of the Δpfl mutant. In compensation reactions, serine and threonine were consumed better by the Δpfl mutant than by the wild type, suggesting that their catabolism contributes to the refilling of formyl-tetrahydrofolate, which acts as a donor of formyl groups in, e.g., purine and protein biosynthesis. This notion was supported by reduced production of formylated peptides by the Δpfl mutant compared to that of the parental strain, as demonstrated by weaker formyl-peptide receptor 1 (FPR1)-mediated activation of leukocytes with the mutant. FPR1 stimulation could also be restored either by addition of formate or by complementation of the mutation. Furthermore, arginine consumption and arc operon transcription were increased in the Δpfl mutant. Unlike what occurred with the investigated anaerobic conditions, a biofilm is distinguished by nutrient, oxygen, and pH gradients, and we thus assume that Pfl plays a significant role in the anaerobic layer of a biofilm. Fdh might be critical in (micro)aerobic layers, as formate oxidation is correlated with the generation of NADH/H(+), whose regeneration requires respiration.

  18. 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.

  19. 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.

  20. Production of superoxide/hydrogen peroxide by the mitochondrial 2-oxoadipate dehydrogenase complex.

    Science.gov (United States)

    Goncalves, Renata L S; Bunik, Victoria I; Brand, Martin D

    2016-02-01

    In humans, mutations in dehydrogenase E1 and transketolase domain containing 1 (DHTKD1) are associated with neurological abnormalities and accumulation of 2-oxoadipate, 2-aminoadipate, and reactive oxygen species. The protein encoded by DHTKD1 has sequence and structural similarities to 2-oxoglutarate dehydrogenase, and the 2-oxoglutarate dehydrogenase complex can produce superoxide/H2O2 at high rates. The DHTKD1 enzyme is hypothesized to catalyze the oxidative decarboxylation of 2-oxoadipate, a shared intermediate of the degradative pathways for tryptophan, lysine and hydroxylysine. Here, we show that rat skeletal muscle mitochondria can produce superoxide/H2O2 at high rates when given 2-oxoadipate. We identify the putative mitochondrial 2-oxoadipate dehydrogenase complex as one of the sources and characterize the conditions that favor its superoxide/H2O2 production. Rates increased at higher NAD(P)H/NAD(P)(+) ratios and were higher at each NAD(P)H/NAD(P)(+) ratio when 2-oxoadipate was present, showing that superoxide/H2O2 was produced during the forward reaction from 2-oxoadipate, but not in the reverse reaction from NADH in the absence of 2-oxoadipate. The maximum capacity of the 2-oxoadipate dehydrogenase complex for production of superoxide/H2O2 is comparable to that of site IF of complex I, and seven, four and almost two-fold lower than the capacities of the 2-oxoglutarate, pyruvate and branched-chain 2-oxoacid dehydrogenase complexes, respectively. Regulation by ADP and ATP of H2O2 production driven by 2-oxoadipate was very different from that driven by 2-oxoglutarate, suggesting that site AF of the 2-oxoadipate dehydrogenase complex is a new source of superoxide/H2O2 associated with the NADH isopotential pool in mitochondria.

  1. Simultaneous immobilization of dehydrogenases on polyvinylidene difluoride resin after separation by non-denaturing two-dimensional electrophoresis

    Energy Technology Data Exchange (ETDEWEB)

    Shimazaki, Youji [Graduate School of Science and Engineering (Science Section) and Venture Business Laboratory, Ehime University, Bunkyo-cho 2-5, Matsuyama City 790-8577 (Japan)], E-mail: yoji@dpc.ehime-u.ac.jp; Kadota, Mariko [Faculty of Science, Ehime University, Matsuyama (Japan)

    2008-06-16

    We detected mouse liver malate, sorbitol and aldehyde dehydrogenases by negative staining, analysis of malate and sorbitol dehydrogenase activities using each substrate, and electron transfers including nicotinamide adenine dinucleotide (NAD) and nitroblue tetrazolium in non-denaturing two-dimensional electrophoresis (2-DE) gel. Dehydrogenases were also identified by electrospray ionization tandem mass spectrometry (ESI-MS/MS) after 2-DE separation and protein detection by negative staining. Spots of dehydrogenases separated by 2-DE were excised, and simultaneously transferred and immobilized on polyvinylidene difuoride (PVDF) resin by electrophoresis. The dehydrogenase activities remained intact after immobilization. In conclusion, resin-immobilized dehydrogenases can be simultaneously obtained after separation by non-denaturing 2-DE, detection by negative staining and transferring to resins.

  2. 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.

  3. 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.

  4. 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.

  5. Plasma Lactate Dehydrogenase Levels Predict Mortality in Acute Aortic Syndromes

    Science.gov (United States)

    Morello, Fulvio; Ravetti, Anna; Nazerian, Peiman; Liedl, Giovanni; Veglio, Maria Grazia; Battista, Stefania; Vanni, Simone; Pivetta, Emanuele; Montrucchio, Giuseppe; Mengozzi, Giulio; Rinaldi, Mauro; Moiraghi, Corrado; Lupia, Enrico

    2016-01-01

    Abstract In acute aortic syndromes (AAS), organ malperfusion represents a key event impacting both on diagnosis and outcome. Increased levels of plasma lactate dehydrogenase (LDH), a biomarker of malperfusion, have been reported in AAS, but the performance of LDH for the diagnosis of AAS and the relation of LDH with outcome in AAS have not been evaluated so far. This was a bi-centric prospective diagnostic accuracy study and a cohort outcome study. From 2008 to 2014, patients from 2 Emergency Departments suspected of having AAS underwent LDH assay at presentation. A final diagnosis was obtained by aortic imaging. Patients diagnosed with AAS were followed-up for in-hospital mortality. One thousand five hundred seventy-eight consecutive patients were clinically eligible, and 999 patients were included in the study. The final diagnosis was AAS in 201 (20.1%) patients. Median LDH was 424 U/L (interquartile range [IQR] 367–557) in patients with AAS and 383 U/L (IQR 331–460) in patients with alternative diagnoses (P < 0.001). Using a cutoff of 450 U/L, the sensitivity of LDH for AAS was 44% (95% confidence interval [CI] 37–51) and the specificity was 73% (95% CI 69–76). Overall in-hospital mortality for AAS was 23.8%. Mortality was 32.6% in patients with LDH ≥ 450 U/L and 16.8% in patients with LDH < 450 U/L (P = 0.006). Following stratification according to LDH quartiles, in-hospital mortality was 12% in the first (lowest) quartile, 18.4% in the second quartile, 23.5% in the third quartile, and 38% in the fourth (highest) quartile (P = 0.01). LDH ≥ 450 U/L was further identified as an independent predictor of death in AAS both in univariate and in stepwise logistic regression analyses (odds ratio 2.28, 95% CI 1.11–4.66; P = 0.025), in addition to well-established risk markers such as advanced age and hypotension. Subgroup analysis showed excess mortality in association with LDH ≥ 450 U/L in elderly, hemodynamically stable

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

    Directory of Open Access Journals (Sweden)

    Ma YM

    2016-04-01

    Full Text Available Yu-mei Ma,1 Shan Zhao2 1Department of Pathology, 2Department of Cancer Second Division, The Second Hospital of Hebei Medical University, Shijiazhuang City, People’s Republic of China Abstract: Aldehyde dehydrogenase 1 (ALDH1 activity has been used as a functional stem cell marker to isolate cancer stem cells in different cancer types, including ovarian cancer. However, which ALDH1’s isoenzymes are contributing to ALDH1 activity in ovarian cancer remains elusive. In addition, the prognostic value of an individual ALDH1 isoenzyme in ovarian cancer is not clear. Thus, we accessed the prognostic value of ALDH1 isoenzymes in ovarian cancer patients through the “Kaplan–Meier plotter” online database, which can be used to determine the effect of the genes on ovarian cancer prognosis. We found that high mRNA expression of five ALDH1 isoenzymes, such as ALDH1A1, ALDH1A2, ALDH1A3, ALDH1B1, and ALDH1L1, was not correlated with overall survival (OS for all 1,306 ovarian cancer patients. In addition, all five of the ALDH1 isoenzymes’ high mRNA expression was found to be uncorrelated with OS in serous cancer or endometrioid cancer patients. However, ALDH1A3’s high mRNA expression is associated with worse OS in grade II ovarian cancer patients, hazard ratio (HR 1.53 (1.14–2.07, P=0.005. ALDH1A2’s high mRNA expression is significantly associated with worse OS in TP53 wild-type ovarian cancer patients, HR 2.86 (1.56–5.08, P=0.00036. In addition, ALDH1A3’s high mRNA expression is significantly associated with better OS in TP53 wild-type ovarian cancer patients, HR 0.56 (0.32–1.00, P=0.04. Our results indicate that although ALDH1 isoenzyme mRNA might not be a prognostic marker for overall ovarian cancer patients, some isoenzymes, such as ALDH1A2 and ALDH1A3, might be a good prognostic marker for some types of ovarian cancer patients. Keywords: ALDH1, cancer stem cell, prognosis, KM plotter, hazard ratio

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

    Science.gov (United States)

    Tolstrup, Janne Schurmann; Nordestgaard, Børge Grønne; Rasmussen, Søren; Tybjaerg-Hansen, Anne; Grønbaek, Morten

    2008-06-01

    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 with the ADH1B.1/1 genotype compared to men with the ADH1B.1/2 genotype. Furthermore, 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.1/1 genotype. Results for ADH1B and ADH1C genotypes among men and women were similar. Finally, because slow ADH1B alcohol degradation is found in more than 90% of the white population compared to less than 10% of East Asians, the population attributable risk of heavy drinking and alcoholism by ADH1B.1/1 genotype was 67 and 62% among the white population compared with 9 and 24% among the East Asian population.

  8. 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.

  9. 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

  10. 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

  11. 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.

  12. 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.

  13. Isolation and biochemical characterization of a glucose dehydrogenase from a hay infusion metagenome.

    Science.gov (United States)

    Basner, Alexander; Antranikian, Garabed

    2014-01-01

    Glucose hydrolyzing enzymes are essential to determine blood glucose level. A high-throughput screening approach was established to identify NAD(P)-dependent glucose dehydrogenases for the application in test stripes and the respective blood glucose meters. In the current report a glucose hydrolyzing enzyme, derived from a metagenomic library by expressing recombinant DNA fragments isolated from hay infusion, was characterized. The recombinant clone showing activity on glucose as substrate exhibited an open reading frame of 987 bp encoding for a peptide of 328 amino acids. The isolated enzyme showed typical sequence motifs of short-chain-dehydrogenases using NAD(P) as a co-factor and had a sequence similarity between 33 and 35% to characterized glucose dehydrogenases from different Bacillus species. The identified glucose dehydrogenase gene was expressed in E. coli, purified and subsequently characterized. The enzyme, belonging to the superfamily of short-chain dehydrogenases, shows a broad substrate range with a high affinity to glucose, xylose and glucose-6-phosphate. Due to its ability to be strongly associated with its cofactor NAD(P), the enzyme is able to directly transfer electrons from glucose oxidation to external electron acceptors by regenerating the cofactor while being still associated to the protein.

  14. Isolation and biochemical characterization of a glucose dehydrogenase from a hay infusion metagenome.

    Directory of Open Access Journals (Sweden)

    Alexander Basner

    Full Text Available Glucose hydrolyzing enzymes are essential to determine blood glucose level. A high-throughput screening approach was established to identify NAD(P-dependent glucose dehydrogenases for the application in test stripes and the respective blood glucose meters. In the current report a glucose hydrolyzing enzyme, derived from a metagenomic library by expressing recombinant DNA fragments isolated from hay infusion, was characterized. The recombinant clone showing activity on glucose as substrate exhibited an open reading frame of 987 bp encoding for a peptide of 328 amino acids. The isolated enzyme showed typical sequence motifs of short-chain-dehydrogenases using NAD(P as a co-factor and had a sequence similarity between 33 and 35% to characterized glucose dehydrogenases from different Bacillus species. The identified glucose dehydrogenase gene was expressed in E. coli, purified and subsequently characterized. The enzyme, belonging to the superfamily of short-chain dehydrogenases, shows a broad substrate range with a high affinity to glucose, xylose and glucose-6-phosphate. Due to its ability to be strongly associated with its cofactor NAD(P, the enzyme is able to directly transfer electrons from glucose oxidation to external electron acceptors by regenerating the cofactor while being still associated to the protein.

  15. Isolation and characterization of an inducible NAD-dependent butyraldehyde dehydrogenase from clostridium acetobutylicum

    Energy Technology Data Exchange (ETDEWEB)

    Schreiber, W.; Duerre, P. [Universitaet Ulm (Germany)

    1996-12-31

    A NAD-dependent butyraldehyde dehydrogenase (BAD) has been purified from C. acetobutylicum DSM 792 and DSM 173 1. This key enzyme of butanol production, catalyzing the conversion of butyryl-CoA to butyraldehyde, was induced shortly before the onset of butanol production and proved to be oxygen-sensitive. A one step purification procedure on reactive green 19 allowed to purify the enzyme to homogeneity. The purified protein was found to be extremely unstable and could only partially be stabilized by addition of mercaptoethanol and storage below -20{degrees}C. The enzyme subunit had a molecular mass of 39.5 kDa. In the reverse reaction (butyryl-CoA-forming) the apparent pH optimum was 9.75 and Vmax was significantly higher with butyraldehyde and propionaldehyde than with acetaldehyde. BAD could also use NADP+, but NAD+ was the preferred coenzyme for the reverse reaction. The N-terminal amino acid sequence of the C. acetobutylicurn DSM 792 protein showed high homology to glyceraldehyde-3-phosphate dehydrogenases (GAP), especially to the protein of C. pasteurianum. Genomic libraries of C. acetobutylicum DSM 792 were screened by hybridization using PCR-generated heterologous probes encoding the gap gene of C. pasteurianum. Sequence analysis of the positive clones revealed high homology, but no identity to the N-terminal amino acid sequence of the butyraldehyde dehydrogenase. Thus, BAD from C. acetobutylicum is distinctly different from other reported aldehyde dehydrogenases with butyraldehyde dehydrogenase activity.

  16. Analysis of isocitrate dehydrogenase-1/2 gene mutations in gliomas

    Institute of Scientific and Technical Information of China (English)

    YU Lei; QI Song-tao; LI Zhi-yong

    2010-01-01

    Objective To highlight recent researches which may show promise for histomolecular classification and new treatments for gliomas.Data sources All articles cited in this review were mainly searched from PubMed, which were published in English from 1996 to 2010.Study selection Original articles and critical reviews selected were relevant to the isocitrate dehydrogenase-1/2 mutation in gliomas and other tumors.Results Extraordinary high rates of somatic mutations in isocitrate dehydrogenase-1/2 occur in the majority of World Health Organization grade Ⅱ and grade Ⅲ gliomas as well as grade Ⅳ secondary glioblastomas. Isocitrate dehydrogenase-1/2 mutations are associated with younger age at diagnosis and a better prognosis in patients with mutated tumors. The functional role of isocitrate dehydrogenase-1/2 mutations in the pathogenesis of gliomas is still unclear.Conclusion Isocitrate dehydrogenase-1/2 mutations define a specific subtype of gliomas and may have great significance in the diagnosis, prognosis, and treatment of patients with these tumors.

  17. 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.

  18. Selective n-butanol production by Clostridium sp. MTButOH1365 during continuous synthesis gas fermentation due to expression of synthetic thiolase, 3-hydroxy butyryl-CoA dehydrogenase, crotonase, butyryl-CoA dehydrogenase, butyraldehyde dehydrogenase, and NAD-dependent butanol dehydrogenase.

    Science.gov (United States)

    Berzin, Vel; Tyurin, Michael; Kiriukhin, Michael

    2013-02-01

    Acetogen Clostridum sp. MT1962 produced 287 mM acetate (p < 0.005) and 293 mM ethanol (p < 0.005) fermenting synthesis gas blend 60% CO and 40% H₂ in single-stage continuous fermentation. This strain was metabolically engineered to the biocatalyst Clostridium sp. MTButOH1365. The engineered biocatalyst lost production of ethanol and acetate while initiated the production of 297 mM of n-butanol (p < 0.005). The metabolic engineering comprised Cre-lox66/lox71-based elimination of phosphotransacetylase and acetaldehyde dehydrogenase along with integration to chromosome synthetic thiolase, 3-hydroxy butyryl-CoA dehydrogenase, crotonase, butyryl-CoA dehydrogenase, butyraldehyde dehydrogenase, and NAD-dependent butanol dehydrogenase. This is the first report on elimination of acetate and ethanol production genes and expression of synthetic gene cluster encoding n-butanol biosynthesis pathway in acetogen biocatalyst for selective fuel n-butanol production with no antibiotic support for the introduced genes.

  19. Construction of Mutant Glucose Oxidases with Increased Dye-Mediated Dehydrogenase Activity

    Directory of Open Access Journals (Sweden)

    Koji Sode

    2012-11-01

    Full Text Available Mutagenesis studies on glucose oxidases (GOxs were conducted to construct GOxs with reduced oxidase activity and increased dehydrogenase activity. We focused on two representative GOxs, of which crystal structures have already been reported—Penicillium amagasakiense GOx (PDB ID; 1gpe and Aspergillus niger GOx (PDB ID; 1cf3. We constructed oxygen-interacting structural models for GOxs, and predicted the residues responsible for oxidative half reaction with oxygen on the basis of the crystal structure of cholesterol oxidase as well as on the fact that both enzymes are members of the glucose/methanol/choline (GMC oxidoreductase family. Rational amino acid substitution resulted in the construction of an engineered GOx with drastically decreased oxidase activity and increased dehydrogenase activity, which was higher than that of the wild-type enzyme. As a result, the dehydrogenase/oxidase ratio of the engineered enzyme was more than 11-fold greater than that of the wild-type enzyme. These results indicate that alteration of the dehydrogenase/oxidase activity ratio of GOxs is possible by introducing a mutation into the putative functional residues responsible for oxidative half reaction with oxygen of these enzymes, resulting in a further increased dehydrogenase activity. This is the first study reporting the alteration of GOx electron acceptor preference from oxygen to an artificial electron acceptor.

  20. 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.

  1. Oxidative DNA damage induced by benz[a]anthracene dihydrodiols in the presence of dihydrodiol dehydrogenase.

    Science.gov (United States)

    Seike, Kazuharu; Murata, Mariko; Hirakawa, Kazutaka; Deyashiki, Yoshihiro; Kawanishi, Shosuke

    2004-11-01

    Tobacco smoke and polluted air are risk factors for lung cancer and contain many kinds of polycyclic aromatic hydrocarbons (PAHs) including benzo[a]pyrene (B[a]P) and benz[a]anthracene (BA). BA, as well as B[a]P, is assessed as probably carcinogenic to humans (IARC group 2A). BA is metabolized to several dihydrodiols. Dihydrodiol dehydrogenase (DD), a member of the aldo-keto reductase superfamily, catalyzes NAD(P)+-linked oxidation of dihydrodiols of aromatic hydrocarbons to corresponding catechols. To clarify the role of DD on PAH carcinogenesis, we examined oxidative DNA damage induced by trans-dihydrodiols of BA and B[a]P treated with DD using 32P-5'-end-labeled DNA fragments obtained from the human p53 tumor suppressor gene. In addition, we investigated the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), an indicator of oxidative DNA damage, in calf thymus DNA by using HPLC with an electrochemical detector. DD-catalyzed BA-1,2-dihydrodiol caused Cu(II)-mediated DNA damage including 8-oxodG formation in the presence of NAD+. BA-1,2-dihydrodiol induced a Fpg sensitive and piperidine labile G lesion at the 5'-ACG-3' sequence complementary to codon 273 of the human p53 tumor suppressor gene, which is known as a hotspot. DNA damage was inhibited by catalase and bathocuproine, suggesting the involvement of H2O2 and Cu(I). The observation of NADH production by UV-visible spectroscopy suggested that DD catalyzed BA-1,2-dihydrodiol most efficiently to the corresponding catechol among the PAH-dihydrodiols tested. A time-of-flight mass spectroscopic study showed that the catechol form of BA-1,2-dihydrodiol formed after DD treatment. In conclusion, BA-1,2-dihydrodiol can induce DNA damage more efficiently than B[a]P-7,8-dihydrodiol and other BA-dihydrodiols in the presence of DD. The reaction mechanism on oxidative DNA damage may be explained by theoretical calculations with an enthalpy change of dihydrodiols and oxidation potential of their catechol forms. DD

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

    Energy Technology Data Exchange (ETDEWEB)

    Doherty, R.E.; Haywood-Small, S.L. [Biomedical Research Centre, Sheffield Hallam University, Sheffield S1 1WB (United Kingdom); Sisley, K. [Department of Oncology, Academic Unit of Ophthalmology and Orthopties, University of Sheffield, Sheffield S10 2RX (United Kingdom); Cross, N.A., E-mail: n.cross@shu.ac.uk [Biomedical Research Centre, Sheffield Hallam University, Sheffield S1 1WB (United Kingdom)

    2011-11-04

    Highlights: Black-Right-Pointing-Pointer Isolated ALDH{sup Hi} PC3 cells preferentially form primitive holoclone-type colonies. Black-Right-Pointing-Pointer Primitive holoclone colonies are predominantly ALDH{sup Lo} but contain rare ALDH{sup Hi} cells. Black-Right-Pointing-Pointer Holoclone-forming cells are not restricted to the ALDH{sup Hi} population. Black-Right-Pointing-Pointer ALDH phenotypic plasticity occurs in PC3 cells (ALDH{sup Lo} to ALDH{sup Hi} and vice versa). Black-Right-Pointing-Pointer ALDH{sup Hi} 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 ALDH{sup Lo} cells in experimental models. However it is unclear whether all CSC-like cells are within the ALDH{sup Hi} population, or whether all ALDH{sup Hi} 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 ALDH{sup Hi} cells enriches for the most primitive holoclone population, however holoclone formation is not restricted to ALDH{sup Hi} cells. In addition, we show that ALDH activity undergoes phenotypic plasticity, since the ALDH{sup Lo} population can develop ALDH{sup Hi} populations comparable to parental cells within 2 weeks in culture. Furthermore, we show that the majority of ALDH{sup Hi} cells are found within the least primitive paraclone population, which is circumvented by culturing PC3 cells as spheroids in

  3. Integration of Inhibition Kinetics and Molecular Dynamics Simulations: A Urea-Mediated Folding Study on Acetaldehyde Dehydrogenase 1.

    Science.gov (United States)

    Xu, Yingying; Lee, Jinhyuk; Lü, Zhi-Rong; Mu, Hang; Zhang, Qian; Park, Yong-Doo

    2016-07-01

    Understanding the mechanism of acetaldehyde dehydrogenase 1 (ALDH1) folding is important because this enzyme is directly involved in several types of cancers and other diseases. We investigated the urea-mediated unfolding of ALDH1 by integrating kinetic inhibition studies with computational molecular dynamics (MD) simulations. Conformational changes in the enzyme structure were also analyzed using intrinsic and 1-anilinonaphthalene-8-sulfonate (ANS)-binding fluorescence measurements. Kinetic studies revealed that the direct binding of urea to ALDH1 induces inactivation of ALDH1 in a manner of mixed-type inhibition. Tertiary structural changes associated with regional hydrophobic exposure of the active site were observed. The urea binding regions on ALDH1 were predicted by docking simulations and were partly shared with active site residues of ALDH1 and with interface residues of the oligomerization domain for tetramer formation. The docking results suggest that urea prevents formation of the ALDH1 normal shape for the tetramer state as well as entrance of the substrate into the active site. Our study provides insight into the structural changes that accompany urea-mediated unfolding of ALDH1 and the catalytic role associated with conformational changes.

  4. 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.

  5. 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.

  6. 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 cosmological simulation, Illustris, follow a tight relation between star formation rate and stellar mass. This relation agrees well with the observed relation at a redshift of z = 0 and z = 4, but at intermediate redshifts of z ' 2 the normalisation is lower than in real observations. This is highlighted...

  7. 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%.

  8. Crystallization and initial X-ray diffraction analysis of human pyruvate dehydrogenase

    Science.gov (United States)

    Ciszak, E.; Korotchkina, L. G.; Hong, Y. S.; Joachimiak, A.; Patel, M. S.

    2001-01-01

    Human pyruvate dehydrogenase (E1) is a component enzyme of the pyruvate dehydrogenase complex. The enzyme catalyzes the irreversible decarboxylation of pyruvic acid and the rate-limiting reductive acetylation of the lipoyl moiety linked to the dihydrolipoamide acetyltransferase component of the pyruvate dehydrogenase complex. E1 is an alpha(2)beta(2) tetramer ( approximately 154 kDa). Crystals of this recombinant enzyme have been grown in polyethylene glycol 3350 using a vapor-diffusion method at 295 K. The crystals are characterized as orthorhombic, space group P2(1)2(1)2(1), with unit-cell parameters a = 64.2, b = 126.9, c = 190.2 A. Crystals diffracted to a minimum d spacing of 2.5 A. The asymmetric unit contains one alpha(2)beta(2) tetrameric E1 assembly; self-rotation function analysis showed a pseudo-twofold symmetry relating the two alphabeta dimers.

  9. 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.

  10. 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......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......), tetradecadienoic acid, 14:2(n-6), and hexadecadienoic acid, 16:2(n-6). Palmitoyl-CoA and behenoyl-CoA dehydrogenase in fibroblasts were deficient. Muscle VLCAD activity was very low. DNA analysis revealed compound heterozygosity for two missense mutations in the VLCAD gene. The relatively mild clinical course may...

  11. 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.

  12. Relayed 13C magnetization transfer: Detection of malate dehydrogenase reaction in vivo

    Science.gov (United States)

    Yang, Jehoon; Shen, Jun

    2007-02-01

    Malate dehydrogenase catalyzes rapid interconversion between dilute metabolites oxaloacetate and malate. Both oxaloacetate and malate are below the detection threshold of in vivo MRS. Oxaloacetate is also in rapid exchange with aspartate catalyzed by aspartate aminotransferase, the latter metabolite is observable in vivo using 13C MRS. We hypothesized that the rapid turnover of oxaloacetate can effectively relay perturbation of magnetization between malate and aspartate. Here, we report indirect observation of the malate dehydrogenase reaction by saturating malate C2 resonance at 71.2 ppm and detecting a reduced aspartate C2 signal at 53.2 ppm due to relayed magnetization transfer via oxaloacetate C2 at 201.3 ppm. Using this strategy the rate of the cerebral malate dehydrogenase reaction was determined to be 9 ± 2 μmol/g wet weight/min (means ± SD, n = 5) at 11.7 Tesla in anesthetized adult rats infused with [1,6- 13C 2]glucose.

  13. 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.

  14. Structural Insights into l-Tryptophan Dehydrogenase from a Photoautotrophic Cyanobacterium, Nostoc punctiforme.

    Science.gov (United States)

    Wakamatsu, Taisuke; Sakuraba, Haruhiko; Kitamura, Megumi; Hakumai, Yuichi; Fukui, Kenji; Ohnishi, Kouhei; Ashiuchi, Makoto; Ohshima, Toshihisa

    2017-01-15

    l-Tryptophan dehydrogenase from Nostoc punctiforme NIES-2108 (NpTrpDH), despite exhibiting high amino acid sequence identity (>30%)/homology (>50%) with NAD(P)(+)-dependent l-Glu/l-Leu/l-Phe/l-Val dehydrogenases, exclusively catalyzes reversible oxidative deamination of l-Trp to 3-indolepyruvate in the presence of NAD(+) Here, we determined the crystal structure of the apo form of NpTrpDH. The structure of the NpTrpDH monomer, which exhibited high similarity to that of l-Glu/l-Leu/l-Phe dehydrogenases, consisted of a substrate-binding domain (domain I, residues 3 to 133 and 328 to 343) and an NAD(+)/NADH-binding domain (domain II, residues 142 to 327) separated by a deep cleft. The apo-NpTrpDH existed in an open conformation, where domains I and II were apart from each other. The subunits dimerized themselves mainly through interactions between amino acid residues around the β-1 strand of each subunit, as was observed in the case of l-Phe dehydrogenase. The binding site for the substrate l-Trp was predicted by a molecular docking simulation and validated by site-directed mutagenesis. Several hydrophobic residues, which were located in the active site of NpTrpDH and possibly interacted with the side chain of the substrate l-Trp, were arranged similarly to that found in l-Leu/l-Phe dehydrogenases but fairly different from that of an l-Glu dehydrogenase. Our crystal structure revealed that Met-40, Ala-69, Ile-74, Ile-110, Leu-288, Ile-289, and Tyr-292 formed a hydrophobic cluster around the active site. The results of the site-directed mutagenesis experiments suggested that the hydrophobic cluster plays critical roles in protein folding, l-Trp recognition, and catalysis. Our results provide critical information for further characterization and engineering of this enzyme.

  15. Homology modelling and docking analysis of L-lactate dehydrogenase from Streptococcus thermopilus

    Directory of Open Access Journals (Sweden)

    Vukić Vladimir R.

    2016-01-01

    Full Text Available The aim of this research was to create a three-dimensional model of L-lactate dehydrogenase from the main yoghurt starter culture - Streptococcus thermopilus, to analyse its structural features and investigate substrate binding in the active site. NCBI BlastP was used against the Protein Data Bank database in order to identify the template for construction of homology models. Multiple sequence alignment was performed using the program MUSCULE within the UGENE 1.11.3 program. Homology models were constructed using the program Modeller v. 9.17. The obtained 3D model was verified by Ramachandran plots. Molecular docking simulations were performed using the program Surflex-Dock. The highest sequence similarity was observed with L-lactate dehydrogenase from Lactobacillus casei subsp. casei, with 69% identity. Therefore, its structure (PDB ID: 2ZQY:A was selected as a modelling template for homology modelling. Active residues are by sequence similarity predicted: S. thermophilus - HIS181 and S. aureus - HIS179. Binding energy of pyruvate to L-lactate dehydrogenase of S. thermopilus was - 7.874 kcal/mol. Pyruvate in L-lactate dehydrogenase of S. thermopilus makes H bonds with catalytic HIS181 (1.9 Å, as well as with THR235 (3.6 Å. Although our results indicate similar position of substrates between L-lactate dehydrogenase of S. thermopilus and S. aureus, differences in substrate distances and binding energy values could influence the reaction rate. Based on these results, the L-lactate dehydrogenase model proposed here could be used as a guide for further research, such as transition states of the reaction through molecular dynamics. [Projekat Ministarstva nauke Republike Srbije, br. III 46009

  16. Production of superoxide/H2O2 by dihydroorotate dehydrogenase in rat skeletal muscle mitochondria.

    Science.gov (United States)

    Hey-Mogensen, Martin; Goncalves, Renata L S; Orr, Adam L; Brand, Martin D

    2014-07-01

    Dehydrogenases that use ubiquinone as an electron acceptor, including complex I of the respiratory chain, complex II, and glycerol-3-phosphate dehydrogenase, are known to be direct generators of superoxide and/or H2O2. Dihydroorotate dehydrogenase oxidizes dihydroorotate to orotate and reduces ubiquinone to ubiquinol during pyrimidine metabolism, but it is unclear whether it produces superoxide and/or H2O2 directly or does so only indirectly from other sites in the electron transport chain. Using mitochondria isolated from rat skeletal muscle we establish that dihydroorotate oxidation leads to superoxide/H2O2 production at a fairly high rate of about 300pmol H2O2·min(-1)·mg protein(-1) when oxidation of ubiquinol is prevented and complex II is uninhibited. This H2O2 production is abolished by brequinar or leflunomide, known inhibitors of dihydroorotate dehydrogenase. Eighty percent of this rate is indirect, originating from site IIF of complex II, because it can be prevented by malonate or atpenin A5, inhibitors of complex II. In the presence of inhibitors of all known sites of superoxide/H2O2 production (rotenone to inhibit sites in complex I (site IQ and, indirectly, site IF), myxothiazol to inhibit site IIIQo in complex III, and malonate plus atpenin A5 to inhibit site IIF in complex II), dihydroorotate dehydrogenase generates superoxide/H2O2, at a small but significant rate (23pmol H2O2·min(-1)·mg protein(-1)), from the ubiquinone-binding site. We conclude that dihydroorotate dehydrogenase can generate superoxide and/or H2O2 directly at low rates and is also capable of indirect production at higher rates from other sites through its ability to reduce the ubiquinone pool.

  17. 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...

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

    Directory of Open Access Journals (Sweden)

    Peng Giia-Sheun

    2009-01-01

    Full Text Available 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; these polymorphisms have been shown to be the important genetic determinants in ethanol metabolism and alcoholism. Here, we briefly review recent advances in genomic studies of human ADH/ALDH families and alcoholism, with an emphasis on the pharmacogenetic consequences of venous blood acetaldehyde in the different ALDH2 genotypes following the intake of various doses of ethanol. This paper illustrates a paradigmatic example of phenotypic verifications in a protective disease gene for substance abuse.

  19. 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.

  20. [Isoformes of Malate Dehydrogenase from Rhodovulum Steppense A-20s Grown Chemotrophically under Aerobic Condtions].

    Science.gov (United States)

    Eprintsev, A T; Falaleeva, M I; Lyashchenko, M S; Gataullinaa, M O; Kompantseva, E I

    2016-01-01

    Three malate dehydrogenase isoforms (65-, 60-, and 71-fold purifications) with specific activities of 4.23, 3.88, and 4.56 U/mg protein were obtained in an electrophoretically homogenous state from Rhodovulum steppense bacteria strain A-20s chemotropically grown under aerobic conditions. The physicochemical and kinetic properties of malate dehydrogenase isoforms were determined. The molecular weight and the Michaelis constants were determined; the effect of hydrogen ions on the forward and reverse MDH reaction was studied. The results of the study demonstrated that the enzyme consists of subunits; the molecular weight of subunits was determined by SDS-PAGE.

  1. [Physicochemical, catalytic, and regulatory properties of malate dehydrogenase from Rhodovulum steppense bacteria, strain A-20s].

    Science.gov (United States)

    Eprintsev, A T; Falaleeva, M I; Parfenova, I V; Liashchenko, M S; Kompantseva, E I; Tret'iakova, A Iu

    2014-01-01

    The physicochemical, regulatory, and kinetic properties of malate dehydrogenase (EC 1.1.1.37) from haloalkaliphilic purple nonsulfur Rhodovulum steppense bacteria, strain A-20s, were studied. The malate dehydrogenase (MDH) preparation with a specific activity of 0.775 ± 0.113 U/mg protein was obtained in an electrophoretically homogeneous state using multistep purification. Using homogenous preparations, the molecular weight and the Michaelis constant of the enzyme were determined; the effects of metal ions, the temperature effect, and the thermal stability of the MDH were studied. The dimer structure of the enzyme was demonstrated by DS-Na-electrophoresis.

  2. NAD(+)-linked alcohol dehydrogenase 1 regulates methylglyoxal concentration in Candida albicans.

    Science.gov (United States)

    Kwak, Min-Kyu; Ku, MyungHee; Kang, Sa-Ouk

    2014-04-02

    We purified a fraction that showed NAD(+)-linked methylglyoxal dehydrogenase activity, directly catalyzing methylglyoxal oxidation to pyruvate, which was significantly increased in glutathione-depleted Candida albicans. It also showed NADH-linked methylglyoxal-reducing activity. The fraction was identified as a NAD(+)-linked alcohol dehydrogenase (ADH1) through mass spectrometric analyses. In ADH1-disruptants of both the wild type and glutathione-depleted cells, the intracellular methylglyoxal concentration increased significantly; defects in growth, differentiation, and virulence were observed; and G2-phase arrest was induced.

  3. 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....

  4. 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

  5. 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.

  6. The diagnostic value of alcohol dehydrogenase (ADH) isoenzymes and aldehyde dehydrogenase (ALDH) measurement in the sera of patients with brain tumor

    Science.gov (United States)

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

    2017-01-01

    Introduction Alcohol dehydrogenase (ADH) isoenzymes and aldehyde dehydrogenase (ALDH) exist in the brain. Alcohol dehydrogenase and ALDH are also present in brain tumor cells. Moreover, the activity of class I isoenzymes was significantly higher in cancer than healthy brain cells. The activity of these enzymes in tumor tissue is reflected in the serum and could thus be helpful for diagnostics of brain neoplasms. The aim of this study was to investigate the potential role of ADH and ALDH as markers for brain tumors. Material and methods Serum samples were taken for routine biochemical investigation from 115 patients suffering from brain tumors (65 glioblastomas, 50 meningiomas). For the measurement of the activity of class I and II ADH isoenzymes and ALDH activity, fluorometric methods were used. The total ADH activity and activity of class III and IV isoenzymes were measured by the photometric method. Results There was a significant increase in the activity of ADH I isoenzyme and ADH total in the sera of brain tumor patients compared to the controls. The diagnostic sensitivity for ADH I was 78%, specificity 85%, and positive and negative predictive values were 86% and 76% respectively. The sensitivity and specificity of ADH I increased with the stage of the carcinoma. Area under receiver-operating characteristic curve for ADH I was 0.71. Conclusions The results suggest a potential role for ADH I as a marker for brain tumor. PMID:28261287

  7. Galaxy Formation

    DEFF Research Database (Denmark)

    Sparre, Martin

    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 cosmological simulation, Illustris, follow a tight relation between star formation rate and stellar mass. This relation agrees well with the observed relation at a redshift of z = 0 and z = 4, but at intermediate redshifts of z ' 2 the normalisation is lower than in real observations. This is highlighted...... of GRB host galaxies is affected by the fact that GRBs appear mainly to happen in low-metallicity galaxies. Solving this problem will make it possible to derive the total cosmic star formation rate more reliably from number counts of GRBs....

  8. 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...

  9. Structure and Function of Plasmodium falciparum malate dehydrogenase: Role of Critical Amino Acids in C-substrate Binding Procket

    Science.gov (United States)

    Malaria parasite thrives on anaerobic fermentation of glucose for energy. Earlier studies from our lab have demonstrated that a cytosolic malate dehydrogenase (PfMDH) with striking similarity to lactate dehydrogenase (PfLDH) might complement PfLDH function in Plasmodium falciparum. The N-terminal g...

  10. A rapid procedure for the in situ assay of periplasmic, PQQ-dependent methanol dehydrogenase in intact single bacterial colonies.

    Science.gov (United States)

    Vemuluri, Venkata Ramana; Shaw, Shreya; Autenrieth, Caroline; Ghosh, Robin

    2017-03-23

    Mechanistic details of methanol oxidation catalyzed by the periplasmically-located pyrroloquinoline quinone-dependent methanol dehydrogenase of methylotrophs can be elucidated using site-directed mutants. Here, we present an in situ colony assay of methanol dehydrogenase, which allows robotic screening of large populations of intact small colonies, and regrowth of colonies for subsequent analysis.

  11. 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

  12. Functional and structural characterization of a synthetic peptide representing the N-terminal domain of prokaryotic pyruvate dehydrogenase

    NARCIS (Netherlands)

    Hengeveld, A.F.; Mierlo, van C.P.M.; Hooven, van den H.W.; Visser, A.J.W.G.; Kok, de A.

    2002-01-01

    A synthetic peptide (Nterm-E1p) is used to characterize the structure and function of the N-terminal region (amino acid residues 4-45) of the pyruvate dehydrogenase component (E1p) from the pyruvate dehydrogenase multienzyme complex (PDHC) from Azotobacter vinelandii. Activity and binding studies es

  13. Hippocampal formation

    NARCIS (Netherlands)

    Cappaert, N.L.M.; van Strien, N.M.; Witter, M.P.; Paxinos, G.

    2015-01-01

    The hippocampal formation and parahippocampal region are prominent components of the rat nervous system and play a crucial role in learning, memory, and spatial navigation. Many new details regarding the entorhinal cortex have been discovered since the previous edition, and the growing interest in t

  14. Highly stereoselective biosynthesis of (R)-α-hydroxy carboxylic acids through rationally re-designed mutation of D-lactate dehydrogenase.

    Science.gov (United States)

    Zheng, Zhaojuan; Sheng, Binbin; Gao, Chao; Zhang, Haiwei; Qin, Tong; Ma, Cuiqing; Xu, Ping

    2013-12-02

    An NAD-dependent D-lactate dehydrogenase (D-nLDH) of Lactobacillus bulgaricus ATCC 11842 was rationally re-designed for asymmetric reduction of a homologous series of α-keto carboxylic acids such as phenylpyruvic acid (PPA), α-ketobutyric acid, α-ketovaleric acid, β-hydroxypyruvate. Compared with wild-type D-nLDH, the Y52L mutant D-nLDH showed elevated activities toward unnatural substrates especially with large substitutes at C-3. By the biocatalysis combined with a formate dehydrogenase for in situ generation of NADH, the corresponding (R)-α-hydroxy carboxylic acids could be produced at high yields and highly optical purities. Taking the production of chiral (R)-phenyllactic acid (PLA) from PPA for example, 50 mM PPA was completely reduced to (R)-PLA in 90 min with a high yield of 99.0% and a highly optical purity (>99.9% e.e.) by the coupling system. The results presented in this work suggest a promising alternative for the production of chiral α-hydroxy carboxylic acids.

  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. The Glutamate Dehydrogenase Pathway and Its Roles in Cell and Tissue Biology in Health and Disease

    Science.gov (United States)

    Plaitakis, Andreas; Kalef-Ezra, Ester; Kotzamani, Dimitra; Zaganas, Ioannis; Spanaki, Cleanthe

    2017-01-01

    Glutamate dehydrogenase (GDH) is a hexameric enzyme that catalyzes the reversible conversion of glutamate to α-ketoglutarate and ammonia while reducing NAD(P)+ to NAD(P)H. It is found in all living organisms serving both catabolic and anabolic reactions. In mammalian tissues, oxidative deamination of glutamate via GDH generates α-ketoglutarate, which is metabolized by the Krebs cycle, leading to the synthesis of ATP. In addition, the GDH pathway is linked to diverse cellular processes, including ammonia metabolism, acid-base equilibrium, redox homeostasis (via formation of fumarate), lipid biosynthesis (via oxidative generation of citrate), and lactate production. While most mammals possess a single GDH1 protein (hGDH1 in the human) that is highly expressed in the liver, humans and other primates have acquired, via duplication, an hGDH2 isoenzyme with distinct functional properties and tissue expression profile. The novel hGDH2 underwent rapid evolutionary adaptation, acquiring unique properties that enable enhanced enzyme function under conditions inhibitory to its ancestor hGDH1. These are thought to provide a biological advantage to humans with hGDH2 evolution occurring concomitantly with human brain development. hGDH2 is co-expressed with hGDH1 in human brain, kidney, testis and steroidogenic organs, but not in the liver. In human cerebral cortex, hGDH1 and hGDH2 are expressed in astrocytes, the cells responsible for removing and metabolizing transmitter glutamate, and for supplying neurons with glutamine and lactate. In human testis, hGDH2 (but not hGDH1) is densely expressed in the Sertoli cells, known to provide the spermatids with lactate and other nutrients. In steroid producing cells, hGDH1/2 is thought to generate reducing equivalents (NADPH) in the mitochondria for the biosynthesis of steroidal hormones. Lastly, up-regulation of hGDH1/2 expression occurs in cancer, permitting neoplastic cells to utilize glutamine/glutamate for their growth. In

  17. The Glutamate Dehydrogenase Pathway and Its Roles in Cell and Tissue Biology in Health and Disease

    Directory of Open Access Journals (Sweden)

    Andreas Plaitakis

    2017-02-01

    Full Text Available Glutamate dehydrogenase (GDH is a hexameric enzyme that catalyzes the reversible conversion of glutamate to α-ketoglutarate and ammonia while reducing NAD(P+ to NAD(PH. It is found in all living organisms serving both catabolic and anabolic reactions. In mammalian tissues, oxidative deamination of glutamate via GDH generates α-ketoglutarate, which is metabolized by the Krebs cycle, leading to the synthesis of ATP. In addition, the GDH pathway is linked to diverse cellular processes, including ammonia metabolism, acid-base equilibrium, redox homeostasis (via formation of fumarate, lipid biosynthesis (via oxidative generation of citrate, and lactate production. While most mammals possess a single GDH1 protein (hGDH1 in the human that is highly expressed in the liver, humans and other primates have acquired, via duplication, an hGDH2 isoenzyme with distinct functional properties and tissue expression profile. The novel hGDH2 underwent rapid evolutionary adaptation, acquiring unique properties that enable enhanced enzyme function under conditions inhibitory to its ancestor hGDH1. These are thought to provide a biological advantage to humans with hGDH2 evolution occurring concomitantly with human brain development. hGDH2 is co-expressed with hGDH1 in human brain, kidney, testis and steroidogenic organs, but not in the liver. In human cerebral cortex, hGDH1 and hGDH2 are expressed in astrocytes, the cells responsible for removing and metabolizing transmitter glutamate, and for supplying neurons with glutamine and lactate. In human testis, hGDH2 (but not hGDH1 is densely expressed in the Sertoli cells, known to provide the spermatids with lactate and other nutrients. In steroid producing cells, hGDH1/2 is thought to generate reducing equivalents (NADPH in the mitochondria for the biosynthesis of steroidal hormones. Lastly, up-regulation of hGDH1/2 expression occurs in cancer, permitting neoplastic cells to utilize glutamine/glutamate for their growth

  18. The Glutamate Dehydrogenase Pathway and Its Roles in Cell and Tissue Biology in Health and Disease.

    Science.gov (United States)

    Plaitakis, Andreas; Kalef-Ezra, Ester; Kotzamani, Dimitra; Zaganas, Ioannis; Spanaki, Cleanthe

    2017-02-08

    Glutamate dehydrogenase (GDH) is a hexameric enzyme that catalyzes the reversible conversion of glutamate to α-ketoglutarate and ammonia while reducing NAD(P)⁺ to NAD(P)H. It is found in all living organisms serving both catabolic and anabolic reactions. In mammalian tissues, oxidative deamination of glutamate via GDH generates α-ketoglutarate, which is metabolized by the Krebs cycle, leading to the synthesis of ATP. In addition, the GDH pathway is linked to diverse cellular processes, including ammonia metabolism, acid-base equilibrium, redox homeostasis (via formation of fumarate), lipid biosynthesis (via oxidative generation of citrate), and lactate production. While most mammals possess a single GDH1 protein (hGDH1 in the human) that is highly expressed in the liver, humans and other primates have acquired, via duplication, an hGDH2 isoenzyme with distinct functional properties and tissue expression profile. The novel hGDH2 underwent rapid evolutionary adaptation, acquiring unique properties that enable enhanced enzyme function under conditions inhibitory to its ancestor hGDH1. These are thought to provide a biological advantage to humans with hGDH2 evolution occurring concomitantly with human brain development. hGDH2 is co-expressed with hGDH1 in human brain, kidney, testis and steroidogenic organs, but not in the liver. In human cerebral cortex, hGDH1 and hGDH2 are expressed in astrocytes, the cells responsible for removing and metabolizing transmitter glutamate, and for supplying neurons with glutamine and lactate. In human testis, hGDH2 (but not hGDH1) is densely expressed in the Sertoli cells, known to provide the spermatids with lactate and other nutrients. In steroid producing cells, hGDH1/2 is thought to generate reducing equivalents (NADPH) in the mitochondria for the biosynthesis of steroidal hormones. Lastly, up-regulation of hGDH1/2 expression occurs in cancer, permitting neoplastic cells to utilize glutamine/glutamate for their growth. In

  19. Activation of Human Salivary Aldehyde Dehydrogenase by Sulforaphane: Mechanism and Significance

    Science.gov (United States)

    Alam, Md. Fazle; Laskar, Amaj Ahmed; Maryam, Lubna

    2016-01-01

    Cruciferous vegetables contain the bio-active compound sulforaphane (SF) which has been reported to protect individuals against various diseases by a number of mechanisms, including activation of the phase II detoxification enzymes. In this study, we show that the extracts of five cruciferous vegetables that we commonly consume and SF activate human salivary aldehyde dehydrogenase (hsALDH), which is a very important detoxifying enzyme in the mouth. Maximum activation was observed at 1 μg/ml of cabbage extract with 2.6 fold increase in the activity. There was a ~1.9 fold increase in the activity of hsALDH at SF concentration of ≥ 100 nM. The concentration of SF at half the maximum response (EC50 value) was determined to be 52 ± 2 nM. There was an increase in the Vmax and a decrease in the Km of the enzyme in the presence of SF. Hence, SF interacts with the enzyme and increases its affinity for the substrate. UV absorbance, fluorescence and CD studies revealed that SF binds to hsALDH and does not disrupt its native structure. SF binds with the enzyme with a binding constant of 1.23 x 107 M-1. There is one binding site on hsALDH for SF, and the thermodynamic parameters indicate the formation of a spontaneous strong complex between the two. Molecular docking analysis depicted that SF fits into the active site of ALDH3A1, and facilitates the catalytic mechanism of the enzyme. SF being an antioxidant, is very likely to protect the catalytic Cys 243 residue from oxidation, which leads to the increase in the catalytic efficiency and hence the activation of the enzyme. Further, hsALDH which is virtually inactive towards acetaldehyde exhibited significant activity towards it in the presence of SF. It is therefore very likely that consumption of large quantities of cruciferous vegetables or SF supplements, through their activating effect on hsALDH can protect individuals who are alcohol intolerant against acetaldehyde toxicity and also lower the risk of oral cancer

  20. The mitochondrial monoamine oxidase-aldehyde dehydrogenase pathway: a potential site of action of daidzin.

    Science.gov (United States)

    Rooke, N; Li, D J; Li, J; Keung, W M

    2000-11-02

    Recent studies showed that daidzin suppresses ethanol intake in ethanol-preferring laboratory animals. In vitro, it potently and selectively inhibits the mitochondrial aldehyde dehydrogenase (ALDH-2). Further, it inhibits the conversion of monoamines such as serotonin (5-HT) and dopamine (DA) into their respective acid metabolites, 5-hydroxyindole-3-acetic acid (5-HIAA) and 3,4-dihydroxyphenylacetic acid (DOPAC) in isolated hamster or rat liver mitochondria. Studies on the suppression of ethanol intake and inhibition of 5-HIAA (or DOPAC) formation by six structural analogues of daidzin suggested a potential link between these two activities. This, together with the finding that daidzin does not affect the rates of mitochondria-catalyzed oxidative deamination of these monoamines, raised the possibility that the ethanol intake-suppressive (antidipsotropic) action of daidzin is not mediated by the monoamines but rather by their reactive biogenic aldehyde intermediates such as 5-hydroxyindole-3-acetaldehyde (5-HIAL) and/or 3,4-dihydroxyphenylacetaldehyde (DOPAL) which accumulate in the presence of daidzin. To further evaluate this possibility, we synthesized more structural analogues of daidzin and tested and compared their antidipsotropic activities in Syrian golden hamsters with their effects on monoamine metabolism in isolated hamster liver mitochondria using 5-HT as the substrate. Effects of daidzin and its structural analogues on the activities of monoamine oxidase (MAO) and ALDH-2, the key enzymes involved in 5-HT metabolism in the mitochondria, were also examined. Results from these studies reveal a positive correlation between the antidipsotropic activities of these analogues and their abilities to increase 5-HIAL accumulation during 5-HT metabolism in isolated hamster liver mitochondria. Daidzin analogues that potently inhibit ALDH-2 but have no or little effect on MAO are most antidipsotropic, whereas those that also potently inhibit MAO exhibit little, if

  1. Disruption of the 11-cis-Retinol Dehydrogenase Gene Leads to Accumulation of cis-Retinols and cis-Retinyl Esters

    OpenAIRE

    Driessen, Carola A. G. G.; Winkens, Huub J.; Hoffmann, Kirstin; Kuhlmann, Leonoor D.; Janssen, Bert P. M.; van Vugt, Anke H M; Van Hooser, J. Preston; Wieringa, B. E.; Deutman, August F; Palczewski, Krzysztof; Ruether, Klaus; Janssen, Jacques J. M.

    2000-01-01

    To elucidate the possible role of 11-cis-retinol dehydrogenase in the visual cycle and/or 9-cis-retinoic acid biosynthesis, we generated mice carrying a targeted disruption of the 11-cis-retinol dehydrogenase gene. Homozygous 11-cis-retinol dehydrogenase mutants developed normally, including their retinas. There was no appreciable loss of photoreceptors. Recently, mutations in the 11-cis-retinol dehydrogenase gene in humans have been associated with fundus albipunctatus. In 11-cis-retinol deh...

  2. Production and characterization of a thermostable L-threonine dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus

    NARCIS (Netherlands)

    Machielsen, M.P.; Oost, van der J.

    2006-01-01

    The gene encoding a threonine dehydrogenase (TDH) has been identified in the hyperthermophilic archaeon Pyrococcus furiosus. The Pf-TDH protein has been functionally produced in Escherichia coli and purified to homogeneity. The enzyme has a tetrameric conformation with a molecular mass of ¿ 155 kDa.

  3. 5FU and oxaliplatin-containing chemotherapy in two dihydropyrimidine dehydrogenase-deficient patients

    NARCIS (Netherlands)

    Reerink, O; Mulder, NH; Szabo, BG; Hospers, GAP

    2004-01-01

    Patients with a germline mutation leading to a deficiency of the dihydropyrimidine dehydrogenase (DPD) enzyme are at risk from developing severe toxicity on the administration of 5FU-containing chemotherapy. We report on the implications of this inborn genetic error in two patients who received 5FU

  4. 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

  5. Fuel utilization in patients with very long-chain acyl-coa dehydrogenase deficiency

    DEFF Research Database (Denmark)

    ØRngreen, Mette C; Nørgaard, Mette; Sacchetti, Massimo

    2004-01-01

    Fuel utilization in two adult patients with the myopathic form of very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency and five healthy subjects was investigated with stable isotopes during exercise at 50% of VO2max. The findings indicate that residual VLCAD activity in the patients...

  6. 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…

  7. CRYSTAL-STRUCTURE OF AN ELECTRON-TRANSFER COMPLEX BETWEEN METHYLAMINE DEHYDROGENASE AND AMICYANIN

    NARCIS (Netherlands)

    CHEN, LY; DURLEY, R; POLIKS, BJ; HAMADA, K; CHEN, ZW; MATHEWS, FS; DAVIDSON, VL; SATOW, Y; HUIZINGA, E; VELLIEUX, FMD; HOL, WGJ

    1992-01-01

    The crystal structure of the complex between the quinoprotein methylamine dehydrogenase (MADH) and the type I blue copper protein amicyanin, both from Paracoccus denitrificans, has been determined at 2.5-angstrom resolution using molecular replacement. The search model was MADH from Thiobacillus ver

  8. Alcohol consumption and type 2 diabetes - Influence of genetic variation in alcohol dehydrogenase

    NARCIS (Netherlands)

    Beulens, J.W.J.; Rimm, E.B.; Hendriks, H.F.J.; Hu, F.B.; Manson, J.E.; Hunter, D.J.; Mukamal, K.J.

    2007-01-01

    OBJECTIVE-We sought to investigate whether a polymorphism I in the alcohol dehydrogenase 1c (ADH1C) gene modifies the association between alcohol consumption and type 2 diabetes. RESEARCH DESIGN AND METHODS-In nested case-control studies of 640 women with incident diabetes and 1,000 control subjects

  9. Alcohol consumption and type 2 diabetes: Influence of genetic variation in alcohol dehydrogenase

    NARCIS (Netherlands)

    Beulens, J.W.J.; Rimm, E.B.; Hendriks, H.F.J.; Hu, F.B.; Manson, J.E.; Hunter, D.J.; Mukamal, K.J.

    2007-01-01

    OBJECTIVE - We sought to investigate whether a polymorphism in the alcohol dehydrogenase 1c (ADH1C) gene modifies the association between alcohol consumption and type 2 diabetes. RESEARCH DESIGN AND METHODS - In nested case-control studies of 640 women with incident diabetes and 1,000 control subjec

  10. 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.

  11. Purification, characterization, and cloning of a bifunctional molybdoenzyme with hydratase and alcohol dehydrogenase activity

    NARCIS (Netherlands)

    Jin, J.; Straathof, A.J.J.; Pinkse, M.W.H.; Hanefeld, U.

    2010-01-01

    A bifunctional hydratase/alcohol dehydrogenase was isolated from the cyclohexanol degrading bacterium Alicycliphilus denitrificans DSMZ 14773. The enzyme catalyzes the addition of water to α,β-unsaturated carbonyl compounds and the subsequent alcohol oxidation. The purified enzyme showed three subun

  12. Binding of inhibiting adducts of ketones and NAD(+) to alcohol dehydrogenase from Drosophila melanogaster

    NARCIS (Netherlands)

    Smilda, T; Jekel, PA; Bruining, MAM; Beintema, JJ

    1998-01-01

    Drosophila alcohol dehydrogenase (DADH) can be converted with NAD(+) and ketone into more negatively charged isoforms. Completely modified ADH isoforms are inactive, but activity is regained after native polyacrylamide gel electrophoresis depending on the ketone used. When unmodified ADH is incubate

  13. 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

  14. Statistical Measure of a Gene Evolution The Case of Glyceraldehyde-3-Phosphate Dehydrogenase Gene

    CERN Document Server

    Chattopadhyay, S; Chakrabarti, J; Chattopadhyay, Sujay; Sahoo, Satyabrata; Chakrabarti, Jayprokas

    2000-01-01

    The enzyme Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) catalyses the decomposition of glucose. The gene that produces the GAPDH is therefore present in a wide class of organisms. We show that for this gene the average value of the fluctuations in nucleotide distribution in the codons, normalized to strand bias, provides a reasonable measure of how the gene has evolved in time.

  15. Relevance of expanded neonatal screening of medium-chain acyl co-a dehydrogenase deficiency

    DEFF Research Database (Denmark)

    Couce, M L; Castiñeiras, D E; Moure, J D;

    2011-01-01

    Neonatal screening of medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is of major importance due to the significant morbidity and mortality in undiagnosed patients. MCADD screening has been performed routinely in Galicia since July 2000, and until now 199,943 newborns have been screened. W...

  16. A population-based study of high-grade gliomas and mutated isocitrate dehydrogenase 1

    DEFF Research Database (Denmark)

    Dahlrot, Rikke H; Kristensen, Bjarne W; Hjelmborg, Jacob;

    2012-01-01

    High-grade gliomas have a dismal prognosis, and prognostic factors are needed to optimize treatment algorithms. In this study we identified clinical prognostic factors as well as the prognostic value of isocitrate dehydrogenase 1 (IDH1) status in a population-based group of patients with high...

  17. Heteroexpression and characterization of a monomeric isocitrate dehydrogenase from the multicellular prokaryote Streptomyces avermitilis MA-4680.

    Science.gov (United States)

    Wang, Ao; Cao, Zheng-Yu; Wang, Peng; Liu, Ai-Min; Pan, Wei; Wang, Jie; Zhu, Guo-Ping

    2011-08-01

    A monomeric NADP-dependent isocitrate dehydrogenase from the multicellular prokaryote Streptomyces avermitilis MA-4680 (SaIDH) was heteroexpressed in Escherichia coli, and the His-tagged enzyme was further purified to homogeneity. The molecular weight of SaIDH was about 80 kDa which is typical for monomeric isocitrate dehydrogenases. Structure-based sequence alignment reveals that the deduced amino acid sequence of SaIDH shows high sequence identity with known momomeric isocitrate dehydrogenase, and the coenzyme, substrate and metal ion binding sites are completely conserved. The optimal pH and temperature of SaIDH were found to be pH 9.4 and 45°C, respectively. Heat-inactivation studies showed that heating for 20 min at 50°C caused a 50% loss in enzymatic activity. In addition, SaIDH was absolutely specific for NADP+ as electron acceptor. Apparent Km values were 4.98 μM for NADP+ and 6,620 μM for NAD+, respectively, using Mn2+ as divalent cation. The enzyme performed a 33,000-fold greater specificity (kcat/Km) for NADP+ than NAD+. Moreover, SaIDH activity was entirely dependent on the presence of Mn2+ or Mg2+, but was strongly inhibited by Ca2+ and Zn2+. Taken together, our findings implicate the recombinant SaIDH is a divalent cation-dependent monomeric isocitrate dehydrogenase which presents a remarkably high cofactor preference for NADP+.

  18. Crystallization and preliminary X-ray diffraction of malate dehydrogenase from Plasmodium falciparum

    NARCIS (Netherlands)

    Wrenger, Carsten; Mueller, Ingrid B.; Butzloff, Sabine; Jordanova, Rositsa; Lunev, Sergey; Groves, Matthew R.

    2012-01-01

    The expression, purification, crystallization and preliminary X-ray diffraction characterization of malate dehydrogenase (MDH) from the malarial parasite Plasmodium falciparum (PfMDH) are reported. In order to gain a deeper understanding of the function and role of PfMDH, the protein was purified to

  19. 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.

  20. 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

  1. 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....

  2. 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.

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

    DEFF Research Database (Denmark)

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

    2006-01-01

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

  4. 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...

  5. 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...

  6. 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).

  7. 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…

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

    DEFF Research Database (Denmark)

    Nellemann, Birgitte; Vendelbo, Mikkel H; Nielsen, Thomas S

    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....

  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. 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...

  11. 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

  12. Electron transfer between a quinohemoprotein alcohol dehydrogenase and an electrode via a redox polymer network

    NARCIS (Netherlands)

    Stigter, E.C.A.; Jong, G.A.H. de; Jongejan, J.A.; Duine, J.A.; Lugt, J.P. van der; Somers, W.A.C.

    1996-01-01

    A quinohemoprotein alcohol dehydrogenase (QH-EDH) from Comamonas testosteroni was immobilized on an electrode in a redox polymer network consisting of a polyvinylpyridine partially N-complexed with osmiumbis-(bipyridine)chloride. The enzyme effectively transfers electrons to the electrode via the po

  13. 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

  14. 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

  15. 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.

  16. Cloud Formation

    Science.gov (United States)

    Graham, Mark Talmage

    2004-05-01

    Cloud formation is crucial to the heritage of modern physics, and there is a rich literature on this important topic. In 1927, Charles T.R. Wilson was awarded the Nobel Prize in physics for applications of the cloud chamber.2 Wilson was inspired to study cloud formation after working at a meteorological observatory on top of the highest mountain in Scotland, Ben Nevis, and testified near the end of his life, "The whole of my scientific work undoubtedly developed from the experiments I was led to make by what I saw during my fortnight on Ben Nevis in September 1894."3 To form clouds, Wilson used the sudden expansion of humid air.4 Any structure the cloud may have is spoiled by turbulence in the sudden expansion, but in 1912 Wilson got ion tracks to show up by using strobe photography of the chamber immediately upon expansion.5 In the interim, Millikan's study in 1909 of the formation of cloud droplets around individual ions was the first in which the electron charge was isolated. This study led to his famous oil drop experiment.6 To Millikan, as to Wilson, meteorology and physics were professionally indistinct. With his meteorological physics expertise, in WWI Millikan commanded perhaps the first meteorological observation and forecasting team essential to military operation in history.7 But even during peacetime meteorology is so much of a concern to everyone that a regular news segment is dedicated to it. Weather is the universal conversation topic, and life on land could not exist as we know it without clouds. One wonders then, why cloud formation is never covered in physics texts.

  17. A novel bio-electronic tongue using different cellobiose dehydrogenases to resolve mixtures of various sugars and interfering analytes.

    Science.gov (United States)

    Cipri, Andrea; Schulz, Christopher; Ludwig, Roland; Gorton, Lo; Del Valle, Manel

    2016-05-15

    A novel application of cellobiose dehydrogenase (CDH) as sensing element for a Bioelectronic Tongue (BioET) system has been tested. In this work CDHs from various fungi, which exhibit different substrate specificities, were used to discriminate between lactose and glucose in presence of the interfering matrix compound Ca(2+) in various mixtures. This work exploits the advantage of an electronic tongue system with practically zero pre-treatment of samples and operation at low voltages in a direct electron transfer mode. The Artificial Neural Network (ANN) used in the BioET system to interpret the voltammetric data was able to provide a correct prediction of the concentrations of the analytes considered. Correlation coefficients in the comparison of obtained vs. expected concentrations were highly significant, especially for lactose (R(2)=0.975) and Ca(2+) (R(2)=0.945). This BioET application has a high potential especially for the food and dairy industry and also, if further miniaturised in screen printed format, for its in-situ use.

  18. Aldehyde dehydrogenase-expressing colon stem cells contribute to tumorigenesis in the transition from colitis to cancer.

    Science.gov (United States)

    Carpentino, Joseph E; Hynes, Mark J; Appelman, Henry D; Zheng, Tong; Steindler, Dennis A; Scott, Edward W; Huang, Emina H

    2009-10-15

    Patients with chronic ulcerative colitis are at increased risk of developing colorectal cancer. Although current hypotheses suggest that sporadic colorectal cancer is due to inability to control cancer stem cells, the cancer stem cell hypothesis has not yet been validated in colitis-associated cancer. Furthermore, the identification of the colitis to cancer transition is challenging. We recently showed that epithelial cells with the increased expression of aldehyde dehydrogenase in sporadic colon cancer correlate closely with tumor-initiating ability. We sought to determine whether ALDH can be used as a marker to isolate tumor-initiating populations from patients with chronic ulcerative colitis. We used fluorescence-activated cell sorting to identify precursor colon cancer stem cells from colitis patients and report both their transition to cancerous stem cells in xenografting studies as well as their ability to generate spheres in vitro. Similar to sporadic colon cancer, these colitis-derived tumors were capable of propagation as sphere cultures. However, unlike the origins of sporadic colon cancer, the primary colitic tissues did not express any histologic evidence of dysplasia. To elucidate a potential mechanism for our findings, we compared the stroma of these different environments and determined that at least one paracrine factor is up-regulated in the inflammatory and malignant stroma compared with resting, normal stroma. These data link colitis and cancer identifying potential tumor-initiating cells from colitic patients, suggesting that sphere and/or xenograft formation will be useful to survey colitic patients at risk of developing cancer.

  19. Alpha-ketoglutarate reduces ethanol toxicity in Drosophila melanogaster by enhancing alcohol dehydrogenase activity and antioxidant capacity.

    Science.gov (United States)

    Bayliak, Maria M; Shmihel, Halyna V; Lylyk, Maria P; Storey, Kenneth B; Lushchak, Volodymyr I

    2016-09-01

    Ethanol at low concentrations (alcohol dehydrogenase (ADH) activity as compared with those reared on control diet or diet with ethanol only. Native gel electrophoresis data suggested that this combination diet might promote post-translational modifications of ADH protein with the formation of a highly active ADH form. The ethanol-containing diet led to significantly higher levels of triacylglycerides stored in adult flies, and this parameter was not altered by AKG supplement. The influence of diet on antioxidant defenses was also assessed. In ethanol-fed flies, catalase activity was higher in males and the levels of low molecular mass thiols were unchanged in both sexes compared to control values. Feeding on a mixture of AKG and ethanol did not affect catalase activity but caused a higher level of low molecular mass thiols compared to ethanol-fed flies. It can be concluded that both a stimulation of some components of antioxidant defense and the increase in ADH activity may be responsible for the protective effects of AKG diet supplementation in combination with ethanol. The results suggest that AKG might be useful as a treatment option to neutralize toxic effects of excessive ethanol intake and to improve the physiological state of D. melanogaster and other animals, potentially including humans.

  20. 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}.

  1. The driver and passenger effects of isocitrate dehydrogenase 1 and 2 mutations in oncogenesis and survival prolongation.

    Science.gov (United States)

    Molenaar, Remco J; Radivoyevitch, Tomas; Maciejewski, Jaroslaw P; van Noorden, Cornelis J F; Bleeker, Fonnet E

    2014-12-01

    Mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) are key events in the development of glioma, acute myeloid leukemia (AML), chondrosarcoma, intrahepatic cholangiocarcinoma (ICC), and angioimmunoblastic T-cell lymphoma. They also cause D-2-hydroxyglutaric aciduria and Ollier and Maffucci syndromes. IDH1/2 mutations are associated with prolonged survival in glioma and in ICC, but not in AML. The reason for this is unknown. In their wild-type forms, IDH1 and IDH2 convert isocitrate and NADP(+) to α-ketoglutarate (αKG) and NADPH. Missense mutations in the active sites of these enzymes induce a neo-enzymatic reaction wherein NADPH reduces αKG to D-2-hydroxyglutarate (D-2HG). The resulting D-2HG accumulation leads to hypoxia-inducible factor 1α degradation, and changes in epigenetics and extracellular matrix homeostasis. Such mutations also imply less NADPH production capacity. Each of these effects could play a role in cancer formation. Here, we provide an overview of the literature and discuss which downstream molecular effects are likely to be the drivers of the oncogenic and survival-prolonging properties of IDH1/2 mutations. We discuss interactions between mutant IDH1/2 inhibitors and conventional therapies. Understanding of the biochemical consequences of IDH1/2 mutations in oncogenesis and survival prolongation will yield valuable information for rational therapy design: it will tell us which oncogenic processes should be blocked and which "survivalogenic" effects should be retained.

  2. Characterization of NADP(+)-dependent isocitrate dehydrogenase isozymes from a psychrophilic bacterium, Colwellia psychrerythraea strain 34H.

    Science.gov (United States)

    Suzuki, Kaori; Takada, Yasuhiro

    2016-08-01

    NADP(+)-dependent isocitrate dehydrogenase (IDH) isozymes of a psychrophilic bacterium, Colwellia psychrerythraea strain 34H, were characterized. The coexistence of monomeric and homodimeric IDHs in this bacterium was confirmed by Western blot analysis, the genes encoding two monomeric (IDH-IIa and IDH-IIb) and one dimeric (IDH-I) IDHs were cloned and overexpressed in Escherichia coli, and the three IDH proteins were purified. Both of the purified IDH-IIa and IDH-IIb were found to be cold-adapted enzymes while the purified IDH-I showed mesophilic properties. However, the specific activities of IDH-IIa and IDH-IIb were lower even at low temperatures than that of IDH-I. Therefore, IDH-I was suggested to be important for the growth of this bacterium. The results of colony formation of E. coli transformants carrying the respective IDH genes and IDH activities in their crude extracts indicated that the expression of the IDH-IIa gene is cold-inducible in the E. coli cells.

  3. 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.

  4. 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.

  5. 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

  6. Mutational Analyses of Glucose Dehydrogenase and Glucose-6-Phosphate Dehydrogenase Genes in Pseudomonas fluorescens Reveal Their Effects on Growth and Alginate Production.

    Science.gov (United States)

    Maleki, Susan; Mærk, Mali; Valla, Svein; Ertesvåg, Helga

    2015-05-15

    The biosynthesis of alginate has been studied extensively due to the importance of this polymer in medicine and industry. Alginate is synthesized from fructose-6-phosphate and thus competes with the central carbon metabolism for this metabolite. The alginate-producing bacterium Pseudomonas fluorescens relies on the Entner-Doudoroff and pentose phosphate pathways for glucose metabolism, and these pathways are also important for the metabolism of fructose and glycerol. In the present study, the impact of key carbohydrate metabolism enzymes on growth and alginate synthesis was investigated in P. fluorescens. Mutants defective in glucose-6-phosphate dehydrogenase isoenzymes (Zwf-1 and Zwf-2) or glucose dehydrogenase (Gcd) were evaluated using media containing glucose, fructose, or glycerol. Zwf-1 was shown to be the most important glucose-6-phosphate dehydrogenase for catabolism. Both Zwf enzymes preferred NADP as a coenzyme, although NAD was also accepted. Only Zwf-2 was active in the presence of 3 mM ATP, and then only with NADP as a coenzyme, indicating an anabolic role for this isoenzyme. Disruption of zwf-1 resulted in increased alginate production when glycerol was used as the carbon source, possibly due to decreased flux through the Entner-Doudoroff pathway rendering more fructose-6-phosphate available for alginate biosynthesis. In alginate-producing cells grown on glucose, disruption of gcd increased both cell numbers and alginate production levels, while this mutation had no positive effect on growth in a non-alginate-producing strain. A possible explanation is that alginate synthesis might function as a sink for surplus hexose phosphates that could otherwise be detrimental to the cell.

  7. Electrogenerated chemiluminescence biosensor with alcohol dehydrogenase and tris(2,2'-bipyridyl)ruthenium (II) immobilized in sol-gel hybrid material.

    Science.gov (United States)

    Xu, Zhiai; Guo, Zhihui; Dong, Shaojun

    2005-09-15

    An ethanol biosensor based on electrogenerated chemiluminescence detection was developed. Electrogenerated chemiluminescence reagent tris(2,2'-bipyridyl)ruthenium (II) and alcohol dehydrogenase were immobilized in the same sol-gel hybrid film. The copolymer poly(vinyl alcohol) with 4-vinylpyridine and cation exchanger Nafion were incorporated into sol-gel film to provide the microenvironment for retaining the activity of enzyme and immobilize tris(2,2'-bipyridyl)ruthenium (II). The design was simpler than the previous two-layer format. The experimental conditions, such as scan rate, pH and concentration of the cofactor were investigated. The intensity of electrogenerated chemiluminescence increased linearly with ethanol concentration from 2.5x10(-5) to 5.0x10(-2) M and detection limit was 1.0x10(-5) M. The prepared biosensor exhibited high sensitivity, wide linear range and good stability.

  8. Marked differences in drug-induced methemoglobinemia in sheep are not due to RBC glucose-6-phosphate dehydrogenase, reduced glutathione, or methemoglobin reductase activity

    Energy Technology Data Exchange (ETDEWEB)

    Martin, D.G.; Guertler, A.T.; Lagutchik, M.S.; Woodard, C.L.; Leonard, D.A.

    1993-05-13

    Benzocaine is a commonly used topical anesthetic that is structurally similar to current candidates for cyanide prophylaxis. Benzocaine induces profound methemoglobinemia in some sheep but not others. After topical benzocaine administration certain sheep respond to form MHb (elevated MHb 16-50% after a 56-280 mg dose, a 2-10 second spray with benzocine), while other phenotypically similar sheep fail to significantly form MHb (less than a 2% increase from baseline). Deficiencies in Glucose-6-phosphate dehydrogenase (G-6-PD), reduced glutathione (GSH), and MHb reductase increase the susceptibility to methemoglobinemia in man and animals. Sheep are used as a model for G-6-PD deficiency in man, and differences in this enzyme level could cause the variable response seen in these sheep. Similarly, differences in GSH and MHb reductase could be responsible for the observed differences in MHb formation.

  9. 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...... an overview of cement production, selected cement properties, and clinker phase relations. An extended summary of laboratory-scale investigations on clinkerization reactions, the most important reactions in cement production, is provided. Clinker formations by solid state reactions, solid−liquid and liquid...

  10. The separate roles of PQQ and apo-enzyme syntheses in the regulation of glucose dehydrogenase activity in Klebsiella pneumoniae NCTC 418.

    Science.gov (United States)

    Hommes, R W; Herman, P T; Postma, P W; Tempest, D W; Neijssel, O M

    1989-01-01

    No holoenzyme pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase and only very low apoenzyme levels could be detected in cells of Klebsiella pneumoniae, growing anaerobically, or carrying out a fumarate or nitrate respiration. Low glucose dehydrogenase activity in some aerobic glucose-excess cultures of K. pneumoniae (ammonia or sulphate limitation) was increased significantly by addition of PQQ, whereas in cells already possessing a high glucose dehydrogenase activity (phosphate or potassium limitation) extra PQQ had almost no effect. These observations indicate that the glucose dehydrogenase activity in K. pneumoniae is modulated by both PQQ synthesis and synthesis of the glucose dehydrogenase apo-enzyme.

  11. 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.

  12. 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.

  13. 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...

  14. [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.

  15. 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.

  16. 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.

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

    Directory of Open Access Journals (Sweden)

    Tripty A. Hirani

    2011-01-01

    Full Text Available We have developed an in vitro system for detailed analysis of reversible phosphorylation of the plant mitochondrial pyruvate dehydrogenase complex, comprising recombinant Arabidopsis thaliana α2β2-heterotetrameric pyruvate dehydrogenase (E1 plus A. thaliana E1-kinase (AtPDK. Upon addition of MgATP, Ser292, which is located within the active-site loop structure of E1α, is phosphorylated. In addition to Ser292, Asp295 and Gly297 are highly conserved in the E1α active-site loop sequences. Mutation of Asp295 to Ala, Asn, or Leu greatly reduced phosphorylation of Ser292, while mutation of Gly297 had relatively little effect. Quantitative two-hybrid analysis was used to show that mutation of Asp295 did not substantially affect binding of AtPDK to E1α. When using pyruvate as a variable substrate, the Asp295 mutant proteins had modest changes in kcat, Km, and kcat/Km values. Therefore, we propose that Asp295 plays an important role in stabilizing the active-site loop structure, facilitating transfer of the γ-phosphate from ATP to the Ser residue at regulatory site one of E1α.

  18. Galaxy Formation

    CERN Document Server

    Longair, Malcolm S

    2008-01-01

    This second edition of Galaxy Formation is an up-to-date text on astrophysical cosmology, expounding the structure of the classical cosmological models from a contemporary viewpoint. This forms the background to a detailed study of the origin of structure and galaxies in the Universe. The derivations of many of the most important results are derived by simple physical arguments which illuminate the results of more advanced treatments. A very wide range of observational data is brought to bear upon these problems, including the most recent results from WMAP, the Hubble Space Telescope, galaxy surveys like the Sloan Digital Sky Survey and the 2dF Galaxy Redshift Survey, studies of Type 1a supernovae, and many other observations.

  19. 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.

  20. 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.

  1. Proline dehydrogenase is a positive regulator of cell death in different kingdoms.

    Science.gov (United States)

    Cecchini, Nicolás M; Monteoliva, Mariela I; Alvarez, María E

    2011-08-01

    Proline dehydrogenase (ProDH) catalyzes the flavin-dependent oxidation of Pro into Δ1-pyrroline-5-carboxylate (P5C). This is the first of the two enzymatic reactions that convert proline (Pro) into glutamic acid (Glu). The P5C thus produced is non-enzymatically transformed into glutamate semialdehyde (GSA), which acts as a substrate of P5C dehydrogenase (P5CDH) to generate Glu. Activation of ProDH can generate different effects depending on the behaviour of other enzymes of this metabolism. Under different conditions it can generate toxic levels of P5C, alter the cellular redox homeostasis and even produce reactive oxygen species (ROS). Recent studies indicate that in Arabidopsis, the enzyme potentiates the oxidative burst and cell death associated to the Hypersensitive Responses (HR). Interestingly, activation of ProDH can also produce harmful effects in other organisms, suggesting that the enzyme may play a conserved role in the control of cell death.

  2. Expression, Purification, Crystallization And Preliminary X-Ray Studies of Histamine Dehydrogenase From Nocardioides Simplex

    Energy Technology Data Exchange (ETDEWEB)

    Reed, T.M.; Hirakawa, H.; Mure, M.; Scott, E.E.; Limburg, J.

    2009-05-21

    Histamine dehydrogenase (HADH) from Nocardioides simplex catalyzes the oxidative deamination of histamine to produce imidazole acetaldehyde and an ammonium ion. HADH is functionally related to trimethylamine dehydrogenase (TMADH), but HADH has strict substrate specificity towards histamine. HADH is a homodimer, with each 76 kDa subunit containing two redox cofactors: a [4Fe-4S] cluster and an unusual covalently bound flavin mononucleotide, 6-S-cysteinyl-FMN. In order to understand the substrate specificity of HADH, it was sought to determine its structure by X-ray crystallography. This enzyme has been expressed recombinantly in Escherichia coli and successfully crystallized in two forms. Diffraction data were collected to 2.7 {angstrom} resolution at the SSRL synchrotron with 99.7% completeness. The crystals belonged to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 101.14, b = 107.03, c = 153.35 {angstrom}.

  3. Nucleotide sequence of yeast GDH1 encoding nicotinamide adenine dinucleotide phosphate-dependent glutamate dehydrogenase.

    Science.gov (United States)

    Moye, W S; Amuro, N; Rao, J K; Zalkin, H

    1985-07-15

    The yeast GDH1 gene encodes NADP-dependent glutamate dehydrogenase. This gene was isolated by complementation of an Escherichia coli glutamate auxotroph. NADP-dependent glutamate dehydrogenase was overproduced 6-10-fold in Saccharomyces cerevisiae bearing GDH1 on a multicopy plasmid. The nucleotide sequence of the 1362-base pair coding region and 5' and 3' flanking sequences were determined. Transcription start sites were located by S1 nuclease mapping. Regulation of GDH1 was not maintained when the gene was present on a multicopy plasmid. Protein secondary structure predictions identified a region with potential to form the dinucleotide-binding domain. The amino acid sequences of the yeast and Neurospora crassa enzymes are 63% conserved. Unlike the N. crassa gene, yeast GDH1 has no introns.

  4. 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.

  5. 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.

  6. Peroxisomal NADP-isocitrate dehydrogenase is required for Arabidopsis stomatal movement.

    Science.gov (United States)

    Leterrier, Marina; Barroso, Juan B; Valderrama, Raquel; Begara-Morales, Juan C; Sánchez-Calvo, Beatriz; Chaki, Mounira; Luque, Francisco; Viñegla, Benjamin; Palma, José M; Corpas, Francisco J

    2016-03-01

    Peroxisomes are subcellular organelles characterized by a simple morphological structure but have a complex biochemical machinery involved in signaling processes through molecules such as hydrogen peroxide (H2O2) and nitric oxide (NO). Nicotinamide adenine dinucleotide phosphate (NADPH) is an essential component in cell redox homeostasis, and its regeneration is critical for reductive biosynthesis and detoxification pathways. Plants have several NADPH-generating dehydrogenases, with NADP-isocitrate dehydrogenase (NADP-ICDH) being one of these enzymes. Arabidopsis contains three genes that encode for cytosolic, mitochondrial/chloroplastic, and peroxisomal NADP-ICDH isozymes although the specific function of each of these remains largely unknown. Using two T-DNA insertion lines of the peroxisomal NADP-ICDH designated as picdh-1 and picdh-2, the data show that the peroxisomal NADP-ICDH is involved in stomatal movements, suggesting that peroxisomes are a new element in the signaling network of guard cells.

  7. Activity of the mitochondrial pyruvate dehydrogenase complex in plants is stimulated in the presence of malate.

    Science.gov (United States)

    Igamberdiev, Abir U; Lernmark, Ulrikа; Gardeström, Per

    2014-11-01

    The effect of malate on the steady-state activity of the pea (Pisum sativum L.) and barley (Hordeum vulgare L.) leaf pyruvate dehydrogenase complex (PDC) has been studied in isolated mitochondria. The addition of malate was found to be stimulatory for the mitochondrial PDC, however there was no stimulation of chloroplast PDC. The stimulation was saturated below 1mM malate and was apparently related to а partially activated complex, which activity increased in the presence of malate by about twofold. Malate also reversed the reduction of PDC activity in the presence of glycine. Based on the obtained kinetic data, we suggest that the effect of malate is rather not a direct activation of PDC but involves the establishment of NAD-malate dehydrogenase equilibrium, decreasing concentration of NADH and relieving its inhibitory effect of PDC.

  8. A α-glycerophosphate dehydrogenase is present in Trypanosoma cruzi glycosomes

    Directory of Open Access Journals (Sweden)

    JL Concepcion

    2001-07-01

    Full Text Available α-glycerophosphate dehydrogenase (α-GPDH-EC.1.1.1.8 has been considered absent in Trypanosoma cruzi in contradiction with all other studied trypanosomatids. After observing that the sole malate dehydrogenase can not maintain the intraglycosomal redox balance, GPDH activity was looked for and found, although in very variable levels, in epimastigotes extracts. GPDH was shown to be exclusively located in the glycosome of T. cruzi by digitonin treatment and isopycnic centrifugation. Antibody against T. brucei GPDH showed that this enzyme seemed to be present in an essentially inactive form at the beginning of the epimastigotes growth. GPDH is apparently linked to a salicylhydroxmic-sensitive glycerophosphate reoxidizing system and plays an essential role in the glycosome redox balance.

  9. Isolation and characterization of an apple cytosolic malate dehydrogenase gene reveal its function in malate synthesis.

    Science.gov (United States)

    Yao, Yu-Xin; Li, Ming; Zhai, Heng; You, Chun-Xiang; Hao, Yu-Jin

    2011-03-15

    Cytosolic NAD-dependent malate dehydrogenase (cyMDH) is an enzyme crucial for malate synthesis in the cytosol. The apple MdcyMDH gene (GenBank Accession No. DQ221207) encoding the cyMDH enzyme in apple was cloned and functionally characterized. The protein was subcellularly localized to the cytoplasm and plasma membrane. Based on kinetic parameters, it mainly catalyzes the reaction from oxalacetic acid (OAA) to malate in vitro. The expression level of MdcyMDH was positively correlated with malate dehydrogenase (MDH) activity throughout fruit development, but not with malate content, especially in the ripening apple fruit. MdcyMDH overexpression contributed to malate accumulation in the apple callus and tomato. Taken together, our results support the involvement of MdcyMDH directly in malate synthesis and indirectly in malate accumulation through the regulation of genes/enzymes associated with malate degradation and transportation, gluconeogenesis and the tricarboxylic acid cycle.

  10. 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.

  11. Alpha-hydroxybutyrate dehydrogenase activity in sex-linked muscular dystrophy.

    Science.gov (United States)

    Johnston, H A; Wilkinson, J H; Withycombe, W A; Raymond, S

    1966-05-01

    In two families with severe sex-linked muscular dystrophy, high levels of alpha-hydroxybutyrate dehydrogenase (HBD), lactate dehydrogenase (LD), aspartate transaminase (AspT), aldolase, and creatine phosphokinase (CPK) were found in the sera of three young affected males. In both families the mother had a raised level of HBD activity. Four sisters of the three affected boys had raised serum enzyme levels, and they are regarded as presumptive carriers of the disease. Biopsy specimens of dystrophic muscle had LD and HBD contents which were significantly lower than those of control specimens, while the HBD/LD ratios were markedly greater. Muscle from two unaffected members of the same family also exhibited high ratios, indicating the presence of the electrophoretically fast LD isoenzymes, and this was confirmed by acrylamide-gel electrophoresis.

  12. Interaction of thiamin diphosphate with phosphorylated and dephosphorylated mammalian pyruvate dehydrogenase complex.

    Science.gov (United States)

    Liu, Xiaoqing; Bisswanger, Hans

    2005-01-01

    Kinetic and binding studies were carried out on substrate and cofactor interaction with the pyruvate dehydrogenase complex from bovine heart. Fluoropyruvate and pyruvamide, previously described as irreversible and allosteric inhibitors, respectively, are strong competitive inhibitors with respect to pyruvate. Binding of thiamin diphosphate was used to study differences between the active dephosphorylated and inactive phosphorylated enzyme states by spectroscopic methods. The change in both the intrinsic tryptophan fluorescence and the fluorescence of the 6-bromoacetyl-2-dimethylaminonaphthalene-labelled enzyme complex produced on addition of the cofactor showed similar binding behaviour for both enzyme forms, with slightly higher affinity for the phosphorylated form. Changes in the CD spectrum, especially the negative Cotton effect at 330 nm as a function of cofactor concentration, both in the absence and presence of pyruvate, also revealed no drastic differences between the two enzyme forms. Thus, inactivation of the enzyme activity of the pyruvate dehydrogenase complex is not caused by impeding the binding of substrate or cofactor.

  13. 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...... and a history of seizures, who was found to excrete increased amounts of 2-methylbutyryl glycine in the urine. The SBCAD gene was examined with sequence analysis. His development was assessed with psychometric testing before and after a trial with low protein diet. RESULTS: We found homozygosity for A > G...... 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...

  14. Disruption of the NADPH-dependent glutamate dehydrogenase affects the morphology of two industrial strains of Penicillium chrysogenum

    DEFF Research Database (Denmark)

    Thykær, Jette; Kildegaard, Kanchana Rueksomtawin; Noorman, H.

    2009-01-01

    New morphological aspects of Penicillium chrysogenum were found during physiological characterisation of two NADPH-dependent glutamate dehydrogenase mutant strains. A morphological characterisation of the previously constructed strains, together with the two beta-lactam producing industrial recip...

  15. Class 2 aldehyde dehydrogenase. Characterization of the hamster enzyme, sensitive to daidzin and conserved within the family of multiple forms.

    Science.gov (United States)

    Hjelmqvist, L; Lundgren, R; Norin, A; Jörnvall, H; Vallee, B; Klyosov, A; Keung, W M

    1997-10-13

    Mitochondrial (class 2) hamster aldehyde dehydrogenase has been purified and characterized. Its primary structure has been determined and correlated with the tertiary structure recently established for this class from another species. The protein is found to represent a constant class within a complex family of multiple forms. Variable segments that occur in different species correlate with non-functional segments, in the same manner as in the case of the constant class of alcohol dehydrogenases (class III type) of another protein family, but distinct from the pattern of the corresponding variable enzymes. Hence, in both these protein families, overall variability and segment architectures behave similarly, with at least one 'constant' form in each case, class III in the case of alcohol dehydrogenases, and at least class 2 in the case of aldehyde dehydrogenases.

  16. Marked and variable inhibition by chemical fixation of cytochrome oxidase and succinate dehydrogenase in single motoneurons

    Science.gov (United States)

    Chalmers, G. R.; Edgerton, V. R.

    1989-01-01

    The effect of tissue fixation on succinate dehydrogenase and cytochrome oxidase activity in single motoneurons of the rat was demonstrated using a computer image processing system. Inhibition of enzyme activity by chemical fixation was variable, with some motoneurons being affected more than others. It was concluded that quantification of enzymatic activity in chemically fixed tissue provides an imprecise estimate of enzyme activities found in fresh-frozen tissues.

  17. Purification of methanol dehydrogenase from mouth methylotrophic bacteria of tropical region

    OpenAIRE

    Waturangi, D.; Marko, N.; M. T. Suhartono2)

    2011-01-01

    Aims: Purification of methanol dehydrogenase (MDH) from methylotrophic bacteria was conducted to obtain pure enzyme for further research and industrial applications due to the enzyme’s unique activity that catalyzes oxidation of methanol as an important carbon source in methylotrophic bacteria.Methodology and Results: The enzyme was screened from methylotrophic bacteria isolated from human mouth. Purification of this enzyme was conducted using ammonium sulphate precipitation followed by catio...

  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. 5FU and oxaliplatin-containing chemotherapy in two dihydropyrimidine dehydrogenase-deficient patients.

    Science.gov (United States)

    Reerink, O; Mulder, N H; Szabo, B G; Hospers, G A P

    2004-01-01

    Patients with a germline mutation leading to a deficiency of the dihydropyrimidine dehydrogenase (DPD) enzyme are at risk from developing severe toxicity on the administration of 5FU-containing chemotherapy. We report on the implications of this inborn genetic error in two patients who received 5FU and oxaliplatin. A possible co-medication effect of oxaliplatin is considered, as are the consequences of screening for DPD deficiency.

  20. Isolation and Biochemical Characterization of a Glucose Dehydrogenase from a Hay Infusion Metagenome

    OpenAIRE

    Alexander Basner; Garabed Antranikian

    2014-01-01

    Glucose hydrolyzing enzymes are essential to determine blood glucose level. A high-throughput screening approach was established to identify NAD(P)-dependent glucose dehydrogenases for the application in test stripes and the respective blood glucose meters. In the current report a glucose hydrolyzing enzyme, derived from a metagenomic library by expressing recombinant DNA fragments isolated from hay infusion, was characterized. The recombinant clone showing activity on glucose as substrate ex...

  1. 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, i...... for the association between alcohol drinking habits and the risk of developing acute coronary syndrome, if any, is very limited....

  2. [Alanine dehydrogenase of the cyanobacterium Plectonema boryanum in the early period of cyanophage LPP-3 development].

    Science.gov (United States)

    Perepelitsa, S I; Koltukova, N V; Mendzhul, M I

    1995-01-01

    It has been studied how reproduction of LPP-3 in Plectonema boryanum cells influences the alanine dehydrogenase activity. It has been found that immediately after the virus adsorption the enzyme activity falls by 50% and the anabolic reaction is blocked. Physicochemical properties of the enzyme vary as well. An infected cell has one isoenzyme-octamer with pl 9.1-9.2, pH-optimum by action 9-10, molecular weight about 27 kDa.

  3. Mitochondrial malate dehydrogenase lowers leaf respiration and alters photorespiration and plant growth in Arabidopsis.

    Science.gov (United States)

    Tomaz, Tiago; Bagard, Matthieu; Pracharoenwattana, Itsara; Lindén, Pernilla; Lee, Chun Pong; Carroll, Adam J; Ströher, Elke; Smith, Steven M; Gardeström, Per; Millar, A Harvey

    2010-11-01

    Malate dehydrogenase (MDH) catalyzes a reversible NAD(+)-dependent-dehydrogenase reaction involved in central metabolism and redox homeostasis between organelle compartments. To explore the role of mitochondrial MDH (mMDH) in Arabidopsis (Arabidopsis thaliana), knockout single and double mutants for the highly expressed mMDH1 and lower expressed mMDH2 isoforms were constructed and analyzed. A mmdh1mmdh2 mutant has no detectable mMDH activity but is viable, albeit small and slow growing. Quantitative proteome analysis of mitochondria shows changes in other mitochondrial NAD-linked dehydrogenases, indicating a reorganization of such enzymes in the mitochondrial matrix. The slow-growing mmdh1mmdh2 mutant has elevated leaf respiration rate in the dark and light, without loss of photosynthetic capacity, suggesting that mMDH normally uses NADH to reduce oxaloacetate to malate, which is then exported to the cytosol, rather than to drive mitochondrial respiration. Increased respiratory rate in leaves can account in part for the low net CO(2) assimilation and slow growth rate of mmdh1mmdh2. Loss of mMDH also affects photorespiration, as evidenced by a lower postillumination burst, alterations in CO(2) assimilation/intercellular CO(2) curves at low CO(2), and the light-dependent elevated concentration of photorespiratory metabolites. Complementation of mmdh1mmdh2 with an mMDH cDNA recovered mMDH activity, suppressed respiratory rate, ameliorated changes to photorespiration, and increased plant growth. A previously established inverse correlation between mMDH and ascorbate content in tomato (Solanum lycopersicum) has been consolidated in Arabidopsis and may potentially be linked to decreased galactonolactone dehydrogenase content in mitochondria in the mutant. Overall, a central yet complex role for mMDH emerges in the partitioning of carbon and energy in leaves, providing new directions for bioengineering of plant growth rate and a new insight into the molecular mechanisms

  4. 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.

  5. Coulometric bioelectrocatalytic reactions based on NAD-dependent dehydrogenases in tricarboxylic acid cycle

    Energy Technology Data Exchange (ETDEWEB)

    Fukuda, Jun [Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502 (Japan); Tsujimura, Seiya [Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502 (Japan)], E-mail: seiya@kais.kyoto-u.ac.jp; Kano, Kenji [Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502 (Japan)], E-mail: kkano@kais.kyoto-u.ac.jp

    2008-12-30

    This paper describes the characterization of mediated electro-enzymatic electrolysis systems based on NAD-dependent dehydrogenase reactions in the tricarboxylic acid (TCA) cycle. A micro-bulk electrolysis system with a carbon felt anode immersed in an electrolysis solution with a value of about 10 {mu}L was constructed for coulometric analysis of the substrate oxidation. Diaphorase (DI) was used to couple the NAD-dependent dehydrogenase reaction with the anode reaction of a suitable redox mediator. We focused on three types of NAD-dependant dehydrogenases reactions in this research: (1) isocitrate oxidation, in which the standard Gibbs energy change ({delta}G{sup o}') is negative; (2) {alpha}-ketoglutarate oxidation, which involves an electrochemically active coenzyme A (CoA); and (3) malate oxidation, which is thermodynamically unfavorable because of a large positive {delta}G{sup o}' value. The complete electrolysis of isocitrate was easily achieved, supporting the effective re-oxidation of NADH in the diaphorase-catalyzed electrochemical reaction. CoA was unfavorably oxidized at the electrodes in the presence of some mediators. The electrocatalytic oxidation of CoA was suppressed and the quantitative electrochemical oxidation of {alpha}-ketoglutarate was achieved by selecting a suitable mediator with negligibly slow electron transfer kinetics with CoA. The uphill malate oxidation was susceptible to product inhibition in the bioelectrochemical system, although NADH generated in the malate dehydrogenase reaction was immediately oxidized in the electrochemical system. The inhibition was successfully suppressed by linking citrate synthase to quench oxaloacetate and to make the total {delta}G{sup o}' value negative.

  6. Role of Alanine Dehydrogenase of Mycobacterium tuberculosis during Recovery from Hypoxic Nonreplicating Persistence.

    Directory of Open Access Journals (Sweden)

    Michelle M Giffin

    Full Text Available Mycobacterium tuberculosis can maintain a nonreplicating persistent state in the host for decades, but must maintain the ability to efficiently reactivate and produce active disease to survive and spread in a population. Among the enzymes expressed during this dormancy is alanine dehydrogenase, which converts pyruvate to alanine, and glyoxylate to glycine concurrent with the oxidation of NADH to NAD. It is involved in the metabolic remodeling of M. tuberculosis through its possible interactions with both the glyoxylate and methylcitrate cycle. Both mRNA levels and enzymatic activities of isocitrate lyase, the first enzyme of the glyoxylate cycle, and alanine dehydrogenase increased during entry into nonreplicating persistence, while the gene and activity for the second enzyme of the glyoxylate cycle, malate synthase were not. This could suggest a shift in carbon flow away from the glyoxylate cycle and instead through alanine dehydrogenase. Expression of ald was also induced in vitro by other persistence-inducing stresses such as nitric oxide, and was expressed at high levels in vivo during the initial lung infection in mice. Enzyme activity was maintained during extended hypoxia even after transcription levels decreased. An ald knockout mutant of M. tuberculosis showed no reduction in anaerobic survival in vitro, but resulted in a significant lag in the resumption of growth after reoxygenation. During reactivation the ald mutant had an altered NADH/NAD ratio, and alanine dehydrogenase is proposed to maintain the optimal NADH/NAD ratio during anaerobiosis in preparation of eventual regrowth, and during the initial response during reoxygenation.

  7. Role of apoptosis-inducing factor, proline dehydrogenase, and NADPH oxidase in apoptosis and oxidative stress

    Directory of Open Access Journals (Sweden)

    Becker DF

    2012-02-01

    Full Text Available Sathish Kumar Natarajan, Donald F BeckerDepartment of Biochemistry and Redox Biology Center, University of Nebraska-Lincoln, Lincoln, NEAbstract: Flavoproteins catalyze a variety of reactions utilizing flavin mononucleotide or flavin adenine dinucleotide as cofactors. The oxidoreductase properties of flavoenzymes implicate them in redox homeostasis, oxidative stress, and various cellular processes, including programmed cell death. Here we explore three critical flavoproteins involved in apoptosis and redox signaling, ie, apoptosis-inducing factor (AIF, proline dehydrogenase, and NADPH oxidase. These proteins have diverse biochemical functions and influence apoptotic signaling by unique mechanisms. The role of AIF in apoptotic signaling is two-fold, with AIF changing intracellular location from the inner mitochondrial membrane space to the nucleus upon exposure of cells to apoptotic stimuli. In the mitochondria, AIF enhances mitochondrial bioenergetics and complex I activity/assembly to help maintain proper cellular redox homeostasis. After translocating to the nucleus, AIF forms a chromatin degrading complex with other proteins, such as cyclophilin A. AIF translocation from the mitochondria to the nucleus is triggered by oxidative stress, implicating AIF as a mitochondrial redox sensor. Proline dehydrogenase is a membrane-associated flavoenzyme in the mitochondrion that catalyzes the rate-limiting step of proline oxidation. Upregulation of proline dehydrogenase by the tumor suppressor, p53, leads to enhanced mitochondrial reactive oxygen species that induce the intrinsic apoptotic pathway. NADPH oxidases are a group of enzymes that generate reactive oxygen species for oxidative stress and signaling purposes. Upon activation, NADPH oxidase 2 generates a burst of superoxide in neutrophils that leads to killing of microbes during phagocytosis. NADPH oxidases also participate in redox signaling that involves hydrogen peroxide-mediated activation of

  8. Pyruvate dehydrogenase complex and nicotinamide nucleotide transhydrogenase constitute an energy consuming redox circuit

    OpenAIRE

    2015-01-01

    Cellular proteins rely on reversible redox reactions to establish and maintain biological structure and function. How redox catabolic (NAD+:NADH) and anabolic (NADP+:NADPH) processes integrate during metabolism to maintain cellular redox homeostasis however is unknown. The present work identifies a continuously cycling, mitochondrial membrane potential-dependent redox circuit between the pyruvate dehydrogenase complex (PDHC) and nicotinamide nucleotide transhydrogenase (NNT). PDHC is shown to...

  9. Choline dehydrogenase interacts with SQSTM1/p62 to recruit LC3 and stimulate mitophagy

    OpenAIRE

    Park, Sungwoo; Choi, Seon-Guk; Yoo, Seung-Min; Son, Jin H.; Jung, Yong-Keun

    2014-01-01

    CHDH (choline dehydrogenase) is an enzyme catalyzing the dehydrogenation of choline to betaine aldehyde in mitochondria. Apart from this well-known activity, we report here a pivotal role of CHDH in mitophagy. Knockdown of CHDH expression impairs CCCP-induced mitophagy and PARK2/parkin-mediated clearance of mitochondria in mammalian cells, including HeLa cells and SN4741 dopaminergic neuronal cells. Conversely, overexpression of CHDH accelerates PARK2-mediated mitophagy. CHDH is found on both...

  10. 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.

  11. Triazole inhibitors of Cryptosporidium parvum inosine 5′-monophosphate dehydrogenase

    OpenAIRE

    Maurya, Sushil K.; Gollapalli, Deviprasad R.; Kirubakaran, Sivapriya; Zhang, Minjia; Johnson, Corey R.; Benjamin, Nicole N.; Hedstrom, Lizbeth; Gregory D Cuny

    2009-01-01

    Cryptosporidium parvum is an important human pathogen and potential bioterrorism agent. This protozoan parasite cannot salvage guanine or guanosine and therefore relies on inosine 5′-monophosphate dehydrogenase (IMPDH) for biosynthesis of guanine nucleotides and hence for survival. Since C. parvum IMPDH is highly divergent from the host counterpart, selective inhibitors could potentially be used to treat cryptosporidiosis with minimal effects on its mammalian host. A series of 1,2,3-triazole ...

  12. Succinate dehydrogenase activity and soma size of motoneurons innervating different portions of the rat tibialis anterior

    Science.gov (United States)

    Ishihara, A.; Roy, R. R.; Edgerton, V. R.

    1995-01-01

    The spatial distribution, soma size and oxidative enzyme activity of gamma and alpha motoneurons innervating muscle fibres in the deep (away from the surface of the muscle) and superficial (close to the surface of the muscle) portions of the tibialis anterior in normal rats were determined. The deep portion had a higher percentage of high oxidative fibres than the superficial portion of the muscle. Motoneurons were labelled by retrograde neuronal transport of fluorescent tracers: Fast Blue and Nuclear Yellow were injected into the deep portion and Nuclear Yellow into the superficial portion of the muscle. Therefore, motoneurons innervating the deep portion were identified by both a blue fluorescent cytoplasm and a golden-yellow fluorescent nucleus, while motoneurons innervating the superficial portion were identified by only a golden-yellow fluorescent nucleus. After staining for succinate dehydrogenase activity on the same section used for the identification of the motoneurons, soma size and succinate dehydrogenase activity of the motoneurons were measured. The gamma and alpha motoneurons innervating both the deep and superficial portions were located primarily at L4 and were intermingled within the same region of the dorsolateral portion of the ventral horn in the spinal cord. Mean soma size was similar for either gamma or alpha motoneurons in the two portions of the muscle. The alpha motoneurons innervating the superficial portion had a lower mean succinate dehydrogenase activity than those innervating the deep portion of the muscle. An inverse relationship between soma size and succinate dehydrogenase activity of alpha, but not gamma, motoneurons innervating both the deep and superficial portions was observed. Based on three-dimensional reconstructions within the spinal cord, there were no apparent differences in the spatial distribution of the motoneurons, either gamma or alpha, associated with the deep and superficial compartments of the muscle. The data

  13. Daidzin inhibits mitochondrial aldehyde dehydrogenase and suppresses ethanol intake of Syrian golden hamsters

    OpenAIRE

    Keung, Wing Ming; Klyosov, Anatole A; Vallee, Bert L.

    1997-01-01

    Daidzin is the major active principle in extracts of radix puerariae, a traditional Chinese medication that suppresses the ethanol intake of Syrian golden hamsters. It is the first isoflavone recognized to have this effect. Daidzin is also a potent and selective inhibitor of human mitochondrial aldehyde dehydrogenase (ALDH-2). To establish a link between these two activities, we have tested a series of synthetic structural analogs of daidzin. The results demonstrate a direct correlation betwe...

  14. 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...

  15. 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.

  16. In Situ lactate dehydrogenase activiy-a novel renal cortical imaging biomarker of tubular injury?

    DEFF Research Database (Denmark)

    Nielsen, Per Mose; Laustsen, Christoffer; Bertelsen, Lotte Bonde;

    , apoptosis and inflammation. Lactate dehydrogenase (LDH) activity has previously been suggested as a renal tubular injury marker, but has a major limitation in the sense that it can only be measured in terminal kidneys. By the use of a hyperpolarized [1-13C]pyruvate magnetic resonance imaging (MRI) approach...... to monitor metabolic changes, we here investigate LDH activity and renal metabolism after IRI. This procedure gives a novel non-invasive method for investigation renal tissue injury in concern with IRI....

  17. Effects of some drugs on human erythrocyte glucose 6-phosphate dehydrogenase: an in vitro study.

    Science.gov (United States)

    Akkemik, Ebru; Budak, Harun; Ciftci, Mehmet

    2010-12-01

    Inhibitory effects of some drugs on glucose 6-phosphate dehydrogenase from the erythrocytes of human have been investigated. For this purpose, at the beginning, erythrocyte glucose 6-phosphate dehydrogenase was purified 2256 times in a yield of 44.22% by using ammonium sulphate precipitation and 2', 5'-ADP Sepharose 4B affinity gel. Temperature of +4°C was maintained during the purification process. Enzyme activity was determined with the Beutler method by using a spectrophotometer at 340 nm. This method was utilized for all kinetic studies. Ketotifen, dacarbazine, thiocolchicoside, meloxicam, methotrexate, furosemide, olanzapine, methylprednizolone acetate, paricalcitol, ritodrine hydrochloride, and gadobenate-dimeglumine were used as drugs. All the drugs indicated the inhibitory effects on the enzyme. Ki constants for glucose 6-phosphate dehydrogenase were found by means of Lineweaver-Burk graphs. While methylprednizolone acetate showed competitive inhibition, the others displayed non-competitive inhibition. In addition, IC(50) values of the drugs were determined by plotting Activity% vs [I].

  18. Mycofactocin-associated mycobacterial dehydrogenases with non-exchangeable NAD cofactors

    Science.gov (United States)

    Haft, Daniel H.; Pierce, Phillip G.; Mayclin, Stephen J.; Sullivan, Amy; Gardberg, Anna S.; Abendroth, Jan; Begley, Darren W.; Phan, Isabelle Q.; Staker, Bart L.; Myler, Peter J.; Marathias, Vasilios M.; Lorimer, Donald D.; Edwards, Thomas E.

    2017-01-01

    During human infection, Mycobacterium tuberculosis (Mtb) survives the normally bacteriocidal phagosome of macrophages. Mtb and related species may be able to combat this harsh acidic environment which contains reactive oxygen species due to the mycobacterial genomes encoding a large number of dehydrogenases. Typically, dehydrogenase cofactor binding sites are open to solvent, which allows NAD/NADH exchange to support multiple turnover. Interestingly, mycobacterial short chain dehydrogenases/reductases (SDRs) within family TIGR03971 contain an insertion at the NAD binding site. Here we present crystal structures of 9 mycobacterial SDRs in which the insertion buries the NAD cofactor except for a small portion of the nicotinamide ring. Line broadening and STD-NMR experiments did not show NAD or NADH exchange on the NMR timescale. STD-NMR demonstrated binding of the potential substrate carveol, the potential product carvone, the inhibitor tricyclazol, and an external redox partner 2,6-dichloroindophenol (DCIP). Therefore, these SDRs appear to contain a non-exchangeable NAD cofactor and may rely on an external redox partner, rather than cofactor exchange, for multiple turnover. Incidentally, these genes always appear in conjunction with the mftA gene, which encodes the short peptide MftA, and with other genes proposed to convert MftA into the external redox partner mycofactocin. PMID:28120876

  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. Comparative enzymology-new insights from studies of an "old" enzyme, lactate dehydrogenase.

    Science.gov (United States)

    Storey, Kenneth B

    2016-09-01

    Comparative enzymology explores the molecular mechanisms that alter the properties of enzymes to best fit and adapt them to the biotic demands and abiotic stresses that affect the cellular environment in which these protein catalysts function. For many years, comparative enzymology was primarily concerned with analyzing enzyme functional properties (e.g. substrate affinities, allosteric effectors, responses to temperature or pH, stabilizers, denaturants, etc.) in order to determine how enzyme properties were optimized to function under changing conditions. More recently it became apparent that posttranslational modifications of enzymes play a huge role in metabolic regulation. At first, such modifications appeared to target just crucial regulatory enzymes but recent work is showing that many dehydrogenases are also targets of posttranslational modification leading to substantial changes in enzyme properties. The present article focuses in particular on lactate dehydrogenase (LDH) showing that stress-induced changes in enzyme properties can be linked with reversible posttranslational modifications; e.g. changes in the phosphorylation state of LDH occur in response to dehydration stress in frogs and anoxia exposure of turtles and snails. Furthermore, these studies show that LDH is also a target of other posttranslational modifications including acetylation, methylation and ubiquitination that change in response to anoxia or dehydration stress. Selected new methods for exploring posttranslational modifications of dehydrogenases are discussed and new challenges for the future of comparative enzymology are presented that will help to achieve a deeper understanding of biochemical adaptation through enzyme regulation.

  1. Protein S-glutathionylation alters superoxide/hydrogen peroxide emission from pyruvate dehydrogenase complex.

    Science.gov (United States)

    O'Brien, Marisa; Chalker, Julia; Slade, Liam; Gardiner, Danielle; Mailloux, Ryan J

    2017-05-01

    Pyruvate dehydrogenase (Pdh) is a vital source of reactive oxygen species (ROS) in several different tissues. Pdh has also been suggested to serve as a mitochondrial redox sensor. Here, we report that O2(•-)/ H2O2 emission from pyruvate dehydrogenase (Pdh) is altered by S-glutathionylation. Glutathione disulfide (GSSG) amplified O2(•-)/ H2O2 production by purified Pdh during reverse electron transfer (RET) from NADH. Thiol oxidoreductase glutaredoxin-2 (Grx2) reversed these effects confirming that Pdh is a target for S-glutathionylation. S-glutathionylation had the opposite effect during forward electron transfer (FET) from pyruvate to NAD(+) lowering O2(•-)/ H2O2 production. Immunoblotting for protein glutathione mixed disulfides (PSSG) following diamide treatment confirmed that purified Pdh can be S-glutathionylated. Similar observations were made with mouse liver mitochondria. S-glutathionylation catalysts diamide and disulfiram significantly reduced pyruvate or 2-oxoglutarate driven O2(•-)/ H2O2 production in liver mitochondria, results that were confirmed using various Pdh, 2-oxoglutarate dehydrogenase (Ogdh), and respiratory chain inhibitors. Immunoprecipitation of Pdh and Ogdh confirmed that either protein can be S-glutathionylated by diamide and disulfiram. Collectively, our results demonstrate that the S -glutathionylation of Pdh alters the amount of ROS formed by the enzyme complex. We also confirmed that Ogdh is controlled in a similar manner. Taken together, our results indicate that the redox sensing and ROS forming properties of Pdh and Ogdh are linked to S-glutathionylation.

  2. 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.

  3. Structural and functional analysis of betaine aldehyde dehydrogenase from Staphylococcus aureus.

    Science.gov (United States)

    Halavaty, Andrei S; Rich, Rebecca L; Chen, Chao; Joo, Jeong Chan; Minasov, George; Dubrovska, Ievgeniia; Winsor, James R; Myszka, David G; Duban, Mark; Shuvalova, Ludmilla; Yakunin, Alexander F; Anderson, Wayne F

    2015-05-01

    When exposed to high osmolarity, methicillin-resistant Staphylococcus aureus (MRSA) restores its growth and establishes a new steady state by accumulating the osmoprotectant metabolite betaine. Effective osmoregulation has also been implicated in the acquirement of a profound antibiotic resistance by MRSA. Betaine can be obtained from the bacterial habitat or produced intracellularly from choline via the toxic betaine aldehyde (BA) employing the choline dehydrogenase and betaine aldehyde dehydrogenase (BADH) enzymes. Here, it is shown that the putative betaine aldehyde dehydrogenase SACOL2628 from the early MRSA isolate COL (SaBADH) utilizes betaine aldehyde as the primary substrate and nicotinamide adenine dinucleotide (NAD(+)) as the cofactor. Surface plasmon resonance experiments revealed that the affinity of NAD(+), NADH and BA for SaBADH is affected by temperature, pH and buffer composition. Five crystal structures of the wild type and three structures of the Gly234Ser mutant of SaBADH in the apo and holo forms provide details of the molecular mechanisms of activity and substrate specificity/inhibition of this enzyme.

  4. α -Ketoglutarate accumulation is not dependent on isocitrate dehydrogenase activity during tellurite detoxification in Escherichia coli.

    Science.gov (United States)

    Reinoso, Claudia A; Appanna, Vasu D; Vásquez, Claudio C

    2013-01-01

    Tellurite is toxic to most microorganisms because of its ability to generate oxidative stress. However, the way in which tellurite interferes with cellular processes is not fully understood to date. In this line, it was previously shown that tellurite-exposed cells displayed reduced activity of the α-ketoglutarate dehydrogenase complex (α-KGDH), which resulted in α-ketoglutarate (α-KG) accumulation. In this work, we assessed if α-KG accumulation in tellurite-exposed E. coli could also result from increased isocitrate dehydrogenase (ICDH) and glutamate dehydrogenase (GDH) activities, both enzymes involved in α-KG synthesis. Unexpectedly both activities were found to decrease in the presence of the toxicant, an observation that seems to result from the decreased transcription of icdA and gdhA genes (encoding ICDH and GDH, resp.). Accordingly, isocitrate levels were found to increase in tellurite-exposed E. coli. In the presence of the toxicant, cells lacking icdA or gdhA exhibited decreased reactive oxygen species (ROS) levels and higher tellurite sensitivity as compared to the wild type strain. Finally, a novel branch activity of ICDH as tellurite reductase is presented.

  5. 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.

  6. Escherichia coli D-malate dehydrogenase, a generalist enzyme active in the leucine biosynthesis pathway.

    Science.gov (United States)

    Vorobieva, Anastassia A; Khan, Mohammad Shahneawz; Soumillion, Patrice

    2014-10-17

    The enzymes of the β-decarboxylating dehydrogenase superfamily catalyze the oxidative decarboxylation of D-malate-based substrates with various specificities. Here, we show that, in addition to its natural function affording bacterial growth on D-malate as a carbon source, the D-malate dehydrogenase of Escherichia coli (EcDmlA) naturally expressed from its chromosomal gene is capable of complementing leucine auxotrophy in a leuB(-) strain lacking the paralogous isopropylmalate dehydrogenase enzyme. To our knowledge, this is the first example of an enzyme that contributes with a physiologically relevant level of activity to two distinct pathways of the core metabolism while expressed from its chromosomal locus. EcDmlA features relatively high catalytic activity on at least three different substrates (L(+)-tartrate, D-malate, and 3-isopropylmalate). Because of these properties both in vivo and in vitro, EcDmlA may be defined as a generalist enzyme. Phylogenetic analysis highlights an ancient origin of DmlA, indicating that the enzyme has maintained its generalist character throughout evolution. We discuss the implication of these findings for protein evolution.

  7. Pcal_1699, an extremely thermostable malate dehydrogenase from hyperthermophilic archaeon Pyrobaculum calidifontis.

    Science.gov (United States)

    Gharib, Ghazaleh; Rashid, Naeem; Bashir, Qamar; Gardner, Qura-Tul Ann Afza; Akhtar, Muhammad; Imanaka, Tadayuki

    2016-01-01

    Two malate dehydrogenase homologs, Pcal_0564 and Pcal_1699, have been found in the genome of Pyrobaculum calidifontis. The gene encoding Pcal_1699 consisted of 927 nucleotides corresponding to a polypeptide of 309 amino acids. To examine the properties of Pcal_1699, the structural gene was cloned, expressed in Escherichia coli and the purified gene product was characterized. Pcal_1699 was NADH specific enzyme exhibiting a high malate dehydrogenase activity (886 U/mg) at optimal pH (10) and temperature (90 °C). Unfolding studies suggested that urea could not induce complete unfolding and inactivation of Pcal_1699 even at a final concentration of 8 M; however, in the presence of 4 M guanidine hydrochloride enzyme structure was unfolded with complete loss of enzyme activity. Thermostability experiments revealed that Pcal_1699 is the most thermostable malate dehydrogenase, reported to date, retaining more than 90 % residual activity even after heating for 6 h in boiling water.

  8. NADP-Dependent Aldehyde Dehydrogenase from Archaeon Pyrobaculum sp.1860: Structural and Functional Features

    Directory of Open Access Journals (Sweden)

    Ekaterina Yu. Bezsudnova

    2016-01-01

    Full Text Available We present the functional and structural characterization of the first archaeal thermostable NADP-dependent aldehyde dehydrogenase AlDHPyr1147. In vitro, AlDHPyr1147 catalyzes the irreversible oxidation of short aliphatic aldehydes at 60–85°С, and the affinity of AlDHPyr1147 to the NADP+ at 60°С is comparable to that for mesophilic analogues at 25°С. We determined the structures of the apo form of AlDHPyr1147 (3.04 Å resolution, three binary complexes with the coenzyme (1.90, 2.06, and 2.19 Å, and the ternary complex with the coenzyme and isobutyraldehyde as a substrate (2.66 Å. The nicotinamide moiety of the coenzyme is disordered in two binary complexes, while it is ordered in the ternary complex, as well as in the binary complex obtained after additional soaking with the substrate. AlDHPyr1147 structures demonstrate the strengthening of the dimeric contact (as compared with the analogues and the concerted conformational flexibility of catalytic Cys287 and Glu253, as well as Leu254 and the nicotinamide moiety of the coenzyme. A comparison of the active sites of AlDHPyr1147 and dehydrogenases characterized earlier suggests that proton relay systems, which were previously proposed for dehydrogenases of this family, are blocked in AlDHPyr1147, and the proton release in the latter can occur through the substrate channel.

  9. 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.

  10. 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.

  11. Changes in cinnamyl alcohol dehydrogenase activities from sugarcane cultivars inoculated with Sporisorium scitamineum sporidia.

    Science.gov (United States)

    Santiago, Rocío; Alarcón, Borja; de Armas, Roberto; Vicente, Carlos; Legaz, María Estrella

    2012-06-01

    This study describes a method for determining cinnamyl alcohol dehydrogenase activity in sugarcane stems using reverse phase (RP) high-performance liquid chromatography to elucidate their possible lignin origin. Activity is assayed using the reverse mode, the oxidation of hydroxycinnamyl alcohols into hydroxycinnamyl aldehydes. Appearance of the reaction products, coniferaldehyde and sinapaldehyde is determined by measuring absorbance at 340 and 345 nm, respectively. Disappearance of substrates, coniferyl alcohol and sinapyl alcohol is measured at 263 and 273 nm, respectively. Isocratic elution with acetonitrile:acetic acid through an RP Mediterranea sea C18 column is performed. As case examples, we have examined two different cultivars of sugarcane; My 5514 is resistant to smut, whereas B 42231 is susceptible to the pathogen. Inoculation of sugarcane stems elicits lignification and produces significant increases of coniferyl alcohol dehydrogenase (CAD) and sinapyl alcohol dehydrogenase (SAD). Production of lignin increases about 29% in the resistant cultivar and only 13% in the susceptible cultivar after inoculation compared to uninoculated plants. Our results show that the resistance of My 5514 to smut is likely derived, at least in part, to a marked increase of lignin concentration by the activation of CAD and SAD.

  12. 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.

  13. Human placental glucose dehydrogenase: IEF polymorphism in two Italian populations and enzyme activity in the six common phenotypes.

    Science.gov (United States)

    Scacchi, R; Corbo, R M; Calzolari, E; Laconi, G; Palmarino, R; Lucarelli, P

    1985-01-01

    Glucose dehydrogenase (hexose-6-phosphate dehydrogenase) has been assayed qualitatively and quantitatively in more than 600 human placentae collected in two Italian populations. The gene frequencies for GDH1, GDH2 and GDH3 were, respectively, 0.66, 0.21 and 0.12 in Continental Italy and 0.65, 0.23 and 0.12 in Sardinia. Among the six common phenotypes there was no difference in catalytic activity.

  14. 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.

  15. Physiological regulation of isocitrate dehydrogenase and the role of 2-oxoglutarate in Prochlorococcus sp. strain PCC 9511.

    Science.gov (United States)

    Domínguez-Martín, María Agustina; López-Lozano, Antonio; Diez, Jesús; Gómez-Baena, Guadalupe; Rangel-Zúñiga, Oriol Alberto; García-Fernández, José Manuel

    2014-01-01

    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. 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.

  17. 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

  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. Global Analysis of Mannitol 2-Dehydrogenase in Lactobacillus reuteri CRL 1101 during Mannitol Production through Enzymatic, Genetic and Proteomic Approaches.

    Science.gov (United States)

    Ortiz, Maria Eugenia; Bleckwedel, Juliana; Fadda, Silvina; Picariello, Gianluca; Hebert, Elvira M; Raya, Raúl R; Mozzi, Fernanda

    2017-01-01

    Several plants, fungi, algae, and certain bacteria produce mannitol, a polyol derived from fructose. Mannitol has multiple industrial applications in the food, pharmaceutical, and medical industries, being mainly used as a non-metabolizable sweetener in foods. Many heterofermentative lactic acid bacteria synthesize mannitol when an alternative electron acceptor such as fructose is present in the medium. In previous work, we reported the ability of Lactobacillus reuteri CRL 1101 to efficiently produce mannitol from sugarcane molasses as carbon source at constant pH of 5.0; the activity of the enzyme mannitol 2-dehydrogenase (MDH) responsible for the fructose conversion into mannitol being highest during the log cell growth phase. Here, a detailed assessment of the MDH activity and relative expression of the mdh gene during the growth of L. reuteri CRL 1101 in the presence of fructose is presented. It was observed that MDH was markedly induced by the presence of fructose. A direct correlation between the maximum MDH enzyme activity and a high level of mdh transcript expression during the log-phase of cells grown in a fructose-containing chemically defined medium was detected. Furthermore, two proteomic approaches (2DE and shotgun proteomics) applied in this study confirmed the inducible expression of MDH in L. reuteri. A global study of the effect of fructose on activity, mdh gene, and protein expressions of MDH in L. reuteri is thus for the first time presented. This work represents a deep insight into the polyol formation by a Lactobacillus strain with biotechnological potential in the nutraceutics and pharmaceutical areas.

  2. 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.

  3. Purification and characterization of xylitol dehydrogenase with l-arabitol dehydrogenase activity from the newly isolated pentose-fermenting yeast Meyerozyma caribbica 5XY2.

    Science.gov (United States)

    Sukpipat, Wiphat; Komeda, Hidenobu; Prasertsan, Poonsuk; Asano, Yasuhisa

    2017-01-01

    Meyerozyma caribbica strain 5XY2, which was isolated from an alcohol fermentation starter in Thailand, was found to catabolize l-arabinose as well as d-glucose and d-xylose. The highest production amounts of ethanol from d-glucose, xylitol from d-xylose, and l-arabitol from l-arabinose were 0.45 g/g d-glucose, 0.60 g/g d-xylose, and 0.61 g/g l-arabinose with 21.7 g/L ethanol, 20.2 g/L xylitol, and 30.3 g/l l-arabitol, respectively. The enzyme with l-arabitol dehydrogenase (LAD) activity was purified from the strain and found to exhibit broad specificity to polyols, such as xylitol, d-sorbitol, ribitol, and l-arabitol. Xylitol was the preferred substrate with Km=16.1 mM and kcat/Km=67.0 min(-1)mM(-1), while l-arabitol was also a substrate for the enzyme with Km=31.1 mM and kcat/Km=6.5 min(-1) mM(-1). Therefore, this enzyme from M. caribbica was named xylitol dehydrogenase (McXDH). McXDH had an optimum temperature and pH at 40°C and 9.5, respectively. The McXDH gene included a coding sequence of 1086 bp encoding a putative 362 amino acid protein of 39 kDa with an apparent homopentamer structure. Native McXDH and recombinant McXDH exhibited relative activities toward l-arabitol of approximately 20% that toward xylitol, suggesting the applicability of this enzyme with the functions of XDH and LAD to the development of pentose-fermenting Saccharomyces cerevisiae.

  4. 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.

  5. Comparative genomics of aldehyde dehydrogenase 5a1 (succinate semialdehyde dehydrogenase and accumulation of gamma-hydroxybutyrate associated with its deficiency

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    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.

  6. 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.

  7. 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.

  8. The PduQ enzyme is an alcohol dehydrogenase used to recycle NAD+ internally within the Pdu microcompartment of Salmonella enterica.

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    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.

  9. Recovery of electric energy from formate by using a recombinant strain of Escherichia coli.

    Science.gov (United States)

    Ojima, Yoshihiro; Kawata, Teruyoshi; Matsuo, Nahoko; Nishinoue, Yosuke; Taya, Masahito

    2014-10-01

    Recombinant Escherichia coli cells were applied for the recovery of electric energy from formate. Initially, the fdh gene, which encodes formate dehydrogenase (FDH) of Mycobacterium vaccae, was introduced into E. coli cells to allow efficient degradation of formate. The constructed microbial fuel cell (MFC) with E. coli BW25113 cells carrying fdh gene showed appreciable generation of current density in the presence of formate as a substrate. Current density and polarization curves revealed that the performance of MFC under examined conditions was limited by the electron transfer from bulk liquid to the electrode surface; accordingly, agitation resulted in an increase in the current density and achieved a coulombic efficiency of 21.7 % on the basis of formate consumed. Thus, gene recombination enables E. coli cells to utilize formate as a fuel for MFC.

  10. Soil dehydrogenase in a land degradation-rehabilitation gradient: observations from a savanna site with a wet/dry seasonal cycle

    OpenAIRE

    Doi, Ryoichi; Ranamukhaarachchi, Senaratne Leelananda

    2013-01-01

    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 moistu...

  11. The human L-threonine 3-dehydrogenase gene is an expressed pseudogene

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    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.

  12. 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.

  13. Engineering of alanine dehydrogenase from Bacillus subtilis for novel cofactor specificity.

    Science.gov (United States)

    Lerchner, Alexandra; Jarasch, Alexander; Skerra, Arne

    2016-09-01

    The l-alanine dehydrogenase of Bacillus subtilis (BasAlaDH), which is strictly dependent on NADH as redox cofactor, efficiently catalyzes the reductive amination of pyruvate to l-alanine using ammonia as amino group donor. To enable application of BasAlaDH as regenerating enzyme in coupled reactions with NADPH-dependent alcohol dehydrogenases, we alterated its cofactor specificity from NADH to NADPH via protein engineering. By introducing two amino acid exchanges, D196A and L197R, high catalytic efficiency for NADPH was achieved, with kcat /KM  = 54.1 µM(-1)  Min(-1) (KM  = 32 ± 3 µM; kcat  = 1,730 ± 39 Min(-1) ), almost the same as the wild-type enzyme for NADH (kcat /KM  = 59.9 µM(-1)  Min(-1) ; KM  = 14 ± 2 µM; kcat  = 838 ± 21 Min(-1) ). Conversely, recognition of NADH was much diminished in the mutated enzyme (kcat /KM  = 3 µM(-1)  Min(-1) ). BasAlaDH(D196A/L197R) was applied in a coupled oxidation/transamination reaction of the chiral dicyclic dialcohol isosorbide to its diamines, catalyzed by Ralstonia sp. alcohol dehydrogenase and Paracoccus denitrificans ω-aminotransferase, thus allowing recycling of the two cosubstrates NADP(+) and l-Ala. An excellent cofactor regeneration with recycling factors of 33 for NADP(+) and 13 for l-Ala was observed with the engineered BasAlaDH in a small-scale biocatalysis experiment. This opens a biocatalytic route to novel building blocks for industrial high-performance polymers.

  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. Acute and chronic effects of diazinon on the activities of three dehydrogenases in the digestive system of a freshwater teleost fish Channa punctatus.

    Science.gov (United States)

    Sastry, K V; Malik, P V

    1982-01-01

    The effect of acute exposure to LC50 for 96 h (3.1 mg/l) and chronic exposure to a sublethal concentration (0.31 mg/l) of diazinon has been studied in the liver, stomach, intestine and pyloric ceca of a freshwater teleost fish, Channa punctatus. In acute exposure succinate dehydrogenase (SDH) activity was elevated in intestine and pyloric ceca. No alteration was noted in lactate dehydrogenase activity but pyruvate dehydrogenase was inhibited in pyloric ceca. Chronic exposure resulted in inhibition of the activities of the three dehydrogenases in all the four parts at both intervals.

  16. 20 alpha-hydroxysteroid dehydrogenase expression in a murine virus-induced myeloproliferative syndrome.

    Science.gov (United States)

    Marcovistz, R; Le Bousse-Kerdiles, M C; Maillere, B; Smadja-Joffe, F; Poirrier, V; Jasmin, C

    1991-11-01

    The myeloproliferative sarcoma virus (MPSV) infection in DBA/2 mice leads to important quantitative and qualitative changes in their hemopoiesis. These findings suggest a disturbance in the production and action of a certain hemopoietic factor similar to IL3. Here, we show that the level of the 20 alpha-hydroxysteroid dehydrogenase (20 alpha-SDH) expression, which can be induced by IL3, is dramatically increased in spleen and thymus of MPSV-infected mice. Our results suggest that quantification of 20 alpha-SDH activity can be used to indicate abnormal production of a growth factor similar to IL3 in hemopoietic system diseases.

  17. Retinol dehydrogenase-10 regulates pancreas organogenesis and endocrine cell differentiation via paracrine retinoic acid signalling

    DEFF Research Database (Denmark)

    Arregi, Igor; Climent, Maria; Iliev, Dobromir;

    2016-01-01

    Vitamin A-derived retinoic acid (RA) signals are critical for the development of several organs, including the pancreas. However, the tissue-specific control of RA synthesis in organ and cell lineage development has only poorly been addressed in vivo. Here we show that Retinol dehydrogenase-10 (Rdh...... and a hypoplastic ventral pancreas with retarded tubulogenesis and branching. Conditional disruption of Rdh10 from the endoderm caused increased mortality, reduced body weight and lowered blood glucose levels after birth. Endodermal Rdh10 deficiency led to a smaller dorsal pancreas with a reduced density of early...

  18. Cariogenicity of a lactate dehydrogenase-deficient mutant of Streptococcus mutans serotype c in gnotobiotic rats.

    OpenAIRE

    FitzGerald, R.J.; Adams, B. O.; Sandham, H. J.; Abhyankar, S

    1989-01-01

    A lactate dehydrogenase-deficient (Ldh-) mutant of a human isolate of Streptococcus mutans serotype c was tested in a gnotobiotic rat caries model. Compared with the wild-type Ldh-positive (Ldh+) strains, it was significantly (alpha less than or equal to 0.005) less cariogenic in experiments with two different sublines of Sprague-Dawley rats. The Ldh- mutant strain 044 colonized the oral cavity of the test animals to the same extent as its parent strain 041, although its initial implantation ...

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

    Science.gov (United States)

    Touma, E; Rashed, M; Vianey-Saban, C; Sakr, A; Divry, P; Gregersen, N; Andresen, B

    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 long 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.

 PMID:11124787

  20. Evidence of redox imbalance in a patient with succinic semialdehyde dehydrogenase deficiency

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    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.