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Sample records for alcohol dehydrogenase inhibition

  1. Inhibition by ethanol, acetaldehyde and trifluoroethanol of reactions catalysed by yeast and horse liver alcohol dehydrogenases.

    PubMed Central

    Dickenson, C J; Dickinson, F M

    1978-01-01

    1. Produced inhibition by ethanol of the acetaldehyde-NADH reaction, catalysed by the alcohol dehydrogenases from yeast and horse liver, was studied at 25 degrees C and pH 6-9. 2. The results with yeast alcohol dehydrogenase are generally consistent with the preferred-pathway mechanism proposed previously [Dickenson & Dickinson (1975) Biochem. J. 147, 303-311]. The observed hyperbolic inhibition by ethanol of the maximum rate of acetaldehyde reduction confirms the existence of the alternative pathway involving an enzyme-ethanol complex. 3. The maximum rate of acetaldehyde reduction with horse liver alcohol dehydrogenase is also subject to hyperbolic inhibition by ethanol. 4. The measured inhibition constants for ethanol provide some of the information required in the determination of the dissociation constant for ethanol from the active ternary complex. 5. Product inhibition by acetaldehyde of the ethanol-NAD+ reaction with yeast alcohol dehydrogenase was examined briefly. The results are consistent with the proposed mechanism. However, the nature of the inhibition of the maximum rate cannot be determined within the accessible range of experimental conditions. 6. Inhibition of yeast alcohol dehydrogenase by trifluoroethanol was studied at 25 degrees C and pH 6-10. The inhibition was competitive with respect to ethanol in the ethanol-NAD+ reaction. Estimates were made of the dissociation constant for trifluoroethanol from the enzyme-NAD+-trifluoroethanol complex in the range pH6-10. PMID:208509

  2. Alcohol Dehydrogenase from Methylobacterium organophilum

    PubMed Central

    Wolf, H. J.; Hanson, R. S.

    1978-01-01

    The alcohol dehydrogenase from Methylobacterium organophilum, a facultative methane-oxidizing bacterium, has been purified to homogeneity as indicated by sodium dodecyl sulfate-gel electrophoresis. It has several properties in common with the alcohol dehydrogenases from other methylotrophic bacteria. The active enzyme is a dimeric protein, both subunits having molecular weights of about 62,000. The enzyme exhibits broad substrate specificity for primary alcohols and catalyzes the two-step oxidation of methanol to formate. The apparent Michaelis constants of the enzyme are 2.9 × 10−5 M for methanol and 8.2 × 10−5 M for formaldehyde. Activity of the purified enzyme is dependent on phenazine methosulfate. Certain characteristics of this enzyme distinguish it from the other alcohol dehydrogenases of other methylotrophic bacteria. Ammonia is not required for, but stimulates the activity of newly purified enzyme. An absolute dependence on ammonia develops after storage of the purified enzyme. Activity is not inhibited by phosphate. The fluorescence spectrum of the enzyme indicates that it and the cofactor associated with it may be chemically different from the alcohol dehydrogenases from other methylotrophic bacteria. The alcohol dehydrogenases of Hyphomicrobium WC-65, Pseudomonas methanica, Methylosinus trichosporium, and several facultative methylotrophs are serologically related to the enzyme purified in this study. The enzymes of Rhodopseudomonas acidophila and of organisms of the Methylococcus group did not cross-react with the antiserum prepared against the alcohol dehydrogenase of M. organophilum. Images PMID:80974

  3. Nitric oxide inhibition of alcohol dehydrogenase in fresh-cut apples ( Malus domestica Borkh).

    PubMed

    Amissah, Joris Gerald Niilante; Hotchkiss, Joseph H; Watkins, Chris B

    2013-11-20

    The effects of nitric oxide (NO) and nitrite treatment on alcohol dehydrogenase activity and the shelf life of apple tissue were investigated. Fresh-cut apple slices were stored for 2 days at 6 °C in 0.25-1% NO (v/v, balance N2) or 100% N2 atmospheres. Slices were also treated with 1% NO or 2 mM sodium nitrite (NaNO2) for 20 min, stored for 6 weeks in 100% N2 at 6 °C, and analyzed for acetaldehyde, ethanol, and ethyl acetate accumulation, firmness, and color. Compared with N2 or deionized water controls, treatment with 1% NO or 2 mM NaNO2 inhibited ethanol accumulation, whereas that of acetaldehyde increased. Ethyl acetate accumulation was inhibited only by NO. Slice firmness was not affected by NO or NaNO2 treatment, but slices were darker than the untreated controls. NO and nitrite may extend the shelf life of fresh-cut produce with low concentrations of phenolic compounds.

  4. 11β-hydroxysteroid dehydrogenase inhibition as a new potential therapeutic target for alcohol abuse

    PubMed Central

    Sanna, P P; Kawamura, T; Chen, J; Koob, G F; Roberts, A J; Vendruscolo, L F; Repunte-Canonigo, V

    2016-01-01

    The identification of new and more effective treatments for alcohol abuse remains a priority. Alcohol intake activates glucocorticoids, which have a key role in alcohol's reinforcing properties. Glucocorticoid effects are modulated in part by the activity of 11β-hydroxysteroid dehydrogenases (11β-HSD) acting as pre-receptors. Here, we tested the effects on alcohol intake of the 11β-HSD inhibitor carbenoxolone (CBX, 18β-glycyrrhetinic acid 3β-O-hemisuccinate), which has been extensively used in the clinic for the treatment of gastritis and peptic ulcer and is active on both 11β-HSD1 and 11β-HSD2 isoforms. We observed that CBX reduces both baseline and excessive drinking in rats and mice. The CBX diastereomer 18α-glycyrrhetinic acid 3β-O-hemisuccinate (αCBX), which we found to be selective for 11β-HSD2, was also effective in reducing alcohol drinking in mice. Thus, 11β-HSD inhibitors may be a promising new class of candidate alcohol abuse medications, and existing 11β-HSD inhibitor drugs may be potentially re-purposed for alcohol abuse treatment. PMID:26978742

  5. Inhibition of human alcohol and aldehyde dehydrogenases by cimetidine and assessment of its effects on ethanol metabolism.

    PubMed

    Lai, Ching-Long; Li, Yeung-Pin; Liu, Chiu-Ming; Hsieh, Hsiu-Shan; Yin, Shih-Jiun

    2013-02-25

    Previous studies have reported that cimetidine, an H2-receptor antagonist used to treat gastric and duodenal ulcers, can inhibit alcohol dehydrogenases (ADHs) and ethanol metabolism. Human alcohol dehydrogenases and aldehyde dehydrogenases (ALDHs), the principal enzymes responsible for metabolism of ethanol, are complex enzyme families that exhibit functional polymorphisms among ethnic groups and distinct tissue distributions. We investigated the inhibition by cimetidine of alcohol oxidation by recombinant human ADH1A, ADH1B1, ADH1B2, ADH1B3, ADH1C1, ADH1C2, ADH2, and ADH4, and aldehyde oxidation by ALDH1A1 and ALDH2 at pH 7.5 and a cytosolic NAD(+) concentration. Cimetidine acted as competitive or noncompetitive inhibitors for the ADH and ALDH isozymes/allozymes with near mM inhibition constants. The metabolic interactions between cimetidine and ethanol/acetaldehyde were assessed by computer simulation using the inhibition equations and the determined kinetic constants. At therapeutic drug levels (0.015 mM) and physiologically relevant concentrations of ethanol (10 mM) and acetaldehyde (10 μM) in target tissues, cimetidine could weakly inhibit (<5%) the activities of ADH1B2 and ADH1B3 in liver, ADH2 in liver and small intestine, ADH4 in stomach, and ALDH1A1 in the three tissues, but not significantly affect ADH1A, ADH1B1, ADH1C1/2, or ALDH2. At higher drug levels, which may accumulate in cells (0.2 mM), the activities of the weakly-inhibited enzymes may be decreased more significantly. The quantitative effects of cimetidine on metabolism of ethanol and other physiological substrates of ADHs need further investigation.

  6. Inhibition of human alcohol and aldehyde dehydrogenases by acetaminophen: Assessment of the effects on first-pass metabolism of ethanol.

    PubMed

    Lee, Yung-Pin; Liao, Jian-Tong; Cheng, Ya-Wen; Wu, Ting-Lun; Lee, Shou-Lun; Liu, Jong-Kang; Yin, Shih-Jiun

    2013-11-01

    Acetaminophen is one of the most widely used over-the-counter analgesic, antipyretic medications. Use of acetaminophen and alcohol are commonly associated. Previous studies showed that acetaminophen might affect bioavailability of ethanol by inhibiting gastric alcohol dehydrogenase (ADH). However, potential inhibitions by acetaminophen of first-pass metabolism (FPM) of ethanol, catalyzed by the human ADH family and by relevant aldehyde dehydrogenase (ALDH) isozymes, remain undefined. ADH and ALDH both exhibit racially distinct allozymes and tissue-specific distribution of isozymes, and are principal enzymes responsible for ethanol metabolism in humans. In this study, we investigated acetaminophen inhibition of ethanol oxidation with recombinant human ADH1A, ADH1B1, ADH1B2, ADH1B3, ADH1C1, ADH1C2, ADH2, and ADH4, and inhibition of acetaldehyde oxidation with recombinant human ALDH1A1 and ALDH2. The investigations were done at near physiological pH 7.5 and with a cytoplasmic coenzyme concentration of 0.5 mM NAD(+). Acetaminophen acted as a noncompetitive inhibitor for ADH enzymes, with the slope inhibition constants (Kis) ranging from 0.90 mM (ADH2) to 20 mM (ADH1A), and the intercept inhibition constants (Kii) ranging from 1.4 mM (ADH1C allozymes) to 19 mM (ADH1A). Acetaminophen exhibited noncompetitive inhibition for ALDH2 (Kis = 3.0 mM and Kii = 2.2 mM), but competitive inhibition for ALDH1A1 (Kis = 0.96 mM). The metabolic interactions between acetaminophen and ethanol/acetaldehyde were assessed by computer simulation using inhibition equations and the determined kinetic constants. At therapeutic to subtoxic plasma levels of acetaminophen (i.e., 0.2-0.5 mM) and physiologically relevant concentrations of ethanol (10 mM) and acetaldehyde (10 μm) in target tissues, acetaminophen could inhibit ADH1C allozymes (12-26%) and ADH2 (14-28%) in the liver and small intestine, ADH4 (15-31%) in the stomach, and ALDH1A1 (16-33%) and ALDH2 (8.3-19%) in all 3 tissues. The

  7. Oxidation of methanol, ethylene glycol, and isopropanol with human alcohol dehydrogenases and the inhibition by ethanol and 4-methylpyrazole.

    PubMed

    Lee, Shou-Lun; Shih, Hsuan-Ting; Chi, Yu-Chou; Li, Yeung-Pin; Yin, Shih-Jiun

    2011-05-30

    Human alcohol dehydrogenases (ADHs) include multiple isozymes with broad substrate specificity and ethnic distinct allozymes. ADH catalyzes the rate-limiting step in metabolism of various primary and secondary aliphatic alcohols. The oxidation of common toxic alcohols, that is, methanol, ethylene glycol, and isopropanol by the human ADHs remains poorly understood. Kinetic studies were performed in 0.1M sodium phosphate buffer, at pH 7.5 and 25°C, containing 0.5 mM NAD(+) and varied concentrations of substrate. K(M) values for ethanol with recombinant human class I ADH1A, ADH1B1, ADH1B2, ADH1B3, ADH1C1, and ADH1C2, and class II ADH2 and class IV ADH4 were determined to be in the range of 0.12-57 mM, for methanol to be 2.0-3500 mM, for ethylene glycol to be 4.3-2600mM, and for isopropanol to be 0.73-3400 mM. ADH1B3 appeared to be inactive toward ethylene glycol, and ADH2 and ADH4, inactive with methanol. The variations for V(max) for the toxic alcohols were much less than that of the K(M) across the ADH family. 4-Methylpyrazole (4MP) was a competitive inhibitor with respect to ethanol for ADH1A, ADH1B1, ADH1B2, ADH1C1 and ADH1C2, and a noncompetitive inhibitor for ADH1B3, ADH2 and ADH4, with the slope inhibition constants (K(is)) for the whole family being 0.062-960 μM and the intercept inhibition constants (K(ii)), 33-3000 μM. Computer simulation studies using inhibition equations in the presence of alternate substrate ethanol and of dead-end inhibitor 4MP with the determined corresponding kinetic parameters for ADH family, indicate that the oxidation of the toxic alcohols up to 50mM are largely inhibited by 20 mM ethanol or by 50 μM 4MP with some exceptions. The above findings provide an enzymological basis for clinical treatment of methanol and ethylene glycol poisoning by 4MP or ethanol with pharmacogenetic perspectives.

  8. Michael hydratase alcohol dehydrogenase or just alcohol dehydrogenase?

    PubMed Central

    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 spectrophotometric assay and an activity staining in a native gel of the dehydrogenase. New insights in the recently discovered organocatalytic Michael addition of water led to the conclusion that the previously performed experiments to identify MhyADH as a bi-functional enzyme and their results need to be reconsidered and the reliability of the methodology used needs to be critically evaluated. PMID:24949265

  9. Kinetic and mechanistic studies of methylated liver alcohol dehydrogenase.

    PubMed Central

    Tsai, C S

    1978-01-01

    Reductive methylation of lysine residues activates liver alcohol dehydrogenase in the oxidation of primary alcohols, but decreases the activity of the enzyme towards secondary alcohols. The modification also desensitizes the dehydrogenase to substrate inhibition at high alcohol concentrations. Steady-state kinetic studies of methylated liver alcohol dehydrogenase over a wide range of alcohol concentrations suggest that alcohol oxidation proceeds via a random addition of coenzyme and substrate with a pathway for the formation of the productive enzyme-NADH-alcohol complex. To facilitate the analyses of the effects of methylation on liver alcohol dehydrogenase and factors affecting them, new operational kinetic parameters to describe the results at high substrate concentration were introduced. The changes in the dehydrogenase activity on alkylation were found to be associated with changes in the maximum velocities that are affected by the hydrophobicity of alkyl groups introduced at lysine residues. The desensitization of alkylated liver alcohol dehydrogenase to substrate inhibition is identified with a decrease in inhibitory Michaelis constants for alcohols and this is favoured by the steric effects of substituents at the lysine residues. PMID:697732

  10. Inhibitory effect of disulfiram (Antabuse) on alcohol dehydrogenase activity.

    PubMed

    Carper, W R; Dorey, R C; Beber, J H

    1987-10-01

    We investigated the effect of disulfiram (Antabuse) on the activity of alcohol dehydrogenase (EC 1.1.1.1) in vitro. We observed a time-dependent inhibition of this dehydrogenase by disulfiram and diethyldithiocarbamate similar to that obtained for aldehyde dehydrogenase (EC 1.2.1.3). These results suggest a possible explanation for various side effects observed in the clinical use of Antabuse.

  11. Inhibition of human alcohol and aldehyde dehydrogenases by aspirin and salicylate: assessment of the effects on first-pass metabolism of ethanol.

    PubMed

    Lee, Shou-Lun; Lee, Yung-Pin; Wu, Min-Li; Chi, Yu-Chou; Liu, Chiu-Ming; Lai, Ching-Long; Yin, Shih-Jiun

    2015-05-01

    Previous studies have reported that aspirin significantly reduced the first-pass metabolism (FPM) of ethanol in humans thereby increasing adverse effects of alcohol. The underlying causes, however, remain poorly understood. Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH), principal enzymes responsible for metabolism of ethanol, are complex enzyme families that exhibit functional polymorphisms among ethnic groups and distinct tissue distributions. We investigated the inhibition profiles by aspirin and its major metabolite salicylate of ethanol oxidation by recombinant human ADH1A, ADH1B1, ADH1B2, ADH1B3, ADH1C1, ADH1C2, ADH2, and ADH4, and acetaldehyde oxidation by ALDH1A1 and ALDH2, at pH 7.5 and 0.5 mM NAD(+). Competitive inhibition pattern was found to be a predominant type among the ADHs and ALDHs studied, although noncompetitive and uncompetitive inhibitions were also detected in a few cases. The inhibition constants of salicylate for the ADHs and ALDHs were considerably lower than that of aspirin with the exception of ADH1A that can be ascribed to a substitution of Ala-93 at the bottom of substrate pocket as revealed by molecular docking experiments. Kinetic inhibition equation-based simulations show at higher therapeutic levels of blood plasma salicylate (1.5 mM) that the decrease of activities at 2-10 mM ethanol for ADH1A/ADH2 and ADH1B2/ADH1B3 are predicted to be 75-86% and 31-52%, respectively, and that the activity decline for ALDH1A1 and ALDH2 at 10-50 μM acetaldehyde to be 62-73%. Our findings suggest that salicylate may substantially inhibit hepatic FPM of alcohol at both the ADH and ALDH steps when concurrent intaking aspirin.

  12. [Effects of H2-blockers on alcohol dehydrogenase (ADH) activity].

    PubMed

    Jelski, Wojciech; Orywal, Karolina; Szmitkowski, Maciej

    2008-12-01

    First-pass metabolism (FPM) of alcohol is demonstrated by lower blood alcohol concentrations after oral than intravenous administration of the same dose. FPM occurs predominantly in the stomach and has been attributed to class IV of alcohol dehydrogenase (ADH) isoenzyme localizated in the gastric mucosa. A number of factors that influence on gastric ADH activity and thereby modulate FPM have been identified. These include age, sex, ethnicity, concentrations and amounts of alcohol consumed and drugs. Several H2-receptor antagonists, including cimetidine and ranitidine, inhibit gastric ADH activity and reduce FPM, resulting in higher blood alcohol concentrations after H2-blockers administration.

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

    PubMed

    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.

  14. Fundamental molecular differences between alcohol dehydrogenase classes.

    PubMed Central

    Danielsson, O; Atrian, S; Luque, T; Hjelmqvist, L; Gonzàlez-Duarte, R; Jörnvall, H

    1994-01-01

    Two types of alcohol dehydrogenase in separate protein families are the "medium-chain" zinc enzymes (including the classical liver and yeast forms) and the "short-chain" enzymes (including the insect form). Although the medium-chain family has been characterized in prokaryotes and many eukaryotes (fungi, plants, cephalopods, and vertebrates), insects have seemed to possess only the short-chain enzyme. We have now also characterized a medium-chain alcohol dehydrogenase in Drosophila. The enzyme is identical to insect octanol dehydrogenase. It is a typical class III alcohol dehydrogenase, similar to the corresponding human form (70% residue identity), with mostly the same residues involved in substrate and coenzyme interactions. Changes that do occur are conservative, but Phe-51 is of functional interest in relation to decreased coenzyme binding and increased overall activity. Extra residues versus the human enzyme near position 250 affect the coenzyme-binding domain. Enzymatic properties are similar--i.e., very low activity toward ethanol (Km beyond measurement) and high selectivity for formaldehyde/glutathione (S-hydroxymethylglutathione; kcat/Km = 160,000 min-1.mM-1). Between the present class III and the ethanol-active class I enzymes, however, patterns of variability differ greatly, highlighting fundamentally separate molecular properties of these two alcohol dehydrogenases, with class III resembling enzymes in general and class I showing high variation. The gene coding for the Drosophila class III enzyme produces an mRNA of about 1.36 kb that is present at all developmental stages of the fly, compatible with the constitutive nature of the vertebrate enzyme. Taken together, the results bridge a previously apparent gap in the distribution of medium-chain alcohol dehydrogenases and establish a strictly conserved class III enzyme, consistent with an important role for this enzyme in cellular metabolism. Images PMID:8197167

  15. Inhibition of alcohol dehydrogenase after 2-propanol exposure in different geographic races of Drosophila mojavensis: lack of evidence for selection at the Adh-2 locus.

    PubMed

    Pfeiler, Edward; Reed, Laura K; Markow, Therese A

    2005-03-15

    High frequencies of the fast allele of alcohol dehydrogenase-2 (Adh-2F) are found in populations of Drosophila mojavensis that inhabit the Baja California peninsula (race BII) whereas the slow allele (Adh-2S) predominates at most other localities within the species' geographic range. Race BII flies utilize necrotic tissue of pitaya agria cactus (Stenocereus gummosus) which contains high levels of 2-propanol, whereas flies from most other localities utilize different cactus hosts in which 2-propanol levels are low. To test if 2-propanol acts as a selective force on Adh-2 genotype, or whether some other yet undetermined genetic factor is responsible, mature males of D. mojavensis lines derived from the Grand Canyon (race A) and Santa Catalina Island (race C), each with individuals homozygous for Adh-2F and Adh-2S, were exposed to 2-propanol for 24 h and ADH-2 specific activity was then determined on each genotype. Flies from five other localities homozygous for either the fast or slow allele also were examined. Results for all reported races of D. mojavensis were obtained. 2-propanol exposure inhibited ADH-2 specific activity in both genotypes from all localities, but inhibition was significantly less in two populations of race BII flies homozygous for Adh-2F. When F/F and S/S genotypes in flies from the same locality were compared, both genotypes showed high 2-propanol inhibition that was not statistically different, indicating that the F/F genotype alone does not provide a benefit against the inhibitory effects of 2-propanol. ADH-1 activity in female ovaries was inhibited less by 2-propanol than ADH-2. These results do not support the hypothesis that 2-propanol acts as a selective factor favoring the Adh-2F allele.

  16. Ethanol oxidation and the inhibition by drugs in human liver, stomach and small intestine: Quantitative assessment with numerical organ modeling of alcohol dehydrogenase isozymes.

    PubMed

    Chi, Yu-Chou; Lee, Shou-Lun; Lai, Ching-Long; Lee, Yung-Pin; Lee, Shiao-Pieng; Chiang, Chien-Ping; Yin, Shih-Jiun

    2016-10-25

    Alcohol dehydrogenase (ADH) is the principal enzyme responsible for metabolism of ethanol. Human ADH constitutes a complex isozyme family with striking variations in kinetic function and tissue distribution. Liver and gastrointestinal tract are the major sites for first-pass metabolism (FPM). Their relative contributions to alcohol FPM and degrees of the inhibitions by aspirin and its metabolite salicylate, acetaminophen and cimetidine remain controversial. To address this issue, mathematical organ modeling of ethanol-oxidizing activities in target tissues and that of the ethanol-drug interactions were constructed by linear combination of the corresponding numerical rate equations of tissue constituent ADH isozymes with the documented isozyme protein contents, kinetic parameters for ethanol oxidation and the drug inhibitions of ADH isozymes/allozymes that were determined in 0.1 M sodium phosphate at pH 7.5 and 25 °C containing 0.5 mM NAD(+). The organ simulations reveal that the ADH activities in mucosae of the stomach, duodenum and jejunum with ADH1C*1/*1 genotype are less than 1%, respectively, that of the ADH1B*1/*1-ADH1C*1/*1 liver at 1-200 mM ethanol, indicating that liver is major site of the FPM. The apparent hepatic KM and Vmax for ethanol oxidation are simulated to be 0.093 ± 0.019 mM and 4.0 ± 0.1 mmol/min, respectively. At 95% clearance in liver, the logarithmic average sinusoidal ethanol concentration is determined to be 0.80 mM in accordance with the flow-limited gradient perfusion model. The organ simulations indicate that higher therapeutic acetaminophen (0.5 mM) inhibits 16% of ADH1B*1/*1 hepatic ADH activity at 2-20 mM ethanol and that therapeutic salicylate (1.5 mM) inhibits 30-31% of the ADH1B*2/*2 activity, suggesting potential significant inhibitions of ethanol FPM in these allelotypes. The result provides systematic evaluations and predictions by computer simulation on potential ethanol FPM in target tissues and hepatic

  17. Molecular characterization of benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II of Acinetobacter calcoaceticus.

    PubMed Central

    Gillooly, D J; Robertson, A G; Fewson, C A

    1998-01-01

    The nucleotide sequences of xylB and xylC from Acinetobacter calcoaceticus, the genes encoding benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II, were determined. The complete nucleotide sequence indicates that these two genes form part of an operon and this was supported by heterologous expression and physiological studies. Benzaldehyde dehydrogenase II is a 51654 Da protein with 484 amino acids per subunit and it is typical of other prokaryotic and eukaryotic aldehyde dehydrogenases. Benzyl alcohol dehydrogenase has a subunit Mr of 38923 consisting of 370 amino acids, it stereospecifically transfers the proR hydride of NADH, and it is a member of the family of zinc-dependent long-chain alcohol dehydrogenases. The enzyme appears to be more similar to animal and higher-plant alcohol dehydrogenases than it is to most other microbial alcohol dehydrogenases. Residue His-51 of zinc-dependent alcohol dehydrogenases is thought to be necessary as a general base for catalysis in this category of alcohol dehydrogenases. However, this residue was found to be replaced in benzyl alcohol dehydrogenase from A. calcoaceticus by an isoleucine, and the introduction of a histidine residue in this position did not alter the kinetic coefficients, pH optimum or substrate specificity of the enzyme. Other workers have shown that His-51 is also absent from the TOL-plasmid-encoded benzyl alcohol dehydrogenase of Pseudomonas putida and so these two closely related enzymes presumably have a catalytic mechanism that differs from that of the archetypal zinc-dependent alcohol dehydrogenases. PMID:9494109

  18. Fast internal dynamics in alcohol dehydrogenase

    SciTech Connect

    Monkenbusch, M.; Stadler, A. Biehl, R.; Richter, D.; Ollivier, J.; Zamponi, M.

    2015-08-21

    Large-scale domain motions in alcohol dehydrogenase (ADH) have been observed previously by neutron spin-echo spectroscopy (NSE). We have extended the investigation on the dynamics of ADH in solution by using high-resolution neutron time-of-flight (TOF) and neutron backscattering (BS) spectroscopy in the incoherent scattering range. The observed hydrogen dynamics were interpreted in terms of three mobility classes, which allowed a simultaneous description of the measured TOF and BS spectra. In addition to the slow global protein diffusion and domain motions observed by NSE, a fast internal process could be identified. Around one third of the protons in ADH participate in the fast localized diffusive motion. The diffusion coefficient of the fast internal motions is around two third of the value of the surrounding D{sub 2}O solvent. It is tempting to associate the fast internal process with solvent exposed amino acid residues with dangling side chains.

  19. Mammalian class IV alcohol dehydrogenase (stomach alcohol dehydrogenase): structure, origin, and correlation with enzymology.

    PubMed Central

    Parés, X; Cederlund, E; Moreno, A; Hjelmqvist, L; Farrés, J; Jörnvall, H

    1994-01-01

    The structure of a mammalian class IV alcohol dehydrogenase has been determined by peptide analysis of the protein isolated from rat stomach. The structure indicates that the enzyme constitutes a separate alcohol dehydrogenase class, in agreement with the distinct enzymatic properties; the class IV enzyme is somewhat closer to class I (the "classical" liver alcohol dehydrogenase; approximately 68% residue identities) than to the other classes (II, III, and V; approximately 60% residue identities), suggesting that class IV might have originated through duplication of an early vertebrate class I gene. The activity of the class IV protein toward ethanol is even higher than that of the classical liver enzyme. Both Km and kcat values are high, the latter being the highest of any class characterized so far. Structurally, these properties are correlated with replacements at the active site, affecting both substrate and coenzyme binding. In particular, Ala-294 (instead of valine) results in increased space in the middle section of the substrate cleft, Gly-47 (instead of a basic residue) results in decreased charge interactions with the coenzyme pyrophosphate, and Tyr-363 (instead of a basic residue) may also affect coenzyme binding. In combination, these exchanges are compatible with a promotion of the off dissociation and an increased turnover rate. In contrast, residues at the inner part of the substrate cleft are bulky, accounting for low activity toward secondary alcohols and cyclohexanol. Exchanges at positions 259-261 involve minor shifts in glycine residues at a reverse turn in the coenzyme-binding fold. Clearly, class IV is distinct in structure, ethanol turnover, stomach expression, and possible emergence from class I. PMID:8127901

  20. Yeast Alcohol Dehydrogenase Structure and Catalysis

    PubMed Central

    2015-01-01

    Yeast (Saccharomyces cerevisiae) alcohol dehydrogenase I (ADH1) is the constitutive enzyme that reduces acetaldehyde to ethanol during the fermentation of glucose. ADH1 is a homotetramer of subunits with 347 amino acid residues. A structure for ADH1 was determined by X-ray crystallography at 2.4 Å resolution. The asymmetric unit contains four different subunits, arranged as similar dimers named AB and CD. The unit cell contains two different tetramers made up of “back-to-back” dimers, AB:AB and CD:CD. The A and C subunits in each dimer are structurally similar, with a closed conformation, bound coenzyme, and the oxygen of 2,2,2-trifluoroethanol ligated to the catalytic zinc in the classical tetrahedral coordination with Cys-43, Cys-153, and His-66. In contrast, the B and D subunits have an open conformation with no bound coenzyme, and the catalytic zinc has an alternative, inverted coordination with Cys-43, Cys-153, His-66, and the carboxylate of Glu-67. The asymmetry in the dimeric subunits of the tetramer provides two structures that appear to be relevant for the catalytic mechanism. The alternative coordination of the zinc may represent an intermediate in the mechanism of displacement of the zinc-bound water with alcohol or aldehyde substrates. Substitution of Glu-67 with Gln-67 decreases the catalytic efficiency by 100-fold. Previous studies of structural modeling, evolutionary relationships, substrate specificity, chemical modification, and site-directed mutagenesis are interpreted more fully with the three-dimensional structure. PMID:25157460

  1. Hepatic alcohol dehydrogenase activity in alcoholic subjects with and without liver disease.

    PubMed Central

    Vidal, F; Perez, J; Morancho, J; Pinto, B; Richart, C

    1990-01-01

    Alcohol dehydrogenase activity was measured in samples of liver tissue from a group of alcoholic and non-alcoholic subjects to determine whether decreased liver alcohol dehydrogenase activity is a consequence of ethanol consumption or liver damage. The alcoholic patients were classified further into the following groups: control subjects with no liver disease (group 1), subjects with non-cirrhotic liver disease (group 2), and subjects with cirrhotic liver disease (group 3). The non-alcoholic subjects were also divided, using the same criteria, into groups 4, 5, and 6, respectively. The analysis of the results showed no significant differences when mean alcohol dehydrogenase activities of alcoholic and non-alcoholic patients with similar degrees of liver pathology were compared (groups 1 v 4, 2 v 5, and 3 v 6. Alcohol dehydrogenase activity was, however, severely reduced in patients with liver disease compared with control subjects. Our findings suggest that alcohol consumption does not modify hepatic alcohol dehydrogenase activity. The reduction in specific alcohol dehydrogenase activity in alcoholic liver disease is a consequence of liver damage. PMID:2379876

  2. Contribution of liver alcohol dehydrogenase to metabolism of alcohols in rats.

    PubMed

    Plapp, Bryce V; Leidal, Kevin G; Murch, Bruce P; Green, David W

    2015-06-05

    The kinetics of oxidation of various alcohols by purified rat liver alcohol dehydrogenase (ADH) were compared with the kinetics of elimination of the alcohols in rats in order to investigate the roles of ADH and other factors that contribute to the rates of metabolism of alcohols. Primary alcohols (ethanol, 1-propanol, 1-butanol, 2-methyl-1-propanol, 3-methyl-1-butanol) and diols (1,3-propanediol, 1,3-butanediol, 1,4-butanediol, 1,5-pentanediol) were eliminated in rats with zero-order kinetics at doses of 5-20 mmol/kg. Ethanol was eliminated most rapidly, at 7.9 mmol/kgh. Secondary alcohols (2-propanol-d7, 2-propanol, 2-butanol, 3-pentanol, cyclopentanol, cyclohexanol) were eliminated with first order kinetics at doses of 5-10 mmol/kg, and the corresponding ketones were formed and slowly eliminated with zero or first order kinetics. The rates of elimination of various alcohols were inhibited on average 73% (55% for 2-propanol to 90% for ethanol) by 1 mmol/kg of 4-methylpyrazole, a good inhibitor of ADH, indicating a major role for ADH in the metabolism of the alcohols. The Michaelis kinetic constants from in vitro studies (pH 7.3, 37 °C) with isolated rat liver enzyme were used to calculate the expected relative rates of metabolism in rats. The rates of elimination generally increased with increased activity of ADH, but a maximum rate of 6±1 mmol/kg h was observed for the best substrates, suggesting that ADH activity is not solely rate-limiting. Because secondary alcohols only require one NAD(+) for the conversion to ketones whereas primary alcohols require two equivalents of NAD(+) for oxidation to the carboxylic acids, it appears that the rate of oxidation of NADH to NAD(+) is not a major limiting factor for metabolism of these alcohols, but the rate-limiting factors are yet to be identified.

  3. [Effect Of Polyelectrolytes on Catalytic Activity of Alcohol Dehydrogenase].

    PubMed

    Dubrovsky, A V; Musina, E V; Kim, A L; Tikhonenko, S A

    2016-01-01

    Fluorescent and optical spectroscopy were used to study the interaction of alcohol dehydrogenase (ADH) with negatively charged polystyrene sulfonate (PSS) and dextran sulfate (DS), as well as positively charged poly(diallyldimethylammonium) (PDADMA). As found, DS and PDADMA did not affect the structural and catalytic enzyme properties. In contrast, PSS slightly decreased the protein self-fluorescence over 1 h of incubation, which is associated with partial destruction of its quaternary (globular) structure. Investigation of the ADH activity with and without PSS showed its dependency on the incubation time and the PSS presence. Sodium chloride (2.0 M and 0.2 M) or ammonium sulfate (0.1 M) added to the reaction mixture did not completely protect the enzyme quaternary structure from the PSS action. However ammonium sulfate or 0.2 M sodium chloride stabilized the enzyme and partially inhibited the negative PSS effect.

  4. "Enzymogenesis": classical liver alcohol dehydrogenase origin from the glutathione-dependent formaldehyde dehydrogenase line.

    PubMed Central

    Danielsson, O; Jörnvall, H

    1992-01-01

    Analysis of the activity and structure of lower vertebrate alcohol dehydrogenases reveals that relationships between the classical liver and yeast enzymes need not be continuous. Both the ethanol activity of class I-type alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) and the glutathione-dependent formaldehyde activity of the class III-type enzyme [formaldehyde:NAD+ oxidoreductase (glutathione-formylating), EC 1.2.1.1] are present in liver down to at least the stage of bony fishes (cod liver: ethanol activity, 3.4 units/mg of protein in one enzyme; formaldehyde activity, 4.5 units/mg in the major form of another enzyme). Structural analysis of the latter protein reveals it to be a typical class III enzyme, with limited variation from the mammalian form and therefore with stable activity and structure throughout much of the vertebrate lineage. In contrast, the classical alcohol dehydrogenase (the class I enzyme) appears to be the emerging form, first in activity and later also in structure. The class I activity is present already in the piscine line, whereas the overall structural-type enzyme is not observed until amphibians and still more recent vertebrates. Consequently, the class I/III duplicatory origin appears to have arisen from a functional class III form, not a class I form. Therefore, ethanol dehydrogenases from organisms existing before this duplication have origins separate from those leading to the "classical" liver alcohol dehydrogenases. The latter now often occur in isozyme forms from further gene duplications and have a high rate of evolutionary change. The pattern is, however, not simple and we presently find in cod the first evidence for isozymes also within a class III alcohol dehydrogenase. Overall, the results indicate that both of these classes of vertebrate alcohol dehydrogenase are important and suggest a protective metabolic function for the whole enzyme system. Images PMID:1409630

  5. Mutation of Arg-115 of human class III alcohol dehydrogenase: a binding site required for formaldehyde dehydrogenase activity and fatty acid activation.

    PubMed Central

    Engeland, K; Höög, J O; Holmquist, B; Estonius, M; Jörnvall, H; Vallee, B L

    1993-01-01

    The origin of the fatty acid activation and formaldehyde dehydrogenase activity that distinguishes human class III alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) from all other alcohol dehydrogenases has been examined by site-directed mutagenesis of its Arg-115 residue. The Ala- and Asp-115 mutant proteins were expressed in Escherichia coli and purified by affinity chromatography and ion-exchange HPLC. The activities of the recombinant native and mutant enzymes toward ethanol are essentially identical, but mutagenesis greatly decreases the kcat/Km values for glutathione-dependent formaldehyde oxidation. The catalytic efficiency for the Asp variant is < 0.1% that of the unmutated enzyme, due to both a higher Km and a lower kcat value. As with the native enzyme, neither mutant can oxidize methanol, be saturated by ethanol, or be inhibited by 4-methylpyrazole; i.e., they retain these class III characteristics. In contrast, however, their activation by fatty acids, another characteristic unique to class III alcohol dehydrogenase, is markedly attenuated. The Ala mutant is activated only slightly, but the Asp mutant is not activated at all. The results strongly indicate that Arg-115 in class III alcohol dehydrogenase is a component of the binding site for activating fatty acids and is critical for the binding of S-hydroxymethylglutathione in glutathione-dependent formaldehyde dehydrogenase activity. PMID:8460164

  6. [Thermal stability of lactate dehydrogenase and alcohol dehydrogenase incorporated into highly concentrated gels].

    PubMed

    Kulis, Iu Iu

    1979-03-01

    The rate constants for inactivation of lactate dehydrogenase and alcohol dehydrogenase in solution at 65 degrees C (pH 7,5) are 0,72 and 0,013 min-1, respectively. The enzyme incorporation into acrylamide gels results in immobilized enzymes, whose residual activity is 18--25% of the original one. In 6,7% gels the rate of thermal inactivation for lactate dehydrogenase is decreased nearly 10-fold, whereas the inactivation rate for alcohol dehydrogenase is increased 4,6-fold as compared to the soluble enzymes. In 14% and 40% gels the inactivation constants for lactate dehydrogenase are 6,3.10(-3) and 5,9.10(-4) min-1, respectively. In 60% gels the thermal inactivation of lactate dehydrogenase is decelerated 3600-fold as compared to the native enzyme. The enthalpy and enthropy for the inactivation of the native enzyme are equal to 62,8 kcal/mole and 116,9 cal/(mole.grad.) for the native enzyme and those of gel-incorporated (6,7%) enzyme -- 38,7 kcal/mole and 42 cal/(mole.grad.), respectively. The thermal stability of alcohol dehydrogenase in 60% gels is increased 12-fold. To prevent gel swelling, methacrylic acid and allylamine were added to the matrix, with subsequent treatment by dicyclohexylcarbodiimide. The enzyme activity of the modified gels is 2,7--3% of that for the 6,7% gels. The stability of lactate dehydrogenase in such gels is significantly increased. A mechanism of stabilization of the subunit enzymes in highly concentrated gels is discussed.

  7. Recent advances in biotechnological applications of alcohol dehydrogenases.

    PubMed

    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.

  8. Inhibition of membrane-bound succinate dehydrogenase by disulfiram.

    PubMed

    Jay, D

    1991-04-01

    The effect of disulfiram on succinate oxidase and succinate dehydrogenase activities of beef heart submitochondrial particles was studied. Results show that disulfiram inhibits both functions. Succinate and malonate suppress the inhibitory action of disulfiram when succinate dehydrogenase is stabilized in an active conformation. Disulfiram is not able to inhibit the enzyme when succinate dehydrogenase is inactivated by oxaloacetate. The inhibitory effect of disulfiram is reverted by the addition of dithiothreitol. From these results, it is proposed that disulfiram inhibits the utilization of succinate by a direct modification of an -SH group located in the catalytically active site of succinate dehydrogenase.

  9. Contribution of Liver Alcohol Dehydrogenase to Metabolism of Alcohols in Rats

    PubMed Central

    Plapp, Bryce V.; Leidal, Kevin G.; Murch, Bruce P.; Green, David W.

    2015-01-01

    The kinetics of oxidation of various alcohols by purified rat liver alcohol dehydrogenase (ADH) were compared with the kinetics of elimination of the alcohols in rats in order to investigate the roles of ADH and other factors that contribute to the rates of metabolism of alcohols. Primary alcohols (ethanol, 1-propanol, 1-butanol, 2-methyl-1-propanol, 3-methyl-1-butanol) and diols (1,3-propanediol, 1,3-butanediol, 1,4-butanediol, 1,5-pentanediol) were eliminated in rats with zero-order kinetics at doses of 5–20 mmole/kg. Ethanol was eliminated most rapidly, at 7.9 mmole/kg•h. Secondary alcohols (2-propanol-d7, 2-propanol, 2-butanol, 3-pentanol, cyclopentanol, cyclohexanol) were eliminated with first order kinetics at doses of 5–10 mmole/kg, and the corresponding ketones were formed and slowly eliminated with zero or first order kinetics. The rates of elimination of various alcohols were inhibited on average 73% (55% for 2-propanol to 90% for ethanol) by 1 mmole/kg of 4-methylpyrazole, a good inhibitor of ADH, indicating a major role for ADH in the metabolism of the alcohols. The Michaelis kinetic constants from in vitro studies (pH 7.3, 37 °C) with isolated rat liver enzyme were used to calculate the expected relative rates of metabolism in rats. The rates of elimination generally increased with increased activity of ADH, but a maximum rate of 6 ± 1 mmole/kg•h was observed for the best substrates, suggesting that ADH activity is not solely rate-limiting. Because secondary alcohols only require one NAD+ for the conversion to ketones whereas primary alcohols require two equivalents of NAD+ for oxidation to the carboxylic acids, it appears that the rate of oxidation of NADH to NAD+ is not a major limiting factor for metabolism of these alcohols, but the rate-limiting factors are yet to be identified. PMID:25641189

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

    PubMed

    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.

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

    PubMed

    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.

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

    PubMed

    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 (<100 nm) were engineered from oil-in-water microemulsion precursors using emulsifying wax NF as the oil phase and polyoxyethylene 20-stearyl ether (Brij 78) as the surfactant. Emulsifying wax contains both cetyl and stearyl alcohols. NPs were incubated with the enzyme and NAD+ at 37 degrees C for up to 48 h, and the concentrations of fatty alcohols were quantitatively determined over time by gas chromatography (GC). The concentrations of cetyl alcohol and stearyl alcohol within the NPs decreased to only 10-20% remaining after 15-24 h of incubation. In parallel, NP size, turbidity and the fluorescence intensity of NADH all increased over time. It was concluded that horse liver alcohol dehydrogenase/NAD+ was able to metabolize the fatty alcohols within the NPs, suggesting that NPs made of fatty alcohols may be metabolized in the body via endogenous alcohol dehydrogenase enzyme systems.

  13. Inducible UDP-glucose dehydrogenase from French bean (Phaseolus vulgaris L.) locates to vascular tissue and has alcohol dehydrogenase activity.

    PubMed

    Robertson, D; Smith, C; Bolwell, G P

    1996-01-01

    UDP-glucose dehydrogenase is responsible for channelling UDP-glucose into the pool of UDP-sugars utilized in the synthesis of wall matrix polysaccharides and glycoproteins. It has been purified to homogeneity from suspension-cultured cells of French bean by a combination of hydrophobic-interaction chromatography, gel filtration and dye-ligand chromatography. The enzyme had a subunit of Mr 40,000. Km values were measured for UDP-glucose as 5.5 +/- 1.4 mM and for NAD+ as 20 +/- 3 microM. It was subject to inhibition by UDP-xylose. UDP-glucose dehydrogenase activity co-purified with alcohol dehydrogenase activity from suspension-cultured cells, elicitor-treated cells and elongating hypocotyls, even when many additional chromatographic steps were employed subsequently. The protein from each source was resolved into virtually identical patterns of isoforms on two-dimensional isoelectric focusing/PAGE. However, a combination of peptide mapping and sequence analysis, gel analysis using activity staining and kinetic analysis suggests that both activities are a function of the same protein. An antibody was raised and used to immunolocalize UDP-glucose dehydrogenase to developing xylem and phloem of French bean hypocotyl. Together with data published previously, these results are consistent with an important role in the regulation of carbon flux into wall matrix polysaccharides.

  14. Inhibition of MMPs by alcohols

    PubMed Central

    Tezvergil-Mutluay, Arzu; Agee, Kelli A.; Hoshika, Tomohiro; Uchiyama, Toshikazu; Tjäderhane, Leo; Breschi, Lorenzo; Mazzoni, Annalisa; Thompson, Jeremy M.; McCracken, Courtney E.; Looney, Stephen W.; Tay, Franklin R.; Pashley, David H.

    2011-01-01

    Objectives While screening the activity of potential inhibitors of matrix metalloproteinases (MMPs), due to the limited water solubility of some of the compounds, they had to be solubilized in ethanol. When ethanol solvent controls were run, they were found to partially inhibit MMPs. Thus, the purpose of this study was to compare the MMP-inhibitory activity of a series of alcohols. Methods The possible inhibitory activity of a series of alcohols was measured against soluble rhMMP-9 and insoluble matrix-bound endogenous MMPs of dentin in completely demineralized dentin. Increasing concentrations (0.17, 0.86, 1.71 and 4.28 moles/L) of a homologous series of alcohols (i.e. methanol, ethanol, propanols, butanols, pentanols, hexanols, the ethanol ester of methacrylic acid, heptanols and octanol) were compared to ethanediol, and propanediol by regression analysis to calculate the molar concentration required to inhibit MMPs by 50% (i.e. the IC50). Results Using two different MMP models, alcohols were shown to inhibit rhMMP-9 and the endogenous proteases of dentin matrix in a dose-dependent manner. The degree of MMP inhibition by alcohols increased with chain length up to 4 methylene groups. Based on the molar concentration required to inhibit rhMMP-9 fifty percent, 2-hydroxyethylmethacrylate (HEMA), 3-hexanol, 3-heptanol and 1-octanol gave the strongest inhibition. Significance The results indicate that alcohols with 4 methylene groups inhibit MMPs more effectively than methanol or ethanol. MMP inhibition was inversely related to the Hoy's solubility parameter for hydrogen bonding forces of the alcohols (i.e. to their hydrophilicity). PMID:21676453

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

    PubMed

    Smilda, T; Kamminga, A H; Reinders, P; Baron, W; van Hylckama Vlieg, J E; Beintema, J J

    2001-05-01

    Enzymic and structural studies on Drosophila alcohol dehydrogenases and other short-chain dehydrogenases/reductases (SDRs) are presented. Like alcohol dehydrogenases from other Drosophila species, the enzyme from D. simulans is more active on secondary than on primary alcohols, although ethanol is its only known physiological substrate. Several secondary alcohols were used to determine the kinetic parameters kcat and Km. The results of these experiments indicate that the substrate-binding region of the enzyme allows optimal binding of a short ethyl side-chain in a small binding pocket, and of a propyl or butyl side-chain in large binding pocket, with stereospecificity for R(-) alcohols. At a high concentration of R(-) alcohols substrate activation occurs. The kcat and Km values determined under these conditions are about two-fold, and two orders of magnitude, respectively, higher than those at low substrate concentrations. Sequence alignment of several SDRs of known, and unknown three-dimensional structures, indicate the presence of several conserved residues in addition to those involved in the catalyzed reactions. Structural roles of these conserved residues could be derived from observations made on superpositioned structures of several SDRs with known structures. Several residues are conserved in tetrameric SDRs, but not in dimeric ones. Two halohydrin-halide-lyases show significant homology with SDRs in the catalytic domains of these enzymes, but they do not have the structural features required for binding NAD+. Probably these lyases descend from an SDR, which has lost the capability to bind NAD+, but the enzyme reaction mechanisms may still be similar.

  16. Multiple alcohol dehydrogenases but no functional acetaldehyde dehydrogenase causing excessive acetaldehyde production from ethanol by oral streptococci.

    PubMed

    Pavlova, Sylvia I; Jin, Ling; Gasparovich, Stephen R; Tao, Lin

    2013-07-01

    Ethanol consumption and poor oral hygiene are risk factors for oral and oesophageal cancers. Although oral streptococci have been found to produce excessive acetaldehyde from ethanol, little is known about the mechanism by which this carcinogen is produced. By screening 52 strains of diverse oral streptococcal species, we identified Streptococcus gordonii V2016 that produced the most acetaldehyde from ethanol. We then constructed gene deletion mutants in this strain and analysed them for alcohol and acetaldehyde dehydrogenases by zymograms. The results showed that S. gordonii V2016 expressed three primary alcohol dehydrogenases, AdhA, AdhB and AdhE, which all oxidize ethanol to acetaldehyde, but their preferred substrates were 1-propanol, 1-butanol and ethanol, respectively. Two additional dehydrogenases, S-AdhA and TdhA, were identified with specificities to the secondary alcohol 2-propanol and threonine, respectively, but not to ethanol. S. gordonii V2016 did not show a detectable acetaldehyde dehydrogenase even though its adhE gene encodes a putative bifunctional acetaldehyde/alcohol dehydrogenase. Mutants with adhE deletion showed greater tolerance to ethanol in comparison with the wild-type and mutant with adhA or adhB deletion, indicating that AdhE is the major alcohol dehydrogenase in S. gordonii. Analysis of 19 additional strains of S. gordonii, S. mitis, S. oralis, S. salivarius and S. sanguinis showed expressions of up to three alcohol dehydrogenases, but none showed detectable acetaldehyde dehydrogenase, except one strain that showed a novel ALDH. Therefore, expression of multiple alcohol dehydrogenases but no functional acetaldehyde dehydrogenase may contribute to excessive production of acetaldehyde from ethanol by certain oral streptococci.

  17. [Class III alcohol dehydrogenase and its role in the human body].

    PubMed

    Jelski, Wojciech; Sani, Tufik Alizade; Szmitkowski, Maciej

    2006-01-01

    Class III alcohol dehydrogenase is composed of two chi subunits, encoded by the ADH5 gene and existing in all tissues examined. It possesses a great ability to metabolize long-chain alcohols, while its capacity to oxidize ethanol is very limited. The amino-acid sequence homology and identical structural and kinetic properties indicate that class III alcohol dehydrogenase and formaldehyde dehydrogenase are identical enzymes. ADH III plays a significant role in the metabolism of formaldehyde in the human body.

  18. Physiological Function of Alcohol Dehydrogenases and Long-Chain (C30) Fatty Acids in Alcohol Tolerance of Thermoanaerobacter ethanolicus

    PubMed Central

    Burdette, D. S.; Jung, S.-H.; Shen, G.-J.; Hollingsworth, R. I.; Zeikus, J. G.

    2002-01-01

    A mutant strain (39E H8) of Thermoanaerobacter ethanolicus that displayed high (8% [vol/vol]) ethanol tolerance for growth was developed and characterized in comparison to the wild-type strain (39E), which lacks alcohol tolerance (<1.5% [vol/vol]). The mutant strain, unlike the wild type, lacked primary alcohol dehydrogenase and was able to increase the percentage of transmembrane fatty acids (i.e., long-chain C30 fatty acids) in response to increasing levels of ethanol. The data support the hypothesis that primary alcohol dehydrogenase functions primarily in ethanol consumption, whereas secondary alcohol dehydrogenase functions in ethanol production. These results suggest that improved thermophilic ethanol fermentations at high alcohol levels can be developed by altering both cell membrane composition (e.g., increasing transmembrane fatty acids) and the metabolic machinery (e.g., altering primary alcohol dehydrogenase and lactate dehydrogenase activities). PMID:11916712

  19. New model for polymerization of oligomeric alcohol dehydrogenases into nanoaggregates.

    PubMed

    Barzegar, Abolfazl; Moosavi-Movahedi, Ali A; Kyani, Anahita; Goliaei, Bahram; Ahmadian, Shahin; Sheibani, Nader

    2010-02-01

    Polymerization and self-assembly of proteins into nanoaggregates of different sizes and morphologies (nanoensembles or nanofilaments) is a phenomenon that involved problems in various neurodegenerative diseases (medicine) and enzyme instability/inactivity (biotechnology). Thermal polymerization of horse liver alcohol dehydrogenase (dimeric) and yeast alcohol dehydrogenase (tetrameric), as biotechnological ADH representative enzymes, was evaluated for the development of a rational strategy to control aggregation. Constructed ADH nuclei, which grew to larger amorphous nanoaggregates, were prevented via high repulsion strain of the net charge values. Good correlation between the variation in scattering and lambda(-2) was related to the amorphousness of the nanoaggregated ADHs, shown by electron microscopic images. Scattering corrections revealed that ADH polymerization was related to the quaternary structural changes, including delocalization of subunits without unfolding, i.e. lacking the 3D conformational and/or secondary-ordered structural changes. The results demonstrated that electrostatic repulsion was not only responsible for disaggregation but also caused a delay in the onset of aggregation temperature, decreasing maximum values of aggregation and amounts of precipitation. Together, our results demonstrate and propose a new model of self-assembly for ADH enzymes based on the construction of nuclei, which grow to formless nanoaggregates with minimal changes in the tertiary and secondary conformations.

  20. Effect of fermented sea tangle on the alcohol dehydrogenase and acetaldehyde dehydrogenase in Saccharomyces cerevisiae.

    PubMed

    Cha, Jae-Young; Jeong, Jae-Jun; Yang, Hyun-Ju; Lee, Bae-Jin; Cho, Young-Su

    2011-08-01

    Sea tangle, a kind of brown seaweed, was fermented with Lactobacillus brevis BJ-20. The gamma-aminobutyric acid (GABA) content in fermented sea tangle (FST) was 5.56% (w/w) and GABA in total free amino acid of FST was 49.5%. The effect of FST on the enzyme activities and mRNA protein expression of alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) involved in alcohol metabolism in Saccharomyces cerevisiae was investigated. Yeast was cultured in YPD medium supplemented with different concentrations of FST powder [0, 0.4, 0.8, and 1.0% (w/v)] for 18 h. FST had no cytotoxic effect on the yeast growth. The highest activities and protein expressions of ADH and ALDH from the cell-free extracts of S. cerevisiae were evident with the 0.4% and 0.8% (w/v) FST-supplemented concentrations, respectively. The highest concentrations of GABA as well as minerals (Zn, Ca, and Mg) were found in the cell-free extracts of S. cerevisiae cultured in medium supplemented with 0.4% (w/v) FST. The levels of GABA, Zn, Ca, and Mg in S. cerevisiae were strongly correlated with the enzyme activities of ADH and ALDH in yeast. These results indicate that FST can enhance the enzyme activities and protein expression of ADH and ALDH in S. cerevisiae.

  1. Amphibian alcohol dehydrogenase, the major frog liver enzyme. Relationships to other forms and assessment of an early gene duplication separating vertebrate class I and class III alcohol dehydrogenases

    SciTech Connect

    Cederlund, E.; Joernvall, H. ); Peralba, J.M.; Pares, X. )

    1991-03-19

    Submammalian alcohol dehydrogenase structures can be used to evaluate the origins and functions of different types of the mammalian enzyme. Two avian forms were recently reported, and the authors now define the major amphibian alcohol dehydrogenase. The enzyme from the liver of the Green frog Rana perezi was purified, carboxymethylated, and submitted to amino acid sequence determination by peptide analysis of six different digest. The protein has a 375-residue subunit and is a class I alcohol dehydrogenase, bridging the gap toward the original separation of the classes that are observable in the human alcohol dehydrogenase system. In relation to the human class I enzyme, the amphibian protein has residue identities exactly halfway (68%) between those for the corresponding avian enzyme (74%) and the human class III enzyme (62%), suggesting an origin of the alcohol dehnydrogenase classes very early in or close to the evolution of the vertebrate line. This conclusion suggests that these enzyme classes are more universal among animals than previously realized and constitutes the first real assessment of the origin of the duplications leading to the alcohol dehydrogenase classes. In conclusion, the amphibian enzyme allows a rough positioning of the divergence of the alcohol dehydrogenase classes, shows that the class I type is widesprread in vertebrates, and functionally conforms with greater variations at the substrate-binding than the coenzyme-binding site.

  2. Alcohol dehydrogenase gene ADH3 activates glucose alcoholic fermentation in genetically engineered Dekkera bruxellensis yeast.

    PubMed

    Schifferdecker, Anna Judith; Siurkus, Juozas; Andersen, Mikael Rørdam; Joerck-Ramberg, Dorte; Ling, Zhihao; Zhou, Nerve; Blevins, James E; Sibirny, Andriy A; Piškur, Jure; Ishchuk, Olena P

    2016-04-01

    Dekkera bruxellensis is a non-conventional Crabtree-positive yeast with a good ethanol production capability. Compared to Saccharomyces cerevisiae, its tolerance to acidic pH and its utilization of alternative carbon sources make it a promising organism for producing biofuel. In this study, we developed an auxotrophic transformation system and an expression vector, which enabled the manipulation of D. bruxellensis, thereby improving its fermentative performance. Its gene ADH3, coding for alcohol dehydrogenase, was cloned and overexpressed under the control of the strong and constitutive promoter TEF1. Our recombinant D. bruxellensis strain displayed 1.4 and 1.7 times faster specific glucose consumption rate during aerobic and anaerobic glucose fermentations, respectively; it yielded 1.2 times and 1.5 times more ethanol than did the parental strain under aerobic and anaerobic conditions, respectively. The overexpression of ADH3 in D. bruxellensis also reduced the inhibition of fermentation by anaerobiosis, the "Custer effect". Thus, the fermentative capacity of D. bruxellensis could be further improved by metabolic engineering.

  3. A new potent inhibitor of horse liver alcohol dehydrogenase: p-methylbenzyl hydroperoxide.

    PubMed

    Skurský, L; Khan, A N; Saleem, M N; al-Tamer, Y Y

    1992-04-01

    A product of p-xylene auto-oxidation, p-methylbenzyl hydroperoxide, acts as a very strong reversible inhibitor of the ethanol dehydrogenating activity of horse liver alcohol dehydrogenase. Concentrations of hydroperoxide as low as that of the enzyme active site (about 10(-8) mol.dm-3) in the assay depresses the activity by 50%. Somewhat less potent is benzyl hydroperoxide (derived from toluene) while the (secondary) hydroperoxide derived from ethylbenzene and tert.butyl hydroperoxide and cumyl hydroperoxide do not inhibit HLAD appreciably.

  4. In vivo regulation of alcohol dehydrogenase and lactate dehydrogenase in Rhizopus oryzae to improve L-lactic acid fermentation.

    PubMed

    Thitiprasert, Sitanan; Sooksai, Sarintip; Thongchul, Nuttha

    2011-08-01

    Rhizopus oryzae is becoming more important due to its ability to produce an optically pure L: -lactic acid. However, fermentation by Rhizopus usually suffers from low yield because of production of ethanol as a byproduct. Limiting ethanol production in living immobilized R. oryzae by inhibition of alcohol dehydrogenase (ADH) was observed in shake flask fermentation. The effects of ADH inhibitors added into the medium on the regulation of ADH and lactate dehydrogenase (LDH) as well as the production of cell biomass, lactic acid, and ethanol were elucidated. 1,2-diazole and 2,2,2-trifluroethanol were found to be the effective inhibitors used in this study. The highest lactic acid yield of 0.47 g/g glucose was obtained when 0.01 mM 2,2,2-trifluoroethanol was present during the production phase of the pregrown R. oryzae. This represents about 38% increase in yield as compared with that from the simple glucose fermentation. Fungal metabolism was suppressed when iodoacetic acid, N-ethylmaleimide, 4,4'-dithiodipyridine, or 4-hydroxymercury benzoic acid were present. Dramatic increase in ADH and LDH activities but slight change in product yields might be explained by the inhibitors controlling enzyme activities at the pyruvate branch point. This showed that in living R. oryzae, the inhibitors regulated the flux through the related pathways.

  5. Cloning, expression, functional validation and modeling of cinnamyl alcohol dehydrogenase isolated from xylem of Leucaena leucocephala.

    PubMed

    Pandey, Brijesh; Pandey, Veda Prakash; Dwivedi, Upendra Nath

    2011-10-01

    A cDNA encoding cinnamyl alcohol dehydrogenase (CAD), catalyzing conversion of cinnamyl aldehydes to corresponding cinnamyl alcohols, was cloned from secondary xylem of Leucaena leucocephala. The cloned cDNA was expressed in Escherichia coli BL21 (DE3) pLysS cells. Temperature and Zn(2+) ion played crucial role in expression and activity of enzyme, such that, at 18°C and at 2 mM Zn(2+) the CAD was maximally expressed as active enzyme in soluble fraction. The expressed protein was purified 14.78-folds to homogeneity on Ni-NTA agarose column with specific activity of 346 nkat/mg protein. The purified enzyme exhibited lowest Km with cinnamyl alcohol (12.2 μM) followed by coniferyl (18.1 μM) and sinapyl alcohol (23.8 μM). Enzyme exhibited high substrate inhibition with cinnamyl (beyond 20 μM) and coniferyl (beyond 100 μM) alcohols. The in silico analysis of CAD protein exhibited four characteristic consensus sequences, GHEXXGXXXXXGXXV; C(100), C(103), C(106), C(114); GXGXXG and C(47), S(49), H(69), L(95), C(163), I(300) involved in catalytic Zn(2+) binding, structural Zn(2+) binding, NADP(+) binding and substrate binding, respectively. Tertiary structure, generated using Modeller 9v5, exhibited a trilobed structure with bulged out structural Zn(2+) binding domain. The catalytic Zn(2+) binding, substrate binding and NADP(+) binding domains formed a pocket protected by two major lobes. The enzyme catalysis, sequence homology and 3-D model, all supported that the cloned CAD belongs to alcohol dehydrogenase family of plants.

  6. Inhibition of membrane-bound succinate dehydrogenase by fluorescamine.

    PubMed

    Jay, D; Jay, E G; Garcia, C

    1993-12-01

    Fluorescamine rapidly inactivated membrane-bound succinate dehydrogenase. The inhibition of the enzyme by this reagent was prevented by succinate and malonate, suggesting that the group modified by fluorescamine was located at the active site. The modification of the active site sulfhydryl group by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) did not alter the inhibitory action of fluorescamine. However, the protective effect of malonate against fluorescamine inhibition was abolished in the enzyme modified at the thiol.

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

    PubMed

    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.

  8. Sequential injection analysis of ethanol using immobilized alcohol dehydrogenase

    SciTech Connect

    Hedenfalk, M.; Mattiasson, B.

    1996-05-01

    A Sequential Injection (SI) system was used to analyze the ethanol concentration in fermentation broth. The method is based on the use of immobilized NAD{sup +} dependent alcohol dehydrogenase. A non-linear standard curve for ethanol (range 0.25-100 mM) was used to determine the concentration in fermentation broth and the results correlated well with HPLC measurements. The assay time was 140 s, 0.5 {mu}mol of cofactor was used for each determination, and the relative standard deviation was less than 6% when analyzing fermentation samples. The assay system is very stable and makes it possible to reduce the cofactor consumption while keeping the system set up simple.

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

    PubMed

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

    1996-07-01

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

  10. Alcohol biosensor based on alcohol dehydrogenase and Meldola Blue immobilized into a carbon paste electrode.

    PubMed

    García Mullor, S; Sánchez-Cabezudo, M; Miranda Ordieres, A J; López Ruiz, B

    1996-05-01

    A yeast alcohol dehydrogenase amperometric carbon paste-based biosensor, with Meldola Blue as a mediator and a dialysis membrane with a very small molecular weight cut-off for protection, is described. The influence of membrane pore size on the stability and overall kinetics of the biosensor is shown using cyclic voltammetry and stationary potential measurements. The operating potential is + 50 mV vs. Ag/AgCl, KCl sat. reference electrode. Application of this device to the determination of ethanol in alcoholic beverages was achieved successfully. In these kinds of samples and at this working potential no interferences were found.

  11. Alcohol dehydrogenase 1B genotype and fetal alcohol syndrome: a HuGE minireview.

    PubMed

    Green, Ridgely Fisk; Stoler, Joan Marilyn

    2007-07-01

    Fetal alcohol syndrome (FAS), 1 of the most common developmental disabilities in the United States, occurs at a rate of 0.5-2.0:1000 live births. Animal model, family, and twin studies suggest a genetic component to FAS susceptibility. Alcohol dehydrogenases (ADHs) catalyze the rate-limiting step in alcohol metabolism. Studies of genetic associations with FAS have focused on the alcohol dehydrogenase 1B (ADH1B) gene, comparing mothers and children with the alleles ADH1B*2 or ADH1B*3, associated with faster ethanol metabolism, with those homozygous for ADH1B*1. While most studies have found a protective effect for genotypes containing ADH1B*2 or ADH1B*3, results have been conflicting, and further investigation into the association between the ADH1B genotype and FAS is needed. Whether increased alcohol intake accounts for the elevated risk reported for the ADH1B*1/ADH1B*1 genotype should be addressed, and future studies would benefit from consistent case definitions, enhanced exposure measurements, larger sample sizes, and careful study design.

  12. Purification of acetaldehyde dehydrogenase and alcohol dehydrogenases from Thermoanaerobacter ethanolicus 39E and characterization of the secondary-alcohol dehydrogenase (2 degrees Adh) as a bifunctional alcohol dehydrogenase--acetyl-CoA reductive thioesterase.

    PubMed

    Burdette, D; Zeikus, J G

    1994-08-15

    The purification and characterization of three enzymes involved in ethanol formation from acetyl-CoA in Thermoanaerobacter ethanolicus 39E (formerly Clostridium thermohydrosulfuricum 39E) is described. The secondary-alcohol dehydrogenase (2 degrees Adh) was determined to be a homotetramer of 40 kDa subunits (SDS/PAGE) with a molecular mass of 160 kDa. The 2 degrees Adh had a lower catalytic efficiency for the oxidation of 1 degree alcohols, including ethanol, than for the oxidation of secondary (2 degrees) alcohols or the reduction of ketones or aldehydes. This enzyme possesses a significant acetyl-CoA reductive thioesterase activity as determined by NADPH oxidation, thiol formation and ethanol production. The primary-alcohol dehydrogenase (1 degree Adh) was determined to be a homotetramer of 41.5 kDa (SDS/PAGE) subunits with a molecular mass of 170 kDa. The 1 degree Adh used both NAD(H) and NADP(H) and displayed higher catalytic efficiencies for NADP(+)-dependent ethanol oxidation and NADH-dependent acetaldehyde (identical to ethanal) reduction than for NADPH-dependent acetaldehyde reduction or NAD(+)-dependent ethanol oxidation. The NAD(H)-linked acetaldehyde dehydrogenase was a homotetramer (360 kDa) of identical subunits (100 kDa) that readily catalysed thioester cleavage and condensation. The 1 degree Adh was expressed at 5-20% of the level of the 2 degrees Adh throughout the growth cycle on glucose. The results suggest that the 2 degrees Adh primarily functions in ethanol production from acetyl-CoA and acetaldehyde, whereas the 1 degree Adh functions in ethanol consumption for nicotinamide-cofactor recycling.

  13. Purification of acetaldehyde dehydrogenase and alcohol dehydrogenases from Thermoanaerobacter ethanolicus 39E and characterization of the secondary-alcohol dehydrogenase (2 degrees Adh) as a bifunctional alcohol dehydrogenase--acetyl-CoA reductive thioesterase.

    PubMed Central

    Burdette, D; Zeikus, J G

    1994-01-01

    The purification and characterization of three enzymes involved in ethanol formation from acetyl-CoA in Thermoanaerobacter ethanolicus 39E (formerly Clostridium thermohydrosulfuricum 39E) is described. The secondary-alcohol dehydrogenase (2 degrees Adh) was determined to be a homotetramer of 40 kDa subunits (SDS/PAGE) with a molecular mass of 160 kDa. The 2 degrees Adh had a lower catalytic efficiency for the oxidation of 1 degree alcohols, including ethanol, than for the oxidation of secondary (2 degrees) alcohols or the reduction of ketones or aldehydes. This enzyme possesses a significant acetyl-CoA reductive thioesterase activity as determined by NADPH oxidation, thiol formation and ethanol production. The primary-alcohol dehydrogenase (1 degree Adh) was determined to be a homotetramer of 41.5 kDa (SDS/PAGE) subunits with a molecular mass of 170 kDa. The 1 degree Adh used both NAD(H) and NADP(H) and displayed higher catalytic efficiencies for NADP(+)-dependent ethanol oxidation and NADH-dependent acetaldehyde (identical to ethanal) reduction than for NADPH-dependent acetaldehyde reduction or NAD(+)-dependent ethanol oxidation. The NAD(H)-linked acetaldehyde dehydrogenase was a homotetramer (360 kDa) of identical subunits (100 kDa) that readily catalysed thioester cleavage and condensation. The 1 degree Adh was expressed at 5-20% of the level of the 2 degrees Adh throughout the growth cycle on glucose. The results suggest that the 2 degrees Adh primarily functions in ethanol production from acetyl-CoA and acetaldehyde, whereas the 1 degree Adh functions in ethanol consumption for nicotinamide-cofactor recycling. Images Figure 1 PMID:8068002

  14. Not only students can express alcohol dehydrogenase: goldfish can too!

    PubMed

    Chamberland, Valérie; Rioux, Pierre

    2010-12-01

    This article describes a novel approach to study the metabolic regulation of the respiratory system in vertebrates that suits physiology lessons for undergraduate students. It consists of an experimental demonstration of the goldfish's (Carassius auratus) adaptations to anoxia. The goldfish is one of the few vertebrates showing strong enzymatic plasticity for the expression of alcohol dehydrogenase (ADH), which allows it to survive long periods of severe anoxia. Therefore, we propose two simple laboratory exercises in which students are first asked to characterize the distribution of ADH isozymes in the goldfish by performing cellulose acetate electrophoresis. The second part of this laboratory lesson is the determination of liver glycogen. To further student comprehension, an interspecies comparative component is integrated, in which the same subjects are studied in an anoxia-sensitive species, the brook charr (Salvelinus fontinalis). ADH in goldfish is restricted to skeletal muscles, where it catalyzes alcoholic fermentation, permitting ethanol excretion through the gills and therefore preventing lactate acidosis caused by sustained glycolysis during anoxia. Electrophoresis also reveals the occurrence of a liver isozyme in the brook charr, which ADH catalyzes in the opposite pathway, allowing the usual ethanol degradation. As for the liver glycogen assay, it shows largely superior content in the goldfish liver compared with the brook charr, providing goldfish with a sustained energy supply during anoxia. The results of this laboratory exercise clearly demonstrate several physiological strategies developed by goldfish to cope with such a crucial environmental challenge as oxygen depletion.

  15. Isolation and partial characterization of the Drosophila alcohol dehydrogenase gene.

    PubMed Central

    Goldberg, D A

    1980-01-01

    The alcohol dehydrogenase (ADH; alcohol: NAD+ oxidoreductase, EC 1.1.1.1) gene (Adh) of Drosophila melanogaster was isolated by utilizing a mutant strain in which the Adh locus is deleted. Adult RNA from wild-type flies was enriched in ADH sequences by gel electrophoresis and then used to prepare labeled cDNA for screening a bacteriophage lambda library of genomic Drosophila DNA. Of the clones that hybridized in the initial screen, one clone was identified that hybridized with labeled cDNA prepared from a wild-type Drosophila strain but did not hybridize with cDNA prepared from an Adh deletion strain. This clone was shown to contain ADH structural gene sequences by three criteria: in situ hybridization, in vitro translation of mRNA selected by hybridization to the cloned DNA, and comparison of the ADH protein sequence with a nucleotide sequence derived from the cloned DNA. Comparison of the restriction site maps from clones of three different wild-type Drosophila strains revealed the presence of a 200-nucleotide sequence in one strain that was absent from the other two strains. The ADH mRNA sequences were located within the cloned DNA by hybridization mapping experiments. Two intervening sequences were identified within Adh by S1 nuclease mapping experiments. Images PMID:6777776

  16. Sequence variation of alcohol dehydrogenase (Adh) paralogs in cactophilic Drosophila.

    PubMed Central

    Matzkin, Luciano M; Eanes, Walter F

    2003-01-01

    This study focuses on the population genetics of alcohol dehydrogenase (Adh) in cactophilic Drosophila. Drosophila mojavensis and D. arizonae utilize cactus hosts, and each host contains a characteristic mixture of alcohol compounds. In these Drosophila species there are two functional Adh loci, an adult form (Adh-2) and a larval and ovarian form (Adh-1). Overall, the greater level of variation segregating in D. arizonae than in D. mojavensis suggests a larger population size for D. arizonae. There are markedly different patterns of variation between the paralogs across both species. A 16-bp intron haplotype segregates in both species at Adh-2, apparently the product of an ancient gene conversion event between the paralogs, which suggests that there is selection for the maintenance of the intron structure possibly for the maintenance of pre-mRNA structure. We observe a pattern of variation consistent with adaptive protein evolution in the D. mojavensis lineage at Adh-1, suggesting that the cactus host shift that occurred in the divergence of D. mojavensis from D. arizonae had an effect on the evolution of the larval expressed paralog. Contrary to previous work we estimate a recent time for both the divergence of D. mojavensis and D. arizonae (2.4 +/- 0.7 MY) and the age of the gene duplication (3.95 +/- 0.45 MY). PMID:12586706

  17. Improved resistance to transition metals of a cobalt-substituted alcohol dehydrogenase 1 from Saccharomyces cerevisiae.

    PubMed

    Cavaletto, M; Pessione, E; Vanni, A; Giunta, C

    2001-11-17

    Cobalt-substituted alcohol dehydrogenase 1 was purified from a yeast culture of Saccharomyces cerevisiae. Its reactivity towards different transition metals was tested and compared with the native zinc enzyme. The cobalt enzyme displayed a catalytic efficiency 100-fold higher than that of the zinc enzyme. Copper, nickel and cadmium exerted a mixed-type inhibition, with a scale of inhibition efficiency: Cu(2+)>Ni(2+)>Cd(2+). In general, a higher resistance of the modified protein to the inhibitory action of transition metals was observed, with two orders of magnitude for copper I(50). The presence of nickel in the complexes enzyme-coenzyme-inhibitor-substrate resulted in a decrease of the ampholytic nature of the catalytic site. On the contrary, cadmium and copper exerted an enhancement of this parameter. Electrostatic or other types of interactions may be involved in conferring a good resistance in the basic pH range, making cobalt enzyme very suitable for biotechnological processes.

  18. Alcohol dehydrogenases and an alcohol oxidase involved in the assimilation of exogenous fatty alcohols in Yarrowia lipolytica.

    PubMed

    Iwama, Ryo; Kobayashi, Satoshi; Ohta, Akinori; Horiuchi, Hiroyuki; Fukuda, Ryouichi

    2015-05-01

    The yeast Yarrowia lipolytica can assimilate hydrophobic substrates, including n-alkanes and fatty alcohols. Here, eight alcohol dehydrogenase genes, ADH1-ADH7 and FADH, and a fatty alcohol oxidase gene, FAO1, were analyzed to determine their roles in the metabolism of hydrophobic substrates. A mutant deleted for all of these genes (ALCY02 strain) showed severely defective growth on fatty alcohols, and enhanced sensitivity to fatty alcohols in glucose-containing media. The ALCY02 strain grew normally on n-tetradecane or n-hexadecane, but exhibited slightly defective growth on n-decane or n-dodecane. It accumulated more 1-dodecanol and less dodecanoic acid than the wild-type strain when n-dodecane was fed. Expression of ADH1, ADH3 or FAO1, but not that of other ADH genes or FADH, in the ALCY02 strain restored its growth on fatty alcohols. In addition, a triple deletion mutant of ADH1, ADH3 and FAO1 showed similarly defective growth on fatty alcohols and on n-dodecane to the ALCY02 strain. Microscopic observation suggests that Adh1p and Adh3p are localized in the cytosol and Fao1p is in the peroxisome. These results suggest that Adh1p, Adh3p and Fao1p are responsible for the oxidation of exogenous fatty alcohols but play less prominent roles in the oxidation of fatty alcohols derived from n-alkanes.

  19. Metformin suppresses gluconeogenesis by inhibiting mitochondrial glycerophosphate dehydrogenase.

    PubMed

    Madiraju, Anila K; Erion, Derek M; Rahimi, Yasmeen; Zhang, Xian-Man; Braddock, Demetrios T; Albright, Ronald A; Prigaro, Brett J; Wood, John L; Bhanot, Sanjay; MacDonald, Michael J; Jurczak, Michael J; Camporez, Joao-Paulo; Lee, Hui-Young; Cline, Gary W; Samuel, Varman T; Kibbey, Richard G; Shulman, Gerald I

    2014-06-26

    Metformin is considered to be one of the most effective therapeutics for treating type 2 diabetes because it specifically reduces hepatic gluconeogenesis without increasing insulin secretion, inducing weight gain or posing a risk of hypoglycaemia. For over half a century, this agent has been prescribed to patients with type 2 diabetes worldwide, yet the underlying mechanism by which metformin inhibits hepatic gluconeogenesis remains unknown. Here we show that metformin non-competitively inhibits the redox shuttle enzyme mitochondrial glycerophosphate dehydrogenase, resulting in an altered hepatocellular redox state, reduced conversion of lactate and glycerol to glucose, and decreased hepatic gluconeogenesis. Acute and chronic low-dose metformin treatment effectively reduced endogenous glucose production, while increasing cytosolic redox and decreasing mitochondrial redox states. Antisense oligonucleotide knockdown of hepatic mitochondrial glycerophosphate dehydrogenase in rats resulted in a phenotype akin to chronic metformin treatment, and abrogated metformin-mediated increases in cytosolic redox state, decreases in plasma glucose concentrations, and inhibition of endogenous glucose production. These findings were replicated in whole-body mitochondrial glycerophosphate dehydrogenase knockout mice. These results have significant implications for understanding the mechanism of metformin's blood glucose lowering effects and provide a new therapeutic target for type 2 diabetes.

  20. Environmental inhibition of 11beta-hydroxysteroid dehydrogenase.

    PubMed

    Reidenberg, M M

    2000-04-03

    Gossypol, a polyphenolic compound from cotton seed, caused hypokalemia in some men receiving it in a trial of its contraceptive activity. Searching for the mechanism for its hypokalemic action led to the observation that it inhibited 11beta-hydroxysteroid dehydrogenase. This would enhance mineralocorticoid effect in the kidney. Many other polyphenols also inhibit this enzyme including those in grapefruit juice. Ingesting 1-2 l of grapefruit juice inhibited this enzyme in two men in a clinical experiment. Tea polyphenols inhibit this enzyme and add to the inhibition caused by gossypol. Men in China have lower serum potassium values than men elsewhere and this is due to the environment, presumably the diet, in China. The importance of dietary and other exogenous inhibitors of this enzyme in electrolyte metabolism remains to be determined.

  1. Cloning and sequencing of the alcohol dehydrogenase II gene from Zymomonas mobilis

    DOEpatents

    Ingram, Lonnie O.; Conway, Tyrrell

    1992-01-01

    The alcohol dehydrogenase II gene from Zymomonas mobilis has been cloned and sequenced. This gene can be expressed at high levels in other organisms to produce acetaldehyde or to convert acetaldehyde to ethanol.

  2. Alcohol dehydrogenase and an inactivator from rice seedlings

    SciTech Connect

    Shimomura, S.; Beevers, H.

    1983-01-01

    Alcohol dehydrogenase (ADH) was measured in the various organs of rice seedlings (Oryza sativa) growing in air. In extracts from ungerminated seeds, the ADH is stable, but in extracts from seedlings more than 2 days old the enzyme initially present loses activity in a time- and temperature-dependent fashion, due to the presence of an inactivating component which increases with age in roots and shoots. The inactivation can be prevented completely by dithiothreitol, and when this is included in the extraction medium the apparent loss of total ADH in roots and shoots with age is not observed. In seedlings grown in N/sub 2/, ADA levels in coleoptile extracts are higher than those in air, the enzyme is stable, and no inactivator can be detected. When seedlings grown for 5 days in air were transferred to N/sub 2/ for 3 days, ADA levels increased and there was a decline in inactivator activity. Transfer back to air after 1 day in N/sub 2/ led to loss of the accumulated ADH and increase in inactivator. These reciprocal changes and the fact that the inactivator is absent from coleoptiles of seedlings grown in N/sub 2/ appear to suggest a regulator role for the inactivator in vivo. However, it is clear that high levels of inactivator and ADH can exist in cells of seedlings grown in air for long periods without loss of enzyme activity, and it is argued that they must normally be separately compartmented.

  3. Genetic control of alcohol dehydrogenase levels in Drosophila.

    PubMed

    Maroni, G

    1978-06-01

    Among the progeny of Drosophila flies heterozygous for two noncomplementing Adh-negative alleles, two individuals were found that had recovered appreciable alcohol dehydrogenase activity, thereby surviving the ethanol medium used as a screen. The most likely explanation is that these Adh-positive flies are the product of intracistronic recombination within the Adh locus. Judging by the distribution of outside markers, one of the crossovers would have been a conventional reciprocal exchange while the other appears to have been an instance of nonreciprocal recombination. The enzymes produced in strains derived from the original survivors can be easily distinguished from wild-type enzymes ADH-S and ADH-F on the basis of their sensitivity to denaturing agents. None of various physical and catalytic properties tested revealed differences between the enzymes of the survivor strains except that in one of them the level of activity is 55--65% of the other. Quantitative immunological determinations of ADH gave estimates of enzyme protein which are proportional to the measured activity levels. These results are interpreted to indicate that different amounts of ADH protein are being accumulated in the two strains.

  4. Mechanistic implications from structures of yeast alcohol dehydrogenase complexed with coenzyme and an alcohol.

    PubMed

    Plapp, Bryce V; Charlier, Henry A; Ramaswamy, S

    2016-02-01

    Yeast alcohol dehydrogenase I is a homotetramer of subunits with 347 amino acid residues, catalyzing the oxidation of alcohols using NAD(+) as coenzyme. A new X-ray structure was determined at 3.0 Å where both subunits of an asymmetric dimer bind coenzyme and trifluoroethanol. The tetramer is a pair of back-to-back dimers. Subunit A has a closed conformation and can represent a Michaelis complex with an appropriate geometry for hydride transfer between coenzyme and alcohol, with the oxygen of 2,2,2-trifluoroethanol ligated at 2.1 Å to the catalytic zinc in the classical tetrahedral coordination with Cys-43, Cys-153, and His-66. Subunit B has an open conformation, and the coenzyme interacts with amino acid residues from the coenzyme binding domain, but not with residues from the catalytic domain. Coenzyme appears to bind to and dissociate from the open conformation. The catalytic zinc in subunit B has an alternative, inverted coordination with Cys-43, Cys-153, His-66 and the carboxylate of Glu-67, while the oxygen of trifluoroethanol is 3.5 Å from the zinc. Subunit B may represent an intermediate in the mechanism after coenzyme and alcohol bind and before the conformation changes to the closed form and the alcohol oxygen binds to the zinc and displaces Glu-67.

  5. Thermostable alcohol dehydrogenase from Thermococcus kodakarensis KOD1 for enantioselective bioconversion of aromatic secondary alcohols.

    PubMed

    Wu, Xi; Zhang, Chong; Orita, Izumi; Imanaka, Tadayuki; Fukui, Toshiaki; Xing, Xin-Hui

    2013-04-01

    A novel thermostable alcohol dehydrogenase (ADH) showing activity toward aromatic secondary alcohols was identified from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1 (TkADH). The gene, tk0845, which encodes an aldo-keto reductase, was heterologously expressed in Escherichia coli. The enzyme was found to be a monomer with a molecular mass of 31 kDa. It was highly thermostable with an optimal temperature of 90°C and a half-life of 4.5 h at 95°C. The apparent K(m) values for the cofactors NAD(P)(+) and NADPH were similar within a range of 66 to 127 μM. TkADH preferred secondary alcohols and accepted various ketones and aldehydes as substrates. Interestingly, the enzyme could oxidize 1-phenylethanol and its derivatives having substituents at the meta and para positions with high enantioselectivity, yielding the corresponding (R)-alcohols with optical purities of greater than 99.8% enantiomeric excess (ee). TkADH could also reduce 2,2,2-trifluoroacetophenone to (R)-2,2,2-trifluoro-1-phenylethanol with high enantioselectivity (>99.6% ee). Furthermore, the enzyme showed high resistance to organic solvents and was particularly highly active in the presence of H2O-20% 2-propanol and H2O-50% n-hexane or n-octane. This ADH is expected to be a useful tool for the production of aromatic chiral alcohols.

  6. Thermostable Alcohol Dehydrogenase from Thermococcus kodakarensis KOD1 for Enantioselective Bioconversion of Aromatic Secondary Alcohols

    PubMed Central

    Wu, Xi; Zhang, Chong; Orita, Izumi; Imanaka, Tadayuki

    2013-01-01

    A novel thermostable alcohol dehydrogenase (ADH) showing activity toward aromatic secondary alcohols was identified from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1 (TkADH). The gene, tk0845, which encodes an aldo-keto reductase, was heterologously expressed in Escherichia coli. The enzyme was found to be a monomer with a molecular mass of 31 kDa. It was highly thermostable with an optimal temperature of 90°C and a half-life of 4.5 h at 95°C. The apparent Km values for the cofactors NAD(P)+ and NADPH were similar within a range of 66 to 127 μM. TkADH preferred secondary alcohols and accepted various ketones and aldehydes as substrates. Interestingly, the enzyme could oxidize 1-phenylethanol and its derivatives having substituents at the meta and para positions with high enantioselectivity, yielding the corresponding (R)-alcohols with optical purities of greater than 99.8% enantiomeric excess (ee). TkADH could also reduce 2,2,2-trifluoroacetophenone to (R)-2,2,2-trifluoro-1-phenylethanol with high enantioselectivity (>99.6% ee). Furthermore, the enzyme showed high resistance to organic solvents and was particularly highly active in the presence of H2O–20% 2-propanol and H2O–50% n-hexane or n-octane. This ADH is expected to be a useful tool for the production of aromatic chiral alcohols. PMID:23354700

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

    PubMed

    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.

  8. Molecular characterization of an aldehyde/alcohol dehydrogenase gene from Clostridium acetobutylicum ATCC 824.

    PubMed Central

    Nair, R V; Bennett, G N; Papoutsakis, E T

    1994-01-01

    A gene (aad) coding for an aldehyde/alcohol dehydrogenase (AAD) was identified immediately upstream of the previously cloned ctfA (J. W. Cary, D. J. Petersen, E. T. Papoutsakis, and G. N. Bennett, Appl. Environ. Microbiol. 56:1576-1583, 1990) of Clostridium acetobutylicum ATCC 824 and sequenced. The 2,619-bp aad codes for a 96,517-Da protein. Primer extension analysis identified two transcriptional start sites 83 and 243 bp upstream of the aad start codon. The N-terminal section of AAD shows homology to aldehyde dehydrogenases of bacterial, fungal, mammalian, and plant origin, while the C-terminal section shows homology to alcohol dehydrogenases of bacterial (which includes three clostridial alcohol dehydrogenases) and yeast origin. AAD exhibits considerable amino acid homology (56% identity) over its entire sequence to the trifunctional protein encoded by adhE from Escherichia coli. Expression of aad from a plasmid in C. acetobutylicum showed that AAD, which appears as a approximately 96-kDa band in denaturing protein gels, provides elevated activities of NADH-dependent butanol dehydrogenase, NAD-dependent acetaldehyde dehydrogenase and butyraldehyde dehydrogenase, and a small increase in NADH-dependent ethanol dehydrogenase. A 957-bp open reading frame that could potentially encode a 36,704-Da protein was identified upstream of aad. Images PMID:8300540

  9. Alcohol dehydrogenase activity in Lactococcus chungangensis: application in cream cheese to moderate alcohol uptake.

    PubMed

    Konkit, Maytiya; Choi, Woo Jin; Kim, Wonyong

    2015-09-01

    Many human gastrointestinal facultative anaerobic and aerobic bacteria possess alcohol dehydrogenase (ADH) activity and are therefore capable of oxidizing ethanol to acetaldehyde. However, the ADH activity of Lactococcus spp., except Lactococcus lactis ssp. lactis, has not been widely determined, though they play an important role as the starter for most cheesemaking technologies. Cheese is a functional food recognized as an aid to digestion. In the current study, the ADH activity of Lactococcus chungangensis CAU 28(T) and 11 reference strains from the genus Lactococcus was determined. Only 5 strains, 3 of dairy origin, L. lactis ssp. lactis KCTC 3769(T), L. lactis ssp. cremoris KCCM 40699(T), and Lactococcus raffinolactis DSM 20443(T), and 2 of nondairy origin, Lactococcus fujiensis NJ317(T) and Lactococcus chungangensis CAU 28(T) KCTC 13185(T), showed ADH activity and possessed the ADH gene. All these strains were capable of making cheese, but the highest level of ADH activity was found in L. chungangensis, with 45.9nmol/min per gram in tryptic soy broth and 65.8nmol/min per gram in cream cheese. The extent that consumption of cheese, following imbibing alcohol, reduced alcohol uptake was observed by following the level of alcohol in the serum of mice. The results show a potential novel benefit of cheese as a dairy functional food.

  10. Characteristics of alcohol dehydrogenases of certain aerobic bacteria representing human colonic flora.

    PubMed

    Nosova, T; Jousimies-Somer, H; Kaihovaara, P; Jokelainen, K; Heine, R; Salaspuro, M

    1997-05-01

    We have recently proposed the existence of a bacteriocolonic pathway for ethanol oxidation [i.e., ethanol is oxidized by alcohol dehydrogenases (ADHs) of intestinal bacteria resulting in high intracolonic levels of reactive and toxic acetaldehyde]. The aim of this in vitro study was to characterize further ADH activity of some aerobic bacteria, representing the normal human colonic flora. These bacteria were earlier shown to possess high cytosolic ADH activities (Escherichia coli IH 133369, Klebsiella pneumoniae IH 35385, Klebsiella oxytoca IH 35339, Pseudomonas aeruginosa IH 35342, and Hafnia alvei IH 53227). ADHs of the tested bacteria strongly preferred NAD as a cofactor. Marked ADH activities were found in all bacteria, even at low ethanol concentrations (1.5 mM) that may occur in the colon due to bacterial fermentation. The Km for ethanol varied from 29.9 mM for K. pneumoniae to 0.06 mM for Hafnia alvei. The inhibition of ADH by 4-methylpyrazole was found to be of the competitive type in 4 of 5 bacteria, and Ki varied from 18.26 +/- 3.3 mM for Escherichia coli to 0.47 +/- 0.13 mM for K. pneumoniae. At pH 7.4, ADH activity was significantly lower than at pH 9.6 in four bacterial strains. ADH of K. oxytoca, however, showed almost equal activities at neutral pH and at 9.6. In conclusion, NAD-linked alcohol dehydrogenases of aerobic colonic bacteria possess low apparent Km's for ethanol. Accordingly, they may oxidize moderate amounts of ethanol ingested during social drinking with nearly maximal velocity. This may result in the marked production of intracolonic acetaldehyde. Kinetic characteristics of the bacterial enzymes may enable some of them to produce acetaldehyde even from endogenous ethanol formed by other bacteria via alcoholic fermentation. The microbial ADHs were inhibited by 4-methylpyrazole by the same competitive inhibition as hepatic ADH, however, with nearly 1000 times lower susceptibility. Individual variations in human colonic flora may thus

  11. Mechanism of protection against alcoholism by an alcohol dehydrogenase polymorphism: development of an animal model.

    PubMed

    Rivera-Meza, Mario; Quintanilla, María Elena; Tampier, Lutske; Mura, Casilda V; Sapag, Amalia; Israel, Yedy

    2010-01-01

    Humans who carry a point mutation in the gene coding for alcohol dehydrogenase-1B (ADH1B*2; Arg47His) are markedly protected against alcoholism. Although this mutation results in a 100-fold increase in enzyme activity, it has not been reported to cause higher levels of acetaldehyde, a metabolite of ethanol known to deter alcohol intake. Hence, the mechanism by which this mutation confers protection against alcoholism is unknown. To study this protective effect, the wild-type rat cDNA encoding rADH-47Arg was mutated to encode rADH-47His, mimicking the human mutation. The mutated cDNA was incorporated into an adenoviral vector and administered to genetically selected alcohol-preferring rats. The V(max) of rADH-47His was 6-fold higher (P<0.001) than that of the wild-type rADH-47Arg. Animals transduced with rAdh-47His showed a 90% (P<0.01) increase in liver ADH activity and a 50% reduction (P<0.001) in voluntary ethanol intake. In animals transduced with rAdh-47His, administration of ethanol (1g/kg) produced a short-lived increase of arterial blood acetaldehyde concentration to levels that were 3.5- to 5-fold greater than those in animals transduced with the wild-type rAdh-47Arg vector or with a noncoding vector. This brief increase (burst) in arterial acetaldehyde concentration after ethanol ingestion may constitute the mechanism by which humans carrying the ADH1B*2 allele are protected against alcoholism.

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

    NASA Astrophysics Data System (ADS)

    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.

  13. Mechanism of aldehyde oxidation catalyzed by horse liver alcohol dehydrogenase.

    PubMed

    Olson, L P; Luo, J; Almarsson, O; Bruice, T C

    1996-07-30

    The mechanism of oxidation of benzaldehyde to benzoic acid catalyzed by horse liver alcohol dehydrogenase (HLADH) has been investigated using the HLADH structure at 2.1 A resolution with NAD+ and pentafluorobenzyl alcohol in the active site [Ramaswamy et al. (1994) Biochemistry 33,5230-5237]. Constructs for molecular dynamics (MD) investigations with HLADH were obtained by a best-fit superimposition of benzaldehyde or its hydrate on the pentafluorobenzyl alcohol bound to the active site Zn(II)ion. Equilibrium bond lengths, angles, and dihedral parameters for Zn(II) bonding residues His67, Cys46, and Cys174 were obtained from small-molecule X-ray crystal structures and an ab initio-derived parameterization of zinc in HLADH [Ryde, U. (1995) Proteins: Struct., Funct., Genet. 21,40-56]. Dynamic simulations in CHARMM were carried out on the following three constructs to 100 ps: (MD1) enzyme with NAD+, benzaldehyde, and zinc-ligated HO-in the active site; (MD2) enzyme with NAD+ and hydrated benzaldehyde monoanion bound to zinc via the pro-R oxygen, with a proton residing on the pro-S oxygen; and (MD3) enzyme with NAD+ and hydrated benzaldehyde monoanion bound to zinc via the pro-S oxygen, with a proton residing on the pro-R oxygen. Analyses were done of 800 sample conformations taken in the last 40 ps of dynamics. Structures from MD1 and MD3 were used to define the initial spatial arrangements of reactive functionalities for semiempirical PM3 calculations. Using PM3, model systems were calculated of ground states and some transition states for aldehyde hydration, hydride transfer, and subsequent proton shuttling. With benzaldehyde and zinc-bound hydroxide ion in the active site, the oxygen of Zn(II)-OH resided at a distance of 2.8-5.5 A from the aldehyde carbonyl carbon during the dynamics simulation. This may be compared to the PM3 transition state for attack of the Zn(II)-OH oxygen on the benzaldehyde carbonyl carbon, which has an O...C distance of 1.877 A. HLADH

  14. Lactate dehydrogenase activity is inhibited by methylmalonate in vitro.

    PubMed

    Saad, Laura O; Mirandola, Sandra R; Maciel, Evelise N; Castilho, Roger F

    2006-04-01

    Methylmalonic acidemia (MMAemia) is an inherited metabolic disorder of branched amino acid and odd-chain fatty acid metabolism, involving a defect in the conversion of methylmalonyl-coenzyme A to succinyl-coenzyme A. Systemic and neurological manifestations in this disease are thought to be associated with the accumulation of methylmalonate (MMA) in tissues and biological fluids with consequent impairment of energy metabolism and oxidative stress. In the present work we studied the effect of MMA and two other inhibitors of mitochondrial respiratory chain complex II (malonate and 3-nitropropionate) on the activity of lactate dehydrogenase (LDH) in tissue homogenates from adult rats. MMA potently inhibited LDH-catalyzed conversion of lactate to pyruvate in liver and brain homogenates as well as in a purified bovine heart LDH preparation. LDH was about one order of magnitude less sensitive to inhibition by MMA when catalyzing the conversion of pyruvate to lactate. Kinetic studies on the inhibition of brain LDH indicated that MMA inhibits this enzyme competitively with lactate as a substrate (K (i)=3.02+/-0.59 mM). Malonate and 3-nitropropionate also strongly inhibited LDH-catalyzed conversion of lactate to pyruvate in brain homogenates, while no inhibition was observed by succinate or propionate, when present in concentrations of up to 25 mM. We propose that inhibition of the lactate/pyruvate conversion by MMA contributes to lactate accumulation in blood, metabolic acidemia and inhibition of gluconeogenesis observed in patients with MMAemia. Moreover, the inhibition of LDH in the central nervous system may also impair the lactate shuttle between astrocytes and neurons, compromising neuronal energy metabolism.

  15. Green tea polyphenols modulate insulin secretion by inhibiting glutamate dehydrogenase.

    PubMed

    Li, Changhong; Allen, Aron; Kwagh, Jae; Doliba, Nicolai M; Qin, Wei; Najafi, Habiba; Collins, Heather W; Matschinsky, Franz M; Stanley, Charles A; Smith, Thomas J

    2006-04-14

    Insulin secretion by pancreatic beta-cells is stimulated by glucose, amino acids, and other metabolic fuels. Glutamate dehydrogenase (GDH) has been shown to play a regulatory role in this process. The importance of GDH was underscored by features of hyperinsulinemia/hyperammonemia syndrome, where a dominant mutation causes the loss of inhibition by GTP and ATP. Here we report the effects of green tea polyphenols on GDH and insulin secretion. Of the four compounds tested, epigallocatechin gallate (EGCG) and epicatechin gallate were found to inhibit GDH with nanomolar ED(50) values and were therefore found to be as potent as the physiologically important inhibitor GTP. Furthermore, we have demonstrated that EGCG inhibits BCH-stimulated insulin secretion, a process that is mediated by GDH, under conditions where GDH is no longer inhibited by high energy metabolites. EGCG does not affect glucose-stimulated insulin secretion under high energy conditions where GDH is probably fully inhibited. We have further shown that these compounds act in an allosteric manner independent of their antioxidant activity and that the beta-cell stimulatory effects are directly correlated with glutamine oxidation. These results demonstrate that EGCG, much like the activator of GDH (BCH), can facilitate dissecting the complex regulation of insulin secretion by pharmacologically modulating the effects of GDH.

  16. Highly selective anti-Prelog synthesis of optically active aryl alcohols by recombinant Escherichia coli expressing stereospecific alcohol dehydrogenase.

    PubMed

    Li, Ming; Nie, Yao; Mu, Xiao Qing; Zhang, Rongzhen; Xu, Yan

    2016-07-03

    Biocatalytic asymmetric synthesis has been widely used for preparation of optically active chiral alcohols as the important intermediates and precursors of active pharmaceutical ingredients. However, the available whole-cell system involving anti-Prelog specific alcohol dehydrogenase is yet limited. A recombinant Escherichia coli system expressing anti-Prelog stereospecific alcohol dehydrogenase from Candida parapsilosis was established as a whole-cell system for catalyzing asymmetric reduction of aryl ketones to anti-Prelog configured alcohols. Using 2-hydroxyacetophenone as the substrate, reaction factors including pH, cell status, and substrate concentration had obvious impacts on the outcome of whole-cell biocatalysis, and xylose was found to be an available auxiliary substrate for intracellular cofactor regeneration, by which (S)-1-phenyl-1,2-ethanediol was achieved with an optical purity of 97%e.e. and yield of 89% under the substrate concentration of 5 g/L. Additionally, the feasibility of the recombinant cells toward different aryl ketones was investigated, and most of the corresponding chiral alcohol products were obtained with an optical purity over 95%e.e. Therefore, the whole-cell system involving recombinant stereospecific alcohol dehydrogenase was constructed as an efficient biocatalyst for highly enantioselective anti-Prelog synthesis of optically active aryl alcohols and would be promising in the pharmaceutical industry.

  17. Comparison of three classes of human liver alcohol dehydrogenase. Emphasis on different substrate binding pockets.

    PubMed

    Eklund, H; Müller-Wille, P; Horjales, E; Futer, O; Holmquist, B; Vallee, B L; Höög, J O; Kaiser, R; Jörnvall, H

    1990-10-24

    Conformational models of the three characterized classes of mammalian liver alcohol dehydrogenase were constructed using computer graphics based on the known three-dimensional structure of the E subunit of the horse enzyme (class I) and the primary structures of the three human enzyme classes. This correlates the substrate-binding pockets of the class I subunits (alpha, beta and gamma in the human enzyme) with those of the class II and III subunits (pi and chi, respectively) for three enzymes that differ in substrate specificity, inhibition pattern and many other properties. The substrate-binding sites exhibit pronounced differences in both shape and properties. Comparing human class I subunits with those of class II and III subunits there are no less than 8 and 10 replacements, respectively, out of 11 residues in the substrate pocket, while in the human class I isozyme variants, only 1-3 of these 11 positions differ. A single residue, Val294, is conserved throughout. The liver alcohol dehydrogenases, with different substrate-specificity pockets, resemble the patterns of other enzyme families such as the pancreatic serine proteases. The inner part of the substrate cleft in the class II and III enzymes is smaller than in the horse class I enzyme, because both Ser48 and Phe93 are replaced by larger residues, Thr and Tyr, respectively. In class II, the residues in the substrate pocket are larger in about half of the positions. It is rich in aromatic residues, four Phe and one Tyr, making the substrate site distinctly smaller than in the class I subunits. In class III, the inner part of the substrate cleft is narrow but the outer part considerably wider and more polar than in the class I and II enzymes. In addition, Ser (or Thr) and Tyr in class II and III instead of His51 may influence proton abstraction/donation at the active site.

  18. Metabolic basis of ethylene glycol monobutyl ether (2-butoxyethanol) toxicity: role of alcohol and aldehyde dehydrogenases

    SciTech Connect

    Ghanayem, B.I.; Burka, L.T.; Matthews, H.B.

    1987-07-01

    2-Butoxyethanol (BE) is a massively produced glycol ether of which more than 230 million pounds was produced in the United States in 1983. It is extensively used in aerosols and cleaning agents intended for household use. This creates a high potential for human exposure during its manufacturing and use. A single exposure of rats to BE causes severe hemolytic anemia accompanied by secondary hemoglobinuria as well as liver and kidney damage. Butoxyacetic acid (BAA) was earlier identified as a urinary metabolite of BE. In addition, we have recently identified two additional urinary metabolites of BE, namely, BE-glucuronide and BE-sulfate conjugates. The current studies were undertaken to investigate the metabolic basis of BE-induced hematotoxicity in male F344 rats. Treatment of rats with pyrazole (alcohol dehydrogenase inhibitor) protected rats against BE-induced hematotoxicity and inhibited BE metabolism to BAA. Pyrazole inhibition of BE metabolism to BAA was accompanied by increased BE metabolism to BE-glucuronide and BE-sulfate as determined by quantitative high-performance liquid chromatography analysis of BE metabolites in urine. There was approximately a 10-fold decrease in the ratio of BAA to BE-glucuronide + BE-sulfate in the urine of rats treated with pyrazole + BE compared to rats treated with BE alone. Pretreatment of rats with cyanamide (aldehyde dehydrogenase inhibitor) also significantly protected rats against BE-induced hematotoxicity and modified BE metabolism in a manner similar to that caused by pyrazole. Administration of equimolar doses of BE, the metabolic intermediate butoxyacetaldehyde, or the ultimate metabolite BAA caused similar hematotoxic effects. Cyanamide also protected rats against butoxyacetaldehyde-induced hematotoxicity.

  19. A Long-Chain Secondary Alcohol Dehydrogenase from Rhodococcus erythropolis ATCC 4277

    PubMed Central

    Ludwig, B.; Akundi, A.; Kendall, K.

    1995-01-01

    A NAD-dependent secondary alcohol dehydrogenase has been purified from the alkane-degrading bacterium, Rhodococcus erythropolis ATCC 4277. The enzyme was found to be active against a broad range of substrates, particularly long-chain secondary aliphatic alcohols. Although optimal activity was observed with linear 2-alcohols containing between 6 and 11 carbon atoms, secondary alcohols as long as 2-tetradecanol were oxidized at 25% of the rate seen with mid-range alcohols. The purified enzyme was specific for the S-(+) stereoisomer of 2-octanol and had a specific activity for 2-octanol of over 200 (mu)mol/min/mg of protein at pH 9 and 37(deg)C, 25-fold higher than that of any previously reported S-(+) secondary alcohol dehydrogenase. Linear primary alcohols containing between 3 and 13 carbon atoms were utilized 20- to 40-fold less efficiently than the corresponding secondary alcohols. The apparent K(infm) value for NAD(sup+) with 2-octanol as the substrate was 260 (mu)M, whereas the apparent K(infm) values for the 2-alcohols ranged from over 5 mM for 2-pentanol to less than 2 (mu)M for 2-tetradecanol. The enzyme showed moderate thermostability (half-life of 4 h at 60(deg)C) and could potentially be useful for the synthesis of optically pure stereoisomers of secondary alcohols. PMID:16535152

  20. Direct Electrochemical Addressing of Immobilized Alcohol Dehydrogenase for the Heterogeneous Bioelectrocatalytic Reduction of Butyraldehyde to Butanol

    PubMed Central

    Schlager, S; Neugebauer, H; Haberbauer, M; Hinterberger, G; Sariciftci, N S

    2015-01-01

    Modified electrodes using immobilized alcohol dehydrogenase enzymes for the efficient electroreduction of butyraldehyde to butanol are presented as an important step for the utilization of CO2-reduction products. Alcohol dehydrogenase was immobilized, embedded in an alginate–silicate hybrid gel, on a carbon felt (CF) electrode. The application of this enzyme to the reduction of an aldehyde to an alcohol with the aid of the coenzyme nicotinamide adenine dinucleotide (NADH), in analogy to the final step in the natural reduction cascade of CO2 to alcohol, has been already reported. However, the use of such enzymatic reductions is limited because of the necessity of providing expensive NADH as a sacrificial electron and proton donor. Immobilization of such dehydrogenase enzymes on electrodes and direct pumping of electrons into the biocatalysts offers an easy and efficient way for the biochemical recycling of CO2 to valuable chemicals or alternative synthetic fuels. We report the direct electrochemical addressing of immobilized alcohol dehydrogenase for the reduction of butyraldehyde to butanol without consumption of NADH. The selective reduction of butyraldehyde to butanol occurs at room temperature, ambient pressure and neutral pH. Production of butanol was detected by using liquid-injection gas chromatography and was estimated to occur with Faradaic efficiencies of around 40 %. PMID:26113881

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

    PubMed

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

    2013-01-08

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

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

    PubMed Central

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

    2013-01-01

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

  3. Disruption of the membrane-bound alcohol dehydrogenase-encoding gene improved glycerol use and dihydroxyacetone productivity in Gluconobacter oxydans.

    PubMed

    Habe, Hiroshi; Fukuoka, Tokuma; Morita, Tomotake; Kitamoto, Dai; Yakushi, Toshiharu; Matsushita, Kazunobu; Sakaki, Keiji

    2010-01-01

    Dihydroxyacetone (DHA) production from glycerol by Gluconobacter oxydans is an industrial form of fermentation, but some problems exist related to microbial DHA production. For example, glycerol inhibits DHA production and affects its biological activity. G. oxydans produces both DHA and glyceric acid (GA) from glycerol simultaneously, and membrane-bound glycerol dehydrogenase and membrane-bound alcohol dehydrogenases are involved in the two reactions, respectively. We discovered that the G. oxydans mutant DeltaadhA, in which the membrane-bound alcohol dehydrogenase-encoding gene (adhA) was disrupted, significantly improved its ability to grow in a higher concentration of glycerol and to produce DHA compared to a wild-type strain. DeltaadhA grew on 220 g/l of initial glycerol and produced 125 g/l of DHA during a 3-d incubation, whereas the wild-type did not. Resting DeltaadhA cells converted 230 g/l of glycerol aqueous solution to 139.7 g/l of DHA during a 3-d incubation. The inhibitory effect of glycerate sodium salt on DeltaadhA was investigated. An increase in the glycerate concentration at the beginning of growth resulted in decreases in both growth and DHA production.

  4. Bradykinetic alcohol dehydrogenases make yeast fitter for growth in the presence of allyl alcohol.

    PubMed

    Plapp, Bryce V; Lee, Ann Ting-I; Khanna, Aditi; Pryor, John M

    2013-02-25

    Previous studies showed that fitter yeast (Saccharomyces cerevisiae) that can grow by fermenting glucose in the presence of allyl alcohol, which is oxidized by alcohol dehydrogenase I (ADH1) to toxic acrolein, had mutations in the ADH1 gene that led to decreased ADH activity. These yeast may grow more slowly due to slower reduction of acetaldehyde and a higher NADH/NAD(+) ratio, which should decrease the oxidation of allyl alcohol. We determined steady-state kinetic constants for three yeast ADHs with new site-directed substitutions and examined the correlation between catalytic efficiency and growth on selective media of yeast expressing six different ADHs. The H15R substitution (a test for electrostatic effects) is on the surface of ADH and has small effects on the kinetics. The H44R substitution (affecting interactions with the coenzyme pyrophosphate) was previously shown to decrease affinity for coenzymes 2-4-fold and turnover numbers (V/Et) by 4-6-fold. The W82R substitution is distant from the active site, but decreases turnover numbers by 5-6-fold, perhaps by effects on protein dynamics. The E67Q substitution near the catalytic zinc was shown previously to increase the Michaelis constant for acetaldehyde and to decrease turnover for ethanol oxidation. The W54R substitution, in the substrate binding site, increases kinetic constants (Ks, by >10-fold) while decreasing turnover numbers by 2-7-fold. Growth of yeast expressing the different ADHs on YPD plates (yeast extract, peptone and dextrose) plus antimycin to require fermentation, was positively correlated with the log of catalytic efficiency for the sequential bi reaction (V1/KiaKb=KeqV2/KpKiq, varying over 4 orders of magnitude, adjusted for different levels of ADH expression) in the order: WT≈H15R>H44R>W82R>E67Q>W54R. Growth on YPD plus 10mM allyl alcohol was inversely correlated with catalytic efficiency. The fitter yeast are "bradytrophs" (slow growing) because the ADHs have decreased catalytic

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

    PubMed

    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.

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

    PubMed

    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)

  7. Functional characterization of cinnamyl alcohol dehydrogenase and caffeic acid O-methyltransferase in Brachypodium distachyon.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lignin is a significant recalcitrant in the conversion of plant biomass to bioethanol. Cinnamyl alcohol dehydrogenase (CAD) and caffeic acid O-methyltransferase (COMT) catalyze key steps in the pathway of lignin monomer biosynthesis. Brown midrib mutants in Zea mays and Sorghum bicolor with impaired...

  8. [Isolation and fermentation conditions of strains producing 1-phenyl-2-amino-ethanol alcohol dehydrogenase].

    PubMed

    Wang, J; Wang, J; Yang, L; Wu, J; Sun, W

    2001-10-01

    A Arachnia sp. P163 producing alcohol dehydrogenase which is able to reduce aminoacetophenone to R-1-phenyl-2-aminoethanol was obtained from soil and cultures. The maximum activity of enzyme was produced by the LB medium containing 1% sodium citrate and peptone, 0.1% phenylaminoethanol as inducer at 30 degrees C for 48 hs.

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

    ERIC Educational Resources Information Center

    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…

  10. Determination of the Subunit Molecular Mass and Composition of Alcohol Dehydrogenase by SDS-PAGE

    ERIC Educational Resources Information Center

    Nash, Barbara T.

    2007-01-01

    SDS-PAGE is a simple, rapid technique that has many uses in biochemistry and is readily adaptable to the undergraduate laboratory. It is, however, a technique prone to several types of procedural pitfalls. This article describes the use of SDS-PAGE to determine the subunit molecular mass and composition of yeast alcohol dehydrogenase employing…

  11. Purification and characterization of a zinc-dependent cinnamyl alcohol dehydrogenase from Leucaena leucocephala, a tree legume.

    PubMed

    Pandey, Brijesh; Pandey, Veda P; Shasany, A K; Dwivedi, U N

    2014-04-01

    A cinnamyl alcohol dehydrogenase (CAD) from the secondary xylem of Leucaena leucocephala has been purified to homogeneity through successive steps of ammonium sulfate fractionation, DEAE cellulose, Sephadex G-75, and Blue Sepharose CL-6B affinity column chromatographies. CAD was purified to 514.2 folds with overall recovery of 13 % and specific activity of 812. 5 nkat/mg. Native and subunit molecular masses of the purified enzyme were found to be ∼76 and ∼38 kDa, respectively, suggesting it to be a homodimer. The enzyme exhibited highest catalytic efficiency (Kcat/Km 3.75 μM(-1) s(-1)) with cinnamyl aldehyde among all the substrates investigated. The pH and temperature optima of the purified CAD were pH 8.8 and 40 °C, respectively. The enzyme activity was enhanced in the presence of 2.0 mM Mg(2+), while Zn(2+) at the same concentration exerted an inhibitory effect. The inclusion of 2.0 mM EDTA in the assay system activated the enzyme. The enzyme was inhibited with caffeic acid and ferulic acid in a concentration-dependent manner, while no inhibition was observed with salicylic acid. Peptide mass analysis of the purified CAD by MALDI-TOF showed a significant homology to alcohol dehydrogenases of MDR superfamily.

  12. Effects of DNA on immunoglobulin production stimulating activity of alcohol dehydrogenase.

    PubMed

    Okamoto, T; Furutani, H; Sasaki, T; Sugahara, T

    1999-09-01

    Alcohol dehydrogenase-I (ADH-I) derived from horse liver stimulated IgM production by human-human hybridoma, HB4C5 cells and lymphocytes. The IPSF activity of ADH-I was suppressed by coexistence of short DNA whose chain length is less than 200 base pairs (bp) and fibrous DNA in a dose-dependent manner. These DNA preparations completely inhibited the IPSF activity at the concentration of 250 mug/ml and 1.0 mg/ml, respectively. DNA sample termed long DNA whose average chain length is 400-7000 bp slightly stimulated IPSF activity at 0.06 mug/ml. However, long DNA suppressed IPSF activity by half at 1.0 mg/ml. The laser confocal microscopic analysis had revealed that ADH-I was incorporated by HB4C5 cells. The uptake of ADH-I was strongly inhibited by short DNA and fibrous DNA. However, long DNA did not suppress the internalization of ADH-I into HB4C5 cells. These findings indicate that short DNA and fibrous DNA depress IPSF activity of ADH-I by inhibiting the internalization of this enzyme. According to the gel-filtration analysis using HPLC, ADH-I did not directly interact with short DNA. It is expected from these findings that short DNA influences HB4C5 cells to suppress the internalization of ADH-I. Moreover, these facts also strongly suggest that ADH-I acts as IPSF after internalization into the cell.

  13. Aldehyde dehydrogenase 2 inhibits inflammatory response and regulates atherosclerotic plaque

    PubMed Central

    Wei, Shu-jian; Zhang, Ming-xiang; Wang, Xu-ping; Yuan, Qiu-huan; Xue, Li; Wang, Jia-li; Cui, Zhao-qiang; Zhang, Yun; Xu, Feng; Chen, Yu-guo

    2016-01-01

    Previous studies demonstrated that aldehyde dehydrogenase 2 (ALDH2) rs671 polymorphism, which eliminates ALDH2 activity down to 1%-6%, is a susceptibility gene for coronary disease. Here we investigated the underlying mechanisms based on our prior clinical and experimental studies. Male apoE−/− mice were transfected with GFP, ALDH2-overexpression and ALDH2-RNAi lentivirus respectively (n=20 each) after constrictive collars were placed around the right common carotid arteries. Consequently, ALDH2 gene silencing led to an increased en face plaque area, more unstable plaque with heavier accumulation of lipids, more macrophages, less smooth muscle cells and collagen, which were associated with aggravated inflammation. However, ALDH2 overexpression displayed opposing effects. We also found that ALDH2 activity decreased in atherosclerotic plaques of human and aged apoE−/− mice. Moreover, in vitro experiments with human umbilical vein endothelial cells further illustrated that, inhibition of ALDH2 activity resulted in elevating inflammatory molecules, an increase of nuclear translocation of NF-κB, and enhanced phosphorylation of NF-κB p65, AP-1 c-Jun, Jun-N terminal kinase and p38 MAPK, while ALDH2 activation could trigger contrary effects. These findings suggested that ALDH2 can influence plaque development and vulnerability, and inflammation via MAPK, NF-κB and AP-1 signaling pathways. PMID:27191745

  14. Ethanol at low concentrations protects glomerular podocytes through alcohol dehydrogenase and 20-HETE.

    PubMed

    McCarthy, Ellen T; Zhou, Jianping; Eckert, Ryan; Genochio, David; Sharma, Rishi; Oni, Olurinde; De, Alok; Srivastava, Tarak; Sharma, Ram; Savin, Virginia J; Sharma, Mukut

    2015-01-01

    Clinical studies suggest cardiovascular and renal benefits of ingesting small amounts of ethanol. Effects of ethanol, role of alcohol dehydrogenase (ADH) or of 20-hydroxyeicosatetraenoic acid (20-HETE) in podocytes of the glomerular filtration barrier have not been reported. We found that mouse podocytes at baseline generate 20-HETE and express ADH but not CYP2e1. Ethanol at high concentrations altered the actin cytoskeleton, induced CYP2e1, increased superoxide production and inhibited ADH gene expression. Ethanol at low concentrations upregulated the expression of ADH and CYP4a12a. 20-HETE, an arachidonic acid metabolite generated by CYP4a12a, blocked the ethanol-induced cytoskeletal derangement and superoxide generation. Ethanol at high concentration or ADH inhibitor increased glomerular albumin permeability in vitro. 20-HETE and its metabolite produced by ADH activity, 20-carboxy-arachidonic acid, protected the glomerular permeability barrier against an ADH inhibitor, puromycin or FSGS permeability factor. We conclude that ADH activity is required for glomerular function, 20-HETE is a physiological substrate of ADH in podocytes and that podocytes are useful biosensors to understand glomeruloprotective effects of ethanol.

  15. Enhanced production of dihydroxyacetone from glycerol by overexpression of glycerol dehydrogenase in an alcohol dehydrogenase-deficient mutant of Gluconobacter oxydans.

    PubMed

    Li, Ming-hua; Wu, Jian; Liu, Xu; Lin, Jin-ping; Wei, Dong-zhi; Chen, Hao

    2010-11-01

    Gluconobacter oxydans can rapidly and incompletely oxidize glycerol to dihydroxyacetone (DHA), a versatile product extensively used in cosmetic, chemical and pharmaceutical industries. To improve DHA production, the glycerol dehydrogenase (GDH) responsible for DHA formation was overexpressed in G. oxydans M5AM, in which the gene coding for the membrane-bound alcohol dehydrogenase (ADH) was interrupted. Real-time PCR and enzyme activity assay revealed that the absence of ADH together with the overexpression of GDH gene resulted in an increased GDH activity in the resulting strain M5AM/GDH, which led to a substantially enhanced production of DHA in a resting cell system. In a batch biotransformation process, M5AM/GDH exhibited a 2.4-fold increased DHA productivity of 2.4g/g CDW/h from 1.0g/g CDW/h, yielding 96g/L DHA from 100g/L glycerol. When 140g/L glycerol was supplied, a final DHA concentration of 134g/L was accumulated within 14h. In four repeated batch runs, 385g DHA over a time period of 34h was achieved from 400g glycerol with an average productivity of 2.2g/g CDW/h. These results indicated that this newly developed strain G. oxydans M5AM/GDH with high productivity and increased tolerance against product inhibition has potential for DHA production in an industrial bioconversion process.

  16. Increasing Anaerobic Acetate Consumption and Ethanol Yields in Saccharomyces cerevisiae with NADPH-Specific Alcohol Dehydrogenase

    PubMed Central

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

    2015-01-01

    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. PMID:26386051

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

    PubMed

    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.

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

    PubMed

    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.

  19. A bifunctional enzyme from Rhodococcus erythropolis exhibiting secondary alcohol dehydrogenase-catalase activities.

    PubMed

    Martinez-Rojas, Enriqueta; Kurt, Tutku; Schmidt, Udo; Meyer, Vera; Garbe, Leif-Alexander

    2014-11-01

    Alcohol dehydrogenases have long been recognized as potential biocatalyst for production of chiral fine and bulk chemicals. They are relevant for industry in enantiospecific production of chiral compounds. In this study, we identified and purified a nicotinamide adenine dinucleotide (NAD)-dependent secondary alcohol dehydrogenase (SdcA) from Rhodococcus erythropolis oxidizing γ-lactols into γ-lactones. SdcA showed broad substrate specificity on γ-lactols; secondary aliphatic alcohols with 8 and 10 carbon atoms were also substrates and oxidized with (2S)-stereospecificity. The enzyme exhibited moderate stability with a half-life of 5 h at 40 °C and 20 days at 4 °C. Mass spectrometric identification revealed high sequence coverage of SdcA amino acid sequence to a highly conserved catalase from R. erythropolis. The corresponding encoding gene was isolated from genomic DNA and subsequently overexpressed in Escherichia coli BL21 DE3 cells. In addition, the recombinant SdcA was purified and characterized in order to confirm that the secondary alcohol dehydrogenase and catalase activity correspond to the same enzyme.

  20. Nitrophenide (Megasul) blocks Eimeria tenella development by inhibiting the mannitol cycle enzyme mannitol-1-phosphate dehydrogenase.

    PubMed

    Allocco, J J; Nare, B; Myers, R W; Feiglin, M; Schmatz, D M; Profous-Juchelka, H

    2001-12-01

    Unsporulated oocysts of the protozoan parasite Eimeria tenella contain high levels of mannitol, which is thought to be the principal energy source for the process of sporulation. Biosynthesis and utilization of this sugar alcohol occurs via a metabolic pathway known as the mannitol cycle. Here, results are presented that suggest that 3-nitrophenyl disulfide (nitrophenide, Megasul), an anticoccidial drug commercially used in the 1950s, inhibits mannitol-1-phosphate dehydrogenase (M1PDH), which catalyzes the committed enzymatic step in the mannitol cycle. Treatment of E. tenella-infected chickens with nitrophenide resulted in a 90% reduction in oocyst shedding. The remaining oocysts displayed significant morphological abnormalities and were largely incapable of further development. Nitrophenide treatment did not affect parasite asexual reproduction, suggesting specificity for the sexual stage of the life cycle. Isolated oocysts from chickens treated with nitrophenide exhibited a dose-dependent reduction in mannitol, suggesting in vivo inhibition of parasite mannitol biosynthesis. Nitrophenide-mediated inhibition of MIPDH was observed in vitro using purified native enzyme. Moreover, MIPDH activity immunoprecipitated from E. tenella-infected cecal tissues was significantly lower in nitrophenide-treated compared with untreated chickens. Western blot analysis and immunohistochemistry showed that parasites from nitrophenide-treated and untreated chickens contained similar enzyme levels. These data suggest that nitrophenide blocks parasite development at the sexual stages by targeting M1PDH. Thus, targeting of the mannitol cycle with drugs could provide an avenue for controlling the spread of E. tenella in commercial production facilities by preventing oocyst shedding.

  1. The vertebrate alcohol dehydrogenase system: variable class II type form elucidates separate stages of enzymogenesis.

    PubMed Central

    Hjelmqvist, L; Estonius, M; Jörnvall, H

    1995-01-01

    A mixed-class alcohol dehydrogenase has been characterized from avian liver. Its functional properties resemble the classical class I type enzyme in livers of humans and animals by exhibiting low Km and kcat values with alcohols (Km = 0.7 mM with ethanol) and low Ki values with 4-methylpyrazole (4 microM). These values are markedly different from corresponding parameters of class II and III enzymes. In contrast, the primary structure of this avian liver alcohol dehydrogenase reveals an overall relationship closer to class II and to some extent class III (69 and 65% residue identities, respectively) than to class I or the other classes of the human alcohol dehydrogenases (52-61%), the presence of an insertion (four positions in a segment close to position 120) as in class II but in no other class of the human enzymes, and the presence of several active site residues considered typical of the class II enzyme. Hence, the avian enzyme has mixed-class properties, being functionally similar to class I, yet structurally similar to class II, with which it also clusters in phylogenetic trees of characterized vertebrate alcohol dehydrogenases. Comparisons reveal that the class II enzyme is approximately 25% more variable than the "variable" class I enzyme, which itself is more variable than the "constant" class III enzyme. The overall extreme, and the unusual chromatographic behavior may explain why the class II enzyme has previously not been found outside mammals. The properties define a consistent pattern with apparently repeated generation of novel enzyme activities after separate gene duplications. Images Fig. 3 PMID:7479907

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

    NASA Astrophysics Data System (ADS)

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

    2015-12-01

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

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

    PubMed

    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.

  4. Acute and chronic ethanol exposure differentially alters alcohol dehydrogenase and aldehyde dehydrogenase activity in the zebrafish liver.

    PubMed

    Tran, Steven; Nowicki, Magda; Chatterjee, Diptendu; Gerlai, Robert

    2015-01-02

    Chronic ethanol exposure paradigms have been successfully used in the past to induce behavioral and central nervous system related changes in zebrafish. However, it is currently unknown whether chronic ethanol exposure alters ethanol metabolism in adult zebrafish. In the current study we examine the effect of acute ethanol exposure on adult zebrafish behavioral responses, as well as alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) activity in the liver. We then examine how two different chronic ethanol exposure paradigms (continuous and repeated ethanol exposure) alter behavioral responses and liver enzyme activity during a subsequent acute ethanol challenge. Acute ethanol exposure increased locomotor activity in a dose-dependent manner. ADH activity was shown to exhibit an inverted U-shaped curve and ALDH activity was decreased by ethanol exposure at all doses. During the acute ethanol challenge, animals that were continuously housed in ethanol exhibited a significantly reduced locomotor response and increased ADH activity, however, ALDH activity did not change. Zebrafish that were repeatedly exposed to ethanol demonstrated a small but significant attenuation of the locomotor response during the acute ethanol challenge but ADH and ALDH activity was similar to controls. Overall, we identified two different chronic ethanol exposure paradigms that differentially alter behavioral and physiological responses in zebrafish. We speculate that these two paradigms may allow dissociation of central nervous system-related and liver enzyme-dependent ethanol induced changes in zebrafish.

  5. Group III alcohol dehydrogenase from Pectobacterium atrosepticum: insights into enzymatic activity and organization of the metal ion-containing region.

    PubMed

    Elleuche, Skander; Fodor, Krisztian; von der Heyde, Amélie; Klippel, Barbara; Wilmanns, Matthias; Antranikian, Garabed

    2014-05-01

    NAD(P)(+)-dependent alcohol dehydrogenases (ADH) are widely distributed in all phyla. These proteins can be assigned to three nonhomologous groups of isozymes, with group III being highly diverse with regards to catalytic activity and primary structure. Members of group III ADHs share a conserved stretch of amino acid residues important for cofactor binding and metal ion coordination, while sequence identities for complete proteins are highly diverse (<20 to >90 %). A putative group III ADH PaYqhD has been identified in BLAST analysis from the plant pathogenic enterobacterium Pectobacterium atrosepticum. The PaYqhD gene was expressed in the heterologous host Escherichia coli, and the recombinant protein was purified in a two-step purification procedure to homogeneity indicating an obligate dimerization of monomers. Four conserved amino acid residues involved in metal ion coordination were substituted with alanine, and their importance for catalytic activity was confirmed by circular dichroism spectrum determination, in vitro, and growth experiments. PaYqhD exhibits optimal activity at 40 °C with short carbon chain aldehyde compounds and NADPH as cofactor indicating the enzyme to be an aldehyde reductase. No oxidative activities towards alcoholic compounds were detectable. EDTA completely inhibited catalytic activity and was fully restored by the addition of Co(2+). Activity measurements together with sequence alignments and structure analysis confirmed that PaYqhD belongs to the butanol dehydrogenase-like enzymes within group III of ADHs.

  6. Aspartate 46, a second sphere ligand to the catalytic zinc, is essential for activity of yeast alcohol dehydrogenase

    SciTech Connect

    Ganzhorn, A.J.; Plapp, B.V.

    1987-05-01

    The crystal structure of horse liver alcohol dehydrogenase (ADH) shows a hydrogen bond between the imidazole of His-67, a ligand to the active site zinc, and the carboxylate of Asp-49. Both residues are conserved in alcohol dehydrogenases. Directed mutagenesis was used to replace the homologous Asp-46 in ADH I from S. cerevisiae with asparagine. The substitution did not alter the overall structure of the enzyme, as judged by CD measurements, but the removal of a negative charge was evident in electrophoresis, and in the absorption and fluorescence spectra. The mutant and wild-type enzymes had similar zinc contents as determined by atomic absorption spectroscopy. Active site titration and steady state kinetics indicated that binding of coenzymes, substrates and substrate analogs is 4-24 fold weaker in the asparagine enzyme. The turnover numbers were reduced by a factor of 70 for ethanol oxidation and 30 for acetaldehyde reduction at pH 7.3, 30/sup 0/C. Dead end inhibition studies and the kinetic isotope effect showed that NAD and ethanol binding follow a rapid equilibrium random mechanism as opposed to the ordered mechanism found for ADH I. They conclude that the carboxyl group of Asp-46 is essential for the electrostatic environment near the active site zinc. Amidation may affect the geometry and/or coordination of the metal complex.

  7. Thermophilic alcohol dehydrogenase from the mesophile Entamoeba histolytica: crystallization and preliminary X-ray characterization.

    PubMed

    Shimon, Linda J W; Peretz, Moshe; Goihberg, Edi; Burstein, Yigal; Frolow, Felix

    2002-03-01

    The tetrameric NADP(+)-dependent secondary alcohol dehydrogenase from Entamoeba histolytica has been crystallized in its apo form. The crystals belong to space group C222(1), with unit-cell parameters a = 76.89, b = 234.24, c = 96.24 A, and diffract to 1.9 A at liquid-nitrogen temperature. Analysis of the Patterson self-rotation function shows that the crystals contain one dimer per asymmetric unit.

  8. Depression of alcohol dehydrogenase activity in rat hepatocyte culture by dihydrotestosterone.

    PubMed

    Mezey, E; Potter, J J; Diehl, A M

    1986-01-15

    Hepatocytes harvested from castrated rats retained a higher alcohol dehydrogenase (EC 1.1.1.1) activity than hepatocytes harvested from normal rats during 7 days of culture. Dihydrotestosterone (1 microM) decreased the enzyme activity, after 2 and 5 days of culture, in hepatocytes from castrated and control animals respectively. Dihydrotestosterone decreased the enzyme activity to similar values in both groups of hepatocytes by the end of 7 days of culture. Testosterone (1 microM) had no effect on the enzyme activity in normal hepatocytes and only a transitory effect in decreasing the enzyme activity in hepatocytes from castrated animals. The increases in alcohol dehydrogenase activity after castration and their suppression by dihydrotestosterone were associated with parallel changes in the rate of ethanol elimination. Additions of substrates of the malate-aspartate shuttle or dinitrophenol did not modify ethanol elimination. These observations indicate that dihydrotestosterone has a direct suppressant effect on hepatocyte alcohol dehydrogenase and that the enzyme activity is a major determinant of the rate of ethanol elimination.

  9. In vivo relationship between monoamine oxidase type B and alcohol dehydrogenase: effects of ethanol and phenylethylamine

    SciTech Connect

    Aliyu, S.U.; Upahi, L.

    1988-01-01

    The role of acute ethanol and phenylethylamine on the brain and platelet monoamine oxidase activities, hepatic cytosolic alcohol dehydrogenase, redox state and motor behavior were studied in male rats. Ethanol on its own decreased the redox couple ratio, as well as, alcohol dehydrogenase activity in the liver while at the same time it increased brain and platelet monoamine oxidase activity due to lower Km with no change in Vmax. The elevation in both brain and platelet MAO activity was associated with ethanol-induced hypomotility in the rats. Co-administration of phenylethylamine and ethanol to the animals, caused antagonism of the ethanol-induced effects described above. The effects of phenylethylamine alone, on the above mentioned biochemical and behavioral indices, are more complex. Phenylethylamine on its own, like ethanol, caused reduction of the cytosolic redox, ratio and elevation of monoamine oxidase activity in the brain and platelets. However, in contrast to ethanol, this monoamine produced hypermotility and activation of the hepatic cytosolic alcohol dehydrogenase activity in the animals.

  10. Asymmetric reduction and oxidation of aromatic ketones and alcohols using W110A secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus.

    PubMed

    Musa, Musa M; Ziegelmann-Fjeld, Karla I; Vieille, Claire; Zeikus, J Gregory; Phillips, Robert S

    2007-01-05

    An enantioselective asymmetric reduction of phenyl ring-containing prochiral ketones to yield the corresponding optically active secondary alcohols was achieved with W110A secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus (W110A TESADH) in Tris buffer using 2-propanol (30%, v/v) as cosolvent and cosubstrate. This concentration of 2-propanol was crucial not only to enhance the solubility of hydrophobic phenyl ring-containing substrates in the aqueous reaction medium, but also to shift the equilibrium in the reduction direction. The resulting alcohols have S-configuration, in agreement with Prelog's rule, in which the nicotinamide-adenine dinucleotide phosphate (NADPH) cofactor transfers its pro-R hydride to the re face of the ketone. A series of phenyl ring-containing ketones, such as 4-phenyl-2-butanone (1a) and 1-phenyl-1,3-butadione (2a), were reduced with good to excellent yields and high enantioselectivities. On the other hand, 1-phenyl-2-propanone (7a) was reduced with lower ee than 2-butanone derivatives. (R)-Alcohols, the anti-Prelog products, were obtained by enantiospecific oxidation of (S)-alcohols through oxidative kinetic resolution of the rac-alcohols using W110A TESADH in Tris buffer/acetone (90:10, v/v).

  11. Ethanol production by the hyperthermophilic archaeon Pyrococcus furiosus by expression of bacterial bifunctional alcohol dehydrogenases.

    PubMed

    Keller, Matthew W; Lipscomb, Gina L; Nguyen, Diep M; Crowley, Alexander T; Schut, Gerrit J; Scott, Israel; Kelly, Robert M; Adams, Michael W W

    2017-02-14

    Ethanol is an important target for the renewable production of liquid transportation fuels. It can be produced biologically from pyruvate, via pyruvate decarboxylase, or from acetyl-CoA, by alcohol dehydrogenase E (AdhE). Thermophilic bacteria utilize AdhE, which is a bifunctional enzyme that contains both acetaldehyde dehydrogenase and alcohol dehydrogenase activities. Many of these organisms also contain a separate alcohol dehydrogenase (AdhA) that generates ethanol from acetaldehyde, although the role of AdhA in ethanol production is typically not clear. As acetyl-CoA is a key central metabolite that can be generated from a wide range of substrates, AdhE can serve as a single gene fuel module to produce ethanol through primary metabolic pathways. The focus here is on the hyperthermophilic archaeon Pyrococcus furiosus, which grows by fermenting sugar to acetate, CO2 and H2 . Previously, by the heterologous expression of adhA from a thermophilic bacterium, P. furiosus was shown to produce ethanol by a novel mechanism from acetate, mediated by AdhA and the native enzyme aldehyde oxidoreductase (AOR). In this study, the AOR gene was deleted from P. furiosus to evaluate ethanol production directly from acetyl-CoA by heterologous expression of the adhE gene from eight thermophilic bacteria. Only AdhEs from two Thermoanaerobacter strains showed significant activity in cell-free extracts of recombinant P. furiosus and supported ethanol production in vivo. In the AOR deletion background, the highest amount of ethanol (estimated 61% theoretical yield) was produced when adhE and adhA from Thermoanaerobacter were co-expressed.

  12. Human liver class I alcohol dehydrogenase gammagamma isozyme: the sole cytosolic 3beta-hydroxysteroid dehydrogenase of iso bile acids.

    PubMed

    Marschall, H U; Oppermann, U C; Svensson, S; Nordling, E; Persson, B; Höög, J O; Jörnvall, H

    2000-04-01

    3beta-Hydroxy (iso) bile acids are formed during enterohepatic circulation from 3alpha-hydroxy bile acids and constitute normal compounds in plasma but are virtually absent in bile. Isoursodeoxycholic acid (isoUDCA) is a major metabolite of UDCA. In a recent study it was found that after administration of isoUDCA, UDCA became the major acid in bile. Thus, epimerization of the 3beta-hydroxy to a 3alpha-hydroxy group, catalyzed by 3beta-hydroxysteroid dehydrogenases (HSD) and 3-oxo-reductases must occur. The present study aims to characterize the human liver bile acid 3beta-HSD. Human liver cytosol and recombinant alcohol dehydrogenase (ADH) betabeta and gammagamma isozymes were subjected to native polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing. Activity staining with oxidized nicotinamide adenine dinucleotide (NAD(+)) or oxidized nicotinamide adenine dinucleotide phosphate (NADP(+)) as cofactors and various iso bile acids as substrates was used to screen for 3beta-HSD activity. Reaction products were identified and quantified by gas chromotography/mass spectrometry (GC/MS). Computer-assisted substrate docking of isoUDCA to the active site of a 3-dimensional model of human class I gammagamma ADH was performed. ADH gammagamma isozyme was identified as the iso bile acid 3beta-HSD present in human liver cytosol, with NAD(+) as a cofactor. Values for k(cat)/K(m) were in the rank order isodeoxycholic acid (isoDCA), isochenodeoxycholic acid (isoCDCA), isoUDCA, and isolithocholic acid (isoLCA) (0.10, 0.09, 0.08, and 0. 05 min(-1) x micromol/L(-1), respectively). IsoUDCA fits as substrate to the 3-dimensional model of the active-site of ADH gammagamma. ADH gammagamma isozyme was defined as the only bile acid 3beta-HSD in human liver cytosol. Hydroxysteroid dehydrogenases are candidates for the binding and transport of 3alpha-hydroxy bile acids. We assume that ADH gammagamma isozyme is involved in cytosolic bile acid binding and transport processes as well.

  13. Isolation and characterization of full-length putative alcohol dehydrogenase genes from polygonum minus

    NASA Astrophysics Data System (ADS)

    Hamid, Nur Athirah Abd; Ismail, Ismanizan

    2013-11-01

    Polygonum minus, locally named as Kesum is an aromatic herb which is high in secondary metabolite content. Alcohol dehydrogenase is an important enzyme that catalyzes the reversible oxidation of alcohol and aldehyde with the presence of NAD(P)(H) as co-factor. The main focus of this research is to identify the gene of ADH. The total RNA was extracted from leaves of P. minus which was treated with 150 μM Jasmonic acid. Full-length cDNA sequence of ADH was isolated via rapid amplification cDNA end (RACE). Subsequently, in silico analysis was conducted on the full-length cDNA sequence and PCR was done on genomic DNA to determine the exon and intron organization. Two sequences of ADH, designated as PmADH1 and PmADH2 were successfully isolated. Both sequences have ORF of 801 bp which encode 266 aa residues. Nucleotide sequence comparison of PmADH1 and PmADH2 indicated that both sequences are highly similar at the ORF region but divergent in the 3' untranslated regions (UTR). The amino acid is differ at the 107 residue; PmADH1 contains Gly (G) residue while PmADH2 contains Cys (C) residue. The intron-exon organization pattern of both sequences are also same, with 3 introns and 4 exons. Based on in silico analysis, both sequences contain "classical" short chain alcohol dehydrogenases/reductases ((c) SDRs) conserved domain. The results suggest that both sequences are the members of short chain alcohol dehydrogenase family.

  14. Structure of daidzin, a naturally occurring anti-alcohol-addiction agent, in complex with human mitochondrial aldehyde dehydrogenase.

    PubMed

    Lowe, Edward D; Gao, Guang-Yao; Johnson, Louise N; Keung, Wing Ming

    2008-08-14

    The ALDH2*2 gene encoding the inactive variant form of mitochondrial aldehyde dehydrogenase (ALDH2) protects nearly all carriers of this gene from alcoholism. Inhibition of ALDH2 has hence become a possible strategy to treat alcoholism. The natural product 7-O-glucosyl-4'-hydroxyisoflavone (daidzin), isolated from the kudzu vine ( Peruraria lobata), is a specific inhibitor of ALDH2 and suppresses ethanol consumption. Daidzin is the active principle in a herbal remedy for "alcohol addiction" and provides a lead for the design of improved ALDH2. The structure of daidzin/ALDH2 in complex at 2.4 A resolution shows the isoflavone moiety of daidzin binding close to the aldehyde substrate-binding site in a hydrophobic cleft and the glucosyl function binding to a hydrophobic patch immediately outside the isoflavone-binding pocket. These observations provide an explanation for both the specificity and affinity of daidzin (IC50 =80 nM) and the affinity of analogues with different substituents at the glucosyl position.

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

    PubMed

    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.

  16. Alcohol Dehydrogenase-1B (rs1229984) and Aldehyde Dehydrogenase-2 (rs671) Genotypes Are Strong Determinants of the Serum Triglyceride and Cholesterol Levels of Japanese Alcoholic Men

    PubMed Central

    Yokoyama, Akira; Yokoyama, Tetsuji; Matsui, Toshifumi; Mizukami, Takeshi; Kimura, Mitsuru; Matsushita, Sachio; Higuchi, Susumu; Maruyama, Katsuya

    2015-01-01

    Background Elevated serum triglyceride (TG) and high-density-lipoprotein cholesterol (HDL-C) levels are common in drinkers. The fast-metabolizing alcohol dehydrogenase-1B encoded by the ADH1B*2 allele (vs. ADH1B*1/*1 genotype) and inactive aldehyde dehydrogenase-2 encoded by the ALDH2*2 allele (vs. ALDH2*1/*1 genotype) modify ethanol metabolism and are prevalent (≈90% and ≈40%, respectively) in East Asians. We attempted to evaluate the associations between the ADH1B and ALDH2 genotypes and lipid levels in alcoholics. Methods The population consisted of 1806 Japanese alcoholic men (≥40 years) who had undergone ADH1B and ALDH2 genotyping and whose serum TG, total cholesterol, and HDL-C levels in the fasting state had been measured within 3 days after admission. Results High serum levels of TG (≥150 mg/dl), HDL-C (>80 mg/dl), and low-density-lipoprotein cholesterol (LDL-C calculated by the Friedewald formula ≥140 mg/dl) were observed in 24.3%, 16.8%, and 15.6%, respectively, of the subjects. Diabetes, cirrhosis, smoking, and body mass index (BMI) affected the serum lipid levels. Multivariate analysis revealed that the presence of the ADH1B*2 allele and the active ALDH2*1/*1 genotype increased the odds ratio (OR; 95% confidence interval) for a high TG level (2.22 [1.67–2.94] and 1.39 [0.99–1.96], respectively), and decreased the OR for a high HDL-C level (0.37 [0.28–0.49] and 0.51 [0.37–0.69], respectively). The presence of the ADH1B*2 allele decreased the OR for a high LDL-C level (0.60 [0.45–0.80]). The ADH1B*2 plus ALDH2*1/*1 combination yielded the highest ORs for high TG levels and lowest OR for a high HDL-C level. The genotype effects were more prominent in relation to the higher levels of TG (≥220 mg/dl) and HDL-C (≥100 mg/dl). Conclusions The fast-metabolizing ADH1B and active ALDH2, and especially a combination of the two were strongly associated with higher serum TG levels and lower serum HDL-C levels of alcoholics. The fast

  17. Alcohol dehydrogenase, SDR and MDR structural stages, present update and altered era.

    PubMed

    Jörnvall, Hans; Landreh, Michael; Östberg, Linus J

    2015-06-05

    It is now about half a century since molecular research on alcohol dehydrogenase (ADH), short-chain dehydrogenase/reductase (SDR) and medium-chain dehydrogenase/reductase (MDR) started. During this time, at least four stages of research can be distinguished, which led to many ADH, SDR and MDR structures from which their origins could be traced. An introductory summary of these stages is given, followed by a current update on the now known structures, including the present pattern of mammalian MDR-ADH enzymes into six classes and their evolutionary relationships. In spite of the wide spread in evolutionary changes from the "constant" class III to the more "variable" other classes, the change in class V (only confirmed as a transcript in humans) and class VI (absent in humans) are also restricted. Such spread in variability is visible also in other dehydrogenases, but not always so restricted in other co-evolving proteins we have studied. Finally, the shift in era of present ADH research is highlighted, as well as levels of likely future continuation.

  18. Ethanol-induced alcohol dehydrogenase E (AdhE) potentiates pneumolysin in Streptococcus pneumoniae.

    PubMed

    Luong, Truc Thanh; Kim, Eun-Hye; Bak, Jong Phil; Nguyen, Cuong Thach; Choi, Sangdun; Briles, David E; Pyo, Suhkneung; Rhee, Dong-Kwon

    2015-01-01

    Alcohol impairs the host immune system, rendering the host more vulnerable to infection. Therefore, alcoholics are at increased risk of acquiring serious bacterial infections caused by Streptococcus pneumoniae, including pneumonia. Nevertheless, how alcohol affects pneumococcal virulence remains unclear. Here, we showed that the S. pneumoniae type 2 D39 strain is ethanol tolerant and that alcohol upregulates alcohol dehydrogenase E (AdhE) and potentiates pneumolysin (Ply). Hemolytic activity, colonization, and virulence of S. pneumoniae, as well as host cell myeloperoxidase activity, proinflammatory cytokine secretion, and inflammation, were significantly attenuated in adhE mutant bacteria (ΔadhE strain) compared to D39 wild-type bacteria. Therefore, AdhE might act as a pneumococcal virulence factor. Moreover, in the presence of ethanol, S. pneumoniae AdhE produced acetaldehyde and NADH, which subsequently led Rex (redox-sensing transcriptional repressor) to dissociate from the adhE promoter. An increase in AdhE level under the ethanol condition conferred an increase in Ply and H2O2 levels. Consistently, S. pneumoniae D39 caused higher cytotoxicity to RAW 264.7 cells than the ΔadhE strain under the ethanol stress condition, and ethanol-fed mice (alcoholic mice) were more susceptible to infection with the D39 wild-type bacteria than with the ΔadhE strain. Taken together, these data indicate that AdhE increases Ply under the ethanol stress condition, thus potentiating pneumococcal virulence.

  19. Bioelectrochemistry of non-covalent immobilized alcohol dehydrogenase on oxidized diamond nanoparticles.

    PubMed

    Nicolau, Eduardo; Méndez, Jessica; Fonseca, José J; Griebenow, Kai; Cabrera, Carlos R

    2012-06-01

    Diamond nanoparticles are considered a biocompatible material mainly due to their non-cytotoxicity and remarkable cellular uptake. Model proteins such as cytochrome c and lysozyme have been physically adsorbed onto diamond nanoparticles, proving it to be a suitable surface for high protein loading. Herein, we explore the non-covalent immobilization of the redox enzyme alcohol dehydrogenase (ADH) from Saccharomyces cerevisiae (E.C.1.1.1.1) onto oxidized diamond nanoparticles for bioelectrochemical applications. Diamond nanoparticles were first oxidized and physically characterized by X-ray diffraction (XRD), FT-IR and TEM. Langmuir isotherms were constructed to investigate the ADH adsorption onto the diamond nanoparticles as a function of pH. It was found that a higher packing density is achieved at the isoelectric point of the enzyme. Moreover, the relative activity of the immobilized enzyme on diamond nanoparticles was addressed under optimum pH conditions able to retain up to 70% of its initial activity. Thereafter, an ethanol bioelectrochemical cell was constructed by employing the immobilized alcohol dehydrogenase onto diamond nanoparticles, this being able to provide a current increment of 72% when compared to the blank solution. The results of this investigation suggest that this technology may be useful for the construction of alcohol biosensors or biofuel cells in the near future.

  20. Properties and evolution of an alcohol dehydrogenase from the Crenarchaeota Pyrobaculum aerophilum.

    PubMed

    Vitale, Annalisa; Rosso, Francesco; Barbarisi, Alfonso; Labella, Tullio; D'Auria, Sabato

    2010-08-01

    The gene encoding a novel alcohol dehydrogenase (ADH) that belongs to the medium chain dehydrogenase/reductase (MDR) superfamily was identified in the hyperthermophilic archaeon, Pyrobaculum aerophilum. The P. aerophilum ADH gene (Pae2687) was over-expressed in Escherichia coli, and the protein (PyAeADHII) was purified to homogeneity and characterized. The PyAeADHII belongs to a medium chain class because its monomer size is 330 residues and even if it is structurally similar to other enzymes belonging to MDR superfamily, it lacks key residues involved in the coordination of the catalytic Zn ion and in the binding of alcoholic substrates typical of other ADHs. Consistently, PyAeADHII does not show activity on a large number of alcohols, aldheydes or ketones. It is active only when alpha-tetralone is used as a substrate. The enzyme has a strict requirement for NADP(H) as the coenzyme and has remarkable thermophilicity, displaying activity at temperatures up to 95 degrees C. The study of the metabolic pathways of P. aerophilum can provide information on the evolution of genes and enzymes and may be crucial for understanding the evolution of eukaryotic cells.

  1. Purification and Characterization of Cinnamyl Alcohol Dehydrogenase Isoforms from the Periderm of Eucalyptus gunnii Hook.

    PubMed Central

    Hawkins, S. W.; Boudet, A. M.

    1994-01-01

    Cinnamyl alcohol dehydrogenase (CAD, EC 1.1.1.195) isoforms were purified from the periderm (containing both suberized and lignified cell layers) of Eucalyptus gunnii Hook stems. Two isoforms (CAD 1P and CAD 2P) were initially characterized, and the major form, CAD 2P, was resolved into three further isoforms by ion-exchange chromatography. Crude extracts contained two aliphatic alcohol dehydrogenases (ADH) and one aromatic ADH, which was later resolved into two further isoforms. Aliphatic ADHs did not use hydroxycinnamyl alcohols as substrates, whereas both aromatic ADH isoforms used coniferyl and sinapyl alcohol as substrates but with a much lower specific activity when compared with benzyl alcohol. The minor form, CAD 1P, was a monomer with a molecular weight of 34,000 that did not co-elute with either aromatic or aliphatic ADH activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis demonstrated that this protein was very similar to another CAD isoform purified from Eucalyptus xylem tissue. CAD 2P had a native molecular weight of approximately 84,000 and was a dimer consisting of two heterogenous subunits (with molecular weights of 42,000 and 44,000). These subunits were differentially combined to give the heterodimer and two homodimers. SDS-PAGE, western blots, and nondenaturing PAGE indicated that the CAD 2P heterodimer was very similar to the main CAD isoform previously purified in our laboratory from differentiating xylem tissue of E. gunnii (D. Goffner, I. Joffroy, J. Grima-Pettenati, C. Halpin, M.E. Knight, W. Schuch, A.M. Boudet [1992] Planta 188: 48-53). Kinetic data indicated that the different CAD 2P isoforms may be implicated in the preferential production of different monolignols used in the synthesis of lignin and/or suberin. PMID:12232063

  2. Cellobionic acid inhibition of cellobiohydrolase I and cellobiose dehydrogenase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    End-product inhibition by cellobiose and glucose is a rate-limiting factor in cellulose hydrolysis by cellulases. While cellobiose and glucose inhibition have been extensively investigated, cellobionate inhibition has been minimally studied despite the discovery that accessory proteins such as cello...

  3. Ethanol Metabolism by HeLa Cells Transduced with Human Alcohol Dehydrogenase Isoenzymes: Control of the Pathway by Acetaldehyde Concentration†

    PubMed Central

    Matsumoto, Michinaga; Cyganek, Izabela; Sanghani, Paresh C.; Cho, Won Kyoo; Liangpunsakul, Suthat; Crabb, David W.

    2010-01-01

    Background Human class I alcohol dehydrogenase 2 isoenzymes (encoded by the ADH1B locus) have large differences in kinetic properties; however, individuals inheriting the alleles for the different isoenzymes exhibit only small differences in alcohol elimination rates. This suggests that other cellular factors must regulate the activity of the isoenzymes. Methods The activity of the isoenzymes expressed from ADH1B*1, ADH1B*2, and ADH1B*3 cDNAs was examined in stably transduced HeLa cell lines, including lines which expressed human low Km aldehyde dehydrogenase (ALDH2). The ability of the cells to metabolize ethanol was compared with that of HeLa cells expressing rat class I ADH (HeLa-rat ADH cells), rat hepatoma (H4IIEC3) cells, and rat hepatocytes. Results The isoenzymes had similar protein half-lives in the HeLa cells. Rat hepatocytes, H4IIEC3 cells, and HeLa-rat ADH cells oxidized ethanol much faster than the cells expressing the ADH1B isoenzymes. This was not explained by high cellular NADH levels or endogenous inhibitors; but rather because the activity of the β1 and β2 ADHs were constrained by the accumulation of acetaldehyde, as shown by the increased rate of ethanol oxidation by cell lines expressing β2 ADH plus ALDH2. Conclusion The activity of the human β2 ADH isoenzyme is sensitive to inhibition by acetaldehyde, which likely limits its activity in vivo. This study emphasizes the importance of maintaining a low steady–state acetaldehyde concentration in hepatocytes during ethanol metabolism. PMID:21166830

  4. The purification and biochemical properties of alcohol dehydrogenase--"fast (Chateau Douglas)" from Drosophila melanogaster.

    PubMed

    Chambers, G K

    1984-06-01

    Alcohol dehydrogenase has been purified from Drosophila melanogaster lines bearing the AdhF, AdhS, and AdhFCh.D. alleles. Biochemical investigations show that the properties of the purified enzymes are very similar to those of crude enzyme extracts except that the pure enzymes are more heat stable. ADH-FCh.D. resembles ADH-S very closely in specific activity, substrate specificity, and a number of kinetic parameters including limiting values for Km(app.) for ethanol. However, it is considerably more heat stable than either of the two common variants. ADH-F differs from ADH-S and ADH-FCh.D. particularly with regard to the rate of oxidation of secondary alcohols. Atomic absorbtion spectroscopy shows that all three allozymes lack zinc or other divalent cations as active-site components. Peptide mapping experiments identify one very active cysteinyl residue; and amide residues in the NAD+ binding domain.

  5. Influence of fermentation conditions on specific activity of the enzymes alcohol and aldehyde dehydrogenase from yeasts.

    PubMed

    Mauricio, J C; Ortega, J M

    1993-01-01

    The effects of anaerobic, semi-aerobic and short aeration fermentation conditions and the addition of ergosterol and oleic acid to musts on the specific activity of alcohol and aldehyde dehydrogenase (ADH and ALDH) from two yeast species, Saccharomyces cerevisiae and Torulaspora delbrueckii, were studied. ADH I biosynthesis only occurred during the first few hours of fermentation. ADH II from S. cerevisiae and ALDH-NADP+ from the two yeast species behaved as constitutive enzymes under all fermentation conditions. ADH II from T. delbrueckii was only synthesized in small amounts, and its activity was always lower than in S. cerevisiae, where it was responsible for the termination of alcoholic fermentation during the steady growth phase.

  6. Characterization of an Arxula adeninivorans alcohol dehydrogenase involved in the metabolism of ethanol and 1-butanol.

    PubMed

    Kasprzak, Jakub; Rauter, Marion; Riechen, Jan; Worch, Sebastian; Baronian, Kim; Bode, Rüdiger; Schauer, Frieder; Kunze, Gotthard

    2016-05-01

    In this study, alcohol dehydrogenase 1 from Arxula adeninivorans (Aadh1p) was identified and characterized. Aadh1p showed activity with short and medium chain length primary alcohols in the forward reaction and their aldehydes in the reverse reaction. Aadh1p has 64% identity with Saccharomyces cerevisiae Adh1p, is localized in the cytoplasm and uses NAD(+) as cofactor. Gene expression analysis showed a low level increase in AADH1 gene expression with ethanol, pyruvate or xylose as the carbon source. Deletion of the AADH1 gene affects growth of the cells with 1-butanol, ethanol and glucose as the carbon source, and a strain which overexpressed the AADH1 gene metabolized 1-butanol more rapidly. An ADH activity assay indicated that Aadh1p is a major enzyme for the synthesis of ethanol and the degradation of 1-butanol in A. adeninivorans.

  7. Characterization of a novel flooding stress-responsive alcohol dehydrogenase expressed in soybean roots.

    PubMed

    Komatsu, Setsuko; Deschamps, Thibaut; Thibaut, Deschamps; Hiraga, Susumu; Kato, Mikio; Chiba, Mitsuru; Hashiguchi, Akiko; Tougou, Makoto; Shimamura, Satoshi; Yasue, Hiroshi

    2011-10-01

    Alcohol dehydrogenase (Adh) is the key enzyme in alcohol fermentation. We analyzed Adh expression in order to clarify the role of Adh of soybeans (Glycine max) to flooding stress. Proteome analysis confirmed that expression of Adh is significantly upregulated in 4-day-old soybean seedlings subjected to 2 days of flooding. Southern hybridization analysis and soybean genome database search revealed that soybean has at least 6 Adh genes. The GmAdh2 gene that responded to flooding was isolated from soybean cultivar Enrei. Adh2 expression was markedly increased 6 h after flooding and decreased 24 h after floodwater drainage. In situ hybridization and Western blot indicated that flooding strongly induces Adh2 expression in RNA and protein levels in the root apical meristem. Osmotic, cold, or drought stress did not induce expression of Adh2. These results indicate that Adh2 is a flooding-response specific soybean gene expressed in root tissue.

  8. Pancreatic injury in hepatic alcohol dehydrogenase-deficient deer mice after subchronic exposure to ethanol

    SciTech Connect

    Kaphalia, Bhupendra S.; Bhopale, Kamlesh K.; Kondraganti, Shakuntala; Wu Hai; Boor, Paul J.; Ansari, G.A. Shakeel

    2010-08-01

    Pancreatitis caused by activation of digestive zymogens in the exocrine pancreas is a serious chronic health problem in alcoholic patients. However, mechanism of alcoholic pancreatitis remains obscure due to lack of a suitable animal model. Earlier, we reported pancreatic injury and substantial increases in endogenous formation of fatty acid ethyl esters (FAEEs) in the pancreas of hepatic alcohol dehydrogenase (ADH)-deficient (ADH{sup -}) deer mice fed 4% ethanol. To understand the mechanism of alcoholic pancreatitis, we evaluated dose-dependent metabolism of ethanol and related pancreatic injury in ADH{sup -} and hepatic ADH-normal (ADH{sup +}) deer mice fed 1%, 2% or 3.5% ethanol via Lieber-DeCarli liquid diet daily for 2 months. Blood alcohol concentration (BAC) was remarkably increased and the concentration was {approx} 1.5-fold greater in ADH{sup -} vs. ADH{sup +} deer mice fed 3.5% ethanol. At the end of the experiment, remarkable increases in pancreatic FAEEs and significant pancreatic injury indicated by the presence of prominent perinuclear space, pyknotic nuclei, apoptotic bodies and dilation of glandular ER were found only in ADH{sup -} deer mice fed 3.5% ethanol. This pancreatic injury was further supported by increased plasma lipase and pancreatic cathepsin B (a lysosomal hydrolase capable of activating trypsinogen), trypsinogen activation peptide (by-product of trypsinogen activation process) and glucose-regulated protein 78 (endoplasmic reticulum stress marker). These findings suggest that ADH-deficiency and high alcohol levels in the body are the key factors in ethanol-induced pancreatic injury. Therefore, determining how this early stage of pancreatic injury advances to inflammation stage could be important for understanding the mechanism(s) of alcoholic pancreatitis.

  9. Pancreatic injury in hepatic alcohol dehydrogenase-deficient deer mice after subchronic exposure to ethanol.

    PubMed

    Kaphalia, Bhupendra S; Bhopale, Kamlesh K; Kondraganti, Shakuntala; Wu, Hai; Boor, Paul J; Ansari, G A Shakeel

    2010-08-01

    Pancreatitis caused by activation of digestive zymogens in the exocrine pancreas is a serious chronic health problem in alcoholic patients. However, mechanism of alcoholic pancreatitis remains obscure due to lack of a suitable animal model. Earlier, we reported pancreatic injury and substantial increases in endogenous formation of fatty acid ethyl esters (FAEEs) in the pancreas of hepatic alcohol dehydrogenase (ADH)-deficient (ADH(-)) deer mice fed 4% ethanol. To understand the mechanism of alcoholic pancreatitis, we evaluated dose-dependent metabolism of ethanol and related pancreatic injury in ADH(-) and hepatic ADH-normal (ADH(+)) deer mice fed 1%, 2% or 3.5% ethanol via Lieber-DeCarli liquid diet daily for 2months. Blood alcohol concentration (BAC) was remarkably increased and the concentration was ∼1.5-fold greater in ADH(-) vs. ADH(+) deer mice fed 3.5% ethanol. At the end of the experiment, remarkable increases in pancreatic FAEEs and significant pancreatic injury indicated by the presence of prominent perinuclear space, pyknotic nuclei, apoptotic bodies and dilation of glandular ER were found only in ADH(-) deer mice fed 3.5% ethanol. This pancreatic injury was further supported by increased plasma lipase and pancreatic cathepsin B (a lysosomal hydrolase capable of activating trypsinogen), trypsinogen activation peptide (by-product of trypsinogen activation process) and glucose-regulated protein 78 (endoplasmic reticulum stress marker). These findings suggest that ADH-deficiency and high alcohol levels in the body are the key factors in ethanol-induced pancreatic injury. Therefore, determining how this early stage of pancreatic injury advances to inflammation stage could be important for understanding the mechanism(s) of alcoholic pancreatitis.

  10. Purification and characterization of a primary-secondary alcohol dehydrogenase from two strains of Clostridium beijerinckii.

    PubMed Central

    Ismaiel, A A; Zhu, C X; Colby, G D; Chen, J S

    1993-01-01

    Two primary alcohols (1-butanol and ethanol) are major fermentation products of several clostridial species. In addition to these two alcohols, the secondary alcohol 2-propanol is produced to a concentration of about 100 mM by some strains of Clostridium beijerinckii. An alcohol dehydrogenase (ADH) has been purified to homogeneity from two strains (NRRL B593 and NESTE 255) of 2-propanol-producing C. beijerinckii. When exposed to air, the purified ADH was stable, whereas the partially purified ADH was inactivated. The ADHs from the two strains had similar structural and kinetic properties. Each had a native M(r) of between 90,000 and 100,000 and a subunit M(r) of between 38,000 and 40,000. The ADHs were NADP(H) dependent, but a low level of NAD(+)-linked activity was detected. They were equally active in reducing aldehydes and 2-ketones, but a much lower oxidizing activity was obtained with primary alcohols than with secondary alcohols. The kcat/Km value for the alcohol-forming reaction appears to be a function of the size of the larger alkyl substituent on the carbonyl group. ADH activities measured in the presence of both acetone and butyraldehyde did not exceed activities measured with either substrate present alone, indicating a common active site for both substrates. There was no similarity in the N-terminal amino acid sequence between that of the ADH and those of fungi and several other bacteria. However, the N-terminal sequence had 67% identity with those of two other anaerobes, Thermoanaerobium brockii and Methanobacterium palustre. Furthermore, conserved glycine and tryptophan residues are present in ADHs of these three anaerobic bacteria and ADHs of mammals and green plants. Images PMID:8349550

  11. Characterization of a Zinc-Containing Alcohol Dehydrogenase with Stereoselectivity from the Hyperthermophilic Archaeon Thermococcus guaymasensis▿

    PubMed Central

    Ying, Xiangxian; Ma, Kesen

    2011-01-01

    An alcohol dehydrogenase (ADH) from hyperthermophilic archaeon Thermococcus guaymasensis was purified to homogeneity and was found to be a homotetramer with a subunit size of 40 ± 1 kDa. The gene encoding the enzyme was cloned and sequenced; this gene had 1,095 bp, corresponding to 365 amino acids, and showed high sequence homology to zinc-containing ADHs and l-threonine dehydrogenases with binding motifs of catalytic zinc and NADP+. Metal analyses revealed that this NADP+-dependent enzyme contained 0.9 ± 0.03 g-atoms of zinc per subunit. It was a primary-secondary ADH and exhibited a substrate preference for secondary alcohols and corresponding ketones. Particularly, the enzyme with unusual stereoselectivity catalyzed an anti-Prelog reduction of racemic (R/S)-acetoin to (2R,3R)-2,3-butanediol and meso-2,3-butanediol. The optimal pH values for the oxidation and formation of alcohols were 10.5 and 7.5, respectively. Besides being hyperthermostable, the enzyme activity increased as the temperature was elevated up to 95°C. The enzyme was active in the presence of methanol up to 40% (vol/vol) in the assay mixture. The reduction of ketones underwent high efficiency by coupling with excess isopropanol to regenerate NADPH. The kinetic parameters of the enzyme showed that the apparent Km values and catalytic efficiency for NADPH were 40 times lower and 5 times higher than those for NADP+, respectively. The physiological roles of the enzyme were proposed to be in the formation of alcohols such as ethanol or acetoin concomitant to the NADPH oxidation. PMID:21515780

  12. Evaluation of the impact of functional diversification on Poaceae, Brassicaceae, Fabaceae, and Pinaceae alcohol dehydrogenase enzymes.

    PubMed

    Thompson, Claudia E; Fernandes, Cláudia L; de Souza, Osmar Norberto; de Freitas, Loreta B; Salzano, Francisco M

    2010-05-01

    The plant alcohol dehydrogenases (ADHs) have been intensively studied in the last years in terms of phylogeny and they have been widely used as a molecular marker. However, almost no information about their three-dimensional structure is available. Several studies point to functional diversification of the ADH, with evidence of its importance, in different organisms, in the ethanol, norepinephrine, dopamine, serotonin, and bile acid metabolism. Computational results demonstrated that in plants these enzymes are submitted to a functional diversification process, which is reinforced by experimental studies indicating distinct enzymatic functions as well as recruitment of specific genes in different tissues. The main objective of this article is to establish a correlation between the functional diversification occurring in the plant alcohol dehydrogenase family and the three-dimensional structures predicted for 17 ADH belonging to Poaceae, Brassicaceae, Fabaceae, and Pinaceae botanical families. Volume, molecular weight and surface areas are not markedly different among them. Important electrostatic and pI differences were observed with the residues responsible for some of them identified, corroborating the function diversification hypothesis. These data furnish important background information for future specific structure-function and evolutionary investigations.

  13. Sequence and structural aspects of the functional diversification of plant alcohol dehydrogenases.

    PubMed

    Thompson, Claudia E; Salzano, Francisco M; de Souza, Osmar Norberto; Freitas, Loreta B

    2007-07-01

    The glycolytic proteins in plants are coded by small multigene families, which provide an interesting contrast to the high copy number of gene families studied to date. The alcohol dehydrogenase (Adh) genes encode glycolytic enzymes that have been characterized in some plant families. Although the amino acid sequences of zinc-containing long-chain ADHs are highly conserved, the metabolic function of this enzyme is variable. They also have different patterns of expression and are submitted to differences in nonsynonymous substitution rates between gene copies. It is possible that the Adh copies have been retained as a consequence of adaptative amino acid replacements which have conferred subtle changes in function. Phylogenetic analysis indicates that there have been a number of separate duplication events within angiosperms, and that genes labeled Adh1, Adh2 and Adh3 in different groups may not be homologous. Nonsynonymous/synonymous ratios yielded no signs of positive selection. However, the coefficients of functional divergence (theta) estimated between the Adh1 and Adh2 gene groups indicate statistically significant site-specific shift of evolutionary rates between them, as well as between those of different botanical families, suggesting that altered functional constraints may have taken place at some amino acid residues after their diversification. The theoretical three-dimensional structure of the alcohol dehydrogenase from Arabis blepharophylla was constructed and verified to be stereochemically valid.

  14. Molecular and physiological aspects of alcohol dehydrogenases in the ethanol metabolism of Saccharomyces cerevisiae.

    PubMed

    de Smidt, Olga; du Preez, James C; Albertyn, Jacobus

    2012-02-01

    The physiological role and possible functional substitution of each of the five alcohol dehydrogenase (Adh) isozymes in Saccharomyces cerevisiae were investigated in five quadruple deletion mutants designated strains Q1-Q5, with the number indicating the sole intact ADH gene. Their growth in aerobic batch cultures was characterised in terms of kinetic and stoichiometric parameters. Cultivation with glucose or ethanol as carbon substrate revealed that Adh1 was the only alcohol dehydrogenase capable of efficiently catalysing the reduction of acetaldehyde to ethanol. The oxidation of produced or added ethanol could also be attributed to Adh1. Growth of strains lacking the ADH1 gene resulted in the production of glycerol as a major fermentation product, concomitant with the production of a significant amount of acetaldehyde. Strains Q2 and Q3, expressing only ADH2 or ADH3, respectively, produced ethanol from glucose, albeit less than strain Q1, and were also able to oxidise added ethanol. Strains Q4 and Q5 grew poorly on glucose and produced ethanol, but were neither able to utilise the produced ethanol nor grow on added ethanol. Transcription profiles of the ADH4 and ADH5 genes suggested that participation of these gene products in ethanol production from glucose was unlikely.

  15. Effect of additives on gas-phase catalysis with immobilised Thermoanaerobacter species alcohol dehydrogenase (ADH T).

    PubMed

    Trivedi, A H; Spiess, A C; Daussmann, T; Büchs, J

    2006-07-01

    This paper presents a strategy for preparing an efficient immobilised alcohol dehydrogenase preparation for a gas-phase reaction. The effects of additives such as buffers and sucrose on the immobilisation efficiency (residual activity and protein loading) and on the gas-phase reaction efficiency (initial reaction rate and half-life) of Thermoanaerobacter sp. alcohol dehydrogenase were studied. The reduction of acetophenone to 1-phenylethanol under in situ cofactor regeneration using isopropanol as co-substrate was used as a model reaction at fixed reaction conditions (temperature and thermodynamic activities). A strongly enhanced thermostability of the enzyme in the gas-phase reaction was achieved when the enzyme was immobilised with 50 mM phosphate buffer (pH 7) containing sucrose five times the protein amount (on weight/weight basis). This resulted in a remarkable productivity of 200 g L(-1) day(-1) even at non-optimised reaction conditions. The interaction of additives with the enzyme and water affects the immobilisation and gas-phase efficiencies of the enzyme. However, it was not possible to predict the effect of additives on the gas-phase reaction efficiency even after knowing their effect on the immobilisation efficiency.

  16. Secondary alcohol dehydrogenase catalyzes the reduction of exogenous acetone to 2-propanol in Trichomonas vaginalis.

    PubMed

    Sutak, Robert; Hrdy, Ivan; Dolezal, Pavel; Cabala, Radomir; Sedinová, Miroslava; Lewin, Joern; Harant, Karel; Müller, Miklos; Tachezy, Jan

    2012-08-01

    Secondary alcohols such as 2-propanol are readily produced by various anaerobic bacteria that possess secondary alcohol dehydrogenase (S-ADH), although production of 2-propanol is rare in eukaryotes. Specific bacterial-type S-ADH has been identified in a few unicellular eukaryotes, but its function is not known and the production of secondary alcohols has not been studied. We purified and characterized S-ADH from the human pathogen Trichomonas vaginalis. The kinetic properties and thermostability of T. vaginalis S-ADH were comparable with bacterial orthologues. The substantial activity of S-ADH in the parasite's cytosol was surprising, because only low amounts of ethanol and trace amounts of secondary alcohols were detected as metabolic end products. However, S-ADH provided the parasite with a high capacity to scavenge and reduce external acetone to 2-propanol. To maintain redox balance, the demand for reducing power to metabolize external acetone was compensated for by decreased cytosolic reduction of pyruvate to lactate and by hydrogenosomal metabolism of pyruvate. We speculate that hydrogen might be utilized to maintain cytosolic reducing power. The high activity of Tv-S-ADH together with the ability of T. vaginalis to modulate the metabolic fluxes indicate efficacious metabolic responsiveness that could be advantageous for rapid adaptation of the parasite to changes in the host environment.

  17. Ethanol utilization regulatory protein: profile alignments give no evidence of origin through aldehyde and alcohol dehydrogenase gene fusion.

    PubMed Central

    Nicholas, H. B.; Persson, B.; Jörnvall, H.; Hempel, J.

    1995-01-01

    The suggestion that the ethanol regulatory protein from Aspergillus has its evolutionary origin in a gene fusion between aldehyde and alcohol dehydrogenase genes (Hawkins AR, Lamb HK, Radford A, Moore JD, 1994, Gene 146:145-158) has been tested by profile analysis with aldehyde and alcohol dehydrogenase family profiles. We show that the degree and kind of similarity observed between these profiles and the ethanol regulatory protein sequence is that expected from random sequences of the same composition. This level of similarity fails to support the suggested gene fusion. PMID:8580855

  18. Increasing the Heme-Dependent Respiratory Efficiency of Lactococcus lactis by Inhibition of Lactate Dehydrogenase

    PubMed Central

    Arioli, Stefania; Zambelli, Daniele; Guglielmetti, Simone; De Noni, Ivano; Pedersen, Martin B.; Pedersen, Per Dedenroth; Dal Bello, Fabio

    2013-01-01

    The discovery of heme-induced respiration in Lactococcus lactis has radically improved the industrial processes used for the biomass production of this species. Here, we show that inhibition of the lactate dehydrogenase activity of L. lactis during growth under respiration-permissive conditions can stimulate aerobic respiration, thereby increasing not only growth efficiency but also the robustness of this organism. PMID:23064338

  19. Comparison of Alcohol Impairment of Behavioral and Attentional Inhibition

    PubMed Central

    Weafer, Jessica; Fillmore, Mark T.

    2012-01-01

    Background Despite the wealth of studies demonstrating the impairing effects of alcohol on behavioral inhibition, less is known regarding effects of the drug on attentional inhibition (i.e., the ability to ignore distracting stimuli in the environment in order to focus attention on relevant information). The current study examined alcohol impairment of both behavioral and attentional inhibition, as well as potential associations between the two mechanisms of inhibitory control. Methods Men (n = 27) and women (n = 21) performed a measure of behavioral inhibition (cued go/no-go task) and a measure of attentional inhibition (delayed ocular return task) following three doses of alcohol: 0.65 g/kg, 0.45 g/kg, and 0.0 g/kg (placebo). Results Alcohol impaired both behavioral and attentional inhibition relative to placebo; however, correlational analyses revealed no associations between measures of behavioral and attentional inhibition following any dose. Additionally, men committed more inhibitory failures on the behavioral inhibition task, whereas women committed more inhibitory failures on the attentional inhibition task. Conclusions These findings suggest that behavioral and attentional inhibition are equally sensitive to the impairing effects of alcohol, yet represent distinct components of inhibitory control. Additionally, the observed gender differences in control of behavior and attention could have important implications regarding negative consequences associated with alcohol-induced disinhibition in men and women. PMID:22673197

  20. Gene cloning and expression of Leifsonia alcohol dehydrogenase (LSADH) involved in asymmetric hydrogen-transfer bioreduction to produce (R)-form chiral alcohols.

    PubMed

    Inoue, Kousuke; Makino, Yoshihide; Dairi, Tohru; Itoh, Nobuya

    2006-02-01

    The gene encoding Leifsonia alcohol dehydrogenase (LSADH), a useful biocatalyst for producing (R)-chiral alcohols, was cloned from the genomic DNA of Leifsonia sp. S749. The gene contained an opening reading frame consisting of 756 nucleotides corresponding to 251 amino acid residues. The subunit molecular weight was calculated to be 24,999, which was consistent with that determined by polyacrylamide gel electrophoresis. The enzyme was expressed in recombinant Escherichia coli cells and purified to homogeneity by three column chromatographies. The predicted amino acid sequence displayed 30-50% homology to known short chain alcohol dehydrogenase/reductases (SDRs); moreover, the NADH-binding site and the three catalytic residues in SDRs were conserved. The recombinant E. coli cells which overexpressed lsadh produced (R)-form chiral alcohols from ketones using 2-propanol as a hydrogen donor with the highest level of productivity ever reported and enantiomeric excess (e.e.).

  1. Phylogeny and structure of the cinnamyl alcohol dehydrogenase gene family in Brachypodium distachyon

    PubMed Central

    Bukh, Christian; Nord-Larsen, Pia Haugaard; Rasmussen, Søren K.

    2012-01-01

    Cinnamyl alcohol dehydrogenase (CAD) catalyses the final step of the monolignol biosynthesis, the conversion of cinnamyl aldehydes to alcohols, using NADPH as a cofactor. Seven members of the CAD gene family were identified in the genome of Brachypodium distachyon and five of these were isolated and cloned from genomic DNA. Semi-quantitative reverse-transcription PCR revealed differential expression of the cloned genes, with BdCAD5 being expressed in all tissues and highest in root and stem while BdCAD3 was only expressed in stem and spikes. A phylogenetic analysis of CAD-like proteins placed BdCAD5 on the same branch as bona fide CAD proteins from maize (ZmCAD2), rice (OsCAD2), sorghum (SbCAD2) and Arabidopsis (AtCAD4, 5). The predicted three-dimensional structures of both BdCAD3 and BdCAD5 resemble that of AtCAD5. However, the amino-acid residues in the substrate-binding domains of BdCAD3 and BdCAD5 are distributed symmetrically and BdCAD3 is similar to that of poplar sinapyl alcohol dehydrogenase (PotSAD). BdCAD3 and BdCAD5 expressed and purified from Escherichia coli both showed a temperature optimum of about 50 °C and molar weight of 49kDa. The optimal pH for the reduction of coniferyl aldehyde were pH 5.2 and 6.2 and the pH for the oxidation of coniferyl alcohol were pH 8 and 9.5, for BdCAD3 and BdCAD5 respectively. Kinetic parameters for conversion of coniferyl aldehyde and coniferyl alcohol showed that BdCAD5 was clearly the most efficient enzyme of the two. These data suggest that BdCAD5 is the main CAD enzyme for lignin biosynthesis and that BdCAD3 has a different role in Brachypodium. All CAD enzymes are cytosolic except for BdCAD4, which has a putative chloroplast signal peptide adding to the diversity of CAD functions. PMID:23028019

  2. PTEN Regulates Glutamine Flux to Pyrimidine Synthesis and Sensitivity to Dihydroorotate Dehydrogenase Inhibition.

    PubMed

    Mathur, Deepti; Stratikopoulos, Elias; Ozturk, Sait; Steinbach, Nicole; Pegno, Sarah; Schoenfeld, Sarah; Yong, Raymund; Murty, Vundavalli V; Asara, John M; Cantley, Lewis C; Parsons, Ramon

    2017-04-01

    Metabolic changes induced by oncogenic drivers of cancer contribute to tumor growth and are attractive targets for cancer treatment. Here, we found that increased growth of PTEN-mutant cells was dependent on glutamine flux through the de novo pyrimidine synthesis pathway, which created sensitivity to the inhibition of dihydroorotate dehydrogenase, a rate-limiting enzyme for pyrimidine ring synthesis. S-phase PTEN-mutant cells showed increased numbers of replication forks, and inhibitors of dihydroorotate dehydrogenase led to chromosome breaks and cell death due to inadequate ATR activation and DNA damage at replication forks. Our findings indicate that enhanced glutamine flux generates vulnerability to dihydroorotate dehydrogenase inhibition, which then causes synthetic lethality in PTEN-deficient cells due to inherent defects in ATR activation. Inhibition of dihydroorotate dehydrogenase could thus be a promising therapy for patients with PTEN-mutant cancers.Significance: We have found a prospective targeted therapy for PTEN-deficient tumors, with efficacy in vitro and in vivo in tumors derived from different tissues. This is based upon the changes in glutamine metabolism, DNA replication, and DNA damage response which are consequences of inactivation of PTENCancer Discov; 7(4); 380-90. ©2017 AACR.See related article by Brown et al., p. 391This article is highlighted in the In This Issue feature, p. 339.

  3. Mutation of Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase at Trp-110 affects stereoselectivity of aromatic ketone reduction.

    PubMed

    Patel, Jay M; Musa, Musa M; Rodriguez, Luis; Sutton, Dewey A; Popik, Vladimir V; Phillips, Robert S

    2014-08-21

    Alcohol dehydrogenases (ADHs) are enzymes that catalyze the reversible reduction of carbonyl compounds to their corresponding alcohols. We have been studying a thermostable, nicotinamide-adenine dinucleotide phosphate (NADP(+))-dependent, secondary ADH from Thermoanaerobacter ethanolicus (TeSADH). In the current work, we expanded our library of TeSADH and adopted the site-saturation mutagenesis approach in creating a comprehensive mutant library at W110. We used phenylacetone as a model substrate to study the effectiveness of our library because this substrate showed low enantioselectivity in our previous work when reduced using W110A TeSADH. Five of the newly designed W110 mutants reduced phenylacetone at >99.9% ee, and two of these mutants exhibit an enantiomeric ratio (E-value) of over 100. These five mutants also reduced 1-phenyl-2-butanone and 4-phenyl-2-butanone to their corresponding (S)-configured alcohols in >99.9% ee. These new mutants of TeSADH will likely have synthetic utility for reduction of aromatic ketones in the future.

  4. DOWNREGULATION OF CINNAMYL-ALCOHOL DEHYDROGENASE IN SWITCHGRASS BY RNA SILENCING RESULTS IN ENHANCED GLUCOSE RELEASE AFTER CELLULASE TREATMENT

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cinnamyl alcohol dehydrogenase (CAD), catalyzes the last step in monolignol biosynthesis and genetic evidence indicates CAD deficiency in grasses both decreases overall lignin, alters lignin structure and increases enzymatic recovery of sugars. To ascertain the effect of CAD downregulation in switch...

  5. Structural insights into substrate specificity and solvent tolerance in alcohol dehydrogenase ADH-'A' from Rhodococcus ruber DSM 44541.

    PubMed

    Karabec, Martin; Łyskowski, Andrzej; Tauber, Katharina C; Steinkellner, Georg; Kroutil, Wolfgang; Grogan, Gideon; Gruber, Karl

    2010-09-14

    The structure of the alcohol dehydrogenase ADH-'A' from Rhodococcus ruber reveals possible reasons for its remarkable tolerance to organic co-solvents and suggests new directions for structure-informed mutagenesis to produce enzymes of altered substrate specificity or improved selectivity.

  6. Mutant alcohol dehydrogenase leads to improved ethanol tolerance in Clostridium thermocellum

    SciTech Connect

    Brown, Steven D; Guss, Adam M; Karpinets, Tatiana V; Parks, Jerry M; Smolin, Nikolai; Yang, Shihui; Land, Miriam L; Klingeman, Dawn Marie; Bhandiwad, Ashwini; Rodriguez, Jr., Miguel; Raman, Babu; Shao, Xiongjun; Mielenz, Jonathan R; Smith, Jeremy C; Keller, Martin; Lynd, Lee R

    2011-01-01

    Clostridium thermocellum is a thermophilic, obligately anaerobic, Gram-positive bacterium that is a candidate microorganism for converting cellulosic biomass into ethanol through consolidated bioprocessing. Ethanol intolerance is an important metric in terms of process economics, and tolerance has often been described as a complex and likely multigenic trait for which complex gene interactions come into play. Here, we resequence the genome of an ethanol-tolerant mutant, show that the tolerant phenotype is primarily due to a mutated bifunctional acetaldehyde-CoA/alcohol dehydrogenase gene (adhE), hypothesize based on structural analysis that cofactor specificity may be affected, and confirm this hypothesis using enzyme assays. Biochemical assays confirm a complete loss of NADH-dependent activity with concomitant acquisition of NADPH-dependent activity, which likely affects electron flow in the mutant. The simplicity of the genetic basis for the ethanol-tolerant phenotype observed here informs rational engineering of mutant microbial strains for cellulosic ethanol production.

  7. 2,2-dipyridyl binding to metal substituted horse liver alcohol dehydrogenase.

    PubMed

    Syvertsen, C; McKinley-McKee, J S

    1984-09-01

    The binding of 2,2-dipyridyl to metal substituted horse liver alcohol dehydrogenase was measured by spectrophotometric titrations. Large changes in the visible absorption spectra were seen for the Co2+, Cu2+ and Ni2+ hybrids upon coordination of 2,2-dipyridyl, due to a change in coordination number. The formation constants for binding to the Co2+ and Cd2+ hybrids are of the order 10(6) M-1, which means that these hybrids have a 500-fold higher affinity for 2,2-dipyridyl than the native Zn2+ enzyme. 2,2-dipyridyl has a 100-fold higher affinity for enzyme bound Cd2+ than for aqueous Cd2+ ions, while for Cu2+ and Zn2+ the opposite is the case. None of the substituted metal ions were removed from the active site during titration with the chelator 2,2-dipyridyl.

  8. Escherichia coli derivatives lacking both alcohol dehydrogenase and phosphotransacetylase grow anaerobically by lactate fermentation.

    PubMed Central

    Gupta, S; Clark, D P

    1989-01-01

    Escherichia coli mutants lacking alcohol dehydrogenase (adh mutants) cannot synthesize the fermentation product ethanol and are unable to grow anaerobically on glucose and other hexoses. Similarly, phosphotransacetylase-negative mutants (pta mutants) neither excrete acetate nor grow anaerobically. However, when a strain carrying an adh deletion was selected for anaerobic growth on glucose, spontaneous pta mutants were isolated. Strains carrying both adh and pta mutations were observed by in vivo nuclear magnetic resonance and shown to produce lactic acid as the major fermentation product. Various combinations of adh pta double mutants regained the ability to grow anaerobically on hexoses, by what amounts to a homolactic fermentation. Unlike wild-type strains, such adh pta double mutants were unable to grow anaerobically on sorbitol or on glucuronic acid. The growth properties of strains carrying various mutations affecting the enzymes of fermentation are discussed in terms of redox balance. PMID:2661531

  9. Escherichia coli derivatives lacking both alcohol dehydrogenase and phosphotransacetylase grow anaerobically by lactate fermentation.

    PubMed

    Gupta, S; Clark, D P

    1989-07-01

    Escherichia coli mutants lacking alcohol dehydrogenase (adh mutants) cannot synthesize the fermentation product ethanol and are unable to grow anaerobically on glucose and other hexoses. Similarly, phosphotransacetylase-negative mutants (pta mutants) neither excrete acetate nor grow anaerobically. However, when a strain carrying an adh deletion was selected for anaerobic growth on glucose, spontaneous pta mutants were isolated. Strains carrying both adh and pta mutations were observed by in vivo nuclear magnetic resonance and shown to produce lactic acid as the major fermentation product. Various combinations of adh pta double mutants regained the ability to grow anaerobically on hexoses, by what amounts to a homolactic fermentation. Unlike wild-type strains, such adh pta double mutants were unable to grow anaerobically on sorbitol or on glucuronic acid. The growth properties of strains carrying various mutations affecting the enzymes of fermentation are discussed in terms of redox balance.

  10. Escherichia coli derivatives lacking both alcohol dehydrogenase and phosphotransacetylase grow anaerobically by lactate fermentation

    SciTech Connect

    Gupta, S.; Clark, D.P. )

    1989-07-01

    Escherichia coli mutants lacking alcohol dehydrogenase (adh mutants) cannot synthesize the fermentation product ethanol and are unable to grow anaerobically on glucose and other hexoses. Similarly, phosphotransacetylase-negative mutants (pta mutants) neither excrete acetate nor grow anaerobically. However, when a strain carrying an adh deletion was selected for anaerobic growth on glucose, spontaneous pta mutants were isolated. Strains carrying both adh and pta mutations were observed by in vivo nuclear magnetic resonance and shown to produce lactic acid as the major fermentation product. Various combinations of adh pta double mutants regained the ability to grow anaerobically on hexoses, by what amounts to a homolactic fermentation. Unlike wild-type strains, such adh pta double mutants were unable to grow anaerobically on sorbitol or on glucuronic acid. The growth properties of strains carrying various mutations affecting the enzymes of fermentation are discussed terms of redox balance.

  11. Correct developmental expression of a cloned alcohol dehydrogenase gene transduced into the Drosophila germ line.

    PubMed

    Goldberg, D A; Posakony, J W; Maniatis, T

    1983-08-01

    We have used P-element-mediated transformation to introduce a cloned Drosophila alcohol dehydrogenase (Adh) gene into the germ line of ADH null flies. Six independent transformants expressing ADH were identified by their acquired resistance to ethanol. Each transformant carries a single copy of the cloned Adh gene in a different chromosomal location. Four of the six transformant lines exhibit normal Adh expression by the following criteria: quantitative levels of ADH enzyme activity in larvae and adults; qualitative tissue specificity; the size of stable Adh mRNA; and the characteristic developmental switch in utilization of two different Adh promoters. The remaining two transformants express ADH enzyme activity with the correct tissue specificity, but at a lower level than wild type. These results demonstrate that an 11.8 kb chromosomal fragment containing the Adh gene includes the cis-acting sequences necessary for its correct developmental expression, and that a variety of chromosomal sites permit proper Adh gene function.

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

    PubMed Central

    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

  13. [Kinetics of the inhibition of succinate dehydrogenase in bull adrenal cortex by malonate and oxaloacetate].

    PubMed

    Mandrik, K A; Vonsovich, V A; Vinogradov, V V

    1983-01-01

    The activity of succinate dehydrogenase from bull adrenal cortex was studied as affected by malonate and oxaloacetate. The both substrate analogs without preincubation (separately and in the mixture) inhibit the enzyme by the competitive type. After a 3 min oxaloacetate preincubation of the enzyme inhibition is of a mixed character. Malonate under these conditions lowers the oxaloacetate effect without changing the type of inhibition. It is supposed that the protective effect is due to a high rate of formation and decay of the enzyme-inhibitory malonate complex.

  14. Inhibitory effects of the dietary flavonoid quercetin on the enzyme activity of zinc(II)-dependent yeast alcohol dehydrogenase: Spectroscopic and molecular docking studies.

    PubMed

    Bhuiya, Sutanwi; Haque, Lucy; Pradhan, Ankur Bikash; Das, Suman

    2017-02-01

    A multispectroscopic exploration was employed to investigate the interaction between the metallo-enzyme alcohol dehydrogenase (ADH) from yeast with bioflavonoid quercetin (QTN). Here, we have characterized the complex formation between QTN and Zn(2+) in aqueous solution and then examined the effect of such complex formation on the enzymatic activity of a zinc(II)-dependent enzyme alcohol dehydrogenase from yeast. We have observed an inhibition of enzymatic activity of ADH in presence of QTN. Enzyme inhibition kinetic experiments revealed QTN as a non-competitive inhibitor of yeast ADH. Perturbation of Circular dichroic (CD) spectrum of ADH in presence of QTN is observed due to the structural changes of ADH on complexation with the above flavonoid. Our results indicate a conformational change of ADH due to removal of Zn(2+) present in the enzyme by QTN. This was further established by molecular modeling study which shows that the flavonoid binds to the Zn(2+) ion which maintains the tertiary structure of the metallo-enzyme. So, QTN abstracts only half of the Zn(2+) ions present in the enzyme i.e. one Zn(2+) ion per monomer. From the present study, the structural alteration and loss of enzymatic activity of ADH are attributed to the complex formation between QTN and Zn(2+).

  15. Alcohol and aldehyde dehydrogenases: structures of the human liver enzymes, functional properties and evolutionary aspects.

    PubMed

    Jörnvall, H; Hempel, J; von Bahr-Lindström, H; Höög, J O; Vallee, B L

    1987-01-01

    All three types of subunit of class I human alcohol dehydrogenase have been analyzed both at the protein and cDNA levels, and the structures of alpha, beta 1, beta 2, gamma 1, and gamma 2 subunits are known. The same applies to class II pi subunits. Extensive protein data are also available for class III chi subunits. In the class I human isozymes, amino acid exchanges occur at 35 positions in total, with 21-28 replacements between any pair of the alpha/beta/gamma chains. These values, compared with those from species differences between the corresponding human and horse enzymes, suggest that isozyme developments in the class I enzyme resulted from separate gene duplications after the divergence of the human and equine evolutionary lines. All subunits exhibit some unique properties, with slightly closer similarity between the human gamma and horse enzyme subunits and somewhat greater deviations towards the human alpha subunit. Differences are large also in segments close to the active site zinc ligands and other functionally important positions. Species differences are distributed roughly equally between the two types of domain in the subunit, whereas isozyme differences are considerably more common in the catalytic than in the coenzyme-binding domain. These facts illustrate a functional divergence among the isozymes but otherwise similar changes during evolution. Polymorphic forms of beta and gamma subunits are characterized by single replacements at one and two positions, respectively, explaining known deviating properties. Class II and class III subunits are considerably more divergent. Their homology with class I isozymes exhibits only 60-65% positional identity. Hence, they reflect further steps towards the development of new enzymes, with variations well above the horse/human species levels, in contrast to the class I forms. Again, functionally important residues are affected, and patterns resembling those previously established for the divergently related

  16. Aliphatic alcohols in spirits inhibit phagocytosis by human monocytes.

    PubMed

    Pál, László; Árnyas, Ervin M; Bujdosó, Orsolya; Baranyi, Gergő; Rácz, Gábor; Ádány, Róza; McKee, Martin; Szűcs, Sándor

    2015-04-01

    A large volume of alcoholic beverages containing aliphatic alcohols is consumed worldwide. Previous studies have confirmed the presence of ethanol-induced immunosuppression in heavy drinkers, thereby increasing susceptibility to infectious diseases. However, the aliphatic alcohols contained in alcoholic beverages might also impair immune cell function, thereby contributing to a further decrease in microbicidal activity. Previous research has shown that aliphatic alcohols inhibit phagocytosis by granulocytes but their effect on human monocytes has not been studied. This is important as they play a crucial role in engulfment and killing of pathogenic microorganisms and a decrease in their phagocytic activity could lead to impaired antimicrobial defence in heavy drinkers. The aim of this study was to measure monocyte phagocytosis following their treatment with those aliphatic alcohols detected in alcoholic beverages. Monocytes were separated from human peripheral blood and phagocytosis of opsonized zymosan particles by monocytes treated with ethanol and aliphatic alcohols individually and in combination was determined. It was shown that these alcohols could suppress the phagocytic activity of monocytes in a concentration-dependent manner and when combined with ethanol, they caused a further decrease in phagocytosis. Due to their additive effects, it is possible that they may inhibit phagocytosis in a clinically meaningful way in alcoholics and episodic heavy drinkers thereby contribute to their increased susceptibility to infectious diseases. However, further research is needed to address this question.

  17. Characterization of alcohol dehydrogenase (ADH12) from Haloarcula marismortui, an extreme halophile from the Dead Sea.

    PubMed

    Timpson, Leanne M; Alsafadi, Diya; Mac Donnchadha, Cillín; Liddell, Susan; Sharkey, Michael A; Paradisi, Francesca

    2012-01-01

    Haloarchaeal alcohol dehydrogenases are of increasing interest as biocatalysts in the field of white biotechnology. In this study, the gene adh12 from the extreme halophile Haloarcula marismortui (HmADH12), encoding a 384 residue protein, was cloned into two vectors: pRV1 and pTA963. The resulting constructs were used to transform host strains Haloferax volcanii (DS70) and (H1209), respectively. Overexpressed His-tagged recombinant HmADH12 was purified by immobilized metal-affinity chromatography (IMAC). The His-tagged protein was visualized by SDS-PAGE, with a subunit molecular mass of 41.6 kDa, and its identity was confirmed by mass spectrometry. Purified HmADH12 catalyzed the interconversion between alcohols and aldehydes and ketones, being optimally active in the presence of 2 M KCl. It was thermoactive, with maximum activity registered at 60°C. The NADP(H) dependent enzyme was haloalkaliphilic for the oxidative reaction with optimum activity at pH 10.0. It favored a slightly acidic pH of 6.0 for catalysis of the reductive reaction. HmADH12 was significantly more tolerant than mesophilic ADHs to selected organic solvents, making it a much more suitable biocatalyst for industrial application.

  18. Isolation of Alcohol Dehydrogenase cDNA and Basal Regulatory Region from Metroxylon sagu

    PubMed Central

    Wee, Ching Ching; Roslan, Hairul Azman

    2012-01-01

    Alcohol dehydrogenase (Adh) is a versatile enzyme involved in many biochemical pathways in plants such as in germination and stress tolerance. Sago palm is plant with much importance to the state of Sarawak as one of the most important crops that bring revenue with the advantage of being able to withstand various biotic and abiotic stresses such as heat, pathogens, and water logging. Here we report the isolation of sago palm Adh cDNA and its putative promoter region via the use of rapid amplification of cDNA ends (RACE) and genomic walking. The isolated cDNA was characterized and determined to be 1464 bp long encoding for 380 amino acids. BLAST analysis showed that the Adh is similar to the Adh1 group with 91% and 85% homology with Elaeis guineensis and Washingtonia robusta, respectively. The putative basal msAdh1 regulatory region was further determined to contain promoter signals of TATA and AGGA boxes and predicted amino acids analyses showed several Adh-specific motifs such as the two zinc-binding domains that bind to the adenosine ribose of the coenzyme and binding to alcohol substrate. A phylogenetic tree was also constructed using the predicted amino acid showed clear separation of Adh from bacteria and clustered within the plant Adh group. PMID:27335670

  19. Pyruvate decarboxylase and alcohol dehydrogenase overexpression in Escherichia coli resulted in high ethanol production and rewired metabolic enzyme networks.

    PubMed

    Yang, Mingfeng; Li, Xuefeng; Bu, Chunya; Wang, Hui; Shi, Guanglu; Yang, Xiushan; Hu, Yong; Wang, Xiaoqin

    2014-11-01

    Pyruvate decarboxylase and alcohol dehydrogenase are efficient enzymes for ethanol production in Zymomonas mobilis. These two enzymes were over-expressed in Escherichia coli, a promising candidate for industrial ethanol production, resulting in high ethanol production in the engineered E. coli. To investigate the intracellular changes to the enzyme overexpression for homoethanol production, 2-DE and LC-MS/MS were performed. More than 1,000 protein spots were reproducibly detected in the gel by image analysis. Compared to the wild-type, 99 protein spots showed significant changes in abundance in the recombinant E. coli, in which 46 were down-regulated and 53 were up-regulated. Most proteins related to tricarboxylic acid cycle, glycerol metabolism and other energy metabolism were up-regulated, whereas proteins involved in glycolysis and glyoxylate pathway were down-regulated, indicating the rewired metabolism in the engineered E. coli. As glycolysis is the main pathway for ethanol production, and it was inhibited significantly in engineered E. coli, further efforts should be directed at minimizing the repression of glycolysis to optimize metabolism network for higher yields of ethanol production.

  20. Thiodiglycol, the hydrolysis product of sulfur mustard: Analysis of in vitro biotransformation by mammalian alcohol dehydrogenases using nuclear magnetic resonance

    SciTech Connect

    Brimfield, A.A.; Hodgson, Ernest

    2006-06-15

    Thiodiglycol (2,2'-bis-hydroxyethylsulfide, TDG), the hydrolysis product of the chemical warfare agent sulfur mustard, has been implicated in the toxicity of sulfur mustard through the inhibition of protein phosphatases in mouse liver cytosol. The absence of any inhibitory activity when TDG was present in assays of pure enzymes, however, led us to investigate the possibility for metabolic activation of TDG to inhibitory compound(s) by cytosolic enzymes. We have successfully shown that mammalian alcohol dehydrogenases (ADH) rapidly oxidize TDG in vitro, but the classic spectrophotometric techniques for following this reaction provided no information on the identity of TDG intermediates and products. The use of proton NMR to monitor the oxidative reaction with structural confirmation by independent synthesis allowed us to establish the ultimate product, 2-hydroxyethylthioacetic acid, and to identify an intermediate equilibrium mixture consisting of 2-hydroxyethylthioacetaldehyde, 2-hydroxyethylthioacetaldehyde hydrate and the cyclic 1,4-oxathian-2-ol. The intermediate nature of this mixture was determined spectrophotometrically when it was shown to drive the production of NADH when added to ADH and NAD.

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

    NASA Technical Reports Server (NTRS)

    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.

  2. In vitro expression of Candida albicans alcohol dehydrogenase genes involved in acetaldehyde metabolism.

    PubMed

    Bakri, M M; Rich, A M; Cannon, R D; Holmes, A R

    2015-02-01

    Alcohol consumption is a risk factor for oral cancer, possibly via its conversion to acetaldehyde, a known carcinogen. The oral commensal yeast Candida albicans may be one of the agents responsible for this conversion intra-orally. The alcohol dehydrogenase (Adh) family of enzymes are involved in acetaldehyde metabolism in yeast but, for C. albicans it is not known which family member is responsible for the conversion of ethanol to acetaldehyde. In this study we determined the expression of mRNAs from three C. albicans Adh genes (CaADH1, CaADH2 and CaCDH3) for cells grown in different culture media at different growth phases by Northern blot analysis and quantitative reverse transcription polymerase chain reaction. CaADH1 was constitutively expressed under all growth conditions but there was differential expression of CaADH2. CaADH3 expression was not detected. To investigate whether CaAdh1p or CaAdh2p can contribute to alcohol catabolism in C. albicans, each gene from the reference strain C. albicans SC5314 was expressed in Saccharomyces cerevisiae. Cell extracts from an CaAdh1p-expressing S. cerevisiae recombinant, but not an CaAdh2p-expressing recombinant, or an empty vector control strain, possessed ethanol-utilizing Adh activity above endogenous S. cerevisiae activity. Furthermore, expression of C. albicans Adh1p in a recombinant S. cerevisiae strain in which the endogenous ScADH2 gene (known to convert ethanol to acetaldehyde in this yeast) had been deleted, conferred an NAD-dependent ethanol-utilizing, and so acetaldehyde-producing, Adh activity. We conclude that CaAdh1p is the enzyme responsible for ethanol use under in vitro growth conditions, and may contribute to the intra-oral production of acetaldehyde.

  3. Molecular cloning and biochemical characterization of two cinnamyl alcohol dehydrogenases from a liverwort Plagiochasma appendiculatum.

    PubMed

    Sun, Yi; Wu, Yifeng; Zhao, Yu; Han, Xiaojuan; Lou, Hongxiang; Cheng, Aixia

    2013-09-01

    Cinnamyl alcohol dehydrogenase (CAD) (EC 1.1.1.195) is a key enzyme in lignin biosynthesis. It catalyzes cinnamyl aldehydes as substrates to form corresponding alcohols, the last step in monolignol biosynthesis. Almost all CAD members of land plants could be divided into three classes according to the phylogenetic analysis, together with gene structure and function. In the present investigation, two cDNAs encoding CADs were obtained from a Chinese liverwort Plagiochasma appendiculatum thallus library and were designated as PaCAD1 and PaCAD2. Phylogenetic analysis showed that PaCAD1 and PaCAD2 belonged to Class II. Full length cDNAs were heterologously expressed in E. coli and the recombinant PaCAD proteins displayed high activity levels using p-coumaryl, caffeyl, coniferyl, 5-hydroxyconiferyl and sinapyl aldehydes as substrates to form corresponding alcohols. The enzyme kinetics results showed that PaCAD1 and PaCAD2 used coniferyl aldehyde as the favourite substrate and showed high catalytic efficiency towards p-coumaryl aldehyde but lowest catalytic efficiency towards 5-hydroxyconiferaldehyde. In accord with the higher lignin content in the thallus than in the callus, the expression level of PaCAD2 was also higher in thallus than in the callus. The expression of PaCAD1 and PaCAD2 was induced by Methyl jasmonic acid (MeJA) treatment. This suggested that these two PaCADs played twin roles in lignin biosynthesis and the defencedefence of abiotic stress in P. appendiculatum. This is the first time that the CADs in liverworts have been functionally characterized.

  4. Structural and biochemical studies of alcohol dehydrogenase isozymes from Kluyveromyces lactis.

    PubMed

    Bozzi, A; Saliola, M; Falcone, C; Bossa, F; Martini, F

    1997-04-25

    The cytosolic and mitochondrial alcohol dehydrogenases from Kluyveromyces lactis (KlADHs) were purified and characterised. Both the N-terminally blocked cytosolic isozymes, KlADH I and KlADH II, were strictly NAD-dependent and exhibited catalytic properties similar to those previously reported for other yeast ADHs. Conversely, the mitochondrial isozymes, KlADH III and KlADH IV, displayed Ala and Asn, respectively, as N-termini and were able to oxidise at an increased rate primary alcohols with aliphatic chains longer than ethanol, such as propanol, butanol, pentanol and hexanol. Interestingly, the mitochondrial KlADHs, at variance with cytosolic isozymes and the majority of ADHs from other sources, were capable of accepting as a cofactor, and in some case almost equally well, either NAD or NADP. Since Asp-223 of horse liver ADH, thought to be responsible for the selection of NAD as coenzyme, is strictly conserved in all the KlADH isozymes, this amino-acid residue should not be considered critical for the coenzyme discrimination with respect to the other residues lining the coenzyme binding pocket of the mitochondrial isozymes. The relatively low specificity of the mitochondrial KlADHs both toward the alcohols and the cofactor could be explained on the basis of an enhanced flexibility of the corresponding catalytic pockets. An involvement of the mitochondrial KlADH isozymes in the physiological reoxidation of the cytosolic NADPH was also hypothesized. Moreover, both cytosolic and KlADH IV isozymes have an additional cysteine, not involved in zinc binding, that could be responsible for the increased activity in the presence of 2-mercaptoethanol.

  5. Aerobic and anaerobic metabolism in oxygen minimum layer fishes: the role of alcohol dehydrogenase.

    PubMed

    Torres, Joseph J; Grigsby, Michelle D; Clarke, M Elizabeth

    2012-06-01

    Zones of minimum oxygen form at intermediate depth in all the world's oceans as a result of global circulation patterns that keep the water at oceanic mid-depths out of contact with the atmosphere for hundreds of years. In areas where primary production is very high, the microbial oxidation of sinking organic matter results in very low oxygen concentrations at mid-depths. Such is the case with the Arabian Sea, with O(2) concentrations reaching zero at 200 m and remaining very low (<0.1 ml O(2)l(-1)) for hundreds of meters below this depth, and in the California borderland, where oxygen levels reach 0.2 ml O(2)l(-1) at 700 m with severely hypoxic (<1.0 ml O(2)l(-1)) waters at depths 300 m above and below that. Despite the very low oxygen, mesopelagic fishes (primarily lanternfishes: Mytophidae) inhabiting the Arabian Sea and California borderland perform a daily vertical migration into the low-oxygen layer, spending daylight hours in the oxygen minimum zone and migrating upward into normoxic waters at night. To find out how fishes were able to survive their daily sojourns into the minimum zone, we tested the activity of four enzymes, one (lactate dehydrogenase, LDH) that served as a proxy for anaerobic glycolysis with a conventional lactate endpoint, a second (citrate synthase, CS) that is indicative of aerobic metabolism, a third (malate dehydrogenase) that functions in the Krebs' cycle and as a bridge linking mitochondrion and cytosol, and a fourth (alcohol dehydrogenase, ADH) that catalyzes the final reaction in a pathway where pyruvate is reduced to ethanol. Ethanol is a metabolic product easily excreted by fish, preventing lactate accumulation. The ADH pathway is rarely very active in vertebrate muscle; activity has previously been seen only in goldfish and other cyprinids capable of prolonged anaerobiosis. Activity of the enzyme suite in Arabian Sea and California fishes was compared with that of ecological analogs in the same family and with the same

  6. NAD-dependent aromatic alcohol dehydrogenase in wheats (Triticum L.) and goatgrasses (Aegilops L.): evolutionary genetics.

    PubMed

    Jaaska, V

    1984-04-01

    Evolutionary electrophoretic variation of a NAD-specific aromatic alcohol dehydrogenase, AADH-E, in wheat and goatgrass species is described and discussed in comparison with a NAD-specific alcohol dehydrogenase (ADH-A) and a NADP-dependent AADH-B studied previously. Cultivated tetraploid emmer wheats (T. turgidum s. l.) and hexaploid bread wheats (T. aestivum s. l.) are all fixed for a heterozygous triplet, E(0.58)/E(0.64). The slowest isoenzyme, E(0.58), is controlled by a homoeoallelic gene on the chromosome arm 6AL of T. aestivum cv. 'Chinese Spring' and is inherent in all diploid wheats, T. monococcum s. Str., T. boeoticum s. l. and T. urartu. The fastest isoenzyme, E(0.64), is presumably controlled by the B- and D-genome homoeoalleles of the bread wheat and is the commonest alloenzyme of diploid goat-grasses, including Ae. speltaides and Ae. tauschii. The tetraploid T. timopheevii s. str. has a particular heterozygous triplet E(0.56)/E(0.71), whereas the hexaploid T. zhukovskyi exhibited polymorphism with electromorphs characteristic of T. timopheevii and T. monococcum. Wild tetraploid wheats, T. dicoccoides and T. araraticum, showed partially homologous intraspecific variation of AADH-E with heterozygous triplets E(0.58)/E(0.64) (the commonest), E(0.58)/E(0.71), E(0.45)/E(0.58), E(0.48)/E(0.58) and E(0.56)/E(0.58) recorded. Polyploid goatgrasses of the D-genome group, excepting Ae. cylindrica, are fixed for the common triplet E(0.58)/E(0.64). Ae. cylindrica and polyploid goatgrasses of the C(u)-genome group, excepting Ae. kotschyi, are homozygous for E(0.64). Ae. kotschyi is exceptional, showing fixed heterozygosity for both AADH-E and ADH-A with unique triplets E(0.56)/E(0.64) and A(0.49)/A(0.56).

  7. Genetic polymorphisms of alcohol dehydrogense-1B and aldehyde dehydrogenase-2, alcohol flushing, mean corpuscular volume, and aerodigestive tract neoplasia in Japanese drinkers.

    PubMed

    Yokoyama, Akira; Mizukami, Takeshi; Yokoyama, Tetsuji

    2015-01-01

    Genetic polymorphisms of alcohol dehydrogenase-1B (ADH1B) and aldehyde dehydrogenase-2 (ALDH2) modulate exposure levels to ethanol/acetaldehyde. Endoscopic screening of 6,014 Japanese alcoholics yielded high detection rates of esophageal squamous cell carcinoma (SCC; 4.1%) and head and neck SCC (1.0%). The risks of upper aerodigestive tract SCC/dysplasia, especially of multiple SCC/dysplasia, were increased in a multiplicative fashion by the presence of a combination of slow-metabolizing ADH1B*1/*1 and inactive heterozygous ALDH2*1/*2 because of prolonged exposure to higher concentrations of ethanol/acetaldehyde. A questionnaire asking about current and past facial flushing after drinking a glass (≈180 mL) of beer is a reliable tool for detecting the presence of inactive ALDH2. We invented a health-risk appraisal (HRA) model including the flushing questionnaire and drinking, smoking, and dietary habits. Esophageal SCC was detected at a high rate by endoscopic mass-screening in high HRA score persons. A total of 5.0% of 4,879 alcoholics had a history of (4.0%) or newly diagnosed (1.0%) gastric cancer. Their high frequency of a history of gastric cancer is partly explained by gastrectomy being a risk factor for alcoholism because of altered ethanol metabolism, e.g., by blood ethanol level overshooting. The combination of H. pylori-associated atrophic gastritis and ALDH2*1/*2 showed the greatest risk of gastric cancer in alcoholics. High detection rates of advanced colorectal adenoma/carcinoma were found in alcoholics, 15.7% of 744 immunochemical fecal occult blood test (IFOBT)-negative alcoholics and 31.5% of the 393 IFOBT-positive alcoholics. Macrocytosis with an MCV≥106 fl increased the risk of neoplasia in the entire aerodigestive tract of alcoholics, suggesting that poor nutrition as well as ethanol/acetaldehyde exposure plays an important role in neoplasia.

  8. A distinct type of alcohol dehydrogenase, adh4+, complements ethanol fermentation in an adh1-deficient strain of Schizosaccharomyces pombe.

    PubMed

    Sakurai, Masao; Tohda, Hideki; Kumagai, Hiromichi; Giga-Hama, Yuko

    2004-03-01

    In the fission yeast Schizosaccharomyces pombe, only one alcohol dehydrogenase gene, adh1(+), has been identified. To elucidate the influence of adh1(+) on ethanol fermentation, we constructed the adh1 null strain (delta adh1). The delta adh1 cells still produced ethanol and grew fermentatively as the wild-type cells. Both DNA microarray and RT-PCR analysis demonstrated that this ethanol production is caused by the enhanced expression of a Saccharomyces cerevisiae ADH4-like gene product (SPAC5H10.06C named adh4(+)). Since the strain lacking both adh1 and adh4 genes (delta adh1 delta adh4) showed non-fermentative retarded growth, only these two ADHs produce ethanol for fermentative growth. This is the first observation that a S. cerevisiae ADH4-like alcohol dehydrogenase functions in yeast ethanol fermentation.

  9. Involvement of AMPK in Alcohol Dehydrogenase Accentuated Myocardial Dysfunction Following Acute Ethanol Challenge in Mice

    PubMed Central

    Guo, Rui; Scott, Glenda I.; Ren, Jun

    2010-01-01

    Objectives Binge alcohol drinking often triggers myocardial contractile dysfunction although the underlying mechanism is not fully clear. This study was designed to examine the impact of cardiac-specific overexpression of alcohol dehydrogenase (ADH) on ethanol-induced change in cardiac contractile function, intracellular Ca2+ homeostasis, insulin and AMP-dependent kinase (AMPK) signaling. Methods ADH transgenic and wild-type FVB mice were acutely challenged with ethanol (3 g/kg/d, i.p.) for 3 days. Oral glucose tolerance test, cardiac AMP/ATP levels, cardiac contractile function, intracellular Ca2+ handling and AMPK signaling (including ACC and LKB1) were examined. Results Ethanol exposure led to glucose intolerance, elevated plasma insulin, compromised cardiac contractile and intracellular Ca2+ properties, downregulated protein phosphatase PP2A subunit and PPAR-γ, as well as phosphorylation of AMPK, ACC and LKB1, all of which except plasma insulin were overtly accentuated by ADH transgene. Interestingly, myocardium from ethanol-treated FVB mice displayed enhanced expression of PP2Cα and PGC-1α, decreased insulin receptor expression as well as unchanged expression of Glut4, the response of which was unaffected by ADH. Cardiac AMP-to-ATP ratio was significantly enhanced by ethanol exposure with a more pronounced increase in ADH mice. In addition, the AMPK inhibitor compound C (10 µM) abrogated acute ethanol exposure-elicited cardiomyocyte mechanical dysfunction. Conclusions In summary, these data suggest that the ADH transgene exacerbated acute ethanol toxicity-induced myocardial contractile dysfunction, intracellular Ca2+ mishandling and glucose intolerance, indicating a role of ADH in acute ethanol toxicity-induced cardiac dysfunction possibly related to altered cellular fuel AMPK signaling cascade. PMID:20585647

  10. Structural basis for substrate specificity differences of horse liver alcohol dehydrogenase isozymes.

    PubMed

    Adolph, H W; Zwart, P; Meijers, R; Hubatsch, I; Kiefer, M; Lamzin, V; Cedergren-Zeppezauer, E

    2000-10-24

    A structure determination in combination with a kinetic study of the steroid converting isozyme of horse liver alcohol dehydrogenase, SS-ADH, is presented. Kinetic parameters for the substrates, 5beta-androstane-3beta,17beta-ol, 5beta-androstane-17beta-ol-3-one, ethanol, and various secondary alcohols and the corresponding ketones are compared for the SS- and EE-isozymes which differ by nine amino acid substitutions and one deletion. Differences in substrate specificity and stereoselectivity are explained on the basis of individual kinetic rate constants for the underlying ordered bi-bi mechanism. SS-ADH was crystallized in complex with 3alpha,7alpha,12alpha-trihydroxy-5beta-cholan -24-acid (cholic acid) and NAD(+), but microspectrophotometric analysis of single crystals proved it to be a mixed complex containing 60-70% NAD(+) and 30-40% NADH. The crystals belong to the space group P2(1) with cell dimensions a = 55.0 A, b = 73.2 A, c = 92.5 A, and beta = 102.5 degrees. A 98% complete data set to 1.54-A resolution was collected at 100 K using synchrotron radiation. The structure was solved by the molecular replacement method utilizing EE-ADH as the search model. The major structural difference between the isozymes is a widening of the substrate channel. The largest shifts in C(alpha) carbon positions (about 5 A) are observed in the loop region, in which a deletion of Asp115 is found in the SS isozyme. SS-ADH easily accommodates cholic acid, whereas steroid substrates of similar bulkiness would not fit into the EE-ADH substrate site. In the ternary complex with NAD(+)/NADH, we find that the carboxyl group of cholic acid ligates to the active site zinc ion, which probably contributes to the strong binding in the ternary NAD(+) complex.

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

    PubMed

    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.

  12. Tyr-94 Phosphorylation Inhibits Pyruvate Dehydrogenase Phosphatase 1 and Promotes Tumor Growth*

    PubMed Central

    Shan, Changliang; Kang, Hee-Bum; Elf, Shannon; Xie, Jianxin; Gu, Ting-Lei; Aguiar, Mike; Lonning, Scott; Hitosugi, Taro; Chung, Tae-Wook; Arellano, Martha; Khoury, Hanna J.; Shin, Dong M.; Khuri, Fadlo R.; Boggon, Titus J.; Fan, Jun

    2014-01-01

    Many cancer cells rely more on aerobic glycolysis (the Warburg effect) than mitochondrial oxidative phosphorylation and catabolize glucose at a high rate. Such a metabolic switch is suggested to be due in part to functional attenuation of mitochondria in cancer cells. However, how oncogenic signals attenuate mitochondrial function and promote the switch to glycolysis remains unclear. We previously reported that tyrosine phosphorylation activates and inhibits mitochondrial pyruvate dehydrogenase kinase (PDK) and phosphatase (PDP), respectively, leading to enhanced inhibitory serine phosphorylation of pyruvate dehydrogenase (PDH) and consequently inhibition of pyruvate dehydrogenase complex (PDC) in cancer cells. In particular, Tyr-381 phosphorylation of PDP1 dissociates deacetylase SIRT3 and recruits acetyltransferase ACAT1 to PDC, resulting in increased inhibitory lysine acetylation of PDHA1 and PDP1. Here we report that phosphorylation at another tyrosine residue, Tyr-94, inhibits PDP1 by reducing the binding ability of PDP1 to lipoic acid, which is covalently attached to the L2 domain of dihydrolipoyl acetyltransferase (E2) to recruit PDP1 to PDC. We found that multiple oncogenic tyrosine kinases directly phosphorylated PDP1 at Tyr-94, and Tyr-94 phosphorylation of PDP1 was common in diverse human cancer cells and primary leukemia cells from patients. Moreover, expression of a phosphorylation-deficient PDP1 Y94F mutant in cancer cells resulted in increased oxidative phosphorylation, decreased cell proliferation under hypoxia, and reduced tumor growth in mice. Together, our findings suggest that phosphorylation at different tyrosine residues inhibits PDP1 through independent mechanisms, which act in concert to regulate PDC activity and promote the Warburg effect. PMID:24962578

  13. Redox Balance in Lactobacillus reuteri DSM20016: Roles of Iron-Dependent Alcohol Dehydrogenases in Glucose/ Glycerol Metabolism.

    PubMed

    Chen, Lu; Bromberger, Paul David; Nieuwenhuiys, Gavin; Hatti-Kaul, Rajni

    2016-01-01

    Lactobacillus reuteri, a heterofermentative bacterium, metabolizes glycerol via a Pdu (propanediol-utilization) pathway involving dehydration to 3-hydroxypropionaldehyde (3-HPA) followed by reduction to 1,3-propandiol (1,3-PDO) with concomitant generation of an oxidized cofactor, NAD+ that is utilized to maintain cofactor balance required for glucose metabolism and even for oxidation of 3-HPA by a Pdu oxidative branch to 3-hydroxypropionic acid (3-HP). The Pdu pathway is operative inside Pdu microcompartment that encapsulates different enzymes and cofactors involved in metabolizing glycerol or 1,2-propanediol, and protects the cells from the toxic effect of the aldehyde intermediate. Since L. reuteri excretes high amounts of 3-HPA outside the microcompartment, the organism is likely to have alternative alcohol dehydrogenase(s) in the cytoplasm for transformation of the aldehyde. In this study, diversity of alcohol dehydrogenases in Lactobacillus species was investigated with a focus on L. reuteri. Nine ADH enzymes were found in L. reuteri DSM20016, out of which 3 (PduQ, ADH6 and ADH7) belong to the group of iron-dependent enzymes that are known to transform aldehydes/ketones to alcohols. L. reuteri mutants were generated in which the three ADHs were deleted individually. The lagging growth phenotype of these deletion mutants revealed that limited NAD+/NADH recycling could be restricting their growth in the absence of ADHs. Notably, it was demonstrated that PduQ is more active in generating NAD+ during glycerol metabolism within the microcompartment by resting cells, while ADH7 functions to balance NAD+/NADH by converting 3-HPA to 1,3-PDO outside the microcompartment in the growing cells. Moreover, evaluation of ADH6 deletion mutant showed strong decrease in ethanol level, supporting the role of this bifuctional alcohol/aldehyde dehydrogenase in ethanol production. To the best of our knowledge, this is the first report revealing both internal and external recycling

  14. Oxidation of Thiodiglycol (2,2’-Thiobis-ethanol) by Alcohol Dehydrogenase: Comparison of Human Isoenzymes

    DTIC Science & Technology

    2000-01-01

    3 . DATES COVERED (From - To) 4. TITLE AND SUBTITLE Oxidation of Thiodiglycol (2,2’-Thiobis-ethanol) by Alcohol Dehydrogenase: Comparison of Human...tion of protein (serine/threonine) phosphatases in tis- sue cytosol by sulfur mustard in vitro [ 3 ]. These en- zymes have been implicated in the...mustard itself [ 3 ]. Unsuccessful attempts to rep- licate the inhibitory effect of TDG on purified prepa- rations of protein phosphatases 1 and 2A (A

  15. Coexpression of pyruvate decarboxylase and alcohol dehydrogenase genes in Lactobacillus brevis.

    PubMed

    Liu, Siqing; Dien, Bruce S; Nichols, Nancy N; Bischoff, Kenneth M; Hughes, Stephen R; Cotta, Michael A

    2007-09-01

    Lactobacillus brevis ATCC367 was engineered to express pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) genes in order to increase ethanol fermentation from biomass-derived residues. First, a Gram-positive Sarcina ventriculi PDC gene (Svpdc) was introduced into L. brevis ATCC 367 to obtain L. brevis bbc03. The SvPDC was detected by immunoblot using an SvPDC oligo peptide antiserum, but no increased ethanol was detected in L. brevis bbc03. Then, an ADH gene from L. brevis (Bradh) was cloned behind the Svpdc gene that generated a pdc/adh-coupled ethanol cassette pBBC04. The pBBC04 restored anaerobic growth and conferred ethanol production of Escheirichia coli NZN111 (a fermentative defective strain incapable of growing anaerobically). Approximately 58 kDa (SvPDC) and 28 kDa (BrADH) recombinant proteins were observed in L. brevis bbc04. These results indicated that the Gram-positive ethanol production genes can be expressed in L. brevis using a Gram-positive promoter and pTRKH2 shuttle vector. This work provides evidence that expressing Gram-positive ethanol genes in pentose utilizing L. brevis will further aid manipulation of this microbe toward biomass to ethanol production.

  16. The Alcohol Dehydrogenase Gene Is Nested in the Outspread Locus of Drosophila Melanogaster

    PubMed Central

    McNabb, S.; Greig, S.; Davis, T.

    1996-01-01

    This report describes the structure and expression of the outspread (osp) gene of Drosophila melanogaster. Previous work showed that chromosomal breakpoints associated with mutations of the osp locus map to both sides of the alcohol dehydrogenase gene (Adh), suggesting that Adh and the adjacent gene Adh(r) are nested in osp. We extended a chromosomal walk and mapped additional osp mutations to define the maximum molecular limit of osp as 119 kb. We identified a 6-kb transcript that hybridizes to osp region DNA and is altered or absent in osp mutants. Accumulation of this RNA peaks during embryonic and pupal periods. The osp cDNAs comprise two distinct classes based on alternative splicing patterns. The 5' end of the longest cDNA was extended by PCR amplification. When hybridized to the osp walk, the 5' extension verifies that Adh and Adh(r) are nested in osp and shows that osp has a transcription unit of >=74 kb. In situ hybridization shows that osp is expressed both maternally and zygotically. In the ovary, osp is transcribed in nurse cells and localized in the oocyte. In embryos, expression is most abundant in the developing visceral and somatic musculature. PMID:8725237

  17. Enhanced Stability and Reusability of Alcohol Dehydrogenase Covalently Immobilized on Magnetic Graphene Oxide Nanocomposites.

    PubMed

    Liu, Liangliang; Yu, Jingang; Chen, Xiaoqing

    2015-02-01

    Graphene oxide (GO) has a unique planar structure and contains many functional groups. As a functional material, it can be functionalized with biomolecules and nanomaterials for various applications. In this study, Magnetic GO (MGO) nanocomposites were synthesized according to covalent binding of amino Fe3O4 nanoparticles onto the GO surface and the as-made nanocomposites were successfully applied as supports for the immobilization of alcohol dehydrogenase (ADH). Compared with free ADH and Fe3O4 nanoparticles immobilized ADH (MNP-ADH), the MGO immobilized ADH (MGO-ADH) exhibited a wider pH stability range and a better thermal stability. Furthermore, the MGO-ADH exhibited better storage stability and reusability than MNP-ADH after recovered by magnetic separations. The MGO-ADH maintained 35.1% activity after 20 days storage and lost about 20.4% activity after ten times usage. The Michaelis constant (Km) of MGO-ADH was close to that of free ADH. The results showed the MGO nanocomposites were appropriate for the immobilization of enzyme. As a novel support, MGO nanocomposites effectively increased the stability of enzyme, allowed the reuse or continuous use of enzymes and therefore improved the potential use in practical.

  18. High current density PQQ-dependent alcohol and aldehyde dehydrogenase bioanodes.

    PubMed

    Aquino Neto, Sidney; Hickey, David P; Milton, Ross D; De Andrade, Adalgisa R; Minteer, Shelley D

    2015-10-15

    In this paper, we explore the bioelectrooxidation of ethanol using pyrroloquinoline quinone (PQQ)-dependent alcohol and aldehyde dehydrogenase (ADH and AldDH) enzymes for biofuel cell applications. The bioanode architectures were designed with both direct electron transfer (DET) and mediated electron transfer (MET) mechanisms employing high surface area materials such as multi-walled carbon nanotubes (MWCNTs) and MWCNT-decorated gold nanoparticles, along with different immobilization techniques. Three different polymeric matrices were tested (tetrabutyl ammonium bromide (TBAB)-modified Nafion; octyl-modified linear polyethyleneimine (C8-LPEI); and cellulose) in the DET studies. The modified Nafion membrane provided the best electrical communication between enzymes and the electrode surface, with catalytic currents as high as 16.8 ± 2.1 µA cm(-2). Then, a series of ferrocene redox polymers were evaluated for MET. The redox polymer 1,1'-dimethylferrocene-modified linear polyethyleneimine (FcMe2-C3-LPEI) provided the best electrochemical response. Using this polymer, the electrochemical assays conducted in the presence of MWCNTs and MWCNTs-Au indicated a Jmax of 781 ± 59 µA cm(-2) and 925 ± 68 µA cm(-2), respectively. Overall, from the results obtained here, DET using the PQQ-dependent ADH and AldDH still lacks high current density, while the bioanodes that operate via MET employing ferrocene-modified LPEI redox polymers show efficient energy conversion capability in ethanol/air biofuel cells.

  19. The alcohol dehydrogenase gene is nested in the outspread locus of Drosophila melanogaster

    SciTech Connect

    McNabb, S.; Greig, S.; Davis, T.

    1996-06-01

    This report describes the structure and expression of the outspread (osp) gene of Drosophila melanogaster. Previous work showed that chromosomal breakpoints associated with mutations of the osp locus map to both sides of the alcohol dehydrogenase gene (Adh), suggesting that Adh and the adjacent gene Adh{sup r} are nested in osp. We extended a chromosomal walk and mapped additional osp mutations to define the maximum molecular limit of osp as 119 kb. We identified a 6-kb transcript that hybridizes to osp region DNA and is altered or absent in osp mutants. Accumulation of this RNA peaks during embryonic and pupal periods. The osp cDNAs comprise two distinct classes based on alternative splicing patterns. The 5{prime} end of the longest cDNA was extended by PCR amplification. When hybridized to the osp walk, the 5{prime} extension verifies that Adh and Adh{sup r} are nested in osp and shows that osp has a transcription unit of {ge}74 kb. In situ hybridization shows that osp is expressed both maternally and zygotically. In the ovary, osp is transcribed in nurse cells and localized in the oocyte. In embryos, expression is most abundant in the developing visceral and somatic musculature. 55 refs., 11 figs., 1 tab.

  20. Two mitochondrial alcohol dehydrogenase activities of Kluyveromyces lactis are differently expressed during respiration and fermentation.

    PubMed

    Saliola, M; Falcone, C

    1995-12-20

    The lactose-utilizing yeast Kluyveromyces lactis is an essentially aerobic organism in which both respiration and fermentation can coexist depending on the sugar concentration. Despite a low fermentative capacity as compared to Saccharomyces cerevisiae, four structural genes encoding alcohol dehydrogenase (ADH) activities are present in this yeast. Two of these activities, namely K1ADH III and K1ADH IV, are located within mitochondria and their presence is dependent on the carbon sources in the medium. In this paper we demonstrate by transcription and activity analysis that KlADH3 is expressed in the presence of low glucose concentrations and in the presence of respiratory carbon sources other than ethanol. Indeed ethanol acts as a strong repressor of this gene. On the other hand, KlADH4 is induced by the presence of ethanol and not by other respiratory carbon sources. We also demonstrate that the presence of KLADH III and KLADH IV in K. lactis cells is dependent on glucose concentration, glucose uptake and the amount of ethanol produced. As a consequence, these activities can be used as markers for the onset of respiratory and fermentative metabolism in this yeast.

  1. Thermal inactivation and conformational lock studies on horse liver alcohol dehydrogenase: structural mechanism.

    PubMed

    Moosavi-Movahedi, Faezeh; Saboury, Ali A; Alijanvand, H Hadi; Bohlooli, M; Salami, M; Moosavi-Movahedi, Ali A

    2013-07-01

    Horse liver alcohol dehydrogenase (HLADH) is a two subunits metal enzyme that has two catalytic sites and two coenzyme domains for each subunit. These subunits are connected together by coenzyme domains. In this study, we investigated the number and sequences of residues that participated in interface locks of HLADH. For this purpose, the kinetics of thermal inactivation of HLADH were studied in a 50 mM pyrophosphate buffer, pH 8.8, using ethanol as a substrate and NAD(+) as a cofactor. The temperature range was between 46°C and 55°C and the conformational lock was developed based on the Poltorak theory and analysis of the curves was done by the conformational lock method for oligomeric enzymes. The conformational lock number of HLADH was 2 when calculated experimentally. The results were confirmed by the Ligplot program computations. Using computational method it was shown that there are two patches binding sites at the interface and they spread over two regions of each chain. In this study we also proposed a thermal denaturation mechanism for HLADH by using different techniques such as UV-Vis fluorescence and circular dichroism (CD) spectroscopy and dynamic light scattering (DLS). The subunits are dissociated and several intermediates appeared during inactivation through increasing the temperature. DLS measurement was performed to study the changes in hydrodynamic radius during thermal inactivation. The three distinct zones that were shown by DLS were also confirmed by fluorescence and CD techniques.

  2. Arabidopsis alcohol dehydrogenase expression in both shoots and roots is conditioned by root growth environment

    NASA Technical Reports Server (NTRS)

    Chung, H. J.; Ferl, R. J.

    1999-01-01

    It is widely accepted that the Arabidopsis Adh (alcohol dehydrogenase) gene is constitutively expressed at low levels in the roots of young plants grown on agar media, and that the expression level is greatly induced by anoxic or hypoxic stresses. We questioned whether the agar medium itself created an anaerobic environment for the roots upon their growing into the gel. beta-Glucuronidase (GUS) expression driven by the Adh promoter was examined by growing transgenic Arabidopsis plants in different growing systems. Whereas roots grown on horizontal-positioned plates showed high Adh/GUS expression levels, roots from vertical-positioned plates had no Adh/GUS expression. Additional results indicate that growth on vertical plates closely mimics the Adh/GUS expression observed for soil-grown seedlings, and that growth on horizontal plates results in induction of high Adh/GUS expression that is consistent with hypoxic or anoxic conditions within the agar of the root zone. Adh/GUS expression in the shoot apex is also highly induced by root penetration of the agar medium. This induction of Adh/GUS in shoot apex and roots is due, at least in part, to mechanisms involving Ca2+ signal transduction.

  3. Computational optimization of AG18051 inhibitor for amyloid-beta binding alcohol dehydrogenase enzyme

    NASA Astrophysics Data System (ADS)

    Marques, Alexandra T.; Antunes, Agostinho; Fernandes, Pedro A.; Ramos, Maria J.

    Amyloid-beta (Abeta) binding alcohol dehydrogenase (ABAD) is a multifunctional enzyme involved in maintaining the homeostasis. The enzyme can also mediate some diseases, including genetic diseases, Alzheimer's disease, and possibly some prostate cancers. Potent inhibitors of ABAD might facilitate a better clarification of the functions of the enzyme under normal and pathogenic conditions and might also be used for therapeutic intervention in disease conditions mediated by the enzyme. The AG18051 is the only presently available inhibitor of ABAD. It binds in the active-site cavity of the enzyme and reacts with the NAD+ cofactor to form a covalent adduct. In this work, we use computational methods to perform a rational optimization of the AG18051 inhibitor, through the introduction of chemical substitutions directed to improve the affinity of the inhibitor to the enzyme. The molecular mechanics-Poisson-Boltzmann surface area methodology was used to predict the relative free binding energy of the different modified inhibitor-NAD-enzyme complexes. We show that it is possible to increase significantly the affinity of the inhibitor to the enzyme with small modifications, without changing the overall structure and ADME (absorption, distribution, metabolism, and excretion) properties of the original inhibitor.

  4. Alcohol and Aldehyde Dehydrogenases Contribute to Sex-Related Differences in Clearance of Zolpidem in Rats

    PubMed Central

    Peer, Cody J.; Strope, Jonathan D.; Beedie, Shaunna; Ley, Ariel M.; Holly, Alesia; Calis, Karim; Farkas, Ronald; Parepally, Jagan; Men, Angela; Fadiran, Emmanuel O.; Scott, Pamela; Jenkins, Marjorie; Theodore, William H.; Sissung, Tristan M.

    2016-01-01

    Objectives: The recommended zolpidem starting dose was lowered in females (5 mg vs. 10 mg) since side effects were more frequent and severe than those of males; the mechanism underlying sex differences in pharmacokinetics (PK) is unknown. We hypothesized that such differences were caused by known sex-related variability in alcohol dehydrogenase (ADH) expression. Methods: Male, female, and castrated male rats were administered 2.6 mg/kg zolpidem, ± disulfiram (ADH/ALDH pathway inhibitor) to compare PK changes induced by sex and gonadal hormones. PK analyses were conducted in rat plasma and rat brain. Key findings: Sex differences in PK were evident: females had a higher CMAX (112.4 vs. 68.1 ug/L) and AUC (537.8 vs. 231.8 h∗ug/L) than uncastrated males. Castration induced an earlier TMAX (0.25 vs. 1 h), greater CMAX (109.1 vs. 68.1 ug/L), and a corresponding AUC increase (339.7 vs. 231.8 h∗ug/L). Administration of disulfiram caused more drastic CMAX and TMAX changes in male vs. female rats that mirrored the effects of castration on first-pass metabolism, suggesting that the observed PK differences may be caused by ADH/ALDH expression. Brain concentrations paralleled plasma concentrations. Conclusion: These findings indicate that sex differences in zolpidem PK are influenced by variation in the expression of ADH/ALDH due to gonadal androgens. PMID:27574509

  5. Molecular control of the induction of alcohol dehydrogenase by ethanol in Drosophila melanogaster larvae

    SciTech Connect

    Kapoun, A.M.; Geer, B.W.; Heinstra, P.W.H. ); Corbin, V. ); McKechnie, S.W. )

    1990-04-01

    The activity of alcohol dehydrogenase, the initial enzyme in the major pathway for ethanol degradation, is induced in Drosophila melanogaster larvae by low concentrations of dietary ethanol. Two lines of evidence indicate that the metabolic products of the ADH pathway for ethanol degradation are not directly involved in the induction of Adh. First, the accumulation of the proximal transcript in Adh{sup n2} larvae was increased when the intracellular level of ethanol was elevated. In addition, the ADH activity, the proximal Adh mRNA, and the intracellular concentration of ethanol were elevated coordinately in wild-type larvae fed hexadeuterated-ethanol, which is metabolized more slowly than normal ethanol.l An examination of P element transformant lines with specific deletions in the 5{prime} regulatory DNA of the Adh gene showed that the DNA sequence between +604 and +634 of the start site of transcription from the distal promoter was essential for this induction. The DNA sequence between {minus}660 and about {minus}5,000 of the distal transcript start site was important for the down-regulation of the induction response.

  6. Crystal structure of the vertebrate NADP(H)-dependent alcohol dehydrogenase (ADH8).

    PubMed

    Rosell, Albert; Valencia, Eva; Parés, Xavier; Fita, Ignacio; Farrés, Jaume; Ochoa, Wendy F

    2003-06-27

    The amphibian enzyme ADH8, previously named class IV-like, is the only known vertebrate alcohol dehydrogenase (ADH) with specificity towards NADP(H). The three-dimensional structures of ADH8 and of the binary complex ADH8-NADP(+) have been now determined and refined to resolutions of 2.2A and 1.8A, respectively. The coenzyme and substrate specificity of ADH8, that has 50-65% sequence identity with vertebrate NAD(H)-dependent ADHs, suggest a role in aldehyde reduction probably as a retinal reductase. The large volume of the substrate-binding pocket can explain both the high catalytic efficiency of ADH8 with retinoids and the high K(m) value for ethanol. Preference of NADP(H) appears to be achieved by the presence in ADH8 of the triad Gly223-Thr224-His225 and the recruitment of conserved Lys228, which define a binding pocket for the terminal phosphate group of the cofactor. NADP(H) binds to ADH8 in an extended conformation that superimposes well with the NAD(H) molecules found in NAD(H)-dependent ADH complexes. No additional reshaping of the dinucleotide-binding site is observed which explains why NAD(H) can also be used as a cofactor by ADH8. The structural features support the classification of ADH8 as an independent ADH class.

  7. Intramolecular electron transport in quinoprotein alcohol dehydrogenase of Acetobacter methanolicus: a redox-titration study

    PubMed

    Frébortova; Matsushita; Arata; Adachi

    1998-01-27

    Quinohemoprotein-cytochrome c complex alcohol dehydrogenase (ADH) of acetic acid bacteria consists of three subunits, of which subunit I contains pyrroloquinoline quinone (PQQ) and heme c, and subunit II contains three heme c components. The PQQ and heme c components are believed to be involved in the intramolecular electron transfer from ethanol to ubiquinone. To study the intramolecular electron transfer in ADH of Acetobacter methanolicus, the redox potentials of heme c components were determined with ADH complex and the isolated subunits I and II of A. methanolicus, as well as hybrid ADH consisting of the subunit I/III complex of Gluconobacter suboxydans ADH and subunit II of A. methanolicus ADH. The redox potentials of hemes c in ADH complex were -130, 49, 188, and 188 mV at pH 7.0 and 24, 187, 190, and 255 mV at pH 4.5. In hybrid ADH, one of these heme c components was largely changed in the redox potential. Reduced ADH was fully oxidized with potassium ferricyanide, while ubiquinone oxidized the enzyme partially. The results indicate that electrons extracted from ethanol at PQQ site are transferred to ubiquinone via heme c in subunit I and two of the three hemes c in subunit II. Copyright 1998 Elsevier Science B.V.

  8. Origin of the human alcohol dehydrogenase system: implications from the structure and properties of the octopus protein.

    PubMed

    Kaiser, R; Fernández, M R; Parés, X; Jörnvall, H

    1993-12-01

    In contrast to the multiplicity of alcohol dehydrogenase in vertebrates, a class III type of the enzyme [i.e., a glutathione-dependent formaldehyde dehydrogenase; formaldehyde; NAD+ oxidoreductase (glutathione-formylating), EC 1.2.1.1.] is the only form detectable in appreciable yield in octopus. It is enzymatically and structurally highly similar to the human class III enzyme, with limited overall residue differences (26%) and only a few conservative residue exchanges at the substrate and coenzyme pockets, reflecting "constant" characteristics of this class over wide time periods. It is distinct from the ethanol-active "variable" class I type of the enzyme (i.e., classical liver alcohol dehydrogenase; alcohol:NAD+ oxidoreductase, EC 1.1.1.1). The residue conservation of class III is also spaced differently from that of class I but is typical of that of proteins in general, emphasizing that class I, with divergence at three functional segments, is the form with deviating properties. In spite of the conservation in class III, surface charges differ considerably. The apparent absence of a class I enzyme in octopus and the constant nature of the class III enzyme support the concept of a duplicative origin of the class I line from the ancient class III form. Still more distant relationships define further enzyme lines that have subunits with other properties.

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

    PubMed

    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.

  10. Origin of the human alcohol dehydrogenase system: implications from the structure and properties of the octopus protein.

    PubMed Central

    Kaiser, R; Fernández, M R; Parés, X; Jörnvall, H

    1993-01-01

    In contrast to the multiplicity of alcohol dehydrogenase in vertebrates, a class III type of the enzyme [i.e., a glutathione-dependent formaldehyde dehydrogenase; formaldehyde; NAD+ oxidoreductase (glutathione-formylating), EC 1.2.1.1.] is the only form detectable in appreciable yield in octopus. It is enzymatically and structurally highly similar to the human class III enzyme, with limited overall residue differences (26%) and only a few conservative residue exchanges at the substrate and coenzyme pockets, reflecting "constant" characteristics of this class over wide time periods. It is distinct from the ethanol-active "variable" class I type of the enzyme (i.e., classical liver alcohol dehydrogenase; alcohol:NAD+ oxidoreductase, EC 1.1.1.1). The residue conservation of class III is also spaced differently from that of class I but is typical of that of proteins in general, emphasizing that class I, with divergence at three functional segments, is the form with deviating properties. In spite of the conservation in class III, surface charges differ considerably. The apparent absence of a class I enzyme in octopus and the constant nature of the class III enzyme support the concept of a duplicative origin of the class I line from the ancient class III form. Still more distant relationships define further enzyme lines that have subunits with other properties. PMID:8248232

  11. Effects of dietary fat on alcohol-pyrazole hepatitis in rats: the pathogenetic role of the nonalcohol dehydrogenase pathway in alcohol-induced hepatic cell injury.

    PubMed

    Takada, A; Matsuda, Y; Takase, S

    1986-08-01

    Rats were fed with two different alcohol-containing (36% of total calories) liquid diets of high fat and low fat (35% and 15% of total calories) with or without 2 mM of pyrazole for 12 weeks. At the 12th week, the serum glutamic oxaloacetic transaminase level was significantly elevated in the alcohol-pyrazole high fat group, but not in the low fat group. Ballooning and necrotic changes of the hepatocytes in the centrolobular area were more prominent in the alcohol-pyrazole high fat group than in the low fat group and alcohol alone groups, indicating that high fat diet accelerates the development of alcohol-pyrazole hepatitis. In the alcohol-pyrazole high fat group, a decrease of hepatic microtubules content and an accumulation of hepatic export proteins in the hepatocytes were found. The protein accumulation was prominent only in the ballooned hepatocytes. Hepatic acetaldehyde levels were significantly higher in the alcohol-pyrazole high fat group than in the alcohol-pyrazole low fat group. These results suggest that the accelerated ethanol metabolism in the nonalcohol dehydrogenase pathway by a high fat diet may play an important role in the development of hepatocytic injuries, by impairing the microtubular function of the hepatocytes.

  12. Gossypol enantiomers potently inhibit human placental 3β-hydroxysteroid dehydrogenase 1 and aromatase activities.

    PubMed

    Dong, Yaoyao; Mao, Baiping; Li, Linxi; Guan, Hongguo; Su, Ying; Li, Xiaoheng; Lian, Qingquan; Huang, Ping; Ge, Ren-Shan

    2016-03-01

    Gossypol is a chemical isolated from cotton seeds. It exists as (+) or (-) enantiomer and has been tested for anticancer, abortion-inducing, and male contraception. Progesterone formed from pregnenolone by 3β-hydroxysteroid dehydrogenase 1 (HSD3B1) and estradiol from androgen by aromatase (CYP19A1) are critical for the maintenance of pregnancy or associated with some cancers. In this study we compared the potencies of (+)- and (-)-gossypol enantiomers in the inhibition of HSD3B1 and aromatase activities as well as progesterone and estradiol production in human placental JEG-3 cells. (+) Gossypol showed potent inhibition on human placental HSD3B1 with IC50 value of 2.3 μM, while (-) gossypol weakly inhibited it with IC50 over 100 μM. In contrast, (-) gossypol moderately inhibited CYP19A1 activity with IC50 of 23 μM, while (+) gossypol had no inhibition when the highest concentration (100 μM) was tested. (+) Gossypol enantiomer competitively inhibited HSD3B1 against substrate pregnenolone and showed mixed mode against NAD(+). (-) Gossypol competitively inhibited CYP19A1 against substrate testosterone. Gossypol enantiomers showed different potency related to their inhibition on human HSD3B1 and CYP19A1. Whether gossypol enantiomer is used alone or in combination relies on its application and beneficial effects.

  13. Isocitrate Dehydrogenase (IDH) Inhibition as Treatment of Myeloid Malignancies: Progress and Future Directions.

    PubMed

    Upadhyay, Vivek A; Brunner, Andrew M; Fathi, Amir T

    2017-03-14

    Isocitrate dehydrogenase (IDH) is an essential metabolic enzyme. Over the last two decades, there has been a growing focus on the metabolic derangements that occur with IDH1 and IDH2 mutations. The altered IDH protein leads to accumulation of 2-hydroxyglutarate (2-HG), a metabolite with oncogenic activity via epigenetic mechanisms. The advent of IDH inhibitors has engendered hope in novel and targeted therapies in IDH1/2 mutant myeloid malignancies. We here summarize the basic physiology of IDH, the metabolic and oncogenic consequences of mutant IDH1/2, and the clinical significance of IDH inhibition in hematologic malignancies. We also discuss completed and ongoing clinical trials focusing on the inhibition of IDH proteins, which have demonstrated preliminary indications of efficacy. The promise of IDH inhibition is now being further investigated as a novel therapeutic approach for AML and other myeloid malignancies.

  14. Alcohols inhibit translation to regulate morphogenesis in C. albicans.

    PubMed

    Egbe, Nkechi E; Paget, Caroline M; Wang, Hui; Ashe, Mark P

    2015-04-01

    Many molecules are secreted into the growth media by microorganisms to modulate the metabolic and physiological processes of the organism. For instance, alcohols like butanol, ethanol and isoamyl alcohol are produced by the human pathogenic fungus, Candida albicans and induce morphological differentiation. Here we show that these same alcohols cause a rapid inhibition of protein synthesis. More specifically, the alcohols target translation initiation, a complex stage of the gene expression process. Using molecular techniques, we have identified the likely translational target of these alcohols in C. albicans as the eukaryotic translation initiation factor 2B (eIF2B). eIF2B is the guanine nucleotide exchange factor for eIF2, which supports the exchange reaction where eIF2.GDP is converted to eIF2.GTP. Even minimal regulation at this step will lead to alterations in the levels of specific proteins that may allow the exigencies of the fungus to be realised. Indeed, similar to the effects of alcohols, a minimal inhibition of protein synthesis with cycloheximide also causes an induction of filamentous growth. These results suggest a molecular basis for the effect of various alcohols on morphological differentiation in C. albicans.

  15. Genistein inhibits activities of methylenetetrahydrofolate reductase and lactate dehydrogenase, enzymes which use NADH as a substrate.

    PubMed

    Grabowski, Michał; Banecki, Bogdan; Kadziński, Leszek; Jakóbkiewicz-Banecka, Joanna; Kaźmierkiewicz, Rajmund; Gabig-Cimińska, Magdalena; Węgrzyn, Grzegorz; Węgrzyn, Alicja; Banecka-Majkutewicz, Zyta

    2015-09-25

    Genistein (5, 7-dihydroxy-3- (4-hydroxyphenyl)-4H-1-benzopyran-4-one) is a natural isoflavone revealing many biological activities. Thus, it is considered as a therapeutic compound in as various disorders as cancer, infections and genetic diseases. Here, we demonstrate for the first time that genistein inhibits activities of bacterial methylenetetrahydrofolate reductase (MetF) and lactate dehydrogenase (LDH). Both enzymes use NADH as a substrate, and results of biochemical as well as molecular modeling studies with MetF suggest that genistein may interfere with binding of this dinucleotide to the enzyme. These results have implications for our understanding of biological functions of genistein and its effects on cellular metabolism.

  16. The Mitochondrial Chaperone TRAP1 Promotes Neoplastic Growth by Inhibiting Succinate Dehydrogenase

    PubMed Central

    Sciacovelli, Marco; Guzzo, Giulia; Morello, Virginia; Frezza, Christian; Zheng, Liang; Nannini, Nazarena; Calabrese, Fiorella; Laudiero, Gabriella; Esposito, Franca; Landriscina, Matteo; Defilippi, Paola; Bernardi, Paolo; Rasola, Andrea

    2013-01-01

    Summary We report that the mitochondrial chaperone TRAP1, which is induced in most tumor types, is required for neoplastic growth and confers transforming potential to noncancerous cells. TRAP1 binds to and inhibits succinate dehydrogenase (SDH), the complex II of the respiratory chain. The respiratory downregulation elicited by TRAP1 interaction with SDH promotes tumorigenesis by priming the succinate-dependent stabilization of the proneoplastic transcription factor HIF1α independently of hypoxic conditions. These findings provide a mechanistic clue to explain the switch to aerobic glycolysis of tumors and identify TRAP1 as a promising antineoplastic target. PMID:23747254

  17. Purification and characterization of alcohol dehydrogenase reducing N-benzyl-3-pyrrolidinone from Geotrichum capitatum.

    PubMed

    Yamada-Onodera, Keiko; Fukui, Masato; Tani, Yoshiki

    2007-02-01

    (S)-N-Benzyl-3-pyrrolidinol is widely used in the synthesis of pharmaceuticals as a chiral building block. We produced 30 mM (S)-N-benzyl-3-pyrrolidinol (enantiometric excess > 99.9%) from the corresponding ketone N-benzyl-3-pyrrolidinone with more than 99.9% yield in 28 h of the resting-cell reaction of Geotrichum capitatum JCM 3908. NAD(+)-dependent alcohol dehydrogenase reducing N-benzyl-3-pyrrolidinone from G. capitatum JCM 3908 was purified to homogeneity by ammonium sulfate fractionation and a series of DEAE-Toyopearl, Butyl-Toyopearl, Superdex 200, and Hydroxyapatite column chromatographies. The results of SDS-PAGE and HPLC showed the enzyme to be a dimer with a molecular mass of 78 kDa. The purified enzyme produced (S)-N-benzyl-3-pyrrolidinol (e.e.>99.9%) from N-benzyl-3-pyrrolidinone. The enzyme reduced 2,3-butanedione, 2-hexanone, cyclohexanone, propionaldehyde, n-butylaldehyde, n-hexylaldehyde, n-octylaldehyde, n-valeraldehyde, and benzylacetone more effectively than it did N-benzyl-3-pyrrolidinone. No activity was detected towards N-benzyl-2-pyrrolidinone or 2-pyrrolidinone. The activity towards (R)-N-benzyl-3-pyrrolidinol was not detected under the assay conditions employed. The oxidizing activity of the enzyme was higher towards 2-propanol, 2-butanol, 2-pentanol, 2-hexanol, 3-hexanol, and 1-phenyl-2-propanol than towards (S)-N-benzyl-3-pyrrolidinol. The K(m) values for N-benzyl-3-pyrrolidinone reduction and (S)-N-benzyl-3-pyrrolidinol oxidation were 0.13 and 8.47 mM, respectively. To our knowledge, this is the first time that an N-benzyl-3-pyrrolidinol/N-benzyl-3-pyrrolidinone oxidoreductase was purified from a eukaryote; moreover, this is the first report of (S)-N-benzyl-3-pyrrolidinol dehydrogenase activity in microorganisms. This enzyme showed features different from those of known prokaryotic N-benzyl-3-pyrrolidinone reductases. This enzyme will be very useful for the production of chiral compounds.

  18. Membrane-bound sugar alcohol dehydrogenase in acetic acid bacteria catalyzes L-ribulose formation and NAD-dependent ribitol dehydrogenase is independent of the oxidative fermentation.

    PubMed

    Adachi, O; Fujii, Y; Ano, Y; Moonmangmee, D; Toyama, H; Shinagawa, E; Theeragool, G; Lotong, N; Matsushita, K

    2001-01-01

    To identify the enzyme responsible for pentitol oxidation by acetic acid bacteria, two different ribitol oxidizing enzymes, one in the cytosolic fraction of NAD(P)-dependent and the other in the membrane fraction of NAD(P)-independent enzymes, were examined with respect to oxidative fermentation. The cytoplasmic NAD-dependent ribitol dehydrogenase (EC 1.1.1.56) was crystallized from Gluconobacter suboxydans IFO 12528 and found to be an enzyme having 100 kDa of molecular mass and 5 s as the sedimentation constant, composed of four identical subunits of 25 kDa. The enzyme catalyzed a shuttle reversible oxidoreduction between ribitol and D-ribulose in the presence of NAD and NADH, respectively. Xylitol and L-arabitol were well oxidized by the enzyme with reaction rates comparable to ribitol oxidation. D-Ribulose, L-ribulose, and L-xylulose were well reduced by the enzyme in the presence of NADH as cosubstrates. The optimum pH of pentitol oxidation was found at alkaline pH such as 9.5-10.5 and ketopentose reduction was found at pH 6.0. NAD-Dependent ribitol dehydrogenase seemed to be specific to oxidoreduction between pentitols and ketopentoses and D-sorbitol and D-mannitol were not oxidized by this enzyme. However, no D-ribulose accumulation was observed outside the cells during the growth of the organism on ribitol. L-Ribulose was accumulated in the culture medium instead, as the direct oxidation product catalyzed by a membrane-bound NAD(P)-independent ribitol dehydrogenase. Thus, the physiological role of NAD-dependent ribitol dehydrogenase was accounted to catalyze ribitol oxidation to D-ribulose in cytoplasm, taking D-ribulose to the pentose phosphate pathway after being phosphorylated. L-Ribulose outside the cells would be incorporated into the cytoplasm in several ways when need for carbon and energy sources made it necessary to use L-ribulose for their survival. From a series of simple experiments, membrane-bound sugar alcohol dehydrogenase was concluded to be

  19. Chronic alcoholism in rats induces a compensatory response, preserving brain thiamine diphosphate, but the brain 2-oxo acid dehydrogenases are inactivated despite unchanged coenzyme levels.

    PubMed

    Parkhomenko, Yulia M; Kudryavtsev, Pavel A; Pylypchuk, Svetlana Yu; Chekhivska, Lilia I; Stepanenko, Svetlana P; Sergiichuk, Andrej A; Bunik, Victoria I

    2011-06-01

    Thiamine-dependent changes in alcoholic brain were studied using a rat model. Brain thiamine and its mono- and diphosphates were not reduced after 20 weeks of alcohol exposure. However, alcoholism increased both synaptosomal thiamine uptake and thiamine diphosphate synthesis in brain, pointing to mechanisms preserving thiamine diphosphate in the alcoholic brain. In spite of the unchanged level of the coenzyme thiamine diphosphate, activities of the mitochondrial 2-oxoglutarate and pyruvate dehydrogenase complexes decreased in alcoholic brain. The inactivation of pyruvate dehydrogenase complex was caused by its increased phosphorylation. The inactivation of 2-oxoglutarate dehydrogenase complex (OGDHC) correlated with a decrease in free thiols resulting from an elevation of reactive oxygen species. Abstinence from alcohol following exposure to alcohol reactivated OGDHC along with restoration of the free thiol content. However, restoration of enzyme activity occurred before normalization of reactive oxygen species levels. Hence, the redox status of cellular thiols mediates the action of oxidative stress on OGDHC in alcoholic brain. As a result, upon chronic alcohol consumption, physiological mechanisms to counteract the thiamine deficiency and silence pyruvate dehydrogenase are activated in rat brain, whereas OGDHC is inactivated due to impaired antioxidant ability.

  20. Engineering of 2,3-butanediol dehydrogenase to reduce acetoin formation by glycerol-overproducing, low-alcohol Saccharomyces cerevisiae.

    PubMed

    Ehsani, Maryam; Fernández, Maria R; Biosca, Josep A; Julien, Anne; Dequin, Sylvie

    2009-05-01

    Engineered Saccharomyces cerevisiae strains overexpressing GPD1, which codes for glycerol-3-phosphate dehydrogenase, and lacking the acetaldehyde dehydrogenase Ald6 display large-scale diversion of the carbon flux from ethanol toward glycerol without accumulating acetate. Although GPD1 ald6 strains have great potential for reducing the ethanol contents in wines, one major side effect is the accumulation of acetoin, having a negative sensory impact on wine. Acetoin is reduced to 2,3-butanediol by the NADH-dependent 2,3-butanediol dehydrogenase Bdh1. In order to investigate the influence of potential factors limiting this reaction, we overexpressed BDH1, coding for native NADH-dependent Bdh1, and the engineered gene BDH1(221,222,223), coding for an NADPH-dependent Bdh1 enzyme with the amino acid changes 221 EIA 223 to 221 SRS 223, in a glycerol-overproducing wine yeast. We have shown that both the amount of Bdh1 and the NADH availability limit the 2,3-butanediol dehydrogenase reaction. During wine fermentation, however, the major limiting factor was the level of synthesis of Bdh1. Consistent with this finding, the overproduction of native or engineered Bdh1 made it possible to redirect 85 to 90% of the accumulated acetoin into 2,3-butanediol, a compound with neutral sensory characteristics. In addition, the production of diacetyl, a compound causing off-flavor in alcoholic beverages, whose production is increased in glycerol-overproducing yeast cells, was decreased by half. The production of higher alcohols and esters, which was slightly decreased or unchanged in GPD1 ald6 cells compared to that in the control cells, was not further modified in BDH1 cells. Overall, rerouting carbons toward glycerol and 2,3-butanediol represents a new milestone in the engineering of a low-alcohol yeast with desirable organoleptic features, permitting the decrease of the ethanol contents in wines by up to 3 degrees.

  1. Alcohol Dehydrogenase in the Diploid Plant STEPHANOMERIA EXIGUA (Compositae): Gene Duplication, Mode of Inheritance and Linkage

    PubMed Central

    Roose, M. L.; Gottlieb, L. D.

    1980-01-01

    Study of the biochemical genetics of alcohol dehydrogenase (ADH) in the annual plant Stephanomeria exigua (Compositae) revealed that the isozymes are specified by a small family of tightly linked structural genes. One set of ADH isozymes (ADH-1) was induced in roots by flooding, and was also expressed in thickened unflooded tap roots, stems, ovaries and seeds. As in other plants, the enzymes are dimeric and form homo- and heterodimers. An electrophoretic survey of ADH-1 phenotypes in two natural populations revealed seven different ADH-1 homodimers in various phenotypes having one to eight enzyme bands. Genetic analysis of segregations from crosses involving 59 plants showed that the ADH-1 isozymes are inherited as a single Mendelian unit, Adh1. Adh1 is polymorphic for forms that specify one, two, or three different ADH-1 subunits (which combine to form homo- and heterodimers), and are expressed co-dominantly in all genotypic combinations. Staining intensity of enzymes extracted from various homozygous and heterozygous plants indicated that the different subunit types specified by Adh1 are produced in approximately equal amounts. These observations suggest that Adh1 is a compound locus consisting of one to several tightly linked (0 recombinants among 579 testcross progeny), coordinately expressed structural genes. The genes in the two triplications also occur in various duplicate complexes and thus could have originated via unequal crossing over. The ADH-2 isozyme found in pollen and seeds is apparently specified by a different gene, Adh2. Adh1 and Adh2 are tightly linked (0 recombinants among 81 testcross progeny). PMID:17249032

  2. Improved paper pulp from plants with suppressed cinnamoyl-CoA reductase or cinnamyl alcohol dehydrogenase.

    PubMed

    O'Connell, Ann; Holt, Karen; Piquemal, Joël; Grima-Pettenati, Jacqueline; Boudet, Alain; Pollet, Brigitte; Lapierre, Catherine; Petit-Conil, Michel; Schuch, Wolfgang; Halpin, Claire

    2002-10-01

    Transgenic plants severely suppressed in the activity of cinnamoyl-CoA reductase were produced by introduction of a partial sense CCR transgene into tobacco. Five transgenic lines with CCR activities ranging from 2 to 48% of wild-type values were selected for further study. Some lines showed a range of aberrant phenotypes including reduced growth, and all had changes to lignin structure making the polymer more susceptible to alkali extraction. The most severely CCR-suppressed line also had significantly decreased lignin content and an increased proportion of free phenolic groups in non-condensed lignin. These changes are likely to make the lignin easier to extract during chemical pulping. Direct Kraft pulping trials confirmed this. More lignin could be removed from the transgenic wood than from wild-type wood at the same alkali charge. A similar improvement in pulping efficiency was recently shown for poplar trees expressing an antisense cinnamyl alcohol dehydrogenase gene. Pulping experiments performed here on CAD-antisense tobacco plants produced near-identical results--the modified lignin was more easily removed during pulping without any adverse effects on the quality of the pulp or paper produced. These results suggest that pulping experiments performed in tobacco can be predictive of the results that will be obtained in trees such as poplar, extending the utility of the tobacco model. On the basis of our results on CCR manipulation in tobacco, we predict that CCR-suppressed trees may show pulping benefits. However, it is likely that CCR-suppression will not be the optimal target for genetic manipulation of pulping character due to the potential associated growth defects.

  3. The Alcohol Dehydrogenase Gene Family in Melon (Cucumis melo L.): Bioinformatic Analysis and Expression Patterns

    PubMed Central

    Jin, Yazhong; Zhang, Chong; Liu, Wei; Tang, Yufan; Qi, Hongyan; Chen, Hao; Cao, Songxiao

    2016-01-01

    Alcohol dehydrogenases (ADH), encoded by multigene family in plants, play a critical role in plant growth, development, adaptation, fruit ripening and aroma production. Thirteen ADH genes were identified in melon genome, including 12 ADHs and one formaldehyde dehydrogenease (FDH), designated CmADH1-12 and CmFDH1, in which CmADH1 and CmADH2 have been isolated in Cantaloupe. ADH genes shared a lower identity with each other at the protein level and had different intron-exon structure at nucleotide level. No typical signal peptides were found in all CmADHs, and CmADH proteins might locate in the cytoplasm. The phylogenetic tree revealed that 13 ADH genes were divided into three groups respectively, namely long-, medium-, and short-chain ADH subfamily, and CmADH1,3-11, which belongs to the medium-chain ADH subfamily, fell into six medium-chain ADH subgroups. CmADH12 may belong to the long-chain ADH subfamily, while CmFDH1 may be a Class III ADH and serve as an ancestral ADH in melon. Expression profiling revealed that CmADH1, CmADH2, CmADH10 and CmFDH1 were moderately or strongly expressed in different vegetative tissues and fruit at medium and late developmental stages, while CmADH8 and CmADH12 were highly expressed in fruit after 20 days. CmADH3 showed preferential expression in young tissues. CmADH4 only had slight expression in root. Promoter analysis revealed several motifs of CmADH genes involved in the gene expression modulated by various hormones, and the response pattern of CmADH genes to ABA, IAA and ethylene were different. These CmADHs were divided into ethylene-sensitive and –insensitive groups, and the functions of CmADHs were discussed. PMID:27242871

  4. Cd-substituted horse liver alcohol dehydrogenase: catalytic site metal coordination geometry and protein conformation.

    PubMed

    Hemmingsen, L; Bauer, R; Bjerrum, M J; Zeppezauer, M; Adolph, H W; Formicka, G; Cedergren-Zeppezauer, E

    1995-05-30

    The coordination geometry of the catalytic site in Cd-substituted horse liver alcohol dehydrogenase (LADH) has been investigated as a function of pH using the method of perturbed angular correlation of gamma-rays (PAC). LADH in solution fully loaded with cadmium, including radioactive 111mCd in the catalytic site [Cd2(111mCd)Cd2LADH], was studied over the pH range 7.9-11.5. Analysis of the PAC spectra showed the ionization of a group with pKa of 11. This pKa value is about 2 pH units higher than that of native zinc-containing LADH. A pKa of 9.6 was found for the binary complex of Cd2(111mCd)Cd2LADH with NAD+. This value is also about 2 pH units higher than that of the binary complex of native zinc-containing enzyme and NAD+. No pH dependency was detected for the binary complex of Cd2(111mCd)Cd2LADH with NADH within the pH range measured (pH 8.3-11.5). Assuming that metal-coordinated water is the ionizing group [Kvassman, J., & Pettersson, G. (1979) Eur. J. Biochem. 100, 115-123], we conclude that the larger ionic radius of Cd(II) relative to Zn(II) in the catalytic site causes the elevated pKa values of metal-bound water. Interpretation of nuclear quadrupole interaction (NQI) parameters derived from PAC spectra is based on the use of the angular overlap model, using the coordinates for the catalytic zinc site from the 1.8 A resolution crystal structure of the ternary complex between LADH, NADH, and dimethyl sulfoxide as a model.(ABSTRACT TRUNCATED AT 250 WORDS)

  5. Complete reversal of coenzyme specificity by concerted mutation of three consecutive residues in alcohol dehydrogenase.

    PubMed

    Rosell, Albert; Valencia, Eva; Ochoa, Wendy F; Fita, Ignacio; Parés, Xavier; Farrés, Jaume

    2003-10-17

    Gastric tissues from amphibian Rana perezi express the only vertebrate alcohol dehydrogenase (ADH8) that is specific for NADP(H) instead of NAD(H). In the crystallographic ADH8-NADP+ complex, a binding pocket for the extra phosphate group of coenzyme is formed by ADH8-specific residues Gly223-Thr224-His225, and the highly conserved Leu200 and Lys228. To investigate the minimal structural determinants for coenzyme specificity, several ADH8 mutants involving residues 223 to 225 were engineered and kinetically characterized. Computer-assisted modeling of the docked coenzymes was also performed with the mutant enzymes and compared with the wild-type crystallographic binary complex. The G223D mutant, having a negative charge in the phosphate-binding site, still preferred NADP(H) over NAD(H), as did the T224I and H225N mutants. Catalytic efficiency with NADP(H) dropped dramatically in the double mutants, G223D/T224I and T224I/H225N, and in the triple mutant, G223D/T224I/H225N (kcat/KmNADPH = 760 mm-1 min-1), as compared with the wild-type enzyme (kcat/KmNADPH = 133330 mm-1 min-1). This was associated with a lower binding affinity for NADP+ and a change in the rate-limiting step. Conversely, in the triple mutant, catalytic efficiency with NAD(H) increased, reaching values (kcat/KmNADH = 155000 mm-1 min-1) similar to those of the wild-type enzyme with NADP(H). The complete reversal of ADH8 coenzyme specificity was therefore attained by the substitution of only three consecutive residues in the phosphate-binding site, an unprecedented achievement within the ADH family.

  6. Expression Pattern of Two Paralogs Encoding Cinnamyl Alcohol Dehydrogenases in Arabidopsis. Isolation and Characterization of the Corresponding Mutants1

    PubMed Central

    Sibout, Richard; Eudes, Aymerick; Pollet, Brigitte; Goujon, Thomas; Mila, Isabelle; Granier, Fabienne; Séguin, Armand; Lapierre, Catherine; Jouanin, Lise

    2003-01-01

    Studying Arabidopsis mutants of the phenylpropanoid pathway has unraveled several biosynthetic steps of monolignol synthesis. Most of the genes leading to monolignol synthesis have been characterized recently in this herbaceous plant, except those encoding cinnamyl alcohol dehydrogenase (CAD). We have used the complete sequencing of the Arabidopsis genome to highlight a new view of the complete CAD gene family. Among nine AtCAD genes, we have identified the two distinct paralogs AtCAD-C and AtCAD-D, which share 75% identity and are likely to be involved in lignin biosynthesis in other plants. Northern, semiquantitative restriction fragment-length polymorphism-reverse transcriptase-polymerase chain reaction and western analysis revealed that AtCAD-C and AtCAD-D mRNA and protein ratios were organ dependent. Promoter activities of both genes are high in fibers and in xylem bundles. However, AtCAD-C displayed a larger range of sites of expression than AtCAD-D. Arabidopsis null mutants (Atcad-D and Atcad-C) corresponding to both genes were isolated. CAD activities were drastically reduced in both mutants, with a higher impact on sinapyl alcohol dehydrogenase activity (6% and 38% of residual sinapyl alcohol dehydrogenase activities for Atcad-D and Atcad-C, respectively). Only Atcad-D showed a slight reduction in Klason lignin content and displayed modifications of lignin structure with a significant reduced proportion of conventional S lignin units in both stems and roots, together with the incorporation of sinapaldehyde structures ether linked at Cβ. These results argue for a substantial role of AtCAD-D in lignification, and more specifically in the biosynthesis of sinapyl alcohol, the precursor of S lignin units. PMID:12805615

  7. Tyr-301 Phosphorylation Inhibits Pyruvate Dehydrogenase by Blocking Substrate Binding and Promotes the Warburg Effect*

    PubMed Central

    Fan, Jun; Kang, Hee-Bum; Shan, Changliang; Elf, Shannon; Lin, Ruiting; Xie, Jianxin; Gu, Ting-Lei; Aguiar, Mike; Lonning, Scott; Chung, Tae-Wook; Arellano, Martha; Khoury, Hanna J.; Shin, Dong M.; Khuri, Fadlo R.; Boggon, Titus J.; Kang, Sumin; Chen, Jing

    2014-01-01

    The mitochondrial pyruvate dehydrogenase complex (PDC) plays a crucial role in regulation of glucose homoeostasis in mammalian cells. PDC flux depends on catalytic activity of the most important enzyme component pyruvate dehydrogenase (PDH). PDH kinase inactivates PDC by phosphorylating PDH at specific serine residues, including Ser-293, whereas dephosphorylation of PDH by PDH phosphatase restores PDC activity. The current understanding suggests that Ser-293 phosphorylation of PDH impedes active site accessibility to its substrate pyruvate. Here, we report that phosphorylation of a tyrosine residue Tyr-301 also inhibits PDH α 1 (PDHA1) by blocking pyruvate binding through a novel mechanism in addition to Ser-293 phosphorylation. In addition, we found that multiple oncogenic tyrosine kinases directly phosphorylate PDHA1 at Tyr-301, and Tyr-301 phosphorylation of PDHA1 is common in EGF-stimulated cells as well as diverse human cancer cells and primary leukemia cells from human patients. Moreover, expression of a phosphorylation-deficient PDHA1 Y301F mutant in cancer cells resulted in increased oxidative phosphorylation, decreased cell proliferation under hypoxia, and reduced tumor growth in mice. Together, our findings suggest that phosphorylation at distinct serine and tyrosine residues inhibits PDHA1 through distinct mechanisms to impact active site accessibility, which act in concert to regulate PDC activity and promote the Warburg effect. PMID:25104357

  8. Tyr-301 phosphorylation inhibits pyruvate dehydrogenase by blocking substrate binding and promotes the Warburg effect.

    PubMed

    Fan, Jun; Kang, Hee-Bum; Shan, Changliang; Elf, Shannon; Lin, Ruiting; Xie, Jianxin; Gu, Ting-Lei; Aguiar, Mike; Lonning, Scott; Chung, Tae-Wook; Arellano, Martha; Khoury, Hanna J; Shin, Dong M; Khuri, Fadlo R; Boggon, Titus J; Kang, Sumin; Chen, Jing

    2014-09-19

    The mitochondrial pyruvate dehydrogenase complex (PDC) plays a crucial role in regulation of glucose homoeostasis in mammalian cells. PDC flux depends on catalytic activity of the most important enzyme component pyruvate dehydrogenase (PDH). PDH kinase inactivates PDC by phosphorylating PDH at specific serine residues, including Ser-293, whereas dephosphorylation of PDH by PDH phosphatase restores PDC activity. The current understanding suggests that Ser-293 phosphorylation of PDH impedes active site accessibility to its substrate pyruvate. Here, we report that phosphorylation of a tyrosine residue Tyr-301 also inhibits PDH α 1 (PDHA1) by blocking pyruvate binding through a novel mechanism in addition to Ser-293 phosphorylation. In addition, we found that multiple oncogenic tyrosine kinases directly phosphorylate PDHA1 at Tyr-301, and Tyr-301 phosphorylation of PDHA1 is common in EGF-stimulated cells as well as diverse human cancer cells and primary leukemia cells from human patients. Moreover, expression of a phosphorylation-deficient PDHA1 Y301F mutant in cancer cells resulted in increased oxidative phosphorylation, decreased cell proliferation under hypoxia, and reduced tumor growth in mice. Together, our findings suggest that phosphorylation at distinct serine and tyrosine residues inhibits PDHA1 through distinct mechanisms to impact active site accessibility, which act in concert to regulate PDC activity and promote the Warburg effect.

  9. Guanine nucleotide binding to the Bateman domain mediates the allosteric inhibition of eukaryotic IMP dehydrogenases

    NASA Astrophysics Data System (ADS)

    Buey, Rubén M.; Ledesma-Amaro, Rodrigo; Velázquez-Campoy, Adrián; Balsera, Mónica; Chagoyen, Mónica; de Pereda, José M.; Revuelta, José L.

    2015-11-01

    Inosine-5'-monophosphate dehydrogenase (IMPDH) plays key roles in purine nucleotide metabolism and cell proliferation. Although IMPDH is a widely studied therapeutic target, there is limited information about its physiological regulation. Using Ashbya gossypii as a model, we describe the molecular mechanism and the structural basis for the allosteric regulation of IMPDH by guanine nucleotides. We report that GTP and GDP bind to the regulatory Bateman domain, inducing octamers with compromised catalytic activity. Our data suggest that eukaryotic and prokaryotic IMPDHs might have developed different regulatory mechanisms, with GTP/GDP inhibiting only eukaryotic IMPDHs. Interestingly, mutations associated with human retinopathies map into the guanine nucleotide-binding sites including a previously undescribed non-canonical site and disrupt allosteric inhibition. Together, our results shed light on the mechanisms of the allosteric regulation of enzymes mediated by Bateman domains and provide a molecular basis for certain retinopathies, opening the door to new therapeutic approaches.

  10. Guanine nucleotide binding to the Bateman domain mediates the allosteric inhibition of eukaryotic IMP dehydrogenases

    PubMed Central

    Buey, Rubén M.; Ledesma-Amaro, Rodrigo; Velázquez-Campoy, Adrián; Balsera, Mónica; Chagoyen, Mónica; de Pereda, José M.; Revuelta, José L.

    2015-01-01

    Inosine-5′-monophosphate dehydrogenase (IMPDH) plays key roles in purine nucleotide metabolism and cell proliferation. Although IMPDH is a widely studied therapeutic target, there is limited information about its physiological regulation. Using Ashbya gossypii as a model, we describe the molecular mechanism and the structural basis for the allosteric regulation of IMPDH by guanine nucleotides. We report that GTP and GDP bind to the regulatory Bateman domain, inducing octamers with compromised catalytic activity. Our data suggest that eukaryotic and prokaryotic IMPDHs might have developed different regulatory mechanisms, with GTP/GDP inhibiting only eukaryotic IMPDHs. Interestingly, mutations associated with human retinopathies map into the guanine nucleotide-binding sites including a previously undescribed non-canonical site and disrupt allosteric inhibition. Together, our results shed light on the mechanisms of the allosteric regulation of enzymes mediated by Bateman domains and provide a molecular basis for certain retinopathies, opening the door to new therapeutic approaches. PMID:26558346

  11. Inhibition of pigeon breast muscle alpha-ketoglutarate dehydrogenase by structural analogs of alpha-ketoglutarate.

    PubMed

    Bunik, V I; Pavlova, O G

    1997-09-01

    Inhibition of alpha-ketoglutarate dehydrogenase (KGD) by dicarboxylates with (oxaloacetate and ketomalonate) and without (malonate, succinate, and glutarate) alpha-keto group was studied. Ketodicarboxylates at low concentrations inhibit KGD in competitive manner. Increase in their concentrations results in appearance of the noncompetitive component. The extent of KGD inhibition by keto dicarboxylates increases with structural similarity of the inhibitor and the substrate, irrespective of preliminary incubation of the enzyme with the inhibitor. This is indicative of blocking the substrate-binding site of KGD by dicarboxylates. In contrast, inhibitory effect of dicarboxylates which contain no keto group increases as their structural similarity with the substrate decreases. Saturation of KGD with dicarboxylates of this type does not completely suppress the enzymatic activity. Alternatively, these analogs display competitive mode of inhibition. Analysis of the data obtained suggests that these dicarboxylates produce catalytically active triple complex keto substrate-KGD-dicarboxylate and that KGD which enters the composition of such a complex inhibits a decreased affinity for the keto substrate as a result of the inhibitor binding.

  12. Kinetics of ethanol inhibition in alcohol fermentation.

    PubMed

    Luong, J H

    1985-03-01

    The inhibitory effect of ethanol on yeast growth and fermentation has been studied for the strain Saccharomyces cerevisiae ATCC No. 4126 under anaerobic batch conditions. The results obtained reveal that there is no striking difference between the response of growth and ethanol fermentation. Two kinetic models are also proposed to describe the kinetic pattern of ethanol inhibition on the specific rates of growth and ethanol fermentation: microi/micro0 = 1 - (P/Pm)alpha (for growth) nui/nu0 = 1 - (P/P'm)beta (for ethanol production). The maximum allowable ethanol concentration above which cells do not grow was predicted to be 112 g/L. The ethanol-producing capability of the cells was completely inhibited at 115 g/L ethanol. The proposed models appear to accurately represent the experimental data obtained in this study and the literature data.

  13. Kinetics of ethanol inhibition in alcohol fermentation

    SciTech Connect

    Luong, J.H.T.

    1985-01-01

    The inhibitory effect of ethanol on yeast growth and fermentation has been studied for the strain Saccharo-myces cerevisiae ATCC No. 4126 under anaerobic batch conditions. The results obtained reveal that there is no striking difference between the response of growth and ethanol fermentation. Two kinetic models are also proposed to describe the kinetic pattern of ethanol inhibition on the specific rates of growth and ethanol fermentation. The maximum allowable ethanol concentration above which cells do not grow was predicted to be 112 g/L. The ethanol-producing capability of the cells was completely inhibited at 115 g/L ethanol. The proposed models appear to accurately represent the experimental data obtained in this study and the literature data.

  14. An enzyme-amplified microtiter plate assay for ethanol: Its application to the detection of peanut ethanol and alcohol dehydrogenase

    SciTech Connect

    Chung, S.Y.; Vercellotti, J.R.; Sanders, T.H.

    1995-12-01

    A calorimetric microliter plate assay for ethanol amplified by aldehyde dehydrogenase (ALDH) was developed. In the assay ethanol from a sample took part in a chain-reaction catalyzed by alcohol dehydrogenase (ADH) and amplified by ALDH in the presence of NAD{sup +}, diaphorase, and p-ibdonitrotetrazolium-violet (INT-violet)(a precursor of red product). The resultant reaction gave a red color, the intensity of which was proportional to the amount of ethanol present. Using the technique, the content of activity from peanuts of differing maturity and curing stages were determined respectively. Data showed that immature peanuts had a higher level of ethanol and a lower ADH activity than mature peanuts, and that the level of ethanol and ADH activity decreased with the curing time. This indicates that peanut maturity and curing have an effect on ethanol. Also, this implies that other peanut volatiles could be affected in the same way as ethanol, a major volatile in peanuts.

  15. Purification and properties of methyl formate synthase, a mitochondrial alcohol dehydrogenase, participating in formaldehyde oxidation in methylotrophic yeasts.

    PubMed Central

    Murdanoto, A P; Sakai, Y; Konishi, T; Yasuda, F; Tani, Y; Kato, N

    1997-01-01

    Methyl formate synthase, which catalyzes methyl formate formation during the growth of methylotrophic yeasts, was purified to homogeneity from methanol-grown Candida boidinii and Pichia methanolica cells. Both purified enzymes were tetrameric, with identical subunits with molecular masses of 42 to 45 kDa, containing two atoms of zinc per subunit. The enzymes catalyze NAD(+)-linked dehydrogenation of the hydroxyl group of the hemiacetal adduct [CH2(OH)OCH3] of methanol and formaldehyde, leading to the formation of a stoichiometric amount of methyl formate. Although neither methanol nor formaldehyde alone acted as a substrate for the enzymes, they showed simple NAD(+)-linked alcohol dehydrogenase activity toward aliphatic long-chain alcohols such as octanol, showing that they belong to the class III alcohol dehydrogenase family. The methyl formate synthase activity of C. boidinii was found in the mitochondrial fraction in subcellular fractionation experiments, suggesting that methyl formate synthase is a homolog of Saccharomyces cerevisiae Adh3p. These results indicate that formaldehyde could be oxidized in a glutathione-independent manner by methyl formate synthase in methylotrophic yeasts. The significance of methyl formate synthase in both formaldehyde resistance and energy metabolism is also discussed. PMID:9143107

  16. Inhibition of branched-chain alpha-ketoacid dehydrogenase kinase by thiamine pyrophosphate at different potassium ionic levels.

    PubMed

    Akita, Keiichi; Fujimura, Yukihiro; Bajotto, Gustavo; Shimomura, Yoshiharu

    2009-05-01

    Inhibition of branched-chain alpha-ketoacid dehydrogenase kinase (BDK) by thiamine pyrophosphate (TPP) was analyzed at two potassium ion (K(+)) concentrations. IC(50) values of 4.6 and 8.0 microM and inhibition constant values of 3.2 and 16.4 microM were obtained in the presence of 20 and 100 mM K(+), respectively. These results suggest that BDK is less sensitive to TPP inhibition under physiological TPP and K(+) concentrations.

  17. Mitochondrial Probe Methyltriphenylphosphonium (TPMP) Inhibits the Krebs Cycle Enzyme 2-Oxoglutarate Dehydrogenase.

    PubMed

    Elkalaf, Moustafa; Tůma, Petr; Weiszenstein, Martin; Polák, Jan; Trnka, Jan

    2016-01-01

    Methyltriphenylphosphonium (TPMP) salts have been widely used to measure the mitochondrial membrane potential and the triphenylphosphonium (TPP+) moiety has been attached to many bioactive compounds including antioxidants to target them into mitochondria thanks to their high affinity to accumulate in the mitochondrial matrix. The adverse effects of these compounds on cellular metabolism have been insufficiently studied and are still poorly understood. Micromolar concentrations of TPMP cause a progressive inhibition of cellular respiration in adherent cells without a marked effect on mitochondrial coupling. In permeabilized cells the inhibition was limited to NADH-linked respiration. We found a mixed inhibition of the Krebs cycle enzyme 2-oxoglutarate dehydrogenase complex (OGDHC) with an estimated IC50 3.93 [3.70-4.17] mM, which is pharmacologically plausible since it corresponds to micromolar extracellular concentrations. Increasing the lipophilic character of the used TPP+ compound further potentiates the inhibition of OGDHC activity. This effect of TPMP on the Krebs cycle ought to be taken into account when interpreting observations on cells and mitochondria in the presence of TPP+ derivatives. Compounds based on or similar to TPP+ derivatives may also be used to alter OGDHC activity for experimental or therapeutic purposes.

  18. Mitochondrial Probe Methyltriphenylphosphonium (TPMP) Inhibits the Krebs Cycle Enzyme 2-Oxoglutarate Dehydrogenase

    PubMed Central

    Elkalaf, Moustafa; Tůma, Petr; Weiszenstein, Martin; Polák, Jan

    2016-01-01

    Methyltriphenylphosphonium (TPMP) salts have been widely used to measure the mitochondrial membrane potential and the triphenylphosphonium (TPP+) moiety has been attached to many bioactive compounds including antioxidants to target them into mitochondria thanks to their high affinity to accumulate in the mitochondrial matrix. The adverse effects of these compounds on cellular metabolism have been insufficiently studied and are still poorly understood. Micromolar concentrations of TPMP cause a progressive inhibition of cellular respiration in adherent cells without a marked effect on mitochondrial coupling. In permeabilized cells the inhibition was limited to NADH-linked respiration. We found a mixed inhibition of the Krebs cycle enzyme 2-oxoglutarate dehydrogenase complex (OGDHC) with an estimated IC50 3.93 [3.70–4.17] mM, which is pharmacologically plausible since it corresponds to micromolar extracellular concentrations. Increasing the lipophilic character of the used TPP+ compound further potentiates the inhibition of OGDHC activity. This effect of TPMP on the Krebs cycle ought to be taken into account when interpreting observations on cells and mitochondria in the presence of TPP+ derivatives. Compounds based on or similar to TPP+ derivatives may also be used to alter OGDHC activity for experimental or therapeutic purposes. PMID:27537184

  19. Myristica fragrans Suppresses Tumor Growth and Metabolism by Inhibiting Lactate Dehydrogenase A.

    PubMed

    Kim, Eun-Yeong; Choi, Hee-Jung; Park, Mi-Ju; Jung, Yeon-Seop; Lee, Syng-Ook; Kim, Keuk-Jun; Choi, Jung-Hye; Chung, Tae-Wook; Ha, Ki-Tae

    2016-01-01

    Most cancer cells predominantly produce ATP by maintaining a high rate of lactate fermentation, rather than by maintaining a comparatively low rate of tricarboxylic acid cycle, i.e., Warburg's effect. In the pathway, the pyruvate produced by glycolysis is converted to lactic acid by lactate dehydrogenase (LDH). Here, we demonstrated that water extracts from the seeds of Myristica fragrans Houtt. (MF) inhibit the in vitro enzymatic activity of LDH. MF effectively suppressed cell growth and the overall Warburg effect in HT29 human colon cancer cells. Although the expression of LDH-A was not changed by MF, both lactate production and LDH activity were decreased in MF-treated cells under both normoxic and hypoxic conditions. In addition, intracellular ATP levels were also decreased by MF treatment, and the uptake of glucose was also reduced by MF treatment. Furthermore, the experiment on tumor growth in the in vivo mice model revealed that MF effectively reduced the growth of allotransplanted Lewis lung carcinoma cells. Taken together, these results suggest that MF effectively inhibits cancer growth and metabolism by inhibiting the activity of LDH, a major enzyme responsible for regulating cancer metabolism. These results implicate MF as a potential candidate for development into a novel drug against cancer through inhibition of LDH activity.

  20. Redox Balance in Lactobacillus reuteri DSM20016: Roles of Iron-Dependent Alcohol Dehydrogenases in Glucose/ Glycerol Metabolism

    PubMed Central

    Bromberger, Paul David; Nieuwenhuiys, Gavin; Hatti-Kaul, Rajni

    2016-01-01

    Lactobacillus reuteri, a heterofermentative bacterium, metabolizes glycerol via a Pdu (propanediol-utilization) pathway involving dehydration to 3-hydroxypropionaldehyde (3-HPA) followed by reduction to 1,3-propandiol (1,3-PDO) with concomitant generation of an oxidized cofactor, NAD+ that is utilized to maintain cofactor balance required for glucose metabolism and even for oxidation of 3-HPA by a Pdu oxidative branch to 3-hydroxypropionic acid (3-HP). The Pdu pathway is operative inside Pdu microcompartment that encapsulates different enzymes and cofactors involved in metabolizing glycerol or 1,2-propanediol, and protects the cells from the toxic effect of the aldehyde intermediate. Since L. reuteri excretes high amounts of 3-HPA outside the microcompartment, the organism is likely to have alternative alcohol dehydrogenase(s) in the cytoplasm for transformation of the aldehyde. In this study, diversity of alcohol dehydrogenases in Lactobacillus species was investigated with a focus on L. reuteri. Nine ADH enzymes were found in L. reuteri DSM20016, out of which 3 (PduQ, ADH6 and ADH7) belong to the group of iron-dependent enzymes that are known to transform aldehydes/ketones to alcohols. L. reuteri mutants were generated in which the three ADHs were deleted individually. The lagging growth phenotype of these deletion mutants revealed that limited NAD+/NADH recycling could be restricting their growth in the absence of ADHs. Notably, it was demonstrated that PduQ is more active in generating NAD+ during glycerol metabolism within the microcompartment by resting cells, while ADH7 functions to balance NAD+/NADH by converting 3-HPA to 1,3-PDO outside the microcompartment in the growing cells. Moreover, evaluation of ADH6 deletion mutant showed strong decrease in ethanol level, supporting the role of this bifuctional alcohol/aldehyde dehydrogenase in ethanol production. To the best of our knowledge, this is the first report revealing both internal and external recycling

  1. Use of a modified alcohol dehydrogenase, ADH1, promoter in construction of diacetyl non-producing brewer's yeast.

    PubMed

    Onnela, M L; Suihko, M L; Penttilä, M; Keränen, S

    1996-08-20

    The bacterial gene, encoding alpha-acetolactate decarboxylase (alpha-ALDC), was expressed in a bottom-fermenting brewer's yeast under the control of a modified Saccharomyces cerevisiae alcohol dehydrogenase (ADH1) promoter which lacks the upstream regions from -800 bp to -1500 bp. In pilot scale brewing conditions, the level of alpha-ALDC produced was high enough to reduce the concentration of diacetyl so that lagering was not required. alpha-ALDC active brewer's yeast strains were also shown to be suitable for high gravity brewing.

  2. Laboratory evolution of Pyrococcus furiosus alcohol dehydrogenase to improve the production of (2S,5S)-hexanediol at moderate temperatures.

    PubMed

    Machielsen, Ronnie; Leferink, Nicole G H; Hendriks, Annemarie; Brouns, Stan J J; Hennemann, Hans-Georg; Daussmann, Thomas; van der Oost, John

    2008-07-01

    There is considerable interest in the use of enantioselective alcohol dehydrogenases for the production of enantio- and diastereomerically pure diols, which are important building blocks for pharmaceuticals, agrochemicals and fine chemicals. Due to the need for a stable alcohol dehydrogenase with activity at low-temperature process conditions (30 degrees C) for the production of (2S,5S)-hexanediol, we have improved an alcohol dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus (AdhA). A stable S-selective alcohol dehydrogenase with increased activity at 30 degrees C on the substrate 2,5-hexanedione was generated by laboratory evolution on the thermostable alcohol dehydrogenase AdhA. One round of error-prone PCR and screening of approximately 1,500 mutants was performed. The maximum specific activity of the best performing mutant with 2,5-hexanedione at 30 degrees C was tenfold higher compared to the activity of the wild-type enzyme. A 3D-model of AdhA revealed that this mutant has one mutation in the well-conserved NADP(H)-binding site (R11L), and a second mutation (A180V) near the catalytic and highly conserved threonine at position 183.

  3. Disruption of lactate dehydrogenase and alcohol dehydrogenase for increased hydrogen production and its effect on metabolic flux in Enterobacter aerogenes.

    PubMed

    Zhao, Hongxin; Lu, Yuan; Wang, Liyan; Zhang, Chong; Yang, Cheng; Xing, Xinhui

    2015-10-01

    Hydrogen production by Enterobacter aerogenes from glucose was enhanced by deleting the targeted ldhA and adh genes responsible for two NADH-consuming pathways which consume most NADH generated from glycolysis. Compared with the wild-type, the hydrogen yield of IAM1183-ΔldhA increased 1.5 fold. Metabolic flux analysis showed both IAM1183-ΔldhA and IAM1183-Δadh exhibited significant changes in flux, including enhanced flux towards the hydrogen generation. The lactate production of IAM1183-ΔldhA significantly decreased by 91.42%, while the alcohol yield of IAM1183-Δadh decreased to 30%. The mutant IAM1183-ΔldhA with better hydrogen-producing performance was selected for further investigation in a 5-L fermentor. The hydrogen production of IAM1183-ΔldhA was 2.3 times higher than the wild-type. Further results from the fermentation process showed that the pH decreased to 5.39 levels, then gradually increased to 5.96, indicating that some acidic metabolites might be degraded or uptaken by cells.

  4. Inhibition of stress mediated cell death by human lactate dehydrogenase B in yeast.

    PubMed

    Sheibani, Sara; Jones, Natalie K; Eid, Rawan; Gharib, Nada; Arab, Nagla T T; Titorenko, Vladimir; Vali, Hojatollah; Young, Paul A; Greenwood, Michael T

    2015-08-01

    We report the identification of human L- lactate dehydrogenase B (LDHB) as a novel Bax suppressor. Yeast heterologously expressing LDHB is also resistant to the lethal effects of copper indicating that it is a general suppressor of stress mediated cell death. To identify potential LDHB targets, LDHB was expressed in yeast mutants defective in apoptosis, necrosis and autophagy. The absence of functional PCD regulators including MCA1, YBH3, cyclophilin (CPR3) and VMA3, as well as the absence of the pro-survival autophagic pathway (ATG1,7) did not interfere with the LDHB mediated protection against copper indicating that LDHB functions independently of known PCD regulators or by simply blocking or stimulating a common PCD promoting or inhibitory pathway. Measurements of lactate levels revealed that short-term copper stress (1.6 mM, 4 h), does not increase intracellular levels of lactate, instead a three-fold increase in extracellular lactate was observed. Thus, yeast cells resemble mammalian cells where different stresses are known to lead to increased lactate production leading to lactic acidosis. In agreement with this, we found that the addition of exogenous lactic acid to growth media was sufficient to induce cell death that could be inhibited by the expression of LDHB. Taken together our results suggest that lactate dehydrogenase is a general suppressor of PCD in yeast.

  5. The Xenopus alcohol dehydrogenase gene family: characterization and comparative analysis incorporating amphibian and reptilian genomes

    PubMed Central

    2014-01-01

    Background The alcohol dehydrogenase (ADH) gene family uniquely illustrates the concept of enzymogenesis. In vertebrates, tandem duplications gave rise to a multiplicity of forms that have been classified in eight enzyme classes, according to primary structure and function. Some of these classes appear to be exclusive of particular organisms, such as the frog ADH8, a unique NADP+-dependent ADH enzyme. This work describes the ADH system of Xenopus, as a model organism, and explores the first amphibian and reptilian genomes released in order to contribute towards a better knowledge of the vertebrate ADH gene family. Results Xenopus cDNA and genomic sequences along with expressed sequence tags (ESTs) were used in phylogenetic analyses and structure-function correlations of amphibian ADHs. Novel ADH sequences identified in the genomes of Anolis carolinensis (anole lizard) and Pelodiscus sinensis (turtle) were also included in these studies. Tissue and stage-specific libraries provided expression data, which has been supported by mRNA detection in Xenopus laevis tissues and regulatory elements in promoter regions. Exon-intron boundaries, position and orientation of ADH genes were deduced from the amphibian and reptilian genome assemblies, thus revealing syntenic regions and gene rearrangements with respect to the human genome. Our results reveal the high complexity of the ADH system in amphibians, with eleven genes, coding for seven enzyme classes in Xenopus tropicalis. Frogs possess the amphibian-specific ADH8 and the novel ADH1-derived forms ADH9 and ADH10. In addition, they exhibit ADH1, ADH2, ADH3 and ADH7, also present in reptiles and birds. Class-specific signatures have been assigned to ADH7, and ancestral ADH2 is predicted to be a mixed-class as the ostrich enzyme, structurally close to mammalian ADH2 but with class-I kinetic properties. Remarkably, many ADH1 and ADH7 forms are observed in the lizard, probably due to lineage-specific duplications. ADH4 is not

  6. The cinnamyl alcohol dehydrogenase gene family in melon (Cucumis melo L.): bioinformatic analysis and expression patterns.

    PubMed

    Jin, Yazhong; Zhang, Chong; Liu, Wei; Qi, Hongyan; Chen, Hao; Cao, Songxiao

    2014-01-01

    Cinnamyl alcohol dehydrogenase (CAD) is a key enzyme in lignin biosynthesis. However, little was known about CADs in melon. Five CAD-like genes were identified in the genome of melons, namely CmCAD1 to CmCAD5. The signal peptides analysis and CAD proteins prediction showed no typical signal peptides were found in all CmCADs and CmCAD proteins may locate in the cytoplasm. Multiple alignments implied that some motifs may be responsible for the high specificity of these CAD proteins, and may be one of the key residues in the catalytic mechanism. The phylogenetic tree revealed seven groups of CAD and melon CAD genes fell into four main groups. CmCAD1 and CmCAD2 belonged to the bona fide CAD group, in which these CAD genes, as representative from angiosperms, were involved in lignin synthesis. Other CmCADs were distributed in group II, V and VII, respectively. Semi-quantitative PCR and real time qPCR revealed differential expression of CmCADs, and CmCAD5 was expressed in different vegetative tissues except mature leaves, with the highest expression in flower, while CmCAD2 and CmCAD5 were strongly expressed in flesh during development. Promoter analysis revealed several motifs of CAD genes involved in the gene expression modulated by various hormones. Treatment of abscisic acid (ABA) elevated the expression of CmCADs in flesh, whereas the transcript levels of CmCAD1 and CmCAD5 were induced by auxin (IAA); Ethylene induced the expression of CmCADs, while 1-MCP repressed the effect, apart from CmCAD4. Taken together, these data suggested that CmCAD4 may be a pseudogene and that all other CmCADs may be involved in the lignin biosynthesis induced by both abiotic and biotic stresses and in tissue-specific developmental lignification through a CAD genes family network, and CmCAD2 may be the main CAD enzymes for lignification of melon flesh and CmCAD5 may also function in flower development.

  7. The Cinnamyl Alcohol Dehydrogenase Gene Family in Melon (Cucumis melo L.): Bioinformatic Analysis and Expression Patterns

    PubMed Central

    Jin, Yazhong; Zhang, Chong; Liu, Wei; Qi, Hongyan; Chen, Hao; Cao, Songxiao

    2014-01-01

    Cinnamyl alcohol dehydrogenase (CAD) is a key enzyme in lignin biosynthesis. However, little was known about CADs in melon. Five CAD-like genes were identified in the genome of melons, namely CmCAD1 to CmCAD5. The signal peptides analysis and CAD proteins prediction showed no typical signal peptides were found in all CmCADs and CmCAD proteins may locate in the cytoplasm. Multiple alignments implied that some motifs may be responsible for the high specificity of these CAD proteins, and may be one of the key residues in the catalytic mechanism. The phylogenetic tree revealed seven groups of CAD and melon CAD genes fell into four main groups. CmCAD1 and CmCAD2 belonged to the bona fide CAD group, in which these CAD genes, as representative from angiosperms, were involved in lignin synthesis. Other CmCADs were distributed in group II, V and VII, respectively. Semi-quantitative PCR and real time qPCR revealed differential expression of CmCADs, and CmCAD5 was expressed in different vegetative tissues except mature leaves, with the highest expression in flower, while CmCAD2 and CmCAD5 were strongly expressed in flesh during development. Promoter analysis revealed several motifs of CAD genes involved in the gene expression modulated by various hormones. Treatment of abscisic acid (ABA) elevated the expression of CmCADs in flesh, whereas the transcript levels of CmCAD1 and CmCAD5 were induced by auxin (IAA); Ethylene induced the expression of CmCADs, while 1-MCP repressed the effect, apart from CmCAD4. Taken together, these data suggested that CmCAD4 may be a pseudogene and that all other CmCADs may be involved in the lignin biosynthesis induced by both abiotic and biotic stresses and in tissue-specific developmental lignification through a CAD genes family network, and CmCAD2 may be the main CAD enzymes for lignification of melon flesh and CmCAD5 may also function in flower development. PMID:25019207

  8. Regulated Expression of Three Alcohol Dehydrogenase Genes in Barley Aleurone Layers 1

    PubMed Central

    Hanson, Andrew D.; Jacobsen, John V.; Zwar, John A.

    1984-01-01

    Three genes specify alcohol dehydrogenase (EC 1.1.1.1.; ADH) enzymes in barley (Hordeum vulgare L.) (Adh 1, Adh 2, and Adh 3). Their polypeptide products (ADH 1, ADH 2, ADH 3) dimerize to give a total of six ADH isozymes which can be resolved by native gel electrophoresis and stained for enzyme activity. Under fully aerobic conditions, aleurone layers of cv Himalaya had a high titer of a single isozyme, the homodimer containing ADH 1 monomers. This isozyme was accumulated by the aleurone tissue during the later part of seed development, and survived seed drying and rehydration. The five other possible ADH isozymes were induced by O2 deficit. The staining of these five isozymes on electrophoretic gels increased progressively in intensity as O2 levels were reduced below 5%, and were most intense at 0% O2. In vivo35S labeling and specific immunoprecipitation of ADH peptides, followed by isoelectric focusing of the ADH peptides in the presence of 8 molar urea (urea-IEF) demonstrated the following. (a) Aleurone layers incubated in air synthesized ADH 1 and a trace of ADH 2; immature layers from developing seeds behaved similarly. (b) At 5% O2, synthesis of ADH 2 increased and ADH 3 appeared. (c) At 2% and 0% O2, the synthesis of all three ADH peptides increased markedly. Cell-free translation of RNA isolated from aleurone layers, followed by immunoprecipitation and urea-IEF of in vitro synthesized ADH peptides, showed that levels of mRNA for all three ADH peptides rose sharply during 1 day of O2 deprivation. Northern hybridizations with a maize Adh 2 cDNA clone established that the clone hybridized with barley mRNA comparable in size to maize Adh 2 mRNA, and that the level of this barley mRNA increased 15- to 20-fold after 1 day at 5% or 2% O2, and about 100-fold after 1 day at 0% O2. We conclude that in aleurone layers, expression of the three barley Adh genes is maximal in the absence of O2, that regulation of mRNA level is likely to be a major controlling factor, and

  9. Manipulating cinnamyl alcohol dehydrogenase (CAD) expression in flax affects fibre composition and properties

    PubMed Central

    2014-01-01

    Background In recent decades cultivation of flax and its application have dramatically decreased. One of the reasons for this is unpredictable quality and properties of flax fibre, because they depend on environmental factors, retting duration and growing conditions. These factors have contribution to the fibre composition, which consists of cellulose, hemicelluloses, lignin and pectin. By far, it is largely established that in flax, lignin reduces an accessibility of enzymes either to pectin, hemicelluloses or cellulose (during retting or in biofuel synthesis and paper production). Therefore, in this study we evaluated composition and properties of flax fibre from plants with silenced CAD (cinnamyl alcohol dehydrogenase) gene, which is key in the lignin biosynthesis. There is evidence that CAD is a useful tool to improve lignin digestibility and/or to lower the lignin levels in plants. Results Two studied lines responded differentially to the introduced modification due to the efficiency of the CAD silencing. Phylogenetic analysis revealed that flax CAD belongs to the “bona-fide” CAD family. CAD down-regulation had an effect in the reduced lignin amount in the flax fibre cell wall and as FT-IR results suggests, disturbed lignin composition and structure. Moreover introduced modification activated a compensatory mechanism which was manifested in the accumulation of cellulose and/or pectin. These changes had putative correlation with observed improved fiber’s tensile strength. Moreover, CAD down-regulation did not disturb at all or has only slight effect on flax plants’ development in vivo, however, the resistance against flax major pathogen Fusarium oxysporum decreased slightly. The modification positively affected fibre possessing; it resulted in more uniform retting. Conclusion The major finding of our paper is that the modification targeted directly to block lignin synthesis caused not only reduced lignin level in fibre, but also affected amount and

  10. Kinetic analysis about the effects of neutral salts on the thermal stability of yeast alcohol dehydrogenase.

    PubMed

    Ikegaya, Kazuo

    2005-03-01

    The effects of salts on the rate constants of inactivation by heat of yeast alcohol dehydrogenase (YADH) at 60.0 degrees C were measured. Different effects were observed at low and high salt concentrations. At high concentrations, some salts had stabilizing effects, while others were destabilizing. The effects of salts in the high concentration range examined can be described as follows: (decreased thermal stability) NaClO(4) < NaI = (C(2)H(5))(4)NBr < NH(4)Br < NaBr = KBr = CsBr = (no addition) < (CH(3))(4)NBr < KCl < KF < Na(2)SO(4) (increased thermal stability). The decreasing effect of NaClO(4) on YADH controlled the thermal stability of the enzyme absolutely and was not compensated by the addition of Na(2)SO(4), a salt which stabilized the enzyme. However, Na(2)SO(4) compensation did occur in response to the decrease in thermal stability caused by (C(2)H(5))(4)NBr. The rate constants of inactivation by heat (k (in)) of the enzyme were measured at various temperatures. Effective values of the thermodynamic activation parameters of thermal inactivation, activation of free energy (DeltaG (double dagger)), activation enthalpy (DeltaH (double dagger)), and activation entropy (DeltaS (double dagger)), were determined. The thermal stability of YADH in 0.8 M Na(2)SO(4) increased more than that of pyruvate kinase from Bacillus stearothermophilus, a moderate thermophile. The changes in the values of DeltaH (double dagger) and DeltaS (double dagger) were great and showed a general compensatory tendency, with the exception of in the case of NaClO(4). The temperature for the general compensation effect (T (c)) was approximately 123 degrees C. With Na(2)SO(4), the thermal stability of YADH at a temperature below T (c) was greater than that in the absence of salt due to the higher values of DeltaH (double dagger) and DeltaS (double dagger), respectively, and thus was an example of low-temperature enzymatic stabilization. With (C(2)H(5))(4)NBr, the thermal stability of YADH

  11. Diversity and Evolutionary Analysis of Iron-Containing (Type-III) Alcohol Dehydrogenases in Eukaryotes

    PubMed Central

    Gaona-López, Carlos; Julián-Sánchez, Adriana

    2016-01-01

    Background Alcohol dehydrogenase (ADH) activity is widely distributed in the three domains of life. Currently, there are three non-homologous NAD(P)+-dependent ADH families reported: Type I ADH comprises Zn-dependent ADHs; type II ADH comprises short-chain ADHs described first in Drosophila; and, type III ADH comprises iron-containing ADHs (FeADHs). These three families arose independently throughout evolution and possess different structures and mechanisms of reaction. While types I and II ADHs have been extensively studied, analyses about the evolution and diversity of (type III) FeADHs have not been published yet. Therefore in this work, a phylogenetic analysis of FeADHs was performed to get insights into the evolution of this protein family, as well as explore the diversity of FeADHs in eukaryotes. Principal Findings Results showed that FeADHs from eukaryotes are distributed in thirteen protein subfamilies, eight of them possessing protein sequences distributed in the three domains of life. Interestingly, none of these protein subfamilies possess protein sequences found simultaneously in animals, plants and fungi. Many FeADHs are activated by or contain Fe2+, but many others bind to a variety of metals, or even lack of metal cofactor. Animal FeADHs are found in just one protein subfamily, the hydroxyacid-oxoacid transhydrogenase (HOT) subfamily, which includes protein sequences widely distributed in fungi, but not in plants), and in several taxa from lower eukaryotes, bacteria and archaea. Fungi FeADHs are found mainly in two subfamilies: HOT and maleylacetate reductase (MAR), but some can be found also in other three different protein subfamilies. Plant FeADHs are found only in chlorophyta but not in higher plants, and are distributed in three different protein subfamilies. Conclusions/Significance FeADHs are a diverse and ancient protein family that shares a common 3D scaffold with a patchy distribution in eukaryotes. The majority of sequenced FeADHs from

  12. Kinetic involvement of acetaldehyde substrate inhibition on the rate equation of yeast aldehyde dehydrogenase.

    PubMed

    Eggert, Matthew W; Byrne, Mark E; Chambers, Robert P

    2012-10-01

    In order to evaluate the effectiveness of aldehyde dehydrogenase (ALDH) from Saccharomyces cerevisiae as a catalyst for the conversion of acetaldehyde into its physiologically and biologically less toxic acetate, the kinetics over broad concentrations were studied to develop a suitable kinetic rate expression. Even with literature accounts of the binding complexations, the yeast ALDH currently lacks a quantitative kinetic rate expression accounting for simultaneous inhibition parameters under higher acetaldehyde concentrations. Both substrate acetaldehyde and product NADH were observed as individual sources of inhibition with the combined effect of a ternary complex of acetaldehyde and the coenzyme leading to experimental rates as little as an eighth of the expected activity. Furthermore, the onset and strength of inhibition from each component were directly affected by the concentration of the co-substrate NAD. While acetaldehyde inhibition of ALDH is initiated below concentrations of 0.05 mM in the presence of 0.5 mM NAD or less, the acetaldehyde inhibition onset shifts to 0.2 mM with as much as 1.6 mM NAD. The convenience of the statistical software package JMP allowed for effective determination of experimental kinetic constants and simplification to a suitable rate expression: v = Vmax(AB)/(K(ia)K(b) + K(b)A + K(a)B + AB + B(2)/K(I-Ald) + B(2)Q/K(I-Ald-NADH) + BQ/K(I-NADH)) where the last three terms represent the inhibition complex terms for acetaldehyde, acetaldehyde-NADH, and NADH, respectively. The corresponding values of K(I-Ald), K(I-Ald-NADH), and K(I-NADH) for yeast ALDH are 2.55, 0.0269, and 0.162 mM at 22 °C and pH 7.8.

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

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed

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

    2016-01-01

    The last decade has witnessed the reawakening of cancer metabolism as a therapeutic target. In particular, inhibition of pyruvate dehydrogenase kinase (PDK) holds remarkable promise. Dichloroacetic acid (DCA), currently undergoing clinical trials, is a unique PDK inhibitor in which it binds to the allosteric pyruvate site of the enzyme. However, the safety of DCA as a drug is compromised by its neurotoxicity, whereas its usefulness as an investigative tool is limited by the high concentrations required to exert observable effects in cell culture. Herein, we report the identification - by making use of saturation-transfer difference NMR spectroscopy, enzymatic assays and computational methods - of furoate and thenoate derivatives as allosteric pyruvate-site-binding PDK2 inhibitors. This work substantiates the pyruvate regulatory pocket as a druggable target.

  15. Aldehyde dehydrogenase inhibition blocks mucosal fibrosis in human and mouse ocular scarring

    PubMed Central

    Ahadome, Sarah D.; Abraham, David J.; Rayapureddi, Suryanarayana; Saw, Valerie P.; Saban, Daniel R.; Calder, Virginia L.; Norman, Jill T.; Ponticos, Markella; Daniels, Julie T.; Dart, John K.

    2016-01-01

    Mucous membrane pemphigoid (MMP) is a systemic mucosal scarring disease, commonly causing blindness, for which there is no antifibrotic therapy. Aldehyde dehydrogenase family 1 (ALDH1) is upregulated in both ocular MMP (OMMP) conjunctiva and cultured fibroblasts. Application of the ALDH metabolite, retinoic acid (RA), to normal human conjunctival fibroblasts in vitro induced a diseased phenotype. Conversely, application of ALDH inhibitors, including disulfiram, to OMMP fibroblasts in vitro restored their functionality to that of normal controls. ALDH1 is also upregulated in the mucosa of the mouse model of scarring allergic eye disease (AED), used here as a surrogate for OMMP, in which topical application of disulfiram decreased fibrosis in vivo. These data suggest that progressive scarring in OMMP results from ALDH/RA fibroblast autoregulation, that the ALDH1 subfamily has a central role in immune-mediated ocular mucosal scarring, and that ALDH inhibition with disulfiram is a potential and readily translatable antifibrotic therapy. PMID:27699226

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

    PubMed

    Keung, W M; Klyosov, A A; Vallee, B L

    1997-03-04

    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 between ALDH-2 inhibition and ethanol intake suppression and raise the possibility that daidzin may, in fact, suppress ethanol intake of golden hamsters by inhibiting ALDH-2. Hamster liver contains not only mitochondrial ALDH-2 but also high concentrations of a cytosolic form, ALDH-1, which is a very efficient catalyst of acetaldehyde oxidation. Further, the cytosolic isozyme is completely resistant to daidzin inhibition. This unusual property of the hamster ALDH-1 isozyme accounts for the fact we previously observed that daidzin can suppress ethanol intake of this species without blocking acetaldehyde metabolism. Thus, the mechanism by which daidzin suppresses ethanol intake in golden hamsters clearly differs from that proposed for the classic ALDH inhibitor disulfiram. We postulate that a physiological pathway catalyzed by ALDH-2, so far undefined, controls ethanol intake of golden hamsters and mediates the antidipsotropic effect of daidzin.

  17. Inhibition of 11β-hydroxysteroid dehydrogenase 2 by the fungicides itraconazole and posaconazole.

    PubMed

    Beck, Katharina R; Bächler, Murielle; Vuorinen, Anna; Wagner, Sandra; Akram, Muhammad; Griesser, Ulrich; Temml, Veronika; Klusonova, Petra; Yamaguchi, Hideaki; Schuster, Daniela; Odermatt, Alex

    2017-04-15

    Impaired 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2)-dependent cortisol inactivation can lead to electrolyte dysbalance, hypertension and cardiometabolic disease. Furthermore, placental 11β-HSD2 essentially protects the fetus from high maternal glucocorticoid levels, and its impaired function has been associated with altered fetal growth and a higher risk for cardio-metabolic diseases in later life. Despite its important role, 11β-HSD2 is not included in current off-target screening approaches. To identify potential 11β-HSD inhibitors among approved drugs, a pharmacophore model was used for virtual screening, followed by biological assessment of selected hits. This led to the identification of several azole fungicides as 11β-HSD inhibitors, showing a significant structure-activity relationship between azole scaffold size, 11β-HSD enzyme selectivity and inhibitory potency. A hydrophobic linker connecting the azole ring to the other, more polar end of the molecule was observed to be favorable for 11β-HSD2 inhibition and selectivity over 11β-HSD1. The most potent 11β-HSD2 inhibition, using cell lysates expressing recombinant human 11β-HSD2, was obtained for itraconazole (IC50 139±14nM), its active metabolite hydroxyitraconazole (IC50 223±31nM) and posaconazole (IC50 460±98nM). Interestingly, experiments with mouse and rat kidney homogenates showed considerably lower inhibitory activity of these compounds towards 11β-HSD2, indicating important species-specific differences. Thus, 11β-HSD2 inhibition by these compounds is likely to be overlooked in preclinical rodent studies. Inhibition of placental 11β-HSD2 by these compounds, in addition to the known inhibition of cytochrome P450 enzymes and P-glycoprotein efflux transport, might contribute to elevated local cortisol levels, thereby affecting fetal programming.

  18. Carbon Dioxide Effects on Ethanol Production, Pyruvate Decarboxylase, and Alcohol Dehydrogenase Activities in Anaerobic Sweet Potato Roots 1

    PubMed Central

    Chang, Ling A.; Hammett, Larry K.; Pharr, David M.

    1983-01-01

    The effect of varied anaerobic atmospheres on the metabolism of sweet potato (Ipomoea batatas [L.] Lam.) roots was studied. The internal gas atmospheres of storage roots changed rapidly when the roots were submerged under water. O2 and N2 gases disappeared quickly and were replaced by CO2. There were no appreciable differences in gas composition among the four cultivars that were studied. Under different anaerobic conditions, ethanol concentration in the roots was highest in a CO2 environment, followed by submergence and a N2 environment in all the cultivars except one. A positive relationship was found between ethanol production and pyruvate decarboxylase activity from both 100% CO2-treated and 100% N2-treated roots. CO2 atmospheres also resulted in higher pyruvate decarboxylase activity than did N2 atmospheres. Concentrations of CO2 were higher within anaerobic roots than those in the ambient anaerobic atmosphere. The level of pyruvate decarboxylase and ethanol in anaerobic roots was proportional to the ambient CO2 concentration. The measurable activity of pyruvate decarboxylase that was present in the roots was about 100 times less than that of alcohol dehydrogenase. Considering these observations, it is suggested that the rate-limiting enzyme for ethanol biosynthesis in sweet potato storage roots under anoxia is likely to be pyruvate decarboxylase rather than alcohol dehydrogenase. PMID:16662798

  19. Yeast and horse liver alcohol dehydrogenases: potential problems in target size analysis and evidence for a monomer active unit

    SciTech Connect

    Suarez, M.D.; Ferguson-Miller, S.

    1987-06-16

    Yeast and horse alcohol dehydrogenases are commonly used as standards for radiation inactivation analysis of proteins, usually assuming that the minimal functional unit corresponds to the physical size in solution, a tetramer (M/sub r/ = 148,000) and a dimer (M/sub r/ = 80,000), respectively. Results described in this paper demonstrate that molecular weight overestimates may be obtained for the yeast protein as a result of its unusual sensitivity to secondary radiation products. Irradiation in the presence of sulhydryl reagents results in a smaller functional size estimate (67,000 +/- 3000) than that obtained in their absence (128,000 +/- 5000), indicating that some sulfhydryl groups in the enzyme may be particularly susceptible to attack by radiolytic species. Analysis of the horse liver enzyme reveals that although it has structural and functional similarities to the yeast protein, it is not as prone to secondary radiation damage and gives a minimal functional size estimate (33,000 +/= 1000) that most closely corresponds to a monomer. Quantitation of disappearance of the protein from a sodium dodecyl sulfate gel as a function of radiation dose also gives a target size (48,000 +/- 3000) in reasonable agreement with the monomer molecular weight. These results indicate that the individual subunits of horse liver alcohol dehydrogenase have independent catalytic capacity and imply that the same may be true for the yeast enzyme.

  20. A wide host-range metagenomic library from a waste water treatment plant yields a novel alcohol/aldehyde dehydrogenase.

    PubMed

    Wexler, Margaret; Bond, Philip L; Richardson, David J; Johnston, Andrew W B

    2005-12-01

    Using DNA obtained from the metagenome of an anaerobic digestor in a waste water treatment plant, we constructed a gene library cloned in the wide host-range cosmid pLAFR3. One cosmid enabled Rhizobium leguminosarum to grow on ethanol as sole carbon and energy source, this being due to the presence of a gene, termed adhEMeta. The AdhEMeta protein most closely resembles the AdhE alcohol dehydrogenase of Clostridium acetobutylicum, where it catalyses the formation of ethanol and butanol in a two-step reductive process. However, cloned adhEMeta did not confer ethanol utilization ability to Escherichia coli or to Pseudomonas aeruginosa, even though it was transcribed in both these hosts. Further, cell-free extracts of E. coli and R. leguminosarum containing cloned adhEMeta had butanol and ethanol dehydrogenase activities when assayed in vitro. In contrast to the well-studied AdhE proteins of C. acetobutylicum and E. coli, the enzyme specified by adhEMeta is not inactivated by oxygen and it enables alcohol to be catabolized. Cloned adhEMeta did, however, confer one phenotype to E. coli. AdhE- mutants of E. coli fail to ferment glucose and introduction of adhEMeta restored the growth of such mutants when grown under fermentative conditions. These observations show that the use of wide host-range vectors enhances the efficacy with which metagenomic libraries can be screened for genes that confer novel functions.

  1. Family history of alcoholism interacts with alcohol to affect brain regions involved in behavioral inhibition

    PubMed Central

    Kareken, David A.; Dzemidzic, Mario; Wetherill, Leah; Eiler, William; Oberlin, Brandon G.; Harezlak, Jaroslaw; Wang, Yang; O’Connor, Sean J.

    2013-01-01

    Rationale Impulsive behavior is associated with both alcohol use disorders and a family history of alcoholism (FHA). One operational definition of impulsive behavior is the stop signal task (SST), which measures the time needed to stop a ballistic hand movement. Objective Employ functional magnetic resonance imaging (fMRI) to study right frontal responses to stop signals in heavy drinking subjects with and without FHA, and as a function of alcohol exposure. Methods Twenty two family history positive (FHP; age = 22.7 years, SD= 1.9) and 18 family history negative (FHN; age = 23.7, SD= 1.8) subjects performed the SST in fMRI in two randomized visits: once during intravenous infusion of alcohol, clamped at a steady-state breath alcohol (BrAC) concentration of 60mg%, and once during infusion of placebo saline. An independent reference group (n= 13, age= 23.7, SD= 1.8) was used to identify a priori right prefrontal regions activated by successful inhibition (Inh) trials, relative to ‘Go’ trials that carried no need for inhibition (Inh > Go). Results FHA interacted with alcohol exposure in right prefrontal cortex, where alcohol reduced [Inh > Go] activation in FHN subjects, but not in FHP subjects. Within this right frontal cortical region, stop signal reaction time (SSRT) also correlated negatively with [Inh > Go] activation, suggesting that the [Inh > Go] activity was related to inhibitory behavior. Conclusions The results are consistent with the low level of response theory (Schuckit, 1980; Quinn & Fromme, 2011), with FHP being less sensitive to alcohol’s effects. PMID:23468100

  2. Elucidating the contributions of multiple aldehyde/alcohol dehydrogenases to butanol and ethanol production in Clostridium acetobutylicum

    PubMed Central

    Dai, Zongjie; Dong, Hongjun; Zhang, Yanping; Li, Yin

    2016-01-01

    Ethanol and butanol biosynthesis in Clostridium acetobutylicum share common aldehyde/alcohol dehydrogenases. However, little is known about the relative contributions of these multiple dehydrogenases to ethanol and butanol production respectively. The contributions of six aldehyde/alcohol dehydrogenases of C. acetobutylicum on butanol and ethanol production were evaluated through inactivation of the corresponding genes respectively. For butanol production, the relative contributions from these enzymes were: AdhE1 > BdhB > BdhA ≈ YqhD > SMB_P058 > AdhE2. For ethanol production, the contributions were: AdhE1 > BdhB > YqhD > SMB_P058 > AdhE2 > BdhA. AdhE1 and BdhB are two essential enzymes for butanol and ethanol production. AdhE1 was relatively specific for butanol production over ethanol, while BdhB, YqhD, and SMB_P058 favor ethanol production over butanol. Butanol synthesis was increased in the adhE2 mutant, which had a higher butanol/ethanol ratio (8.15:1) compared with wild type strain (6.65:1). Both the SMB_P058 mutant and yqhD mutant produced less ethanol without loss of butanol formation, which led to higher butanol/ethanol ratio, 10.12:1 and 10.17:1, respectively. To engineer a more efficient butanol-producing strain, adhE1 could be overexpressed, furthermore, adhE2, SMB_P058, yqhD are promising gene inactivation targets. This work provides useful information guiding future strain improvement for butanol production. PMID:27321949

  3. Protective effects of the alcohol dehydrogenase-ADH1B*3 allele on attention and behavior problems in adolescents exposed to alcohol during pregnancy.

    PubMed

    Dodge, Neil C; Jacobson, Joseph L; Jacobson, Sandra W

    2014-01-01

    Alcohol dehydrogenase is a critical enzyme in the metabolism of alcohol. Expression of three alleles at the ADH1B locus results in enzymes that differ in turnover rate and affinity for alcohol. The ADH1B*3 allele, which appears to be unique to individuals of African descent, is associated with more rapid alcohol metabolism than the more prevalent ADH1B*1 allele. It has been previously demonstrated that the presence of at least one maternal ADH1B*3 allele confers a protective effect against alcohol teratogenicity in infants and children. This study was conducted to determine whether the presence of the ADH1B*3 allele in the mother or child continues to be protective in alcohol-exposed individuals during adolescence. 186 adolescents and 167 mothers participating in a 14-year follow-up of the Detroit Longitudinal Cohort were genotyped for ADH1B alleles. Behavioral reports were obtained from classroom teachers. Frequencies of the ADH1B*3 allele were 17.6% in the mothers and 21.0% in the adolescents, which are consistent with the 15-20% expected for African Americans. Prenatal alcohol exposure was associated with increased attention problems and externalizing behaviors in adolescents born to mothers with two ADH1B*1 alleles but not in those whose mothers had at least one ADH1B*3 allele. A similar pattern was seen in relation to the presence or absence of an ADH1B*3 allele in the adolescent, which may have reflected the presence/absence of the maternal variant. This study is the first to demonstrate that the protective effects of the maternal ADH1B*3 allele continue to be evident during adolescence. These persistent individual differences in vulnerability of offspring to the behavioral effects of fetal alcohol exposure are likely attributable to more rapid metabolism of alcohol that the ADH1B*3 variant confers on the mother, leading to a reduction of the peak blood alcohol concentration to which the fetus is exposed during each drinking episode.

  4. Reconstruction of an Acetogenic 2,3-Butanediol Pathway Involving a Novel NADPH-Dependent Primary-Secondary Alcohol Dehydrogenase

    PubMed Central

    Köpke, Michael; Gerth, Monica L.; Maddock, Danielle J.; Mueller, Alexander P.; Liew, FungMin

    2014-01-01

    Acetogenic bacteria use CO and/or CO2 plus H2 as their sole carbon and energy sources. Fermentation processes with these organisms hold promise for producing chemicals and biofuels from abundant waste gas feedstocks while simultaneously reducing industrial greenhouse gas emissions. The acetogen Clostridium autoethanogenum is known to synthesize the pyruvate-derived metabolites lactate and 2,3-butanediol during gas fermentation. Industrially, 2,3-butanediol is valuable for chemical production. Here we identify and characterize the C. autoethanogenum enzymes for lactate and 2,3-butanediol biosynthesis. The putative C. autoethanogenum lactate dehydrogenase was active when expressed in Escherichia coli. The 2,3-butanediol pathway was reconstituted in E. coli by cloning and expressing the candidate genes for acetolactate synthase, acetolactate decarboxylase, and 2,3-butanediol dehydrogenase. Under anaerobic conditions, the resulting E. coli strain produced 1.1 ± 0.2 mM 2R,3R-butanediol (23 μM h−1 optical density unit−1), which is comparable to the level produced by C. autoethanogenum during growth on CO-containing waste gases. In addition to the 2,3-butanediol dehydrogenase, we identified a strictly NADPH-dependent primary-secondary alcohol dehydrogenase (CaADH) that could reduce acetoin to 2,3-butanediol. Detailed kinetic analysis revealed that CaADH accepts a range of 2-, 3-, and 4-carbon substrates, including the nonphysiological ketones acetone and butanone. The high activity of CaADH toward acetone led us to predict, and confirm experimentally, that C. autoethanogenum can act as a whole-cell biocatalyst for converting exogenous acetone to isopropanol. Together, our results functionally validate the 2,3-butanediol pathway from C. autoethanogenum, identify CaADH as a target for further engineering, and demonstrate the potential of C. autoethanogenum as a platform for sustainable chemical production. PMID:24657865

  5. Inhibition of glyceraldehyde-3-phosphate dehydrogenase in tissues of the rat by acrylamide and related compounds.

    PubMed

    Vyas, I; Lowndes, H E; Howland, R D

    1985-01-01

    In previous investigations acrylamide was found to inhibit several enzymes of glycolysis both in vitro and in vivo. The present study examines the characteristics of the in vitro inhibition of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and compares the in vivo effects of acrylamide on GAPDH activity to other analogs. Inhibition of GAPDH produced by acrylamide was characteristic of an irreversible or slowly reversible mechanism. In vivo, GAPDH activity was determined in sciatic nerve, brain, skeletal muscle and liver after cumulative doses of 250, 350 or 500 mg/kg of acrylamide. Specific activities were significantly lower in extensor muscle and liver after the 250 mg/kg dose. Activities in brain and sciatic nerve tended to be decreased but the differences were not statistically significant. Specific activity of GAPDH was decreased in medulla pons, cerebellum and the rest of the brain after a 350 mg/kg cumulative dose of acrylamide, although protein concentrations were not different from those in controls. The maximum decrease was about 20%. Treatment with acrylamide, methylene-bis-acrylamide (non-neurotoxic), or N-isopropylacrylamide (neurotoxic) significantly decreased the weight of the cortex and associated brain areas as well as general body weights. No signs of developing neuropathy were observed during treatment with methylene-bis-acrylamide to a cumulative dose (8.1 mmoles/kg) equivalent to that of acrylamide causing frank paralysis. Although the compound exhibited some ability to inhibit GAPDH in vitro, no decrease in GAPDH activity was found in rat brain. Treatment with N-isopropylacrylamide resulted in progressive neurologic impairment. After treatment to a cumulative dose of the compound causing a severe hind-limb paralysis (9.2 mmoles/kg), a small but significant decrease in GAPDH was found in the three areas of brain examined.

  6. Reactive oxygen species are involved in arsenic trioxide inhibition of pyruvate dehydrogenase activity.

    PubMed

    Samikkannu, Thangavel; Chen, Chien-Hung; Yih, Ling-Huei; Wang, Alexander S S; Lin, Shu-Yu; Chen, Tsen-Chien; Jan, Kun-Yan

    2003-03-01

    Arsenite was shown to inhibit pyruvate dehydrogenase (PDH) activity through binding to vicinal dithiols in pure enzyme and tissue extract. However, no data are available on how arsenite inhibits PDH activity in human cells. The IC(50) values for arsenic trioxide (As(2)O(3)) to inhibit the PDH activity in porcine heart pure enzyme preparation and in human leukemia cell line HL60 cells were estimated to be 182 and 2 microM, respectively. Thus, As(2)O(3) inactivation of PDH activity was about 90 times more potent in HL60 cells than in purified enzyme preparation. The IC(50) values for As(2)O(3) and phenylarsine oxide to reduce the vicinal thiol content in HL60 cells were estimated to be 81.7 and 1.9 microM, respectively. Thus, As(2)O(3) is a potent PDH inhibitor but a weak vicinal thiol reacting agent in HL60 cells. Antioxidants but not dithiol compounds suppressed As(2)O(3) inhibition of PDH activity in HL60 cells. Conversely, dithiol compounds but not antioxidants suppressed the inhibition of PDH activity by phenylarsine oxide. As(2)O(3) increased H(2)O(2) level in HL60 cells, but this was not observed for phenylarsine oxide. Mitochondrial respiration inhibitors suppressed the As(2)O(3)-induced H(2)O(2) production and As(2)O(3) inhibition of PDH activity. Moreover, metal chelators ameliorated whereas Fenton metals aggravated As(2)O(3) inhibition of PDH activity. Treatment with H(2)O(2) plus Fenton metals also decreased the PDH activity in HL60 cells. Therefore, it seems that As(2)O(3) elevates H(2)O(2) production in mitochondria and this may produce hydroxyl through the Fenton reaction and result in oxidative damage to the protein of PDH. The present results suggest that arsenite may cause protein oxidation to inactivate an enzyme and this can occur at a much lower concentration than arsenite binding directly to the critical thiols.

  7. Electrochemical evidence of self-substrate inhibition as functions regulation for cellobiose dehydrogenase from Phanerochaete chrysosporium.

    PubMed

    Stoica, Leonard; Ruzgas, Tautgirdas; Gorton, Lo

    2009-09-01

    The reaction mechanism of cellobiose dehydrogenase (CDH) from Phanerochaete chrysosporium, adsorbed on graphite electrodes, was investigated by following its catalytic reaction with cellobiose registered in both direct and mediated electron transfer modes between the enzyme and the electrode. A wall-jet flow through amperometric cell housing the CDH-modified graphite electrode was connected to a single line flow injection system. In the present study, it is proven that cellobiose, at concentrations higher than 200 microM, competes for the reduced state of the FAD cofactor and it slows down the transfer of electrons to any 2e(-)/H(+) acceptors or further to the heme cofactor, via the internal electron transfer pathway. Based on and proven by electrochemical results, a kinetic model of substrate inhibition is proposed and supported by the agreement between simulation of plots and experimental data. The implications of this kinetic model, called pseudo-ping-pong mechanism, on the possible functions CDH are also discussed. The enzyme exhibits catalytic activity also for lactose, but in contrast to cellobiose, this sugar does not inhibit the enzyme. This suggests that even if some other substrates are coincidentally oxidized by CDH, however, they do not trigger all the possible natural functions of the enzyme. In this respect, cellobiose is regarded as the natural substrate of CDH.

  8. Evaluation on the inhibition of pyrrol-2-yl ethanone derivatives to lactate dehydrogenase and anticancer activities

    NASA Astrophysics Data System (ADS)

    Lu, Na-Na; Weng, Zhao-Yue; Chen, Qiu-Yun; Boison, Daniel; Xiao, Xin-Xin; Gao, Jing

    2016-08-01

    Lactate dehydrogenase A (LDH-A) is a potentially important metabolic target for the inhibition of the highly activated glycolysis pathway in cancer cells. In order to develop bifunctional compounds as inhibitor of LDH-A and anticancer agents, two pyrrol-2-yl methanone (or ethanone) derivatives (PM1 and PM2) were synthesized and evaluated as inhibitors of LDH-A based on the enzyme assay and cell assay by spectroscopy analysis. Fluorescence and CD spectra results demonstrated that both the change of second structure of LDH-A and the affinity interaction for compounds to LDH-A gave great effect on the activity of LDH-A. In particular, low concentration of compounds (1 μμ-25 μμ) could change the level of pyruvate in cancer cells. Moreover, the in vitro assay results demonstrated that pyrrol-2-yl ethanone derivatives can inhibit the proliferation of cancer cells. Therefore, pyrrol-2-yl ethanone derivatives (PM2) can be both LDH-A inhibitor and anticancer agents.

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

    SciTech Connect

    Short, Duncan M.; Lyon, Robert; Watson, David G.; Barski, Oleg A.; McGarvie, Gail; Ellis, Elizabeth M. . E-mail: Elizabeth.ellis@strath.ac.uk

    2006-01-15

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

  10. Resisting temptation: decreasing alcohol-related affect and drinking behavior by training response inhibition.

    PubMed

    Houben, Katrijn; Nederkoorn, Chantal; Wiers, Reinout W; Jansen, Anita

    2011-07-01

    According to dual-process models, excessive alcohol use emerges when response inhibition ability is insufficient to inhibit automatic impulses to drink alcohol. This study examined whether strengthening response inhibition for alcohol-related cues decreases alcohol intake. Fifty-two heavy drinking students were randomly assigned to one of two conditions: In the beer/no-go condition, participants performed a go/no-go task that consistently paired alcohol-related stimuli with a stopping response, to increase response inhibition for alcohol-related stimuli. In the beer/go condition, in contrast, participants were always required to respond to alcohol-related stimuli during the go/no-go task. Before and after the go/no-go manipulation, we measured weekly alcohol intake and implicit attitudes toward alcohol. In addition, we measured alcohol consumption during a taste test immediately after the go/no-go manipulation. Following the manipulation, participants in the beer/no-go condition demonstrated significantly increased negative implicit attitudes toward alcohol, and a significant reduction in weekly alcohol intake, while participants in the beer/go condition showed a non-significant increase in implicit positive attitudes toward alcohol and a significant increase in weekly alcohol intake. This study demonstrates that repeatedly stopping prepotent responses toward alcohol-related stimuli can be an effective strategy to reduce excessive alcohol use.

  11. Papyriferic acid, an antifeedant triterpene from birch trees, inhibits succinate dehydrogenase from liver mitochondria.

    PubMed

    McLean, Stuart; Richards, Stephen M; Cover, Siow-Leng; Brandon, Sue; Davies, Noel W; Bryant, John P; Clausen, Thomas P

    2009-10-01

    Papyriferic acid (PA) is a triterpene that is secreted by glands on twigs of the juvenile ontogenetic phase of resin producing tree birches (e.g., Betula neoalaskana, B. pendula) and that deters browsing by mammals such as the snowshoe hare (Lepus americanus). We investigated the pharmacology of PA as a first step in understanding its antifeedant effect. After oral administration to rats, PA and several metabolites were found in feces but not urine, indicating that little was absorbed systemically. Metabolism involved various combinations of hydrolysis of its acetyl and malonyl ester groups, and hydroxylation of the terpene moiety. The presence of a malonyl group suggested a possible interaction with succinate dehydrogenase (SDH), a mitochondrial enzyme known to be competitively inhibited by malonic acid. The effect of PA on the oxidation of succinate by SDH was examined in mitochondrial preparations from livers of ox, rabbit, and rat. In all three species, PA was a potent inhibitor of SDH. Kinetic analysis indicated that, unlike malonate, PA acted by an uncompetitive mechanism, meaning that it binds to the enzyme-substrate complex. The hydrolysis product of PA, betulafolienetriol oxide, was inactive on SDH. Overall, the evidence suggests that PA acts as the intact molecule and interacts at a site other than the succinate binding site, possibly binding to the ubiquinone sites on complex II. Papyriferic acid was potent (K(iEIS) ranged from 25 to 45 microM in the three species) and selective, as malate dehydrogenase was unaffected. Although rigorous proof will require further experiments, we have a plausible mechanism for the antifeedant effect of PA: inhibition of SDH in gastrointestinal cells decreases mitochondrial energy production resulting in a noxious stimulus, 5-HT release, and sensations of nausea and discomfort. There is evidence that the co-evolution of birches and hares over a large and geographically-diverse area in Northern Europe and America has

  12. Folate, alcohol, and aldehyde dehydrogenase 2 polymorphism and the risk of oral and pharyngeal cancer in Japanese.

    PubMed

    Matsuo, Keitaro; Rossi, Marta; Negri, Eva; Oze, Isao; Hosono, Satoyo; Ito, Hidemi; Watanabe, Miki; Yatabe, Yasushi; Hasegawa, Yasuhisa; Tanaka, Hideo; Tajima, Kazuo; La Vecchia, Carlo

    2012-03-01

    Folate consumption is inversely associated with the risk of oral and pharyngeal cancer (OPC) and potentially interacts with alcohol drinking in the risk of OPC. Aldehyde dehydrogenase 2 (ALDH2) gene polymorphism is known to interact with alcohol consumption. The aim of this study was to investigate potential interaction between folate, alcohol drinking, and ALDH2 polymorphism in the risk of OPC in a Japanese population. The study group comprised 409 head and neck cancer cases and 1227 age-matched and sex-matched noncancer controls; of these, 251 cases and 759 controls were evaluated for ALDH rs671 polymorphism. Associations were assessed by odds ratios and 95% confidence intervals in multiple logistic regression models. We observed an inverse association between folate consumption and OPC risk. The odds ratio for high folate intake was 0.53 (95% confidence interval: 0.36-0.77) relative to low intake (P trend=0.003). This association was consistent across strata of sex, age, smoking, and ALDH2 genotypes. Interaction between folate consumption, drinking, and ALDH2 genotype was remarkable (three-way interaction, P<0.001). We observed significant interaction among folate, drinking, and ALDH2 genotype in the Japanese population.

  13. Inhibition of etoposide-induced DNA damage and cytotoxicity in L1210 cells by dehydrogenase inhibitors and other agents.

    PubMed

    Wozniak, A J; Glisson, B S; Hande, K R; Ross, W E

    1984-02-01

    The mechanism of action of 4'-demethylepipodophyllotoxin-9-(4,6-O-ethylidene-beta-D-glucopyra noside) (VP-16), an important antitumor agent, is unclear. There is evidence that DNA may be the target of action because VP-16 causes single-strand and double-strand breaks in DNA and produces cytotoxicity over a similar dose range. We have hypothesized that an enzyme system, such as dehydrogenase, catalyzes an oxidation-reduction reaction involving the pendant phenolic group which forms an active metabolite that causes the DNA damage and cytotoxicity. To test our hypothesis, we investigated the effect of disulfiram, an aldehyde dehydrogenase inhibitor, and its metabolite, diethyldithiocarbamate, on VP-16-induced DNA damage in L1210 cells. Using the alkaline elution technique to assay DNA damage, we found that disulfiram and diethyldithiocerbamate inhibited VP-16-induced single-strand breaks. Both compounds were also capable of significantly reducing VP-16-induced cytotoxicity. Oxalic acid, pyrophosphate, and malonic acid, competitive inhibitors of succinate dehydrogenase, and the naturally occurring dehydrogenase substrates, succinic acid, beta-glycerophosphate, and isocitric acid, also blocked the effects of VP-16. Free-radical scavengers were also studied. While sodium benzoate was particularly effective in preventing drug-induced DNA damage and cytotoxicity, a number of other scavengers were not. Our data are consistent with the hypothesis that VP-16 is activated by an enzyme such as a dehydrogenase which transforms it into an active intermediate resulting in DNA damage and, consequently, cell death.

  14. The elementary reactions of the pig heart pyruvate dehydrogenase complex. A study of the inhibition by phosphorylation.

    PubMed

    Walsh, D A; Cooper, R H; Denton, R M; Bridges, B J; Randle, P J

    1976-07-01

    1. A method was devised for preparing pig heart pyruvate dehydrogenase free of thiamin pyrophosphate (TPP), permitting studies of the binding of [35S]TPP to pyruvate dehydrogenase and pyruvate dehydrogenase phosphate. The Kd of TPP for pyruvate dehydrogenase was in the range 6.2-8.2 muM, whereas that for pyruvate dehydrogenase phosphate was approximately 15 muM; both forms of the complex contained about the same total number of binding sites (500 pmol/unit of enzyme). EDTA completely inhibited binding of TPP; sodium pyrophosphate, adenylyl imidodiphosphate and GTP, which are inhibitors (competitive with TPP) of the overall pyruvate dehydrogenase reaction, did not appreciably affect TPP binding. 2. Initial-velocity patterns of the overall pyruvate dehydrogenase reaction obtained with varying TPP, CoA and NAD+ concentrations at a fixed pyruvate concentration were consistent with a sequential three-site Ping Pong mechanism; in the presence of oxaloacetate and citrate synthase to remove acetyl-CoA (an inhibitor of the overall reaction) the values of Km for NAD+ and CoA were 53+/- 5 muM and 1.9+/-0.2 muM respectively. Initial-velocity patterns observed with varying TPP concentrations at various fixed concentrations of pyruvate were indicative of either a compulsory order of addition of substrates to form a ternary complex (pyruvate-Enz-TPP) or a random-sequence mechanism in which interconversion of ternary intermediates is rate-limiting; values of Km for pyruvate and TPP were 25+/-4 muM and 50+/-10 nM respectively. The Kia-TPP (the dissociation constant for Enz-TPP complex calculated from kinetic plots) was close to the value of Kd-TPP (determined by direct binding studies). 3. Inhibition of the overall pyruvate dehydrogenase reaction by pyrophosphate was mixed non-competitive versus pyruvate and competitive versus TPP; however, pyrophosphate did not alter the calculated value for Kia-TPP, consistent with the lack of effect of pyrophosphate on the Kd for TPP. 4

  15. Preparative isolation and analysis of alcohol dehydrogenase inhibitors from Glycyrrhiza uralensis root using ultrafiltration combined with high-performance liquid chromatography and high-speed countercurrent chromatography.

    PubMed

    Chen, Miao; Liu, Liangliang; Chen, Xiaoqing

    2014-07-01

    A simple, rapid, and effective assay based on ultrafiltration combined with high-performance liquid chromatography and high-speed countercurrent chromatography was developed for screening and purifying alcohol dehydrogenase inhibitors from Glycyrrhiza uralensis root extract. Experiments were carried out to optimize binding conditions including alcohol dehydrogenase concentration, incubation time, temperature, and pH. By comparing the chromatograms, three compounds were found possessing alcohol dehydrogenase binding activity in Glycyrrhiza uralensis root. Under the target-guidance of ultrafiltration combined with the high-performance liquid chromatography experiment, liquiritin (1), isoliquiritin (2), and liquiritigenin (3) were separated by high-speed countercurrent chromatography using ethyl acetate/methanol/water (5:1:4) as the solvent system. The alcohol dehydrogenase inhibitory activities of these three isolated compounds were assessed; compound 2 showed strongest inhibitory activity with an IC50 of 8.95 μM. The results of the present study indicated that the combinative method using ultrafiltration, high-performance liquid chromatography and high-speed countercurrent chromatography could be widely applied for the rapid screening and isolation of enzyme inhibitors from complex mixtures.

  16. Characterization of new medium-chain alcohol dehydrogenases adds resolution to duplications of the class I/III and the sub-class I genes.

    PubMed

    Cederlund, Ella; Hedlund, Joel; Hjelmqvist, Lars; Jonsson, Andreas; Shafqat, Jawed; Norin, Annika; Keung, Wing-Ming; Persson, Bengt; Jörnvall, Hans

    2011-05-30

    Four additional variants of alcohol and aldehyde dehydrogenases have been purified and functionally characterized, and their primary structures have been determined. The results allow conclusions about the structural and evolutionary relationships within the large family of MDR alcohol dehydrogenases from characterizations of the pigeon (Columba livia) and dogfish (Scyliorhinus canicula) major liver alcohol dehydrogenases. The pigeon enzyme turns out to be of class I type and the dogfish enzyme of class III type. This result gives a third type of evidence, based on purifications and enzyme characterization in lower vertebrates, that the classical liver alcohol dehydrogenase originated by a gene duplication early in the evolution of vertebrates. It is discernable as the major liver form at about the level in-between cartilaginous and osseous fish. The results also show early divergence within the avian orders. Structures were determined by Edman degradations, making it appropriate to acknowledge the methodological contributions of Pehr Edman during the 65 years since his thesis at Karolinska Institutet, where also the present analyses were performed.

  17. Atomic-Resolution Structures of Horse Liver Alcohol Dehydrogenase with NAD[superscript +] and Fluoroalcohols Define Strained Michaelis Complexes

    SciTech Connect

    Plapp, Bryce V.; Ramaswamy, S.

    2013-01-16

    Structures of horse liver alcohol dehydrogenase complexed with NAD{sup +} and unreactive substrate analogues, 2,2,2-trifluoroethanol or 2,3,4,5,6-pentafluorobenzyl alcohol, were determined at 100 K at 1.12 or 1.14 {angstrom} resolution, providing estimates of atomic positions with overall errors of 0.02 {angstrom}, the geometry of ligand binding, descriptions of alternative conformations of amino acid residues and waters, and evidence of a strained nicotinamide ring. The four independent subunits from the two homodimeric structures differ only slightly in the peptide backbone conformation. Alternative conformations for amino acid side chains were identified for 50 of the 748 residues in each complex, and Leu-57 and Leu-116 adopt different conformations to accommodate the different alcohols at the active site. Each fluoroalcohol occupies one position, and the fluorines of the alcohols are well-resolved. These structures closely resemble the expected Michaelis complexes with the pro-R hydrogens of the methylene carbons of the alcohols directed toward the re face of C4N of the nicotinamide rings with a C-C distance of 3.40 {angstrom}. The oxygens of the alcohols are ligated to the catalytic zinc at a distance expected for a zinc alkoxide (1.96 {angstrom}) and participate in a low-barrier hydrogen bond (2.52 {angstrom}) with the hydroxyl group of Ser-48 in a proton relay system. As determined by X-ray refinement with no restraints on bond distances and planarity, the nicotinamide rings in the two complexes are slightly puckered (quasi-boat conformation, with torsion angles of 5.9{sup o} for C4N and 4.8{sup o} for N1N relative to the plane of the other atoms) and have bond distances that are somewhat different compared to those found for NAD(P){sup +}. It appears that the nicotinamide ring is strained toward the transition state on the path to alcohol oxidation.

  18. Structure-based mutational studies of substrate inhibition of betaine aldehyde dehydrogenase BetB from Staphylococcus aureus.

    PubMed

    Chen, Chao; Joo, Jeong Chan; Brown, Greg; Stolnikova, Ekaterina; Halavaty, Andrei S; Savchenko, Alexei; Anderson, Wayne F; Yakunin, Alexander F

    2014-07-01

    Inhibition of enzyme activity by high concentrations of substrate and/or cofactor is a general phenomenon demonstrated in many enzymes, including aldehyde dehydrogenases. Here we show that the uncharacterized protein BetB (SA2613) from Staphylococcus aureus is a highly specific betaine aldehyde dehydrogenase, which exhibits substrate inhibition at concentrations of betaine aldehyde as low as 0.15 mM. In contrast, the aldehyde dehydrogenase YdcW from Escherichia coli, which is also active against betaine aldehyde, shows no inhibition by this substrate. Using the crystal structures of BetB and YdcW, we performed a structure-based mutational analysis of BetB and introduced the YdcW residues into the BetB active site. From a total of 32 mutations, those in five residues located in the substrate binding pocket (Val288, Ser290, His448, Tyr450, and Trp456) greatly reduced the substrate inhibition of BetB, whereas the double mutant protein H448F/Y450L demonstrated a complete loss of substrate inhibition. Substrate inhibition was also reduced by mutations of the semiconserved Gly234 (to Ser, Thr, or Ala) located in the BetB NAD(+) binding site, suggesting some cooperativity between the cofactor and substrate binding sites. Substrate docking analysis of the BetB and YdcW active sites revealed that the wild-type BetB can bind betaine aldehyde in both productive and nonproductive conformations, whereas only the productive binding mode can be modeled in the active sites of YdcW and the BetB mutant proteins with reduced substrate inhibition. Thus, our results suggest that the molecular mechanism of substrate inhibition of BetB is associated with the nonproductive binding of betaine aldehyde.

  19. Itaconate Links Inhibition of Succinate Dehydrogenase with Macrophage Metabolic Remodeling and Regulation of Inflammation.

    PubMed

    Lampropoulou, Vicky; Sergushichev, Alexey; Bambouskova, Monika; Nair, Sharmila; Vincent, Emma E; Loginicheva, Ekaterina; Cervantes-Barragan, Luisa; Ma, Xiucui; Huang, Stanley Ching-Cheng; Griss, Takla; Weinheimer, Carla J; Khader, Shabaana; Randolph, Gwendalyn J; Pearce, Edward J; Jones, Russell G; Diwan, Abhinav; Diamond, Michael S; Artyomov, Maxim N

    2016-07-12

    Remodeling of the tricarboxylic acid (TCA) cycle is a metabolic adaptation accompanying inflammatory macrophage activation. During this process, endogenous metabolites can adopt regulatory roles that govern specific aspects of inflammatory response, as recently shown for succinate, which regulates the pro-inflammatory IL-1β-HIF-1α axis. Itaconate is one of the most highly induced metabolites in activated macrophages, yet its functional significance remains unknown. Here, we show that itaconate modulates macrophage metabolism and effector functions by inhibiting succinate dehydrogenase-mediated oxidation of succinate. Through this action, itaconate exerts anti-inflammatory effects when administered in vitro and in vivo during macrophage activation and ischemia-reperfusion injury. Using newly generated Irg1(-/-) mice, which lack the ability to produce itaconate, we show that endogenous itaconate regulates succinate levels and function, mitochondrial respiration, and inflammatory cytokine production during macrophage activation. These studies highlight itaconate as a major physiological regulator of the global metabolic rewiring and effector functions of inflammatory macrophages.

  20. Inhibition of the Aldehyde Dehydrogenase 1/2 Family by Psoralen and Coumarin Derivatives.

    PubMed

    Buchman, Cameron D; Hurley, Thomas D

    2017-03-23

    Aldehyde dehydrogenase 2 (ALDH2), one of 19 ALDH superfamily members, catalyzes the NAD(+)-dependent oxidation of aldehydes to their respective carboxylic acids. In this study, we further characterized the inhibition of four psoralen and coumarin derivatives toward ALDH2 and compared them to the ALDH2 inhibitor daidzin for selectivity against five ALDH1/2 isoenzymes. Compound 2 (Ki = 19 nM) binds within the aldehyde-binding site of the free enzyme species of ALDH2. Thirty-three structural analogs were examined to develop a stronger SAR profile. Seven compounds maintained or improved upon the selectivity toward one of the five ALDH1/2 isoenzymes, including compound 36, a selective inhibitor for ALDH2 (Ki = 2.4 μM), and compound 32, which was 10-fold selective for ALDH1A1 (Ki = 1.2 μM) versus ALDH1A2. Further medicinal chemistry on the compounds' basic scaffold could enhance the potency and selectivity for ALDH1A1 or ALDH2 and generate chemical probes to examine the unique and overlapping functions of the ALDH1/2 isoenzymes.

  1. Inhibition of succinate dehydrogenase by malonic acid produces an "excitotoxic" lesion in rat striatum.

    PubMed

    Greene, J G; Porter, R H; Eller, R V; Greenamyre, J T

    1993-09-01

    Excitotoxicity and defects in neuronal energy metabolism have both been implicated in the pathogenesis of neurodegenerative disease. These two mechanisms may be linked through the NMDA receptor, activation of which is dependent on neuronal membrane potential. Because the ability to maintain membrane potential is dependent on neuronal energy metabolism, bioenergetic defects may affect NMDA receptor-mediated excitotoxicity. We now report that reversible inhibition of succinate dehydrogenase (SDH), an enzyme central to both the tricarboxylic acid cycle and the electron transport chain, produces an "excitotoxic" lesion in rat striatum that can be blocked by the NMDA antagonist MK-801. Male Sprague-Dawley rats received intrastriatal stereotaxic injections of the SDH inhibitor malonic acid (1 or 2 mumol) in combination with intraperitoneal injections of vehicle or MK-801 (5 mg/kg) 30 min before and 210 min after malonic acid. Animals were killed 72 h after surgery, and brains were processed for histology, cytochrome oxidase activity, and [3H]MK-801 and [3H]AMPA autoradiography. The higher dose of malonic acid (2 mumol) produced large lesions that were markedly attenuated by treatment with MK-801 (28.1 +/- 3.6 vs. 4.7 +/- 2.6 mm3; p < 0.001). [3H]MK-801 and [3H]AMPA binding were reduced in the lesions by 60 and 63%, respectively. One micromole of malonic acid produced smaller lesions that were almost completely blocked by MK-801 treatment (9.6 +/- 1.3 vs. 0.06 +/- 0.04 mm3; p < 0.0001). The toxic effects of malonic acid were due specifically to inhibition of SDH inasmuch as coinjection of a threefold excess of succinate with the malonic acid blocked the striatal lesions (p < 0.002).(ABSTRACT TRUNCATED AT 250 WORDS)

  2. Antisense inhibition of mitochondrial pyruvate dehydrogenase E1alpha subunit in anther tapetum causes male sterility.

    PubMed

    Yui, Rika; Iketani, Satoru; Mikami, Tetsuo; Kubo, Tomohiko

    2003-04-01

    We hypothesized that cytoplasmic male sterility (CMS) in sugar beet may be the consequence of mitochondrial dysfunctions affecting normal anther development. To test the hypothesis, we attempted to mimic the sugar beet CMS phenotype by inhibiting the expression of mitochondrial pyruvate dehydrogenase (PDH), which is essential for the operation of the tricarboxylic acid (TCA) cycle. Screening with a cDNA library of sugar beet flower buds allowed the identification of two PDH E1alpha subunit genes (bvPDH_E1alpha-1 and bvPDH_E1alpha-2). bvPDH_E1alpha-1 was found to be highly expressed in tap roots, whereas bvPDH_E1alpha-2 was expressed most abundantly in flower buds. Green fluorescent protein (GFP) fusion of bvPDH_E1alpha revealed mitochondrial targeting properties. A 300-bp bvPDH_E1alpha-1 cDNA sequence (from +620 to +926) was connected to a tapetum-specific promoter in the antisense orientation and then introduced into tobacco. Antisense expression of bvPDH_E1alpha-1 resulted in conspicuously decreased endogenous bvPDH_E1alpha-1 transcripts and male sterility. The tapetum in the male-sterile anthers showed swelling or abnormal vacuolation. It is also worth noting that in the sterile anthers, cell organelles, such as elaioplasts, tapetosomes and orbicules were poorly formed and microspores exhibited aberrant exine development. These features are shared by sugar beet CMS. The results thus clearly indicate that inhibition of PDH activity in anther tapetum is sufficient to cause male sterility, a phenocopy of the sugar beet CMS.

  3. The Analysis of Polymorphism of Alcohol Dehydrogenase 3 (ADH3) Gene and Influence of Liver Function Status in Indonesia.

    PubMed

    Suhartini; Mustofa; Nurhantari, Yudha; Rianto, Bambang Udji Djoko

    2017-01-31

    Indonesian culture actually has no historical record of behaviors in consuming alcohol, but there are many recent reports of alcohol abuse among Asian people involving their traditional drink. In genotype studies, the damage of the liver caused by consuming alcohol is influenced by the presence of the polymorphism enzyme gene. The lack of study regarding such topic is a signal to further investigate ADH3 gene distribution and its effect on liver function status. The total of 197 research subjects of Javanese descent received alcohol dehydrogenase 3 (ADH3) genetic polymorphism and liver status tests in the city of Yogyakarta, Indonesian. An analytical study with a cross-sectional design was then conducted on the subjects, with the resulting isolated DNAs amplified through polymerase chain reaction (PCR). The genotype of ADH3 was determined by means of restriction fragment length polymorphism (RFLP) using Ssp1 restricting enzyme. Liver function status was assessed by measuring serum glutamic oxaloacetic transaminase (SGOT), serum glutamic pyruvate transaminase (SGPT) and gamma glutamyl transferase (GGT) using a photometric system. Gene types of ADH3*1 (2.1%), ADH3*2 (82.7%) and ADH3*1/3*2 (15.2%) on the subjects were concluded, finding that there is no difference between the gender. In conclusion most of the ADH3 gene polymorphism of the subjects were ADH3*2 (82.7%). The influence of genetic polymorphisms on the status of liver function in the subjects showed significant difference according to GGT measurement, but the same cannot be said on the other two values measuring SGOT and SGPT.

  4. Starmerella bombicola influences the metabolism of Saccharomyces cerevisiae at pyruvate decarboxylase and alcohol dehydrogenase level during mixed wine fermentation

    PubMed Central

    2012-01-01

    Background The use of a multistarter fermentation process with Saccharomyces cerevisiae and non-Saccharomyces wine yeasts has been proposed to simulate natural must fermentation and to confer greater complexity and specificity to wine. In this context, the combined use of S. cerevisiae and immobilized Starmerella bombicola cells (formerly Candida stellata) was assayed to enhance glycerol concentration, reduce ethanol content and to improve the analytical composition of wine. In order to investigate yeast metabolic interaction during controlled mixed fermentation and to evaluate the influence of S. bombicola on S. cerevisiae, the gene expression and enzymatic activity of two key enzymes of the alcoholic fermentation pathway such as pyruvate decarboxylase (Pdc1) and alcohol dehydrogenase (Adh1) were studied. Results The presence of S. bombicola immobilized cells in a mixed fermentation trial confirmed an increase in fermentation rate, a combined consumption of glucose and fructose, an increase in glycerol and a reduction in the production of ethanol as well as a modification in the fermentation of by products. The alcoholic fermentation of S. cerevisiae was also influenced by S. bombicola immobilized cells. Indeed, Pdc1 activity in mixed fermentation was lower than that exhibited in pure culture while Adh1 activity showed an opposite behavior. The expression of both PDC1 and ADH1 genes was highly induced at the initial phase of fermentation. The expression level of PDC1 at the end of fermentation was much higher in pure culture while ADH1 level was similar in both pure and mixed fermentations. Conclusion In mixed fermentation, S. bombicola immobilized cells greatly affected the fermentation behavior of S. cerevisiae and the analytical composition of wine. The influence of S. bombicola on S. cerevisiae was not limited to a simple additive contribution. Indeed, its presence caused metabolic modifications during S. cerevisiae fermentation causing variation in the gene

  5. The two-step electrochemical oxidation of alcohols using a novel recombinant PQQ alcohol dehydrogenase as a catalyst for a bioanode.

    PubMed

    Takeda, Kouta; Matsumura, Hirotoshi; Ishida, Takuya; Samejima, Masahiro; Igarashi, Kiyohiko; Nakamura, Nobuhumi; Ohno, Hiroyuki

    2013-12-01

    A bioanode has been developed based on the oxidation of ethanol by the recombinant pyrroloquinoline quinone (PQQ) dependent alcohol dehydrogenase from Pseudomonas putidaKT2440 heterologously expressed in Pichia pastoris. The apo form of the recombinant protein (PpADH) was purified and displayed catalytic activity for binding PQQ in the presence of Ca(2+). PpADH exhibited broad substrate specificity towards various alcohols and aldehydes. The Km values for the aldehydes of PpADH were increased compared to those for the alcohols, whereas the kcat values were unaltered. For instance, the Km values at T=298.15K (25 °C) for ethanol and acetaldehyde were 0.21 (± 0.02)mM and 5.8 (± 0.60)mM, respectively. The kcat values for ethanol and acetaldehyde were 24.8 (± 1.2) s(-1) and 31.1 (± 1.2) s(-1), respectively. The aminoferrocene was used as an electron transfer mediator between PpADH and the electrode during electrochemical experiments. The catalytic currents for the oxidation of alcohol and acetaldehyde by PpADH were also observed in this system. The electric charge for the oxidation of ethanol (Q = 2.09 × 10(-3) · C) was increased two-fold compared to that for the oxidation of acetaldehyde (Q = 0.95 × 10(-3) · C), as determined by chronoamperometric measurements. Thus, we have electrochemically demonstrated the two-step oxidation of ethanol to acetate using only PpADH.

  6. Expression of cinnamyl alcohol dehydrogenases and their putative homologues during Arabidopsis thaliana growth and development: lessons for database annotations?

    PubMed

    Kim, Sung-Jin; Kim, Kye-Won; Cho, Man-Ho; Franceschi, Vincent R; Davin, Laurence B; Lewis, Norman G

    2007-07-01

    A major goal currently in Arabidopsis research is determination of the (biochemical) function of each of its approximately 27,000 genes. To date, however, 12% of its genes actually have known biochemical roles. In this study, we considered it instructive to identify the gene expression patterns of nine (so-called AtCAD1-9) of 17 genes originally annotated by The Arabidopsis Information Resource (TAIR) as cinnamyl alcohol dehydrogenase (CAD, EC 1.1.1.195) homologues [see Costa, M.A., Collins, R.E., Anterola, A.M., Cochrane, F.C., Davin, L.B., Lewis N.G., 2003. An in silico assessment of gene function and organization of the phenylpropanoid pathway metabolic networks in Arabidopsis thaliana and limitations thereof. Phytochemistry 64, 1097-1112.]. In agreement with our biochemical studies in vitro [Kim, S.-J., Kim, M.-R., Bedgar, D.L., Moinuddin, S.G.A., Cardenas, C.L., Davin, L.B., Kang, C.-H., Lewis, N.G., 2004. Functional reclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis. Proc. Natl. Acad. Sci. USA 101, 1455-1460.], and analysis of a double mutant [Sibout, R., Eudes, A., Mouille, G., Pollet, B., Lapierre, C., Jouanin, L., Séguin A., 2005. Cinnamyl Alcohol Dehydrogenase-C and -D are the primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis. Plant Cell 17, 2059-2076.], both AtCAD5 (At4g34230) and AtCAD4 (At3g19450) were found to have expression patterns consistent with development/formation of different forms of the lignified vascular apparatus, e.g. lignifying stem tissues, bases of trichomes, hydathodes, abscission zones of siliques, etc. Expression was also observed in various non-lignifying zones (e.g. root caps) indicative of, perhaps, a role in plant defense. In addition, expression patterns of the four CAD-like homologues were investigated, i.e. AtCAD2 (At2g21730), AtCAD3 (At2g21890), AtCAD7 (At4g37980) and AtCAD8 (At4g37990), each of which previously had been demonstrated to have low CAD

  7. Purification and characterization of an NADH-dependent alcohol dehydrogenase from Candida maris for the synthesis of optically active 1-(pyridyl)ethanol derivatives.

    PubMed

    Kawano, Shigeru; Yano, Miho; Hasegawa, Junzo; Yasohara, Yoshihiko

    2011-01-01

    A novel (R)-specific alcohol dehydrogenase (AFPDH) produced by Candida maris IFO10003 was purified to homogeneity by ammonium sulfate fractionation, DEAE-Toyopearl, and Phenyl-Toyopearl, and characterized. The relative molecular mass of the native enzyme was found to be 59,900 by gel filtration, and that of the subunit was estimated to be 28,900 on SDS-polyacrylamide gel electrophoresis. These results suggest that the enzyme is a homodimer. It required NADH as a cofactor and reduced various kinds of carbonyl compounds, including ketones and aldehydes. AFPDH reduced acetylpyridine derivatives, β-keto esters, and some ketone compounds with high enantioselectivity. This is the first report of an NADH-dependent, highly enantioselective (R)-specific alcohol dehydrogenase isolated from a yeast. AFPDH is a very useful enzyme for the preparation of various kinds of chiral alcohols.

  8. Intracerebral Stem Cell Administration Inhibits Relapse-like Alcohol Drinking in Rats.

    PubMed

    Israel, Yedy; Ezquer, Fernando; Quintanilla, María Elena; Morales, Paola; Ezquer, Marcelo; Herrera-Marschitz, Mario

    2017-01-01

    Study describes the blockade of relapse-like alcohol drinking by mesenchymal stem cells (MSCs). High alcohol-intake bred rats consumed alcohol for 3 months and were subjected to repeated alcohol deprivations for 7-14 days, followed by alcohol reaccess. Upon reaccess, animals consumed 2.2 g alcohol/kg in 60 minutes. A single intra-cerebroventricular MSC administration inhibited relapse-like drinking up to 80-85% for 40 days (P < 0.001). An alcohol-use-disorder was prevented.

  9. Stability engineering of the Geobacillus stearothermophilus alcohol dehydrogenase and application for the synthesis of a polyamide 12 precursor.

    PubMed

    Kirmair, Ludwig; Seiler, Daniel Leonard; Skerra, Arne

    2015-12-01

    The thermostable NAD(+)-dependent alcohol dehydrogenase from Geobacillus stearothermophilus (BsADH) was exploited with regard to the biocatalytic synthesis of ω-oxo lauric acid methyl ester (OLAMe), a key intermediate for biobased polyamide 12 production, from the corresponding long-chain alcohol. Recombinant BsADH was produced in Escherichia coli as a homogeneous tetrameric enzyme and showed high activity towards the industrially relevant substrate ω-hydroxy lauric acid methyl ester (HLAMe) with K M = 86 μM and 44 U mg(-1). The equilibrium constant for HLAMe oxidation to the aldehyde (OLAMe) with NAD(+) was determined as 2.16 × 10(-3) from the kinetic parameters of the BsADH-catalyzed forward and reverse reactions. Since BsADH displayed limited stability under oxidizing conditions, the predominant oxidation-prone residue Cys257 was mutated to Leu based on sequence homology with related enzymes and computational simulation. This substitution resulted in an improved BsADH variant exhibiting prolonged stability and an elevated inactivation temperature. Semi-preparative biocatalysis at 60 °C using the stabilized enzyme, employing butyraldehyde for in situ cofactor regeneration with only catalytic amounts of NAD(+), yielded up to 23 % conversion of HLAMe to OLAMe after 30 min. In contrast to other oxidoreductases, no overoxidation to the dodecanoic diacid monomethyl ester was detected. Thus, the mutated BsADH offers a promising biocatalyst for the selective oxidation of fatty alcohols to yield intermediates for industrial polymer production.

  10. Effects of cavities at the nicotinamide binding site of liver alcohol dehydrogenase on structure, dynamics and catalysis.

    PubMed

    Yahashiri, Atsushi; Rubach, Jon K; Plapp, Bryce V

    2014-02-11

    A role for protein dynamics in enzymatic catalysis of hydrogen transfer has received substantial scientific support, but the connections between protein structure and catalysis remain to be established. Valine residues 203 and 207 are at the binding site for the nicotinamide ring of the coenzyme in liver alcohol dehydrogenase and have been suggested to facilitate catalysis with "protein-promoting vibrations" (PPV). We find that the V207A substitution has small effects on steady-state kinetic constants and the rate of hydrogen transfer; the introduced cavity is empty and is tolerated with minimal effects on structure (determined at 1.2 Å for the complex with NAD(+) and 2,3,4,5,6-pentafluorobenzyl alcohol). Thus, no evidence is found to support a role for Val-207 in the dynamics of catalysis. The protein structures and ligand geometries (including donor-acceptor distances) in the V203A enzyme complexed with NAD(+) and 2,3,4,5,6-pentafluorobenzyl alcohol or 2,2,2-trifluoroethanol (determined at 1.1 Å) are very similar to those for the wild-type enzyme, except that the introduced cavity accommodates a new water molecule that contacts the nicotinamide ring. The structures of the V203A enzyme complexes suggest, in contrast to previous studies, that the diminished tunneling and decreased rate of hydride transfer (16-fold, relative to that of the wild-type enzyme) are not due to differences in ground-state ligand geometries. The V203A substitution may alter the PPV and the reorganization energy for hydrogen transfer, but the protein scaffold and equilibrium thermal motions within the Michaelis complex may be more significant for enzyme catalysis.

  11. Ciprofloxacin Therapy Results in Mitigation of ATP Loss after Irradiation Combined with Wound Trauma: Preservation of Pyruvate Dehydrogenase and Inhibition of Pyruvate Dehydrogenase Kinase 1.

    PubMed

    Swift, Joshua M; Smith, Joan T; Kiang, Juliann G

    2015-06-01

    Ionizing radiation exposure combined with wound injury increases animal mortalities than ionizing radiation exposure alone. Ciprofloxacin (CIP) is in the fluroquinolone family of synthetic antibiotic that are available from the strategic national stockpile for emergency use and is known to inhibit bacterial sepsis. The purpose of this study was to evaluate the efficacy of ciprofloxacin as a countermeasure to combined injury mortality and determine the signaling proteins involved in energy machinery. B6D2F1/J female mice were randomly assigned to receive either 9.75 Gy irradiation with Co-60 gamma rays followed by skin wounding (combined injury; CI) or sham procedure (sham). Either ciprofloxacin (90 mg/kg/day) or vehicle (VEH) (water) was administered orally to these mice 2 h after wounding and thereafter daily for 10 days. Determination of tissue adenosine triphosphate (ATP) was conducted, and immunoblotting for signaling proteins involved in ATP machinery was performed. Combined injury resulted in 60% survival after 10 days compared to 100% survival in the sham group. Furthermore, combined injury caused significant reductions of ATP concentrations in ileum, pancreas, brain, spleen, kidney and lung (-25% to -95%) compared to the sham group. Ciprofloxacin administration after combined injury resulted in 100% survival and inhibited reductions in ileum and kidney ATP production. Ileum protein levels of heat-shock protein 70 kDa (HSP-70, a chaperone protein involved in ATP synthesis) and pyruvate dehydrogenase (PDH, an enzyme complex crucial to conversion of pyruvate to acetyl CoA for entrance into TCA cycle) were significantly lower in the CI group (vs. sham group). Using immunoprecipitation and immunoblotting, HSP-70-PDH complex was found to be present in the ileum tissue of CI mice treated with ciprofloxacin. Furthermore, phosphorylation of serine residues of PDH resulting in inactivating PDH enzymatic activity, which occurred after combined injury, was inhibited

  12. Purification and characterization of an anti-Prelog alcohol dehydrogenase from Oenococcus oeni that reduces 2-octanone to (R)-2-octanol.

    PubMed

    Meng, Fantao; Xu, Yan

    2010-04-01

    An anti-Prelog alcohol dehydrogenase from Oenococcus oeni that reduces 2-octanone to (R)-2-octanol was purified by 26-fold to homogeneity. The enzyme had a homodimeric structure consisting of 49 kDa subunits, required NADPH, but not NADH, as a cofactor and was a Zn-independent short-chain dehydrogenase. Aliphatic methyl ketones (chain length > or =6 carbon atoms) and aromatic methyl ketones were the preferred substrates for the enzyme, the best being 2-octanone. Maximum enzyme activity with 2-octanone was at 45 degrees C and at pH 8.0.

  13. Inhibition of 3beta- and 17beta-hydroxysteroid dehydrogenase activities in rat Leydig cells by perfluorooctane acid.

    PubMed

    Zhao, Binghai; Chu, Yanhui; Hardy, Dianne O; Li, Xiao-kun; Ge, Ren-Shan

    2010-01-01

    Perfluorooctane acid (PFOA) is classified as a persistent organic pollutant and as an endocrine disruptor. The mechanism by which PFOA causes reduced testosterone production in males is not known. We tested our hypothesis that PFOA interferes with Leydig cell steroidogenic enzymes by measuring its effect on 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and 17beta-hydroxysteroid dehydrogenase 3 (17beta-HSD3) activities in rat testis microsomes and Leydig cells. The IC(50)s of PFOA and mode of inhibition were assayed. PFOA inhibited microsomal 3beta-HSD with an IC(50) of 53.2+/-25.9 microM and 17beta-HSD3 with an IC(50) 17.7+/-6.8 microM. PFOA inhibited intact Leydig cell 3beta-HSD with an IC(50) of 146.1+/-0.9 microM and 17beta-HSD3 with an IC(50) of 194.8+/-1.0 microM. The inhibitions of 3beta-HSD and 17beta-HSD3 by PFOA were competitive for the substrates. In conclusion, PFOA inhibits 3beta-HSD and 17beta-HSD3 in rat Leydig cells.

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

    SciTech Connect

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

    2009-08-14

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

  15. Nitric oxide inhibits succinate dehydrogenase-driven oxygen consumption in potato tuber mitochondria in an oxygen tension-independent manner.

    PubMed

    Simonin, Vagner; Galina, Antonio

    2013-01-01

    NO (nitric oxide) is described as an inhibitor of plant and mammalian respiratory chains owing to its high affinity for COX (cytochrome c oxidase), which hinders the reduction of oxygen to water. In the present study we show that in plant mitochondria NO may interfere with other respiratory complexes as well. We analysed oxygen consumption supported by complex I and/or complex II and/or external NADH dehydrogenase in Percoll-isolated potato tuber (Solanum tuberosum) mitochondria. When mitochondrial respiration was stimulated by succinate, adding the NO donors SNAP (S-nitroso-N-acetyl-DL-penicillamine) or DETA-NONOate caused a 70% reduction in oxygen consumption rate in state 3 (stimulated with 1 mM of ADP). This inhibition was followed by a significant increase in the Km value of SDH (succinate dehydrogenase) for succinate (Km of 0.77±0.19 to 34.3±5.9 mM, in the presence of NO). When mitochondrial respiration was stimulated by external NADH dehydrogenase or complex I, NO had no effect on respiration. NO itself and DETA-NONOate had similar effects to SNAP. No significant inhibition of respiration was observed in the absence of ADP. More importantly, SNAP inhibited PTM (potato tuber mitochondria) respiration independently of oxygen tensions, indicating a different kinetic mechanism from that observed in mammalian mitochondria. We also observed, in an FAD reduction assay, that SNAP blocked the intrinsic SDH electron flow in much the same way as TTFA (thenoyltrifluoroacetone), a non-competitive SDH inhibitor. We suggest that NO inhibits SDH in its ubiquinone site or its Fe-S centres. These data indicate that SDH has an alternative site of NO action in plant mitochondria.

  16. Substitutions in a flexible loop of horse liver alcohol dehydrogenase hinder the conformational change and unmask hydrogen transfer.

    PubMed

    Ramaswamy, S; Park, D H; Plapp, B V

    1999-10-19

    When horse liver alcohol dehydrogenase binds coenzyme, a rotation of about 10 degrees brings the catalytic domain closer to the coenzyme binding domain and closes the active site cleft. The conformational change requires that a flexible loop containing residues 293-298 in the coenzyme binding domain rearranges so that the coenzyme and some amino acid residues from the catalytic domain can be accommodated. The change appears to control the rate of dissociation of the coenzyme and to be necessary for installation of the proton relay system. In this study, directed mutagenesis produced the activated Gly293Ala/Pro295Thr enzyme. X-ray crystallography shows that the conformations of both free and complexed forms of the mutated enzyme and wild-type apoenzyme are very similar. Binding of NAD(+) and 2,2, 2-trifluoroethanol do not cause the conformational change, but the nicotinamide ribose moiety and alcohol are not in a fixed position. Although the Gly293Ala and Pro295Thr substitutions do not disturb the apoenzyme structure, molecular modeling shows that the new side chains cannot be accommodated in the closed native holoenzyme complex without steric alterations. The mutated enzyme may be active in the "open" conformation. The turnover numbers with ethanol and acetaldehyde increase 1.5- and 5.5-fold, respectively, and dissociation constants for coenzymes and other kinetic constants increase 40-2,000-fold compared to those of the native enzyme. Substrate deuterium isotope effects on the steady state V or V/K(m) parameters of 4-6 with ethanol or benzyl alcohol indicate that hydrogen transfer is a major rate-limiting step in catalysis. Steady state oxidation of benzyl alcohol is most rapid above a pK of about 9 for V and V/K(m) and is 2-fold faster in D(2)O than in H(2)O. The results are consistent with hydride transfer from a ground state zinc alkoxide that forms a low-barrier hydrogen bond with the hydroxyl group of Ser48.

  17. Electron transfer from NADH bound to horse liver alcohol dehydrogenase (NAD+ dependent dehydrogenase): visualisation of the activity in the enzyme crystals and adsorption of formazan derivatives by these crystals.

    PubMed

    Pacaud-Mercier, Karine; Blaghen, Mohamed; Lee, Kang Min; Tritsch, Denis; Biellmann, Jean-François

    2007-02-01

    The crystals of holoenzyme from native and cross-linked alcohol dehydrogenase exhibit electron transfer from NADH to phenazinium methosulfate (PMS), and then to the tetrazolium salt sodium 3,3'-{1-[(phenylamino)carbonyl]-3,4-tetrazolium}-bis(4-methoxy-6-nitro)benzenesulfonate (XXT). The slow dissociation of the cofactor and/or the conformational change associated can now be bypassed. The reduction product, formazan, did not diffuse out of the crystals in buffer and the crystals turned colored. In the presence of dimethyl sulfoxide or dimethoxyethane, the formazan diffused out to the solution. The reaction rates were found to be, respectively, 18% and 15% of the redox reaction rate of ethanol with cinnamaldehyde, close to the activity determined for the enzyme in solution in the presence of dimethoxyethane. The use of system PMS-tetrazolium salt is a useful tool to visualize the activity of dehydrogenases and other electron transferring systems in the crystalline state. The adsorption of formazan by the alcohol dehydrogenase crystals occurs in solution.

  18. The regulation and inhibition of 17beta-hydroxysteroid dehydrogenase in breast cancer.

    PubMed

    Purohit, A; Tutill, H J; Day, J M; Chander, S K; Lawrence, H R; Allan, G M; Fischer, D S; Vicker, N; Newman, S P; Potter, B V L; Reed, M J

    2006-03-27

    17Beta-hydroxysteroid dehydrogenase Type 1 (17beta-HSD1) has a pivotal role in regulating the synthesis of oestradiol (E2) within breast tumours. In whole body studies in postmenopausal women with breast cancer the conversion of oestrone (E1) to E2 (4.4+/-1.1%) was much lower than the inactivation of E2 to E1 (17.3+/-5.0%). In contrast, an examination of in vivo oestrogen metabolism within breast tumours revealed that whereas little metabolism of E2 occurred, E1 was converted to E2 to a much greater extent in malignant (48+/-14%) than in normal (19+/-6%) breast tissue. Findings from these studies originally suggested that oestrogen metabolism within breast tumours may differ from the mainly oxidative direction found in most other body tissues and that the activity of 17beta-HSD1 might be regulated by tumour-derived factors. Several growth factors (e.g. IGF-I, IGF-II) and cytokines (e.g. IL-6, TNFalpha) have now been identified which can markedly stimulate the activity of 17beta-HSD1 and such a mechanism may account for the high concentrations of E2 found in most breast tumours. Cells of the immune system, which can infiltrate breast tumours, are thought to be a major source of the growth factors and cytokines which can modulate 17beta-HSD1 activity. Given the central role that 17beta-HSD1 has in regulating breast tumour E2 concentrations the development of potent inhibitors of this enzyme has recently attracted considerable attention. Our initial studies in this area explored the use of derivatives of E1 as inhibitors, with 2-ethyl- and 2-methoxy E1 being found to inhibit 17beta-HSD1 activity in T-47D breast cancer cells by 96+/-2 and 91+/-1% respectively at 10 microM, but with a lack of specificity. Using the E1 scaffold a number of potent, selective 17beta-HSD1 inhibitors have now been identified including E1- and 2-ethyl-E1 containing a side chain with a m-pyridylmethylamidomethyl functionality extending from the 16beta position of the steroid nucleus. At 10

  19. Aldehyde dehydrogenase activity in Lactococcus chungangensis: Application in cream cheese to reduce aldehyde in alcohol metabolism.

    PubMed

    Konkit, Maytiya; Choi, Woo Jin; Kim, Wonyong

    2016-03-01

    Previous studies have shown that the metabolic capability of colonic microflora may be at least as high as that of the liver or higher than that of the whole human body. Aldehyde dehydrogenase (ALDH) is an enzyme produced by these bacteria that can metabolize acetaldehyde, produce from ethanol to acetate. Lactococcus species, which is commonly used as a starter in dairy products, was recently found to possess the ALDH gene, and the activity of this enzyme was determined. In this study, the ALDH activity of Lactococcus chungangensis CAU 28(T) and 11 other type strains in the genus Lactococcus was studied. Only 5 species, 3 of dairy origin (Lactococcus lactis ssp. lactis KCTC 3769(T), Lactococcus lactis ssp. cremoris KCCM 40699(T), and Lactococcus raffinolactis DSM 20443(T)) and 2 of nondairy origin (Lactococcus fujiensis NJ317(T) and L. chungangensis CAU 28(T)), showed ALDH activity and possessed a gene encoding ALDH. All of these strains were capable of making cream cheese. Among the strains, L. chungangensis produced cream cheese that contained the highest level of ALDH and was found to reduce the level of acetaldehyde in the serum of mice. These results predict a promising role for L. chungangensis CAU28(T) to be used in cheese that can be developed as functional food.

  20. Aldehyde dehydrogenase-2 inhibition blocks remote preconditioning in experimental and human models.

    PubMed

    Contractor, Hussain; Støttrup, Nicolaj B; Cunnington, Colin; Manlhiot, Cedric; Diesch, Jonathan; Ormerod, Julian O M; Jensen, Rebekka; Bøtker, Hans Erik; Redington, Andrew; Schmidt, Michael R; Ashrafian, Houman; Kharbanda, Rajesh K

    2013-05-01

    Mitochondrial aldehyde dehydrogenase-2 (ALDH-2) is involved in preconditioning pathways, but its role in remote ischaemic preconditioning (rIPC) is unknown. We investigated its role in animal and human models of rIPC. (i) In a rabbit model of myocardial infarction, rIPC alone reduced infarct size [69 ± 5.8 % (n = 11) to 40 ± 6.5 % (n = 12), P = 0.019]. However, rIPC protection was lost after pre-treatment with the ALDH-2 inhibitor cyanamide (62 ± 7.6 % controls, n = 10, versus 61 ± 6.9 % rIPC after cyanamide, n = 10, P > 0.05). (ii) In a forearm plethysmography model of endothelial ischaemia-reperfusion injury, 24 individuals of Asian ethnic origin underwent combined rIPC and ischaemia-reperfusion (IR). 11 had wild-type (WT) enzyme and 13 carried the Glu504Lys (ALDH2*2) polymorphism (rendering ALDH-2 functionally inactive). In WT individuals, rIPC protected against impairment of response to acetylcholine (P = 0.9), but rIPC failed to protect carriers of Glu504Lys polymorphism (P = 0.004). (iii) In a second model of endothelial IR injury, 12 individuals participated in a double-blind placebo-controlled crossover study, receiving the ALDH-2 inhibitor disulfiram 600 mg od or placebo for 48 h prior to assessment of flow-mediated dilation (FMD) before and after combined rIPC and IR. With placebo, rIPC was effective with no difference in FMD before and after IR (6.18 ± 1.03 % and 4.76 ± 0.93 % P = 0.1), but disulfiram inhibited rIPC with a reduction in FMD after IR (7.87 ± 1.27 % and 3.05 ± 0.53 %, P = 0.001). This study demonstrates that ALDH-2 is involved in the rIPC pathway in three distinct rabbit and human models. This has potential implications for future clinical studies of remote conditioning.

  1. Cloning and Expression of ntnD, Encoding a Novel NAD(P)+-Independent 4-Nitrobenzyl Alcohol Dehydrogenase from Pseudomonas sp. Strain TW3

    PubMed Central

    James, Keith D.; Hughes, Michelle A.; Williams, Peter A.

    2000-01-01

    Pseudomonas sp. strain TW3 is able to metabolize 4-nitrotoluene to 4-nitrobenzoate and toluene to benzoate aerobically via a route analogous to the upper pathway of the TOL plasmids. We report the cloning and characterization of a benzyl alcohol dehydrogenase gene (ntnD) which encodes the enzyme for the catabolism of 4-nitrobenzyl alcohol and benzyl alcohol to 4-nitrobenzaldehyde and benzaldehyde, respectively. The gene is located downstream of the previously reported ntn gene cluster. NtnD bears no similarity to the analogous TOL plasmid XylB (benzyl alcohol dehydrogenase) protein either in its biochemistry, being NAD(P)+ independent and requiring assay via dye-linked electron transfer, or in its deduced amino acid sequence. It does, however, have significant similarity in its amino acid sequence to other NAD(P)+-independent alcohol dehydrogenases and contains signature patterns characteristic of type III flavin adenine dinucleotide-dependent alcohol oxidases. Reverse transcription-PCR demonstrated that ntnD is transcribed during growth on 4-nitrotoluene, although apparently not as part of the same transcript as the other ntn genes. The substrate specificity of the enzyme expressed from the cloned and overexpressed gene was similar to the activity expressed from strain TW3 grown on 4-nitrotoluene, providing evidence that ntnD is the previously unidentified gene in the pathway of 4-nitrotoluene catabolism. Examination of the 14.8-kb region around the ntn genes suggests that one or more recombination events have been involved in the formation of their current organization. PMID:10809692

  2. Alcohol and aldehyde dehydrogenase from Saccharomyces cerevisiae: specific activity and influence on the production of acetic acid, ethanol and higher alcohols in the first 48 h of fermentation of grape must.

    PubMed

    Millán, C; Mauricio, J C; Ortega, J M

    1990-01-01

    The changes in the specific activity of alcohol dehydrogenase (ADH-I and ADH-II) and aldehyde dehydrogenases [AIDH-NADP+ and AIDH-NAD(P)+] from Saccharomyces cerevisiae during the first 48 h of fermentation of grape must were investigated. The biosynthesis of ADH-I and AIDH-NADP+ took place basically during the adaptation of the yeasts to the must (first 4 h), while that of ADH-II occurred immediately after exponential growth (after 12 h). From the products produced by the yeast, only the specific rate of production of ethanol was found to be directly related to the specific activity of ADH-I.

  3. Downregulation of Cinnamyl-Alcohol Dehydrogenase in Switchgrass by RNA Silencing Results in Enhanced Glucose Release after Cellulase Treatment

    PubMed Central

    Saathoff, Aaron J.; Sarath, Gautam; Chow, Elaine K.; Dien, Bruce S.; Tobias, Christian M.

    2011-01-01

    Cinnamyl alcohol dehydrogenase (CAD) catalyzes the last step in monolignol biosynthesis and genetic evidence indicates CAD deficiency in grasses both decreases overall lignin, alters lignin structure and increases enzymatic recovery of sugars. To ascertain the effect of CAD downregulation in switchgrass, RNA mediated silencing of CAD was induced through Agrobacterium mediated transformation of cv. “Alamo” with an inverted repeat construct containing a fragment derived from the coding sequence of PviCAD2. The resulting primary transformants accumulated less CAD RNA transcript and protein than control transformants and were demonstrated to be stably transformed with between 1 and 5 copies of the T-DNA. CAD activity against coniferaldehyde, and sinapaldehyde in stems of silenced lines was significantly reduced as was overall lignin and cutin. Glucose release from ground samples pretreated with ammonium hydroxide and digested with cellulases was greater than in control transformants. When stained with the lignin and cutin specific stain phloroglucinol-HCl the staining intensity of one line indicated greater incorporation of hydroxycinnamyl aldehydes in the lignin. PMID:21298014

  4. E. coli metabolic protein aldehyde-alcohol dehydrogenase-E binds to the ribosome: a unique moonlighting action revealed

    PubMed Central

    Shasmal, Manidip; Dey, Sandip; Shaikh, Tanvir R.; Bhakta, Sayan; Sengupta, Jayati

    2016-01-01

    It is becoming increasingly evident that a high degree of regulation is involved in the protein synthesis machinery entailing more interacting regulatory factors. A multitude of proteins have been identified recently which show regulatory function upon binding to the ribosome. Here, we identify tight association of a metabolic protein aldehyde-alcohol dehydrogenase E (AdhE) with the E. coli 70S ribosome isolated from cell extract under low salt wash conditions. Cryo-EM reconstruction of the ribosome sample allows us to localize its position on the head of the small subunit, near the mRNA entrance. Our study demonstrates substantial RNA unwinding activity of AdhE which can account for the ability of ribosome to translate through downstream of at least certain mRNA helices. Thus far, in E. coli, no ribosome-associated factor has been identified that shows downstream mRNA helicase activity. Additionally, the cryo-EM map reveals interaction of another extracellular protein, outer membrane protein C (OmpC), with the ribosome at the peripheral solvent side of the 50S subunit. Our result also provides important insight into plausible functional role of OmpC upon ribosome binding. Visualization of the ribosome purified directly from the cell lysate unveils for the first time interactions of additional regulatory proteins with the ribosome. PMID:26822933

  5. Spaceflight exposure effects on transcription, activity, and localization of alcohol dehydrogenase in the roots of Arabidopsis thaliana

    NASA Technical Reports Server (NTRS)

    Porterfield, D. M.; Matthews, S. W.; Daugherty, C. J.; Musgrave, M. E.

    1997-01-01

    Although considerable research and speculation have been directed toward understanding a plant's perception of gravity and the resulting gravitropic responses, little is known about the role of gravity-dependent physical processes in normal physiological function. These studies were conducted to determine whether the roots of plants exposed to spaceflight conditions may be experiencing hypoxia. Arabidopsis thaliana (L.) Heynh. plants were grown in agar medium during 6 or 11 d of spaceflight exposure on shuttle missions STS-54 (CHROMEX-03) and STS-68 (CHROMEX-05), respectively. The analysis included measurement of agar redox potential and root alcohol dehydrogenase (ADH) activity, localization, and expression. ADH activity increased by 89% as a result of spaceflight exposure for both CHROMEX-03 and -05 experiments, and ADH RNase protection assays revealed a 136% increase in ADH mRNA. The increase in ADH activity associated with the spaceflight roots was realized by a 28% decrease in oxygen availability in a ground-based study; however, no reduction in redox potential was observed in measurements of the spaceflight bulk agar. Spaceflight exposure appears to effect a hypoxic response in the roots of agar-grown plants that may be caused by changes in gravity-mediated fluid and/or gas behavior.

  6. Alcohol dehydrogenase activities and ethanol tolerance in Anastrepha (Diptera, Tephritidae) fruit-fly species and their hybrids

    PubMed Central

    2009-01-01

    The ADH (alcohol dehydrogenase) system is one of the earliest known models of molecular evolution, and is still the most studied in Drosophila. Herein, we studied this model in the genus Anastrepha (Diptera, Tephritidae). Due to the remarkable advantages it presents, it is possible to cross species with different Adh genotypes and with different phenotype traits related to ethanol tolerance. The two species studied here each have a different number of Adh gene copies, whereby crosses generate polymorphisms in gene number and in composition of the genetic background. We measured certain traits related to ethanol metabolism and tolerance. ADH specific enzyme activity presented gene by environment interactions, and the larval protein content showed an additive pattern of inheritance, whilst ADH enzyme activity per larva presented a complex behavior that may be explained by epistatic effects. Regression models suggest that there are heritable factors acting on ethanol tolerance, which may be related to enzymatic activity of the ADHs and to larval mass, although a pronounced environmental effect on ethanol tolerance was also observed. By using these data, we speculated on the mechanisms of ethanol tolerance and its inheritance as well as of associated traits. PMID:21637665

  7. [Inheritance and phenotype expression of functional and null alleles of aromatic alcohol dehydrogenase (CAD) in diploid wheats].

    PubMed

    Konovalov, A A; Shundrina, I K; Karpova, E V; Nefedov, A A; Goncharov, N P

    2014-11-01

    Functional F and null 0 alleles of the CAD1 (Aadh1) gene, which controls the biosynthesis of aromatic alcohol dehydrogenase, were studied in hybrids of the diploid wheat T. monococcum L. and Triticum sinskajae A.Filat. et Kurk. The gene CAD1 is located in chromosome 5A and is linked with the awnless gene awnS (La) with a recombination frequency of about 32%. Plants with genotypes FF, F0, and 00 were significantly different in the height and mechanical strength of the stalk (culm). The elastic limit of the culm tissues of plants FF was considerably higher than in 00 plants. F0 heterozygotes had intermediate values. The thickness of the wall of the sclerenchyma was thinner in plants with genotype 00. The chemical structure of lignin of plants with the functional CAD allele contained units of a phloroglucinol series missing in the mutant plants. The CAD genotypes had no effect on the relative content of cellulose and lignin in stalks ofdiploid wheat and insignificantly influenced the ratio of H :G : S units in the lignin structure, as well as some components of extractives.

  8. Isolation and DNA sequence of ADH3, a nuclear gene encoding the mitochondrial isozyme of alcohol dehydrogenase in Saccharomyces cerevisiae.

    PubMed Central

    Young, E T; Pilgrim, D

    1985-01-01

    The Saccharomyces cerevisiae nuclear gene, ADH3, that encodes the mitochondrial alcohol dehydrogenase isozyme ADH III was cloned by virtue of its nucleotide homology to ADH1 and ADH2. Both chromosomal and plasmid-encoded ADH III isozymes were repressed by glucose and migrated heterogeneously on nondenaturing gels. Nucleotide sequence analysis indicated 73 and 74% identity for ADH3 with ADH1 and ADH2, respectively. The amino acid identity between the predicted ADH III polypeptide and ADH I and ADH II was 79 and 80%, respectively. The open reading frame encoding ADH III has a highly basic 27-amino-acid amino-terminal extension relative to ADH I and ADH II. The nucleotide sequence of the presumed leader peptide has a high degree of identity with the untranslated leader regions of ADH1 and ADH2 mRNAs. A strain containing a null allele of ADH3 did not have a detectably altered phenotype. The cloned gene integrated at the ADH3 locus, indicating that this is the structural gene for ADH III. Images PMID:2943982

  9. Molecular dynamics study of zinc binding to cysteines in a peptide mimic of the alcohol dehydrogenase structural zinc site.

    PubMed

    Brandt, Erik G; Hellgren, Mikko; Brinck, Tore; Bergman, Tomas; Edholm, Olle

    2009-02-14

    The binding of zinc (Zn) ions to proteins is important for many cellular events. The theoretical and computational description of this binding (as well as that of other transition metals) is a challenging task. In this paper the binding of the Zn ion to four cysteine residues in the structural site of horse liver alcohol dehydrogenase (HLADH) is studied using a synthetic peptide mimic of this site. The study includes experimental measurements of binding constants, classical free energy calculations from molecular dynamics (MD) simulations and quantum mechanical (QM) electron structure calculations. The classical MD results account for interactions at the molecular level and reproduce the absolute binding energy and the hydration free energy of the Zn ion with an accuracy of about 10%. This is insufficient to obtain correct free energy differences. QM correction terms were calculated from density functional theory (DFT) on small clusters of atoms to include electronic polarisation of the closest waters and covalent contributions to the Zn-S coordination bond. This results in reasonably good agreement with the experimentally measured binding constants and Zn ion hydration free energies in agreement with published experimental values. The study also includes the replacement of one cysteine residue to an alanine. Simulations as well as experiments showed only a small effect of this upon the binding free energy. A detailed analysis indicate that the sulfur is replaced by three water molecules, thereby changing the coordination number of Zn from four (as in the original peptide) to six (as in water).

  10. Alcohol dehydrogenase AdhA plays a role in ethanol tolerance in model cyanobacterium Synechocystis sp. PCC 6803.

    PubMed

    Vidal, Rebeca

    2017-02-03

    The protein AdhA from the cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis) has been previously reported to show alcohol dehydrogenase activity towards ethanol and both NAD and NADP. This protein is currently being used in genetically modified strains of Synechocystis capable of synthesizing ethanol showing the highest ethanol productivities. In the present work, mutant strains of Synechocystis lacking AdhA have been constructed and tested for tolerance to ethanol. The lack of AdhA in the wild-type strain reduces survival to externally added ethanol at lethal concentration of 4% (v/v). On the other hand, the lack of AdhA in an ethanologenic strain diminishes tolerance of cells to internally produced ethanol. It is also shown that light-activated heterotrophic growth (LAHG) of the wild-type strain is impaired in the mutant strain lacking AdhA (∆adhA strain). Photoautotrophic, mixotrophic, and photoheterotrophic growth are not affected in the mutant strain. Based on phenotypic characterization of ∆adhA mutants, the possible physiological function of AdhA in Synechocystis is discussed.

  11. The anaerobic chytridiomycete fungus Piromyces sp. E2 produces ethanol via pyruvate:formate lyase and an alcohol dehydrogenase E.

    PubMed

    Boxma, Brigitte; Voncken, Frank; Jannink, Sander; van Alen, Theo; Akhmanova, Anna; van Weelden, Susanne W H; van Hellemond, Jaap J; Ricard, Guenola; Huynen, Martijn; Tielens, Aloysius G M; Hackstein, Johannes H P

    2004-03-01

    Anaerobic chytridiomycete fungi possess hydrogenosomes, which generate hydrogen and ATP, but also acetate and formate as end-products of a prokaryotic-type mixed-acid fermentation. Notably, the anaerobic chytrids Piromyces and Neocallimastix use pyruvate:formate lyase (PFL) for the catabolism of pyruvate, which is in marked contrast to the hydrogenosomal metabolism of the anaerobic parabasalian flagellates Trichomonas vaginalis and Tritrichomonas foetus, because these organisms decarboxylate pyruvate with the aid of pyruvate:ferredoxin oxidoreductase (PFO). Here, we show that the chytrids Piromyces sp. E2 and Neocallimastix sp. L2 also possess an alcohol dehydrogenase E (ADHE) that makes them unique among hydrogenosome-bearing anaerobes. We demonstrate that Piromyces sp. E2 routes the final steps of its carbohydrate catabolism via PFL and ADHE: in axenic culture under standard conditions and in the presence of 0.3% fructose, 35% of the carbohydrates were degraded in the cytosol to the end-products ethanol, formate, lactate and succinate, whereas 65% were degraded via the hydrogenosomes to acetate and formate. These observations require a refinement of the previously published metabolic schemes. In particular, the importance of the hydrogenase in this type of hydrogenosome has to be revisited.

  12. Investigation of Structural Determinants for the Substrate Specificity in the Zinc-Dependent Alcohol Dehydrogenase CPCR2 from Candida parapsilosis.

    PubMed

    Loderer, Christoph; Dhoke, Gaurao V; Davari, Mehdi D; Kroutil, Wolfgang; Schwaneberg, Ulrich; Bocola, Marco; Ansorge-Schumacher, Marion B

    2015-07-06

    Zinc-dependent alcohol dehydrogenases (ADHs) are a class of enzymes applied in different biocatalytic processes ranging from lab to industrial scale. However, one drawback is the limited substrate range, necessitating a whole array of different ADHs for the relevant substrate classes. In this study, we investigated structural determinants of the substrate spectrum in the zinc-dependent ADH carbonyl reductase 2 from Candida parapsilosis (CPCR2), combining methods of mutational analysis with in silico substrate docking. Assigned active site residues were genetically randomized, and the resulting mutant libraries were screened with a selection of challenging carbonyl substrates. Three variants (C57A, W116K, and L119M) with improved activities toward different substrates were detected at neighboring positions in the active site. Thus, all possible combinations of the mutations were generated and characterized for their substrate specificity, yielding several improved variants. The most interesting were a C57A variant, with a 27-fold increase in specific activity for 4'-acetamidoacetophenone, and the double mutant CPCR2 B16-(C57A, L119M), with a 45-fold improvement in the kcat ⋅KM (-1) value. The obtained variants were further investigated by in silico docking experiments. The results indicate that the mentioned residues are structural determinants of the substrate specificity of CPCR2, being major players in the definition of the active site. Comparison of these results with closely related enzymes suggests that these might even be transferred to other ADHs.

  13. Substitutions at the cofactor phosphate-binding site of a clostridial alcohol dehydrogenase lead to unexpected changes in substrate specificity.

    PubMed

    Maddock, Danielle J; Patrick, Wayne M; Gerth, Monica L

    2015-08-01

    Changing the cofactor specificity of an enzyme from nicotinamide adenine dinucleotide 2'-phosphate (NADPH) to the more abundant NADH is a common strategy for increasing overall enzyme efficiency in microbial metabolic engineering. The aim of this study was to switch the cofactor specificity of the primary-secondary alcohol dehydrogenase from Clostridium autoethanogenum, a bacterium with considerable promise for the bio-manufacturing of fuels and other petrochemicals, from strictly NADPH-dependent to NADH-dependent. We used insights from a homology model to build a site-saturation library focussed on residue S199, the position deemed most likely to disrupt binding of the 2'-phosphate of NADPH. Although the CaADH(S199X) library did not yield any NADH-dependent enzymes, it did reveal that substitutions at the cofactor phosphate-binding site can cause unanticipated changes in the substrate specificity of the enzyme. Using consensus-guided site-directed mutagenesis, we were able to create an enzyme that was stringently NADH-dependent, albeit with a concomitant reduction in activity. This study highlights the role that distal residues play in substrate specificity and the complexity of enzyme-cofactor interactions.

  14. Chaperone activities of bovine and camel beta-caseins: Importance of their surface hydrophobicity in protection against alcohol dehydrogenase aggregation.

    PubMed

    Barzegar, Abolfazl; Yousefi, Reza; Sharifzadeh, Ahmad; Dalgalarrondo, Michèle; Chobert, Jean-Marc; Ganjali, Mohammad Reza; Norouzi, Parviz; Ehsani, Mohammad Reza; Niasari-Naslaji, Amir; Saboury, Ali Akbar; Haertlé, Thomas; Moosavi-Movahedi, Ali Akbar

    2008-05-01

    Beta-casein (beta-CN) showing properties of intrinsically unstructured proteins (IUP) displays many similarities with molecular chaperones and shows anti-aggregation activity in vitro. Chaperone activities of bovine and camel beta-CN were studied using alcohol dehydrogenase (ADH) as a substrate. To obtain an adequate relevant information about the chaperone capacities of studied caseins, three different physical parameters including chaperone constant (k(c), microM(-1)), thermal aggregation constant (k(T), degrees C(-1)) and aggregation rate constant (k(t), min(-1)) were measured. Bovine beta-CN displays greater chaperone activity than camel beta-CN. Fluorescence studies of 8-anilino-1-naphthalenesulfonic acid (ANS) binding demonstrated that bovine beta-CN is doted with larger effective hydrophobic surfaces at all studied temperatures than camel beta-CN. Greater relative hydrophobicity of bovine beta-CN than camel beta-CN may be a factor responsible for stronger interactions of bovine beta-CN with the aggregation-prone pre denatured molecular species of the substrate ADH, which resulted in greater chaperone activity of bovine beta-CN.

  15. Slowed Diffusion and Excluded Volume Both Contribute to the Effects of Macromolecular Crowding on Alcohol Dehydrogenase Steady-State Kinetics.

    PubMed

    Schneider, Samuel H; Lockwood, Schuyler P; Hargreaves, Dominique I; Slade, David J; LoConte, Micaela A; Logan, Bridget E; McLaughlin, Erin E; Conroy, Michael J; Slade, Kristin M

    2015-09-29

    To understand the consequences of macromolecular crowding, studies have largely employed in vitro experiments with synthetic polymers assumed to be both pure and "inert". These polymers alter enzyme kinetics by excluding volume that would otherwise be available to the enzymes, substrates, and products. Presented here is evidence that other factors, in addition to excluded volume, must be considered in the interpretation of crowding studies with synthetic polymers. Dextran has a weaker effect on the Michaelis-Menten kinetic parameters of yeast alcohol dehydrogenase (YADH) than its small molecule counterpart, glucose. For glucose, the decreased Vmax values directly correlate with slower translational diffusion and the decreased Km values likely result from enhanced substrate binding due to YADH stabilization. Because dextran is unable to stabilize YADH to the same extent as glucose, this polymer's ability to decrease Km is potentially due to the nonideality of the solution, a crowding-induced conformational change, or both. Chronoamperometry reveals that glucose and dextran have surprisingly similar ferricyanide diffusion coefficients. Thus, the reduction in Vmax values for glucose is partially offset by an additional macromolecular crowding effect with dextran. Finally, this is the first report that supplier-dependent impurities in dextran affect the kinetic parameters of YADH. Taken together, our results reveal that caution should be used when interpreting results obtained with inert synthetic polymeric agents, as additional effects from the underlying monomer need to be considered.

  16. Aldehyde Dehydrogenase 2 (ALDH2) Polymorphism and the Risk of Alcoholic Liver Cirrhosis among East Asians: A Meta-Analysis

    PubMed Central

    He, Lei; Luo, Hesheng

    2016-01-01

    Purpose The aldehyde dehydrogenase 2 (ALDH2) gene has been implicated in the development of alcoholic liver cirrhosis (ALC) in East Asians. However, the results are inconsistent. In this study, a meta-analysis was performed to assess the associations between the ALDH2 polymorphism and the risk of ALC. Materials and Methods Relevant studies were retrieved by searching PubMed, Web of Science, CNKI, Wanfang and Veipu databases up to January 10, 2015. Pooled odds ratio (OR) and 95% confidence interval (CI) were calculated using either the fixed- or random effects model. Results A total of twelve case-control studies included 1003 cases and 2011 controls were included. Overall, the ALDH2 polymorphism was associated with a decreased risk of ALC (*1/*2 vs. *1/*1: OR=0.78, 95% CI: 0.61–0.99). However, in stratification analysis by country, we failed to detect any association among Chinese, Korean or Japanese populations. Conclusion The pooled evidence suggests that ALDH2 polymorphism may be an important protective factor for ALC in East Asians. PMID:27189280

  17. Spaceflight exposure effects on transcription, activity, and localization of alcohol dehydrogenase in the roots of Arabidopsis thaliana.

    PubMed Central

    Porterfield, D M; Matthews, S W; Daugherty, C J; Musgrave, M E

    1997-01-01

    Although considerable research and speculation have been directed toward understanding a plant's perception of gravity and the resulting gravitropic responses, little is known about the role of gravity-dependent physical processes in normal physiological function. These studies were conducted to determine whether the roots of plants exposed to spaceflight conditions may be experiencing hypoxia. Arabidopsis thaliana (L.) Heynh. plants were grown in agar medium during 6 or 11 d of spaceflight exposure on shuttle missions STS-54 (CHROMEX-03) and STS-68 (CHROMEX-05), respectively. The analysis included measurement of agar redox potential and root alcohol dehydrogenase (ADH) activity, localization, and expression. ADH activity increased by 89% as a result of spaceflight exposure for both CHROMEX-03 and -05 experiments, and ADH RNase protection assays revealed a 136% increase in ADH mRNA. The increase in ADH activity associated with the spaceflight roots was realized by a 28% decrease in oxygen availability in a ground-based study; however, no reduction in redox potential was observed in measurements of the spaceflight bulk agar. Spaceflight exposure appears to effect a hypoxic response in the roots of agar-grown plants that may be caused by changes in gravity-mediated fluid and/or gas behavior. PMID:9085569

  18. Changes in soluble sugar, starch, and alcohol dehydrogenase in Arabidopsis thaliana exposed to N2 diluted atmospheres

    NASA Technical Reports Server (NTRS)

    Porterfield, D. M.; Crispi, M. L.; Musgrave, M. E.

    1997-01-01

    Proper exchange of atmospheric gases is important for normal root and shoot metabolism in plants. This study was conducted to determine how restricted air supply affects foliar carbohydrates, while using the marker enzyme alcohol dehydrogenase (ADH) to report on the oxygenation status of the rootzone. Fourteen-day-old Arabidopsis thaliana (L.) Heynh. plants grown singly in 7-ml tubes containing agarified nutrient medium were placed in coupled Magenta vessels and exposed for six days to either ambient air or one of six different air/nitrogen dilutions. Redox potential of the agar medium was measured immediately after harvesting and freezing leaf tissue, and then root systems were quickly extracted from the agar and frozen for subsequent analyses. Redox potential measurements indicated that this series of gas mixtures produced a transition from hypoxia to anoxia in the root zones. Root ADH activity increased at higher rates as the redox potential neared anoxic levels. In contrast, ADH mRNA expression quickly neared its maximum as the medium became hypoxic and showed little further increase as it became anoxic. Foliar carbohydrate levels increased 1.5- to 2-fold with decreased availability of metabolic gases, with starch increasing at higher concentrations of air than soluble carbohydrate. The results serve as a model for plant performance under microgravity conditions, where absence of convective air movement prevents replenishment of metabolic gases.

  19. Crystal structure of the alcohol dehydrogenase from the hyperthermophilic archaeon Sulfolobus solfataricus at 1.85 A resolution.

    PubMed

    Esposito, Luciana; Sica, Filomena; Raia, Carlo Antonio; Giordano, Antonietta; Rossi, Mosè; Mazzarella, Lelio; Zagari, Adriana

    2002-04-26

    The crystal structure of a medium-chain NAD(H)-dependent alcohol dehydrogenase (ADH) from an archaeon has been solved by multiwavelength anomalous diffraction, using a selenomethionine-substituted enzyme. The protein (SsADH), extracted from the hyperthermophilic organism Sulfolobus solfataricus, is a homo-tetramer with a crystallographic 222 symmetry. Despite the low level of sequence identity, the overall fold of the monomer is similar to that of the other homologous ADHs of known structure. However, a significant difference is the orientation of the catalytic domain relative to the coenzyme-binding domain that results in a larger interdomain cleft. At the bottom of this cleft, the catalytic zinc ion is coordinated tetrahedrally and lacks the zinc-bound water molecule that is usually found in ADH apoform structures. The fourth coordination position is indeed occupied by a Glu residue, as found in bacterial tetrameric ADHs. Other differences are found in the architecture of the substrate pocket whose entrance is more restricted than in other ADHs. SsADH is the first tetrameric ADH X-ray structure containing a second zinc ion playing a structural role. This latter metal ion shows a peculiar coordination, with a glutamic acid residue replacing one of the four cysteine ligands that are highly conserved throughout the structural zinc-containing dimeric ADHs.

  20. Fluoride-containing bioactive glasses inhibit pentose phosphate oxidative pathway and glucose 6-phosphate dehydrogenase activity in human osteoblasts.

    PubMed

    Bergandi, Loredana; Aina, Valentina; Garetto, Stefano; Malavasi, Gianluca; Aldieri, Elisabetta; Laurenti, Enzo; Matera, Lina; Morterra, Claudio; Ghigo, Dario

    2010-02-12

    Bioactive glasses such as Hench's 45S5 (Bioglass) have applications to tissue engineering as well as bone repair, and the insertion of fluoride in their composition has been proposed to enhance their bioactivity. In view of a potential clinical application, we investigated whether fluoride-containing glasses exert toxic effects on human MG-63 osteoblasts, and whether and how fluoride, which is released in the cell culture medium, might play a role in such cytotoxicity. A 24h incubation with 50 microg/ml (12.5 microg/cm(2)) of fluoride-containing bioactive glasses termed HCaCaF(2) (F content: 5, 10 and 15 mol.%) caused the release of lactate dehydrogenase in the extracellular medium (index of cytotoxicity), the accumulation of intracellular malonyldialdehyde (index of lipoperoxidation), and the increase of glutathione consumption. Furthermore, fluoride-containing glasses inhibited the pentose phosphate oxidative pathway and the glucose 6-phosphate dehydrogenase activity. These effects are ascribable to the fluoride content/release of glass powders, since they were mimicked by NaF solutions and were prevented by dimethyl sulfoxide and tempol (two radical scavengers), by superoxide dismutase (a superoxide scavenger), and by glutathione (the most important intracellular antioxidant molecule), but not by apocynin (an inhibitor of NADPH oxidase). The presence of fluoride-containing glasses and NaF caused also the generation of reactive oxygen species, which was prevented by superoxide dismutase and catalase. The data suggest that fluoride released from glasses is the cause of MG-63 cell oxidative damage and is independent of NADPH oxidase activation. Our data provide a new mechanism to explain F(-) ions toxicity: fluoride could trigger, at least in part, an oxidative stress via inhibition of the pentose phosphate oxidative pathway and, in particular, through the oxidative inhibition of glucose 6-phosphate dehydrogenase.

  1. The oxidative fermentation of ethanol in Gluconacetobacter diazotrophicus is a two-step pathway catalyzed by a single enzyme: alcohol-aldehyde Dehydrogenase (ADHa).

    PubMed

    Gómez-Manzo, Saúl; Escamilla, José E; González-Valdez, Abigail; López-Velázquez, Gabriel; Vanoye-Carlo, América; Marcial-Quino, Jaime; de la Mora-de la Mora, Ignacio; Garcia-Torres, Itzhel; Enríquez-Flores, Sergio; Contreras-Zentella, Martha Lucinda; Arreguín-Espinosa, Roberto; Kroneck, Peter M H; Sosa-Torres, Martha Elena

    2015-01-07

    Gluconacetobacter diazotrophicus is a N2-fixing bacterium endophyte from sugar cane. The oxidation of ethanol to acetic acid of this organism takes place in the periplasmic space, and this reaction is catalyzed by two membrane-bound enzymes complexes: the alcohol dehydrogenase (ADH) and the aldehyde dehydrogenase (ALDH). We present strong evidence showing that the well-known membrane-bound Alcohol dehydrogenase (ADHa) of Ga. diazotrophicus is indeed a double function enzyme, which is able to use primary alcohols (C2-C6) and its respective aldehydes as alternate substrates. Moreover, the enzyme utilizes ethanol as a substrate in a reaction mechanism where this is subjected to a two-step oxidation process to produce acetic acid without releasing the acetaldehyde intermediary to the media. Moreover, we propose a mechanism that, under physiological conditions, might permit a massive conversion of ethanol to acetic acid, as usually occurs in the acetic acid bacteria, but without the transient accumulation of the highly toxic acetaldehyde.

  2. Similarity of Escherichia coli propanediol oxidoreductase (fucO product) and an unusual alcohol dehydrogenase from Zymomonas mobilis and Saccharomyces cerevisiae

    SciTech Connect

    Conway, T. ); Ingram, L.O. )

    1989-07-01

    The gene that encodes 1,2-propanediol oxidoreductase (fucO) from Escherichia coli was sequenced. The reading frame specified a protein of 383 amino acids (including the N-terminal methionine), with an aggregate molecular weight of 40,642. The induction of fucO transcription, which occurred in the presence of fucose, was confirmed by Northern blot analysis. In E. coli, the primary fucO transcript was approximately 2.1 kilobases in length. The 5{prime} end of the transcript began more than 0.7 kilobase upstream of the fucO start codon within or beyond the fucA gene. Propanediol oxidoreductase exhibited 41.7% identity with the iron-containing alcohol dehydrogenase II from Zymomonas mobilis and 39.5% identity with ADH4 from Saccharomyces cerevisiae. These three proteins did not share homology with either short-chain or long-chain zinc-containing alcohol dehydrogenase enzymes. We propose that these three unusual alcohol dehydrogenases define a new family of enzymes.

  3. The Oxidative Fermentation of Ethanol in Gluconacetobacter diazotrophicus Is a Two-Step Pathway Catalyzed by a Single Enzyme: Alcohol-Aldehyde Dehydrogenase (ADHa)

    PubMed Central

    Gómez-Manzo, Saúl; Escamilla, José E.; González-Valdez, Abigail; López-Velázquez, Gabriel; Vanoye-Carlo, América; Marcial-Quino, Jaime; de la Mora-de la Mora, Ignacio; Garcia-Torres, Itzhel; Enríquez-Flores, Sergio; Contreras-Zentella, Martha Lucinda; Arreguín-Espinosa, Roberto; Kroneck, Peter M. H.; Sosa-Torres, Martha Elena

    2015-01-01

    Gluconacetobacter diazotrophicus is a N2-fixing bacterium endophyte from sugar cane. The oxidation of ethanol to acetic acid of this organism takes place in the periplasmic space, and this reaction is catalyzed by two membrane-bound enzymes complexes: the alcohol dehydrogenase (ADH) and the aldehyde dehydrogenase (ALDH). We present strong evidence showing that the well-known membrane-bound Alcohol dehydrogenase (ADHa) of Ga. diazotrophicus is indeed a double function enzyme, which is able to use primary alcohols (C2–C6) and its respective aldehydes as alternate substrates. Moreover, the enzyme utilizes ethanol as a substrate in a reaction mechanism where this is subjected to a two-step oxidation process to produce acetic acid without releasing the acetaldehyde intermediary to the media. Moreover, we propose a mechanism that, under physiological conditions, might permit a massive conversion of ethanol to acetic acid, as usually occurs in the acetic acid bacteria, but without the transient accumulation of the highly toxic acetaldehyde. PMID:25574602

  4. Characterization of an Allylic/Benzyl Alcohol Dehydrogenase from Yokenella sp. Strain WZY002, an Organism Potentially Useful for the Synthesis of α,β-Unsaturated Alcohols from Allylic Aldehydes and Ketones

    PubMed Central

    Ying, Xiangxian; Wang, Yifang; Xiong, Bin; Wu, Tingting; Xie, Liping; Yu, Meilan

    2014-01-01

    A novel whole-cell biocatalyst with high allylic alcohol-oxidizing activities was screened and identified as Yokenella sp. WZY002, which chemoselectively reduced the C=O bond of allylic aldehydes/ketones to the corresponding α,β-unsaturated alcohols at 30°C and pH 8.0. The strain also had the capacity of stereoselectively reducing aromatic ketones to (S)-enantioselective alcohols. The enzyme responsible for the predominant allylic/benzyl alcohol dehydrogenase activity was purified to homogeneity and designated YsADH (alcohol dehydrogenase from Yokenella sp.), which had a calculated subunit molecular mass of 36,411 Da. The gene encoding YsADH was subsequently expressed in Escherichia coli, and the purified recombinant YsADH protein was characterized. The enzyme strictly required NADP(H) as a coenzyme and was putatively zinc dependent. The optimal pH and temperature for crotonaldehyde reduction were pH 6.5 and 65°C, whereas those for crotyl alcohol oxidation were pH 8.0 and 55°C. The enzyme showed moderate thermostability, with a half-life of 6.2 h at 55°C. It was robust in the presence of organic solvents and retained 87.5% of the initial activity after 24 h of incubation with 20% (vol/vol) dimethyl sulfoxide. The enzyme preferentially catalyzed allylic/benzyl aldehydes as the substrate in the reduction of aldehydes/ketones and yielded the highest activity of 427 U mg−1 for benzaldehyde reduction, while the alcohol oxidation reaction demonstrated the maximum activity of 79.9 U mg−1 using crotyl alcohol as the substrate. Moreover, kinetic parameters of the enzyme showed lower Km values and higher catalytic efficiency for crotonaldehyde/benzaldehyde and NADPH than for crotyl alcohol/benzyl alcohol and NADP+, suggesting the nature of being an aldehyde reductase. PMID:24509923

  5. Inhibition by N'-nitrosonornicotine of the catalytic activity of glutamate dehydrogenase in alpha-ketoglutarate amination.

    PubMed

    Mao, You-An; Zhong, Ke-Jun; Wei, Wan-Zhi; Wei, Xin-Liang; Lu, Hong-Bing

    2005-02-01

    The effect of N'-nitrosonornicotine (NNN), one of the tobacco-specific nitrosamines, on the catalytic activity of glutamate dehydrogenase (GLDH) in the alpha-ketoglutarate amination, using reduced nicotinamide adenine dinucleotide as coenzyme, was studied by a chronoamperometric method. The maximum reaction rate of the enzyme-catalyzed reaction and the Michaelis-Menten constant, or the apparent Michaelis-Menten constant, were determined in the absence and presence of NNN. NNN remarkably inhibited the bio-catalysis activity of GLDH, and was a reversible competitive inhibitior with K(i), estimated as 199 micromol l(-1) at 25 degrees C and pH 8.0.

  6. PQQ-dependent alcohol dehydrogenase (QEDH) of Pseudomonas aeruginosa is involved in catabolism of acyclic terpenes.

    PubMed

    Chattopadhyay, Ava; Förster-Fromme, Karin; Jendrossek, Dieter

    2010-04-01

    Growth of Pseudomonas aeruginosa on acyclic terpene alcohols such as geraniol depends on the presence of the atuRABCDEFGH gene cluster and a functional acyclic terpene utilisation (Atu) pathway. The proteins encoded by the atu gene cluster are necessary but not sufficient for growth on acyclic terpenes. Comparative 2-dimensional polyacrylamide gel electrophoresis of soluble P. aeruginosa proteins revealed the presence of an additional spot (besides Atu proteins) that is specifically expressed in geraniol cells but is absent in isovalerate-grown cells. The spot was identified as PA1982 gene product a pyrroloquinoline quinone (PQQ) dependent ethanol oxidoreductase (QEDH). Inactivation of PA1982 by insertion mutagenesis resulted in inability of the mutant to utilise ethanol and in reduced growth on geraniol. Growth on ethanol was restored by transferring an intact copy of the PA1982 gene into the mutant. The PA1982 gene product was purified from recombinant Escherichia coli and revealed PQQ-dependent oxidoreductase activity with a variety of substrates including acyclic terpene derivates at comparable V(max)-values. Our results show that QEDH participates in oxidation of acyclic terpene derivates in addition to the well-known function in ethanol metabolism.

  7. Alcohol and aldehyde dehydrogenase polymorphisms and a new strategy for prevention and screening for cancer in the upper aerodigestive tract in East Asians.

    PubMed

    Yokoyama, Akira; Omori, Tai; Yokoyama, Tetsuji

    2010-01-01

    The ethanol in alcoholic beverages and the acetaldehyde associated with alcohol consumption are Group 1 human carcinogens (WHO, International Agency for Research on Cancer). The combination of alcohol consumption, tobacco smoking, the inactive heterozygous aldehyde dehydrogenase-2 genotype (ALDH2*1/*2) and the less-active homozygous alcohol dehydrogenase-1B genotype (ADH1B*1/*1) increases the risk of squamous cell carcinoma (SCC) in the upper aerodigestive tract (UADT) in a multiplicative fashion in East Asians. In addition to being exposed to locally high levels of ethanol, the UADT is exposed to a very high concentration of acetaldehyde from a variety of sources, including that as an ingredient of alcoholic beverages per se and that found in tobacco smoke; acetaldehyde is also produced by salivary microorganisms and mucosal enzymes and is present as blood acetaldehyde. The inefficient degradation of acetaldehyde by weakly expressed ALDH2 in the UADT may be cri! tical to the local accumulation of acetaldehyde, especially in ALDH2*1/*2 carriers. ADH1B*1/*1 carriers tend to experience less intense alcohol flushing and are highly susceptible to heavy drinking and alcoholism. Heavy drinking by persons with the less-active ADH1B*1/*1 leads to longer exposure of the UADT to salivary ethanol and acetaldehyde. The ALDH2*1/*2 genotype is a very strong predictor of synchronous and metachronous multiple SCCs in the UADT. High red cell mean corpuscular volume (MCV), esophageal dysplasia, and melanosis in the UADT, all of which are frequently found in ALDH2*1/*2 drinkers, are useful for identifying high-risk individuals. We invented a simple flushing questionnaire that enables prediction of the ALDH2 phenotype. New health appraisal models that include ALDH2 genotype, the simple flushing questionnaire, or MCV are powerful tools for devising a new strategy for prevention and screening for UADT cancer in East Asians.

  8. Inhibition of Glucose-6-Phosphate Dehydrogenase Could Enhance 1,4-Benzoquinone-Induced Oxidative Damage in K562 Cells

    PubMed Central

    Cao, Meng; Yang, Wenwen; Sun, Fengmei; Xu, Cheng

    2016-01-01

    Benzene is a chemical contaminant widespread in industrial and living environments. The oxidative metabolites of benzene induce toxicity involving oxidative damage. Protecting cells and cell membranes from oxidative damage, glucose-6-phosphate dehydrogenase (G6PD) maintains the reduced state of glutathione (GSH). This study aims to investigate whether the downregulation of G6PD in K562 cell line can influence the oxidative toxicity induced by 1,4-benzoquinone (BQ). G6PD was inhibited in K562 cell line transfected with the specific siRNA of G6PD gene. An empty vector was transfected in the control group. Results revealed that G6PD was significantly upregulated in the control cells and in the cells with inhibited G6PD after they were exposed to BQ. The NADPH/NADP and GSH/GSSG ratio were significantly lower in the cells with inhibited G6PD than in the control cells at the same BQ concentration. The relative reactive oxygen species (ROS) level and DNA oxidative damage were significantly increased in the cell line with inhibited G6PD. The apoptotic rate and G2 phase arrest were also significantly higher in the cells with inhibited G6PD and exposed to BQ than in the control cells. Our results suggested that G6PD inhibition could reduce GSH activity and alleviate oxidative damage. G6PD deficiency is also a possible susceptible risk factor of benzene exposure. PMID:27656260

  9. Helenalin attenuates alcohol-induced hepatic fibrosis by enhancing ethanol metabolism, inhibiting oxidative stress and suppressing HSC activation.

    PubMed

    Lin, Xing; Zhang, Shijun; Huang, Renbin; Wei, Ling; Tan, Shimei; Liang, Shuang; Tian, Yuanchun; Wu, Xiaoyan; Lu, Zhongpeng; Huang, Quanfang

    2014-06-01

    A compound was isolated from Centipeda minima using bioassay-guided screening. The structure of this compound was elucidated based on its spectral data, and it was identified as helenalin. The hepatoprotective effect of helenalin was evaluated using a liver fibrosis model induced by intragastric administration with alcohol within 24 weeks in rats. The results revealed that helenalin significantly prevented alcohol-induced hepatic injury and fibrogenesis, as evidenced by the decrease in serum aminotransferase, the attenuation of histopathological changes, and the inhibition of the hepatic fibrosis indicators, such as hyaluronic acid, type III precollagen, laminin, hydroxyproline and collagen α type I. Mechanistically, studies showed that helenalin expedited ethanol metabolism by enhancing the alcohol and aldehyde dehydrogenase activities. Furthermore, helenalin alleviated lipid peroxidation, recruited the antioxidative defense system, inhibited CYP2E1 activity, and reduced the inflammatory mediators, including TGF-β1, TNF-α, IL-6 and IL-1β and myeloperoxidase, via down-regulation of NF-κB. Helenalin significantly decreased collagen deposition by reducing the profibrotic cytokines like transforming growth factor-β, platelet-derived growth factor-β and connective tissue growth factor, and promoted extracellular matrix degradation by modulating the levels of tissue inhibitor of matrix metalloproteinase-1 and matrix metalloproteinase-9. In addition, helenalin inhibited HSC activation as evidenced by the down-regulation of α-SMA and TGF-β levels. In conclusion, helenalin had a significant protective effect on chronic ethanol-induced hepatic fibrosis and may be a major bioactive ingredient of C. minima.

  10. Establishment of steady-state metabolism of ethanol in perfused rat liver: the quantitative analysis using kinetic mechanism-based rate equations of alcohol dehydrogenase.

    PubMed

    Yao, Chung-Tay; Lai, Ching-Long; Hsieh, Hsiu-Shan; Chi, Chin-Wen; Yin, Shih-Jiun

    2010-09-01

    Alcohol dehydrogenase (ADH) catalyzes oxidation of ingested ethanol to acetaldehyde, the first step in hepatic metabolism. The purpose of this study was to establish an ex vivo rat liver perfusion system under defined and verified steady states with respect to the metabolites and the metabolic rates, and to quantitatively correlate the observed rates with simulations based on the kinetic mechanism-based rate equations of rat liver ADH. Class I ADH1 was isolated from male Sprague-Dawley rats and characterized by steady-state kinetics in the Krebs-Ringer perfusion buffer with supplements. Nonrecirculating liver perfusion with constant input of ethanol at near physiological hepatic blood flow rate was performed in situ. Ethanol and the related metabolites acetaldehyde, acetate, lactate, and pyruvate in perfusates were determined. Results of the initial velocity, product, and dead-end inhibition studies showed that rat ADH1 conformed to the Theorell-Chance Ordered Bi Bi mechanism. Steady-state metabolism of ethanol in the perfused liver maintained up to 3h as evidenced by the steady-state levels of ethanol and metabolites in the effluent, and the steady-state ethanol disappearance rates and acetate production rates. The changes of the metabolic rates were qualitatively and in general quantitatively correlated to the results from simulations with the kinetic rate equations of ADH1 under a wide range of ethanol, in the presence of competitive inhibitor 4-methylpyrazole and of uncompetitive inhibitor isobutyramide. Preliminary flux control analysis estimated that apparent C(ADH)(J) in the perfused liver may approximate 0.7 at constant infusion with 1-2 mM ethanol, suggesting that ADH plays a major but not the exclusive role in governing hepatic ethanol metabolism. The reported steady-state rat liver perfusion system may potentially be applicable to other drug or drug-ethanol interaction studies.

  11. CINNAMYL ALCOHOL DEHYDROGENASE-C and -D Are the Primary Genes Involved in Lignin Biosynthesis in the Floral Stem of ArabidopsisW⃞

    PubMed Central

    Sibout, Richard; Eudes, Aymerick; Mouille, Gregory; Pollet, Brigitte; Lapierre, Catherine; Jouanin, Lise; Séguin, Armand

    2005-01-01

    During lignin biosynthesis in angiosperms, coniferyl and sinapyl aldehydes are believed to be converted into their corresponding alcohols by cinnamyl alcohol dehydrogenase (CAD) and by sinapyl alcohol dehydrogenase (SAD), respectively. This work clearly shows that CAD-C and CAD-D act as the primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis thaliana by supplying both coniferyl and sinapyl alcohols. An Arabidopsis CAD double mutant (cad-c cad-d) resulted in a phenotype with a limp floral stem at maturity as well as modifications in the pattern of lignin staining. Lignin content of the mutant stem was reduced by 40%, with a 94% reduction, relative to the wild type, in conventional β-O-4–linked guaiacyl and syringyl units and incorportion of coniferyl and sinapyl aldehydes. Fourier transform infrared spectroscopy demonstrated that both xylem vessels and fibers were affected. GeneChip data and real-time PCR analysis revealed that transcription of CAD homologs and other genes mainly involved in cell wall integrity were also altered in the double mutant. In addition, molecular complementation of the double mutant by tissue-specific expression of CAD derived from various species suggests different abilities of these genes/proteins to produce syringyl-lignin moieties but does not indicate a requirement for any specific SAD gene. PMID:15937231

  12. Role of glucose-6-phosphate dehydrogenase inhibition in the antiproliferative effects of dehydroepiandrosterone on human breast cancer cells.

    PubMed Central

    Di Monaco, M.; Pizzini, A.; Gatto, V.; Leonardi, L.; Gallo, M.; Brignardello, E.; Boccuzzi, G.

    1997-01-01

    Epidemiological and experimental studies suggest that dehydroepiandrosterone (DHEA) exerts a protective effect against breast cancer. It has been proposed that the non-competitive inhibition of glucose-6-phosphate dehydrogenase (G6PD) contributes to DHEA antitumor action. We evaluated the effects of DHEA on G6PD activity and on the in vitro proliferation of two human breast cancer cell lines, MCF-7 (steroid receptor positive) and MDA-MB-231 (steroid receptor negative), in a serum-free assay. DHEA inhibition of G6PD was only found to occur at concentrations above 10 microM; at these high concentrations, the growth curve was parallel to the enzyme inhibition curve in both cell lines. In contrast, at concentrations in the in vivo breast tissue concentration range, neither cell growth nor enzyme activity was inhibited. The results failed to confirm DHEA's putative anti-tumor action on breast cancer through G6PD inhibition, as the enzyme blockade only becomes apparent at pharmacological concentrations of the steroid. PMID:9052415

  13. Dehydrin, alcohol dehydrogenase, and central metabolite levels are associated with cold tolerance in diploid strawberry (Fragaria spp.).

    PubMed

    Davik, Jahn; Koehler, Gage; From, Britta; Torp, Torfinn; Rohloff, Jens; Eidem, Petter; Wilson, Robert C; Sønsteby, Anita; Randall, Stephen K; Alsheikh, Muath

    2013-01-01

    The use of artificial freezing tests, identification of biomarkers linked to or directly involved in the low-temperature tolerance processes, could prove useful in applied strawberry breeding. This study was conducted to identify genotypes of diploid strawberry that differ in their tolerance to low-temperature stress and to investigate whether a set of candidate proteins and metabolites correlate with the level of tolerance. 17 Fragaria vesca, 2 F. nilgerrensis, 2 F. nubicola, and 1 F. pentaphylla genotypes were evaluated for low-temperature tolerance. Estimates of temperatures where 50 % of the plants survived (LT₅₀) ranged from -4.7 to -12.0 °C between the genotypes. Among the F. vesca genotypes, the LT₅₀ varied from -7.7 °C to -12.0 °C. Among the most tolerant were three F. vesca ssp. bracteata genotypes (FDP821, NCGR424, and NCGR502), while a F. vesca ssp. californica genotype (FDP817) was the least tolerant (LT₅₀) -7.7 °C). Alcohol dehydrogenase (ADH), total dehydrin expression, and content of central metabolism constituents were assayed in select plants acclimated at 2 °C. The LT₅₀ estimates and the expression of ADH and total dehydrins were highly correlated (r(adh) = -0.87, r (dehyd) = -0.82). Compounds related to the citric acid cycle were quantified in the leaves during acclimation. While several sugars and acids were significantly correlated to the LT₅₀ estimates early in the acclimation period, only galactinol proved to be a good LT₅₀ predictor after 28 days of acclimation (r(galact) = 0.79). It is concluded that ADH, dehydrins, and galactinol show great potential to serve as biomarkers for cold tolerance in diploid strawberry.

  14. A Wheat Cinnamyl Alcohol Dehydrogenase TaCAD12 Contributes to Host Resistance to the Sharp Eyespot Disease.

    PubMed

    Rong, Wei; Luo, Meiying; Shan, Tianlei; Wei, Xuening; Du, Lipu; Xu, Huijun; Zhang, Zengyan

    2016-01-01

    Sharp eyespot, caused mainly by the necrotrophic fungus Rhizoctonia cerealis, is a destructive disease in hexaploid wheat (Triticum aestivum L.). In Arabidopsis, certain cinnamyl alcohol dehydrogenases (CADs) have been implicated in monolignol biosynthesis and in defense response to bacterial pathogen infection. However, little is known about CADs in wheat defense responses to necrotrophic or soil-borne pathogens. In this study, we isolate a wheat CAD gene TaCAD12 in response to R. cerealis infection through microarray-based comparative transcriptomics, and study the enzyme activity and defense role of TaCAD12 in wheat. The transcriptional levels of TaCAD12 in sharp eyespot-resistant wheat lines were significantly higher compared with those in susceptible wheat lines. The sequence and phylogenetic analyses revealed that TaCAD12 belongs to IV group in CAD family. The biochemical assay proved that TaCAD12 protein is an authentic CAD enzyme and possesses catalytic efficiencies toward both coniferyl aldehyde and sinapyl aldehyde. Knock-down of TaCAD12 transcript significantly repressed resistance of the gene-silenced wheat plants to sharp eyespot caused by R. cerealis, whereas TaCAD12 overexpression markedly enhanced resistance of the transgenic wheat lines to sharp eyespot. Furthermore, certain defense genes (Defensin, PR10, PR17c, and Chitinase1) and monolignol biosynthesis-related genes (TaCAD1, TaCCR, and TaCOMT1) were up-regulated in the TaCAD12-overexpressing wheat plants but down-regulated in TaCAD12-silencing plants. These results suggest that TaCAD12 positively contributes to resistance against sharp eyespot through regulation of the expression of certain defense genes and monolignol biosynthesis-related genes in wheat.

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

    PubMed

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

    2016-09-01

    Ethanol at low concentrations (<4%) can serve as a food source for fruit fly Drosophila melanogaster, whereas at higher concentrations it may be toxic. In this work, protective effects of dietary alpha-ketoglutarate (AKG) against ethanol toxicity were studied. Food supplementation with 10-mM AKG alleviated toxic effects of 8% ethanol added to food, and improved fly development. Two-day-old adult flies, reared on diet containing both AKG and ethanol, possessed higher 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.

  16. Broadening the cofactor specificity of a thermostable alcohol dehydrogenase using rational protein design introduces novel kinetic transient behavior.

    PubMed

    Campbell, Elliot; Wheeldon, Ian R; Banta, Scott

    2010-12-01

    Cofactor specificity in the aldo-keto reductase (AKR) superfamily has been well studied, and several groups have reported the rational alteration of cofactor specificity in these enzymes. Although most efforts have focused on mesostable AKRs, several putative AKRs have recently been identified from hyperthermophiles. The few that have been characterized exhibit a strong preference for NAD(H) as a cofactor, in contrast to the NADP(H) preference of the mesophilic AKRs. Using the design rules elucidated from mesostable AKRs, we introduced two site-directed mutations in the cofactor binding pocket to investigate cofactor specificity in a thermostable AKR, AdhD, which is an alcohol dehydrogenase from Pyrococcus furiosus. The resulting double mutant exhibited significantly improved activity and broadened cofactor specificity as compared to the wild-type. Results of previous pre-steady-state kinetic experiments suggest that the high affinity of the mesostable AKRs for NADP(H) stems from a conformational change upon cofactor binding which is mediated by interactions between a canonical arginine and the 2'-phosphate of the cofactor. Pre-steady-state kinetics with AdhD and the new mutants show a rich conformational behavior that is independent of the canonical arginine or the 2'-phosphate. Additionally, experiments with the highly active double mutant using NADPH as a cofactor demonstrate an unprecedented transient behavior where the binding mechanism appears to be dependent on cofactor concentration. These results suggest that the structural features involved in cofactor specificity in the AKRs are conserved within the superfamily, but the dynamic interactions of the enzyme with cofactors are unexpectedly complex.

  17. A Wheat Cinnamyl Alcohol Dehydrogenase TaCAD12 Contributes to Host Resistance to the Sharp Eyespot Disease

    PubMed Central

    Rong, Wei; Luo, Meiying; Shan, Tianlei; Wei, Xuening; Du, Lipu; Xu, Huijun; Zhang, Zengyan

    2016-01-01

    Sharp eyespot, caused mainly by the necrotrophic fungus Rhizoctonia cerealis, is a destructive disease in hexaploid wheat (Triticum aestivum L.). In Arabidopsis, certain cinnamyl alcohol dehydrogenases (CADs) have been implicated in monolignol biosynthesis and in defense response to bacterial pathogen infection. However, little is known about CADs in wheat defense responses to necrotrophic or soil-borne pathogens. In this study, we isolate a wheat CAD gene TaCAD12 in response to R. cerealis infection through microarray-based comparative transcriptomics, and study the enzyme activity and defense role of TaCAD12 in wheat. The transcriptional levels of TaCAD12 in sharp eyespot-resistant wheat lines were significantly higher compared with those in susceptible wheat lines. The sequence and phylogenetic analyses revealed that TaCAD12 belongs to IV group in CAD family. The biochemical assay proved that TaCAD12 protein is an authentic CAD enzyme and possesses catalytic efficiencies toward both coniferyl aldehyde and sinapyl aldehyde. Knock-down of TaCAD12 transcript significantly repressed resistance of the gene-silenced wheat plants to sharp eyespot caused by R. cerealis, whereas TaCAD12 overexpression markedly enhanced resistance of the transgenic wheat lines to sharp eyespot. Furthermore, certain defense genes (Defensin, PR10, PR17c, and Chitinase1) and monolignol biosynthesis-related genes (TaCAD1, TaCCR, and TaCOMT1) were up-regulated in the TaCAD12-overexpressing wheat plants but down-regulated in TaCAD12-silencing plants. These results suggest that TaCAD12 positively contributes to resistance against sharp eyespot through regulation of the expression of certain defense genes and monolignol biosynthesis-related genes in wheat. PMID:27899932

  18. Rapid inhibition of pyruvate dehydrogenase: an initiating event in high dietary fat-induced loss of metabolic flexibility in the heart.

    PubMed

    Crewe, Clair; Kinter, Michael; Szweda, Luke I

    2013-01-01

    Cardiac function depends on the ability to switch between fatty acid and glucose oxidation for energy production in response to changes in substrate availability and energetic stress. In obese and diabetic individuals, increased reliance on fatty acids and reduced metabolic flexibility are thought to contribute to the development of cardiovascular disease. Mechanisms by which cardiac mitochondria contribute to diet-induced metabolic inflexibility were investigated. Mice were fed a high fat or low fat diet for 1 d, 1 wk, and 20 wk. Cardiac mitochondria isolated from mice fed a high fat diet displayed a diminished ability to utilize the glycolytically derived substrate pyruvate. This response was rapid, occurring within the first day on the diet, and persisted for up to 20 wk. A selective increase in the expression of pyruvate dehydrogenase kinase 4 and inhibition of pyruvate dehydrogenase are responsible for the rapid suppression of pyruvate utilization. An important consequence is that pyruvate dehydrogenase is sensitized to inhibition when mitochondria respire in the presence of fatty acids. Additionally, increased expression of pyruvate dehydrogenase kinase 4 preceded any observed diet-induced reductions in the levels of glucose transporter type 4 and glycolytic enzymes and, as judged by Akt phosphorylation, insulin signaling. Importantly, diminished insulin signaling evident at 1 wk on the high fat diet did not occur in pyruvate dehydrogenase kinase 4 knockout mice. Dietary intervention leads to a rapid decline in pyruvate dehydrogenase kinase 4 levels and recovery of pyruvate dehydrogenase activity indicating an additional form of regulation. Finally, an overnight fast elicits a metabolic response similar to that induced by high dietary fat obscuring diet-induced metabolic changes. Thus, our data indicate that diet-induced inhibition of pyruvate dehydrogenase may be an initiating event in decreased oxidation of glucose and increased reliance of the heart on

  19. An enantioselective NADP(+)-dependent alcohol dehydrogenase responsible for cooxidative production of (3S)-5-hydroxy-3-methyl-pentanoic acid.

    PubMed

    Takeda, Minoru; Matsumura, Aline Tiemi; Kurosaki, Kaishi; Chhetri, Rajan Thapa; Motomatsu, Shigekazu; Suzuki, Ichiro; Sahabi, Danladi Mahuta

    2016-06-01

    A soil bacterium, Mycobacterium sp. B-009, is able to grow on racemic 1,2-propanediol (PD). The strain was revealed to oxidize 3-methyl-1,5-pentanediol (MPD) to 5-hydroxy-3-methyl-pentanoic acid (HMPA) during growth on PD. MPD was converted into an almost equimolar amount of the S-form of HMPA (S-HMPA) at 72%ee, suggesting the presence of an enantioselective MPD dehydrogenase (MPD-DH). As expected, an NADP(+)-dependent alcohol dehydrogenase, which catalyzes the initial step of MPD oxidation, was detected and purified from the cell-free extract. This enzyme was suggested to be a homodimeric medium-chain alcohol dehydrogenase/reductase (MDR). The catalytic and kinetic parameters indicated that MPD is the most suitable substrate for the enzyme. The enzyme was encoded by a 1047-bp gene (mpd1) and several mycobacterial strains were found to have putative MDR genes similar to mpd1. In a phylogenetic tree, MPD-DH formed an independent clade together with the putative MDR of Mycobacterium neoaurum, which produces opportunistic infections.

  20. 3 Beta-hydroxy-delta 5-steroid dehydrogenase/3-keto-delta 5-steroid isomerase from bovine adrenals: mechanism of inhibition by 3-oxo-4-aza steroids and kinetic mechanism of the dehydrogenase.

    PubMed

    Brandt, M; Levy, M A

    1989-01-10

    Several 3-oxo-4-aza steroids (1) have been identified as inhibitors of the 3 beta-hydroxy-delta 5-steroid dehydrogenase/3-keto-delta 5-steroid isomerase catalyzed conversion of pregnenolone to progesterone. By kinetically decoupling the two enzyme activities isolated from bovine adrenal cortex, it has been demonstrated that inhibition by 1 occurs through interference of both activities. A preferred ordered association of substrates to the 3 beta-hydroxy-delta 5-steroid dehydrogenase in which the cofactor binds prior to steroid was determined by isotope exchange at equilibrium. With this result, the dead-end inhibition patterns of 1 with the dehydrogenase were interpreted to originate from a preferred association of inhibitor within an enzyme ternate containing NADH; this proposal is supported by data from multiple inhibition analysis indicating synergistic binding of NADH and 1. Similarly, inhibition of the 3-keto-delta 5-steroid isomerase by the 3-oxo-4-aza steroids was enhanced in the presence of the positive effector NADH. On the basis of pH profiles upon Vm, Vm/Km, and 1/Ki for both enzyme activities, inhibition is proposed to result from the structural similarity of 1 to intermediate states formed upon enzyme catalysis.

  1. In vivo ethanol elimination in man, monkey and rat: A lack of relationship between the ethanol metabolism and the hepatic activities of alcohol and aldehyde dehydrogenases

    SciTech Connect

    Zorzano, A. ); Herrera, E. )

    1990-01-01

    The in vivo ethanol elimination in human subjects, monkeys and rats was investigated after an oral ethanol dosage. After 0.4 g. ethanol/kg of body weight, ethanol elimination was much slower in human subjects than in monkeys. In order to detect a rise in monkey plasma ethanol concentrations as early as observed in human subjects, ethanol had to be administered at a dose of 3 g/kg body weight. Ethanol metabolism in rats was also much faster than in human subjects. However, human liver showed higher alcohol dehydrogenase activity and higher low Km aldehyde dehydrogenase activity than rat liver. Thus, our data suggest a lack of relationship between hepatic ethanol-metabolizing activities and the in vivo ethanol elimination rate.

  2. Partial oxidative conversion of methane to methanol through selective inhibition of methanol dehydrogenase in methanotrophic consortium from landfill cover soil.

    PubMed

    Han, Ji-Sun; Ahn, Chang-Min; Mahanty, Biswanath; Kim, Chang-Gyun

    2013-11-01

    Using a methanotrophic consortium (that includes Methylosinus sporium NCIMB 11126, Methylosinus trichosporium OB3b, and Methylococcus capsulatus Bath) isolated from a landfill site, the potential for partial oxidation of methane into methanol through selective inhibition of methanol dehydrogenase (MDH) over soluble methane monooxygenase (sMMO) with some selected MDH inhibitors at varied concentration range, was evaluated in batch serum bottle and bioreactor experiments. Our result suggests that MDH activity could effectively be inhibited either at 40 mM of phosphate, 100 mM of NaCl, 40 mM of NH4Cl or 50 μM of EDTA with conversion ratios (moles of CH3OH produced per mole CH4 consumed) of 58, 80, 80, and 43 %, respectively. The difference between extent of inhibition in MDH activity and sMMO activity was significantly correlated (n = 6, p < 0.05) with resultant methane to methanol conversion ratio. In bioreactor study with 100 mM of NaCl, a maximum specific methanol production rate of 9 μmol/mg h was detected. A further insight with qPCR analysis of MDH and sMMO coding genes revealed that the gene copy number continued to increase along with biomass during reactor operation irrespective of presence or absence of inhibitor, and differential inhibition among two enzymes was rather the key for methanol production.

  3. Inhibition of the alpha-ketoglutarate dehydrogenase complex by the myeloperoxidase products, hypochlorous acid and mono-N-chloramine.

    PubMed

    Jeitner, Thomas M; Xu, Hui; Gibson, Gary E

    2005-01-01

    Abstract alpha-Ketoglutarate dehydrogenase (KGDHC) complex activity is diminished in a number of neurodegenerative disorders and its diminution in Alzheimer Disease (AD) is thought to contribute to the major loss of cerebral energy metabolism that accompanies this disease. The loss of KGDHC activity appears to be predominantly due to post-translation modifications. Thiamine deficiency also results in decreased KGDHC activity and a selective neuronal loss. Recently, myeloperoxidase has been identified in the activated microglia of brains from AD patients and thiamine-deficient animals. Myeloperoxidase produces a powerful oxidant, hypochlorous acid that reacts with amines to form chloramines. The aim of this study was to investigate the ability of hypochlorous acid and chloramines to inhibit the activity of KGDHC activity as a first step towards investigating the role of myeloperoxidase in AD. Hypochlorous acid and mono-N-chloramine both inhibited purified and cellular KGDHC and the order of inhibition of the purified complex was hypochlorous acid (1x) > mono-N-chloramine (approximately 50x) > hydrogen peroxide (approximately 1,500). The inhibition of cellular KGDHC occurred with no significant loss of cellular viability at all exposure times that were examined. Thus, hypochlorous acid and chloramines have the potential to inactivate a major target in neurodegeneration.

  4. Inhibition of 15-hydroxyprostaglandin dehydrogenase activity in rabbit gastric antral mucosa by panaxynol isolated from oriental medicines.

    PubMed

    Fujimoto, Y; Sakuma, S; Komatsu, S; Sato, D; Nishida, H; Xiao, Y Q; Baba, K; Fujita, T

    1998-09-01

    Panaxynol is a polyacetylene compound with anti-inflammatory and anti-platelet-aggregatory effects isolated from commonly used oriental medicines. The effects of panaxynol on the activity of prostaglandin-synthesizing and catabolizing enzymes in the rabbit gastric antral mucosa have been examined. At concentrations ranging from 25 to 200 microM panaxynol had no effect on the synthesis of prostaglandins E2, F2alpha and D2 from exogenous arachidonic acid in the microsomal fraction of the gastric mucosa whereas at 25-200 microM it dose-dependently inhibited the activity of 15-hydroxyprostaglandin dehydrogenase (PGDH), which catalyses the initial step of prostaglandin catabolism, in the cytosolic fraction. The concentration required for 50% inhibition (IC50) was approximately 25 microM. Inhibition of PGDH by panaxynol was non-competitive with regard to NAD+ and prostaglandin E2. These results suggest that panaxynol has the potential to inhibit PGDH activity in gastric mucosa, possibly as a result of pharmacological activity.

  5. Probing stereoselectivity and pro-chirality of hydride transfer during short-chain alcohol dehydrogenase activity: a combined quantitative 2H NMR and computational approach.

    PubMed

    Kwiecień, Renata A; Ayadi, Farouk; Nemmaoui, Youssef; Silvestre, Virginie; Zhang, Ben-Li; Robins, Richard J

    2009-02-01

    Different members of the alcohol oxidoreductase family can transfer the hydride of NAD(P)H to either the re- or the si-face of the substrate. The enantioselectivity of transfer is very variable, even for a range of substrates reduced by the same enzyme. Exploiting quantitative isotopic (2)H NMR to measure the transfer of (2)H from NAD(P)(2)H to ethanol, a range of enantiomeric excess between 0.38 and 0.98, depending on the origin of the enzyme and the nature of the cofactor, has been determined. Critically, in no case was only (R)-[1-(2)H]ethanol or (S)-[1-(2)H]ethanol obtained. By calculating the relative energies of the active site models for hydride transfer to the re- or si-face of short-chain aldehydes by alcohol dehydrogenase from Saccharomyces cerevisiae and Lactobacillus brevis, it is shown that the differences in the energy of the systems when the substrate is positioned with the alkyl group in one or the other pocket of the active site could play a role in determining stereoselectivity. These experiments help to provide insight into structural features that influence the potential catalytic flexibility of different alcohol dehydrogenase activities.

  6. Introducing a single secondary alcohol dehydrogenase into butanol-tolerant Clostridium acetobutylicum Rh8 switches ABE fermentation to high level IBE fermentation

    PubMed Central

    2012-01-01

    Background Previously we have developed a butanol tolerant mutant of Clostridium acetobutylicum Rh8, from the wild type strain DSM 1731. Strain Rh8 can tolerate up to 19 g/L butanol, with solvent titer improved accordingly, thus exhibiting industrial application potential. To test if strain Rh8 can be used for production of high level mixed alcohols, a single secondary alcohol dehydrogenase from Clostridium beijerinckii NRRL B593 was overexpressed in strain Rh8 under the control of thl promoter. Results The heterogenous gene sADH was functionally expressed in C. acetobutylicum Rh8. This simple, one-step engineering approach switched the traditional ABE (acetone-butanol-ethanol) fermentation to IBE (isopropanol-butanol-ethanol) fermentation. The total alcohol titer reached 23.88 g/l (7.6 g/l isopropanol, 15 g/l butanol, and 1.28 g/l ethanol) with a yield to glucose of 31.42%. The acid (butyrate and acetate) assimilation rate in isopropanol producing strain Rh8(psADH) was increased. Conclusions The improved butanol tolerance and the enhanced solvent biosynthesis machinery in strain Rh8 is beneficial for production of high concentration of mixed alcohols. Strain Rh8 can thus be considered as a good host for further engineering of solvent/alcohol production. PMID:22742819

  7. Species-specific differences in tissue-specific expression of alcohol dehydrogenase are under the control of complex cis-acting loci: Evidence from Drosophila hybrids

    SciTech Connect

    Ranganayakulu, G.; Reddy, A.R. ); Kirkpatrick, R.B.; Martin, P.F. )

    1991-12-01

    Differences in the expression of alcohol dehydrogenase in the hindgut and testis of adult Drosophila virilis, D. texana, D. novamexicana and D. borealis flies were observed. These heritable differences do not arise due to chromosomal rearrangements, since the polytene chromosome banding patterns did not reveal any such gross chromosomal rearrangements near the Adh locus in any of the tested species. Analysis of the interspecific hybrids revealed that these differences are controlled by complex cis-acting genetic loci. Further, the cis-acting locus controlling the expression of ADH in testis was found to be separable by crossing-over.

  8. The interaction of catalytic metal ions and ionizing groups in equilibrium studies and in transient intermediates of metal-substituted alcohol dehydrogenases.

    PubMed

    Maret, W; Gerber, M; Zeppezauer, M; Dunn, M F

    1985-01-01

    The step of ternary complex interconversion in the reaction catalyzed by horse liver alcohol dehydrogenase has been resolved into five distinct molecular species with the aid of metal-substitution studies in combination with rapid-scanning spectrophotometry. A correlation with electronic absorption spectra at equilibrium provides structural insights into these intermediates. In contrast to NADH, NAD+ only leads to a conformational change of the protein when a negative charge has been created in the vicinity of the catalytic metal ion. This paper presents also a reevaluation of previous assignments of catalytically important groups in the light of some recent results.

  9. Scaffold electrodes based on thioctic acid-capped gold nanoparticles coordinated Alcohol Dehydrogenase and Azure A films for high performance biosensor.

    PubMed

    Gómez-Anquela, C; García-Mendiola, T; Abad, José M; Pita, M; Pariente, F; Lorenzo, E

    2015-12-01

    Nanometric size gold nanoparticles capped with thiotic acid are used to coordinate with the Zn (II) present in the catalytic center of Alcohol Dehydrogenase (ADH). In combination with the NADH oxidation molecular catalyst Azure A, electrografted onto carbon screen-printed electrodes, they are used as scaffold electrodes for the construction of a very efficient ethanol biosensor. The final biosensing device exhibits a highly efficient ethanol oxidation with low overpotential of -0.25 V besides a very good analytical performance with a detection limit of 0.14±0.01 μM and a stable response for more than one month.

  10. Cloning and functional analysis of adhS gene encoding quinoprotein alcohol dehydrogenase subunit III from Acetobacter pasteurianus SKU1108.

    PubMed

    Masud, Uraiwan; Matsushita, Kazunobu; Theeragool, Gunjana

    2010-03-31

    The adhS gene which encodes the smallest subunit, subunit III, of quinoprotein alcohol dehydrogenase (PQQ-ADH) from Acetobacter pasteurianus SKU1108 has been cloned and characterized. The role of this subunit on the function of PQQ-ADH was investigated by construction of adhS gene disruptant and mutants. The adhS gene disruptant completely lost its PQQ-ADH activity and acetate-producing ability but retained acetic acid toleration. In contrast, this disruptant grew well, even better than the wild type, in the ethanol containing medium even though its PQQ-ADH activity and ethanol oxidizing ability was completely lost, while NAD(+)-dependent ADH (NAD(+)-ADH) was induced. Heme staining and immunoblot analysis of both membrane and soluble fractions with anti-ADH subunit III suggested that ethanol did not affect the adhS gene expression but induced PQQ-ADH activity. Over-expressed adhS did not enhance acetic acid production in both the wild type and the adhS disruptant. In addition, deletion analysis of upstream region of adhS gene suggested that its tentative promoter(s) might be located at around 118-268 bp upstream from an initiation codon. Random mutagenesis of adhS gene revealed that complete loss of PQQ-ADH activity and ethanol oxidizing ability were observed in the mutants' lack of the 140 and 73 amino acid residues at the C-terminal, whereas the lack of 22 amino acid residues at the C-terminal affected neither the PQQ-ADH activity nor ethanol oxidizing ability. In addition, some amino acid substitutions such as Leu18Gln, Ala26Val, Val36Ile, Val54Ile, Gly55Asp, Val70Ala and Val107Ala did not show any affect on PQQ-ADH activity and ethanol oxidizing ability. Interestingly, alteration of Thr104Lys led to a complete loss of ethanol oxidizing ability. However, point mutation at the possible promoter region also exhibited low PQQ-ADH activity and ethanol oxidizing ability. This result suggests that 104Thr might be involved in molecular coupling with subunit I in order

  11. Inhibition of mitochondrial 2-oxoglutarate dehydrogenase impairs viability of cancer cells in a cell-specific metabolism-dependent manner

    PubMed Central

    Bunik, Victoria I.; Mkrtchyan, Garik; Grabarska, Aneta; Oppermann, Henry; Daloso, Danilo; Araujo, Wagner L.; Juszczak, Malgorzata; Rzeski, Wojciech; Bettendorff, Lucien; Fernie, Alisdair R.; Meixensberger, Jürgen; Stepulak, Andrzej; Gaunitz, Frank

    2016-01-01

    2-Oxoglutarate dehydrogenase (OGDH) of the tricarboxylic acid (TCA) cycle is often implied to be inactive in cancer, but this was not experimentally tested. We addressed the question through specific inhibition of OGDH by succinyl phosphonate (SP). SP action on different cancer cells was investigated using indicators of cellular viability and reactive oxygen species (ROS), metabolic profiling and transcriptomics. Relative sensitivity of various cancer cells to SP changed with increasing SP exposure and could differ in the ATP- and NAD(P)H-based assays. Glioblastoma responses to SP revealed metabolic sub-types increasing or decreasing cellular ATP/NAD(P)H ratio under OGDH inhibition. Cancer cell homeostasis was perturbed also when viability indicators were SP-resistant, e.g. in U87 and N2A cells. The transcriptomics database analysis showed that the SP-sensitive cells, such as A549 and T98G, exhibit the lowest expression of OGDH compared to other TCA cycle enzymes, associated with higher expression of affiliated pathways utilizing 2-oxoglutarate. Metabolic profiling confirmed the dependence of cellular SP reactivity on cell-specific expression of the pathways. Thus, oxidative decarboxylation of 2-oxoglutarate is significant for the interdependent homeostasis of NAD(P)H, ATP, ROS and key metabolites in various cancer cells. Assessment of cell-specific responses to OGDH inhibition is of diagnostic value for anticancer strategies. PMID:27027236

  12. Monomethylarsonous acid (MMA(III)) and arsenite: LD(50) in hamsters and in vitro inhibition of pyruvate dehydrogenase.

    PubMed

    Petrick, J S; Jagadish, B; Mash, E A; Aposhian, H V

    2001-06-01

    Monomethylarsonous acid (MMA(III)), a metabolite of inorganic arsenic, has received very little attention from investigators of arsenic metabolism in humans. MMA(III), like sodium arsenite, contains arsenic in the +3 oxidation state. Although we have previously demonstrated that it is more toxic than arsenite in cultured Chang human hepatocytes, there are no data showing in vivo toxicity of MMA(III). When MMA(III) or sodium arsenite was administered intraperitoneally to hamsters, the LD(50)s were 29.3 and 112.0 micromol/kg of body wt, respectively. In addition, inhibition of hamster kidney or purified porcine heart pyruvate dehydrogenase (PDH) activity by MMA(III) or arsenite was determined. To inhibit hamster kidney PDH activity by 50%, the concentrations (mean +/- SE) of MMA(III) as methylarsine oxide, MMA(III) as diiodomethylarsine, and arsenite were 59.9 +/- 6.5, 62.0 +/- 1.8, and 115.7 +/- 2.3 microM, respectively. To inhibit activity of purified porcine heart PDH activity by 50%, the concentrations (mean +/- SE) of MMA(III) as methylarsine oxide and arsenite were 17.6 +/- 4.1 and 106.1 +/- 19.8 microM, respectively. These data demonstrate that MMA(III) is more toxic than inorganic arsenite, both in vivo and in vitro, and call into question the hypothesis that methylation of inorganic arsenic is a detoxication process.

  13. Inhibition of snowshoe hare succinate dehydrogenase activity as a mechanism of deterrence for papyriferic acid in birch.

    PubMed

    Forbey, Jennifer Sorensen; Pu, Xinzhu; Xu, Dong; Kielland, Knut; Bryant, John

    2011-12-01

    The plant secondary metabolite papyriferic acid (PA) deters browsing by snowshoe hares (Lepus americanus) on the juvenile developmental stage of the Alaska paper birch (Betula neoalaskana). However, the physiological mechanism that reduces browsing remains unknown. We used pharmacological assays and molecular modeling to test the hypothesis that inhibition of succinate dehydrogenase (SDH) is a mode of action (MOA) of toxicity of PA in snowshoe hares. We tested this hypothesis by measuring the effect of PA on the activity of SDH in liver mitochondria isolated from wild hares. In addition, we used molecular modeling to determine the specific binding site of PA on SDH. We found that PA inhibits SDH from hares by an uncompetitive mechanism in a dose-dependent manner. Molecular modeling suggests that inhibition of SDH is a result of binding of PA at the ubiquinone binding sites in complex II. Our results provide a MOA for toxicity that may be responsible for the concentration-dependent anti-feedant effects of PA. We propose that snowshoe hares reduce the dose-dependent toxic consequences of PA by relying on efflux transporters and metabolizing enzymes that lower systemic exposure to dietary PA.

  14. Potent inhibition of alcohol self-administration in alcohol-preferring rats by a κ-opioid receptor antagonist.

    PubMed

    Cashman, John R; Azar, Marc R

    2014-07-01

    A substituted aryl amide derivative of 6-naltrexamine--17-cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-[(4'-trimethylfluoro)benzamido]morphinan-hydrochloride--(compound 5), previously shown to be a potent κ-opioid receptor antagonist, was used to characterize the physicochemical properties and efficacy to decrease alcohol self-administration in alcohol-preferring rats (P-rats) and binge-like P-rats. Previous studies showed that compounds closely related to compound 5 possessed favorable properties regarding penetration of the blood-brain barrier. Pharmacokinetic studies showed that compound 5 had acceptable bioavailability. In contrast to other κ-receptor antagonists, in particular norbinaltorphimine, compound 5 showed favorable drug-like properties. Based on these findings, further studies were done. Safety studies showed that compound 5 was not hepatotoxic at doses 200-fold greater than an efficacious dose. The effects of compound 5 or naltrexone on the hepatotoxicity of thiobenzamide were investigated. In contrast to naltrexone, which exacerbated thiobenzamide-mediated hepatotoxicity, compound 5 was observed to be hepatoprotective. Based on the physicochemical properties of compound 5, the compound was examined in rat animal models of alcohol self-administration. The inhibition of ethanol self-administration by compound 5 in alcohol-dependent and alcohol-nondependent P-rats trained to self-administer a 10% (w/v) ethanol solution, using operant techniques, showed very potent efficacy (i.e., estimated ED50 values of 4-5 μg/kg). In a binge-like P-rat animal model, inhibition of alcohol self-administration by compound 5 had an estimated ED50 value of 8 μg/kg. The results suggest that compound 5 is a potent drug-like κ-opioid receptor antagonist of utility in alcohol cessation medications development.

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

    PubMed

    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.

  16. Computer-graphics interpretations of residue exchanges between the alpha, beta and gamma subunits of human-liver alcohol dehydrogenase class I isozymes.

    PubMed

    Eklund, H; Horjales, E; Vallee, B L; Jörnvall, H

    1987-09-01

    Three-dimensional models of human alcohol dehydrogenase subunits have been constructed, based on the homologous horse enzyme, with computer graphics. All types of class I subunits (alpha, beta, and gamma) and the major allelic variants (beta 1/beta 2 and gamma 1/gamma 2) have been studied. Residue differences between the E-type subunit of the horse enzyme and any of the subunits of the human isozymes occur at 64 positions, about half of which are isozyme-specific. About two thirds of the substitutions are at the surface and all differences can be accommodated in highly conserved three-dimensional structures. The model of the gamma isozyme is most similar to the crystallographically analyzed horse liver E-type alcohol dehydrogenase, and has all the functional residues identical to those of the E subunit except for one which is slightly smaller: Val-141 in the substrate pocket. The residues involved in coenzyme binding are generally conserved between the horse enzyme and the alpha, beta, and gamma types of the human enzyme. In contrast, single exchanges of these residues are the ones involved in the major allelic differences (beta 1 versus beta 2 and gamma 1 versus gamma 2), which affects the overall rate of alcohol oxidation since NADH dissociation is the rate-determining step. Residue 47 is His in beta 2 and Arg in the beta 1, gamma 1, and gamma 2 subunits, and in horse liver alcohol dehydrogenase. Both His and Arg can make a hydrogen bond to a phosphate oxygen atom of NAD; hence the lower turnover rate of beta 1 apparently derives from a charge effect. The substitution to Gly in the alpha subunit results in one less hydrogen bond in NAD binding, and consequently in rapid dissociation. This may explain why the overall rate is an order of magnitude faster than that of beta 1. The important difference between gamma 1 and gamma 2 is an exchange at position 271 from Arg to Gln which can give a hydrogen bond from Gln in gamma 2 to the adenine of NAD. The tighter binding

  17. Enhancement of cell growth and glycolic acid production by overexpression of membrane-bound alcohol dehydrogenase in Gluconobacter oxydans DSM 2003.

    PubMed

    Zhang, Huan; Shi, Lulu; Mao, Xinlei; Lin, Jinping; Wei, Dongzhi

    2016-11-10

    Membrane-bound alcohol dehydrogenase (mADH) was overexpressed in Gluconobacter oxydans DSM 2003, and the effects on cell growth and glycolic acid production were investigated. The transcription levels of two terminal ubiquinol oxidases (bo3 and bd) in the respiratory chain of the engineered strain G. oxydans-adhABS were up-regulated by 13.4- and 3.8-fold, respectively, which effectively enhanced the oxygen uptake rate, resulting in higher resistance to acid. The cell biomass of G. oxydans-adhABS could increase by 26%-33% when cultivated in a 7L bioreactor. The activities of other major membrane-bound dehydrogenases were also increased to some extent, particularly membrane-bound aldehyde dehydrogenase (mALDH), which is involved in the catalytic oxidation of aldehydes to the corresponding acids and was 1.26-fold higher. Relying on the advantages of the above, G. oxydans-adhABS could produce 73.3gl(-1) glycolic acid after 45h of bioconversion with resting cells, with a molar yield 93.5% and a space-time yield of 1.63gl(-1)h(-1). Glycolic acid production could be further improved by fed-batch fermentation. After 45h of culture, 113.8gl(-1) glycolic acid was accumulated, with a molar yield of 92.9% and a space-time yield of 2.53gl(-1)h(-1), which is the highest reported glycolic acid yield to date.

  18. The TM2 6′ Position of GABAA Receptors Mediates Alcohol Inhibition

    PubMed Central

    Howard, Rebecca J.; Trudell, James R.; Harris, R. Adron

    2012-01-01

    Ionotropic GABAA receptors (GABAARs), which mediate inhibitory neurotransmission in the central nervous system, are implicated in the behavioral effects of alcohol and alcoholism. Site-directed mutagenesis studies support the presence of discrete molecular sites involved in alcohol enhancement and, more recently, inhibition of GABAARs. We used Xenopus laevis oocytes to investigate the 6′ position in the second transmembrane region of GABAARs as a site influencing alcohol inhibition. We asked whether modification of the 6′ position by substitution with larger residues or methanethiol labeling [using methyl methanethiosulfonate (MMTS)] of a substituted cysteine, reduced GABA action and/or blocked further inhibition by alcohols. Labeling of the 6′ position in either α2 or β2 subunits reduced responses to GABA. In addition, methanol and ethanol potentiation increased after MMTS labeling or substitution with tryptophan or methionine, consistent with elimination of an inhibitory site for these alcohols. Specific alcohols, but not the anesthetic etomidate, competed with MMTS labeling at the 6′ position. We verified a role for the 6′ position in previously tested α2β2 as well as more physiologically relevant α2β2γ2s GABAARs. Finally, we built a novel molecular model based on the invertebrate glutamate-gated chloride channel receptor, a GABAAR homolog, revealing that the 6′ position residue faces the channel pore, and modification of this residue alters volume and polarity of the pore-facing cavity in this region. These results indicate that the 6′ positions in both α2 and β2 GABAAR subunits mediate inhibition by short-chain alcohols, which is consistent with the presence of multiple counteracting sites of action for alcohols on ligand-gated ion channels. PMID:22072732

  19. The TM2 6' position of GABA(A) receptors mediates alcohol inhibition.

    PubMed

    Johnson, W David; Howard, Rebecca J; Trudell, James R; Harris, R Adron

    2012-02-01

    Ionotropic GABA(A) receptors (GABA(A)Rs), which mediate inhibitory neurotransmission in the central nervous system, are implicated in the behavioral effects of alcohol and alcoholism. Site-directed mutagenesis studies support the presence of discrete molecular sites involved in alcohol enhancement and, more recently, inhibition of GABA(A)Rs. We used Xenopus laevis oocytes to investigate the 6' position in the second transmembrane region of GABA(A)Rs as a site influencing alcohol inhibition. We asked whether modification of the 6' position by substitution with larger residues or methanethiol labeling [using methyl methanethiosulfonate (MMTS)] of a substituted cysteine, reduced GABA action and/or blocked further inhibition by alcohols. Labeling of the 6' position in either α2 or β2 subunits reduced responses to GABA. In addition, methanol and ethanol potentiation increased after MMTS labeling or substitution with tryptophan or methionine, consistent with elimination of an inhibitory site for these alcohols. Specific alcohols, but not the anesthetic etomidate, competed with MMTS labeling at the 6' position. We verified a role for the 6' position in previously tested α2β2 as well as more physiologically relevant α2β2γ2s GABA(A)Rs. Finally, we built a novel molecular model based on the invertebrate glutamate-gated chloride channel receptor, a GABA(A)R homolog, revealing that the 6' position residue faces the channel pore, and modification of this residue alters volume and polarity of the pore-facing cavity in this region. These results indicate that the 6' positions in both α2 and β2 GABA(A)R subunits mediate inhibition by short-chain alcohols, which is consistent with the presence of multiple counteracting sites of action for alcohols on ligand-gated ion channels.

  20. Immunoresponsive Gene 1 and Itaconate Inhibit Succinate Dehydrogenase to Modulate Intracellular Succinate Levels.

    PubMed

    Cordes, Thekla; Wallace, Martina; Michelucci, Alessandro; Divakaruni, Ajit S; Sapcariu, Sean C; Sousa, Carole; Koseki, Haruhiko; Cabrales, Pedro; Murphy, Anne N; Hiller, Karsten; Metallo, Christian M

    2016-07-01

    Metabolic reprogramming is emerging as a hallmark of the innate immune response, and the dynamic control of metabolites such as succinate serves to facilitate the execution of inflammatory responses in macrophages and other immune cells. Immunoresponsive gene 1 (Irg1) expression is induced by inflammatory stimuli, and its enzyme product cis-aconitate decarboxylase catalyzes the production of itaconate from the tricarboxylic acid cycle. Here we identify an immunometabolic regulatory pathway that links Irg1 and itaconate production to the succinate accumulation that occurs in the context of innate immune responses. Itaconate levels and Irg1 expression correlate strongly with succinate during LPS exposure in macrophages and non-immune cells. We demonstrate that itaconate acts as an endogenous succinate dehydrogenase inhibitor to cause succinate accumulation. Loss of itaconate production in activated macrophages from Irg1(-/-) mice decreases the accumulation of succinate in response to LPS exposure. This metabolic network links the innate immune response and tricarboxylic acid metabolism to function of the electron transport chain.

  1. The Genetics of a Small Autosomal Region of DROSOPHILA MELANOGASTER Containing the Structural Gene for Alcohol Dehydrogenase. II. Lethal Mutations in the Region

    PubMed Central

    Woodruff, R. C.; Ashburner, M.

    1979-01-01

    Forty-seven lethal mutations and alleles of nine visible loci (including alcohol dehydrogenase) have been mapped by both deficiency mapping and, in most cases, by recombination mapping to a small region (34D-35C) of chromosome arm 2L of Drosophila melanogaster. The lethals fall into approximately 21 complementation groups, and we estimate that the total number of lethal plus visible complementation groups within the 34-band deficiency, Df(2L)64j, is approximately 34, a remarkable numerical coincidence. The possible genetic significance of this coincidence is discussed. Lethals mapping close to the structural gene for alcohol dehydrogenase, both distally and proximally, have been identified and will be used for the construction of selective crosses for the study of exchange within this locus. Despite many abnormal cytological features (e.g., ectopic pairing, weak points) region 35 of chromosome arm 2L does not display any unusual genetic features; indeed, in terms of the amount of recombination per band and the average map distance between adjacent loci, this region is similar to that between zeste and white on the X chromosome. PMID:115744

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

    SciTech Connect

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

    1991-04-01

    Zymomonas mobilis genes for pyruvate decarboxylase (pdc) and alcohol dehydrogenase II (adhB) were integrated into the Escherichia coli chromosome within or near the pyruvate formate-lyase gene (pfl). Integration improved the stability of the Z. mobilis genes in E. coli, but further selection was required to increase expression. Spontaneous mutants were selected for resistance to high levels of chloramphenicol that also expressed high levels of the Z. mobilis genes. Analogous mutants were selected for increased expression of alcohol dehydrogenase on aldehyde indicator plates. These mutants were functionally equivalent to the previous plasmid-based strains for the fermentation of xylose and glucose to ethanol. Ethanol concentrations of 54.4 and 41.6 g/liter were obtained from 10% glucose and 8% xylose, respectively. The efficiency of conversion exceeded theoretical limits (0.51 g of ethanol/g of sugar) on the basis of added sugars because of the additional production of ethanol from the catabolism of complex nutrients. Further mutations were introduced to inactivate succinate production (frd) and to block homologous recombination (recA).

  3. Development of an Alcohol Dehydrogenase Biosensor for Ethanol Determination with Toluidine Blue O Covalently Attached to a Cellulose Acetate Modified Electrode

    PubMed Central

    Alpat, Şenol; Telefoncu, Azmi

    2010-01-01

    In this work, a novel voltammetric ethanol biosensor was constructed using alcohol dehydrogenase (ADH). Firstly, alcohol dehydrogenase was immobilized on the surface of a glassy carbon electrode modified by cellulose acetate (CA) bonded to toluidine blue O (TBO). Secondly, the surface was covered by a glutaraldehyde/bovine serum albumin (BSA) cross-linking procedure to provide a new voltammetric sensor for the ethanol determination. In order to fabricate the biosensor, a new electrode matrix containing insoluble Toluidine Blue O (TBO) was obtained from the process, and enzyme/coenzyme was combined on the biosensor surface. The influence of various experimental conditions was examined for the characterization of the optimum analytical performance. The developed biosensor exhibited sensitive and selective determination of ethanol and showed a linear response between 1 × 10−5 M and 4 × 10−4 M ethanol. A detection limit calculated as three times the signal-to-noise ratio was 5.0 × 10−6 M. At the end of the 20th day, the biosensor still retained 50% of its initial activity. PMID:22315566

  4. Modulation of glycerol and ethanol yields during alcoholic fermentation in Saccharomyces cerevisiae strains overexpressed or disrupted for GPD1 encoding glycerol 3-phosphate dehydrogenase.

    PubMed

    Michnick, S; Roustan, J L; Remize, F; Barre, P; Dequin, S

    1997-07-01

    The possibility of the diversion of carbon flux from ethanol towards glycerol in Saccharomyces cerevisiae during alcoholic fermentation was investigated. Variations in the glycerol 3-phosphate dehydrogenase (GPDH) level and similar trends for alcohol dehydrogenase (ADH), pyruvate decarboxylase and glycerol-3-phosphatase were found when low and high glycerol-forming wine yeast strains were compared. GPDH is thus a limiting enzyme for glycerol production. Wine yeast strains with modulated GPD1 (encoding one of the two GPDH isoenzymes) expression were constructed and characterized during fermentation on glucose-rich medium. Engineered strains fermented glucose with a strongly modified [glycerol] : [ethanol] ratio. gpd1delta mutants exhibited a 50% decrease in glycerol production and increased ethanol yield. Overexpression of GPD1 on synthetic must (200 g/l glucose) resulted in a substantial increase in glycerol production ( x 4) at the expense of ethanol. Acetaldehyde accumulated through the competitive regeneration of NADH via GPDH. Accumulation of by-products such as pyruvate, acetate, acetoin, 2,3 butane-diol and succinate was observed, with a marked increase in acetoin production.

  5. Development of an alcohol dehydrogenase biosensor for ethanol determination with toluidine blue O covalently attached to a cellulose acetate modified electrode.

    PubMed

    Alpat, Senol; Telefoncu, Azmi

    2010-01-01

    In this work, a novel voltammetric ethanol biosensor was constructed using alcohol dehydrogenase (ADH). Firstly, alcohol dehydrogenase was immobilized on the surface of a glassy carbon electrode modified by cellulose acetate (CA) bonded to toluidine blue O (TBO). Secondly, the surface was covered by a glutaraldehyde/bovine serum albumin (BSA) cross-linking procedure to provide a new voltammetric sensor for the ethanol determination. In order to fabricate the biosensor, a new electrode matrix containing insoluble Toluidine Blue O (TBO) was obtained from the process, and enzyme/coenzyme was combined on the biosensor surface. The influence of various experimental conditions was examined for the characterization of the optimum analytical performance. The developed biosensor exhibited sensitive and selective determination of ethanol and showed a linear response between 1 × 10(-5) M and 4 × 10(-4) M ethanol. A detection limit calculated as three times the signal-to-noise ratio was 5.0 × 10(-6) M. At the end of the 20(th) day, the biosensor still retained 50% of its initial activity.

  6. Inhibiting 11β-hydroxysteroid dehydrogenase type 1 prevents stress effects on hippocampal synaptic plasticity and impairs contextual fear conditioning.

    PubMed

    Sarabdjitsingh, R Angela; Zhou, Ming; Yau, Joyce L W; Webster, Scott P; Walker, Brian R; Seckl, Jonathan R; Joëls, Marian; Krugers, Harm J

    2014-06-01

    11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) catalyzes intracellular regeneration of corticosterone and cortisol, thereby enhancing glucocorticoid action. Inhibition of 11β-HSD1 reverses the deficits in cognition with aging, a state of elevated glucocorticoid levels. However, any impact of 11β-HSD1 inhibition during high glucocorticoid states in younger animals is unknown. Here we examined whether a single injection of the selective 11β-HSD1 inhibitor UE2316 modifies the effect of stress on hippocampal long-term potentiation and fear conditioning, a learning paradigm that is strongly modulated by glucocorticoids. We found that novelty stress suppresses hippocampal synaptic potentiation. This effect was completely prevented by administration of UE2316 one hour before stress exposure. A single injection of UE2316 also impaired contextual, but not tone-cue-fear conditioning. These observations suggest that local metabolism of glucocorticoids is relevant for the outcome of stress effects on hippocampal synaptic plasticity and contextual fear conditioning. Selective 11β-HSD1 inhibitors may be an interesting new approach to the prevention of trauma-associated psychopathology.

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

    PubMed

    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.

  8. Molecular basis for covalent inhibition of glyceraldehyde-3-phosphate dehydrogenase by a 2-phenoxy-1,4-naphthoquinone small molecule.

    PubMed

    Bruno, Stefano; Uliassi, Elisa; Zaffagnini, Mirko; Prati, Federica; Bergamini, Christian; Amorati, Riccardo; Paredi, Gianluca; Margiotta, Marilena; Conti, Paola; Costi, Maria Paola; Kaiser, Marcel; Cavalli, Andrea; Fato, Romana; Bolognesi, Maria Laura

    2017-01-12

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has recently gained attention as an antiprotozoan and anticancer drug target. We have previously identified 2-phenoxy-1,4-naphthoquinone as an inhibitor of both Trypanosoma brucei and human GAPDH. Herein, through multiple chemical, biochemical, and biological studies, and through the design of analogs, we confirmed the formation of a covalent adduct, we clarified the inhibition mechanism, and we demonstrated antitrypanosomal, antiplasmodial, and cytotoxic activities in cell cultures. The overall results lent support to the hypothesis that 2-phenoxy-1,4-naphthoquinone binds the GAPDH catalytic cysteine covalently through a phenolate displacement mechanism. By investigating the reactivity of 2-phenoxy-1,4-naphthoquinone and its analogs with four GAPDH homologs, we showed that the covalent inhibition is not preceded by the formation of a strong non-covalent complex. However, an up to fivefold difference in inactivation rates among homologs hinted at structural or electrostatic differences of their active sites that could be exploited to further design kinetically selective inhibitors. Moreover, we preliminarily showed that 2-phenoxy-1,4-naphthoquinone displays selectivity for GAPDHs over two other cysteine-dependent enzymes, supporting its suitability as a warhead starting fragment for the design of novel inhibitors.

  9. Aldehyde dehydrogenases inhibition eradicates leukemia stem cells while sparing normal progenitors

    PubMed Central

    Venton, G; Pérez-Alea, M; Baier, C; Fournet, G; Quash, G; Labiad, Y; Martin, G; Sanderson, F; Poullin, P; Suchon, P; Farnault, L; Nguyen, C; Brunet, C; Ceylan, I; Costello, R T

    2016-01-01

    The vast majority of patients with acute myeloid leukemia (AML) achieve complete remission (CR) after standard induction chemotherapy. However, the majority subsequently relapse and die of the disease. A leukemia stem cell (LSC) paradigm has been invoked to explain this failure of CR to reliably translate into cure. Indeed, LSCs are highly enriched in CD34+CD38− leukemic cells that exhibit positive aldehyde dehydrogenase activity (ALDH+) on flow cytometry, these LSCs are resistant to currently existing treatments in AML such as cytarabine and anthracycline that, at the cost of great toxicity on normal cells, are highly active against the leukemic bulk, but spare the LSCs responsible for relapse. To try to combat the LSC population selectively, a well-characterized ALDH inhibitor by the trivial name of dimethyl ampal thiolester (DIMATE) was assessed on sorted CD34+CD38− subpopulations from AML patients and healthy patients. ALDH activity and cell viability were monitored by flow cytometry. From enzyme kinetic studies DIMATE is an active enzyme-dependent, competitive, irreversible inhibitor of ALDH1. On cells in culture, DIMATE is a powerful inhibitor of ALDHs 1 and 3, has a major cytotoxic activity on human AML cell lines. Moreover, DIMATE is highly active against leukemic populations enriched in LSCs, but, unlike conventional chemotherapy, DIMATE is not toxic for healthy hematopoietic stem cells which retained, after treatment, their self-renewing and multi-lineage differentiation capacity in immunodeficient mice, xenografted with human leukemic cells. DIMATE eradicates specifically human AML cells and spares healthy mouse hematologic cells. PMID:27611922

  10. Impact of alcohol use on EEG dynamics of response inhibition: a cotwin control analysis.

    PubMed

    Harper, Jeremy; Malone, Stephen M; Iacono, William G

    2016-11-18

    Research indicates that alcohol misuse is associated with behavioral disinhibition, but the neurophysiological mechanisms governing this relationship remain largely unknown. Recent work suggests that successful inhibition and cognitive control involve electrophysiological theta-band dynamics, including medial frontal cortex (MFC) power enhancement and functional connectivity between the MFC and dorsal prefrontal cortex (dPFC) regions, which may be disrupted by alcohol misuse. In addition, research suggests that, compared to men, women are at heightened risk of experiencing the negative physical and neurocognitive correlates of drinking. The present study tested the hypothesis that alcohol misuse has a deleterious effect on theta-band response inhibition EEG dynamics in a sample of 300 24-year-old same-sex twins. A cotwin control (CTC) design was used to disentangle premorbid risk for alcohol use from the causal effects of alcohol exposure. Drinking was negatively associated with theta-band MFC power and MFC-dPFC connectivity during response inhibition, and this effect was stronger among women. The CTC analysis suggested that, for women, reduced nogo-related theta-band MFC power and MFC-dPFC connectivity were both consistent with the potential deleterious causal effects of alcohol exposure. These findings suggest that diminished theta-band MFC power and MFC-dPFC connectivity may be neurophysiological mechanisms underlying alcohol-related disinhibition. Although preliminary, these results suggest that normative levels of alcohol use during emerging adulthood have potential sex-specific causal effects on response inhibition EEG dynamics, and thus have potentially significant public health implications.

  11. A role for AMPK in the inhibition of glucose-6-phosphate dehydrogenase by polyunsaturated fatty acids

    SciTech Connect

    Kohan, Alison B.; Talukdar, Indrani; Walsh, Callee M.; Salati, Lisa M.

    2009-10-09

    Both polyunsaturated fatty acids and AMPK promote energy partitioning away from energy consuming processes, such as fatty acid synthesis, towards energy generating processes, such as {beta}-oxidation. In this report, we demonstrate that arachidonic acid activates AMPK in primary rat hepatocytes, and that this effect is p38 MAPK-dependent. Activation of AMPK mimics the inhibition by arachidonic acid of the insulin-mediated induction of G6PD. Similar to intracellular signaling by arachidonic acid, AMPK decreases insulin signal transduction, increasing Ser{sup 307} phosphorylation of IRS-1 and a subsequent decrease in AKT phosphorylation. Overexpression of dominant-negative AMPK abolishes the effect of arachidonic acid on G6PD expression. These data suggest a role for AMPK in the inhibition of G6PD by polyunsaturated fatty acids.

  12. A ROLE FOR AMPK IN THE INHIBITION OF GLUCOSE-6-PHOSPHATE DEHYDROGENASE BY POLYUNSATURATED FATTY ACIDS

    PubMed Central

    Kohan, Alison B.; Talukdar, Indrani; Walsh, Callee M.; Salati, Lisa M.

    2009-01-01

    Both polyunsaturated fatty acids and AMPK promote energy partitioning away from energy consuming processes, such as fatty acid synthesis, towards energy generating processes, such as β-oxidation. In this report, we demonstrate that arachidonic acid activates AMPK in primary rat hepatocytes, and that this effect is p38 MAPK-dependent. Activation of AMPK mimics the inhibition by arachidonic acid of the insulin-mediated induction of G6PD. Similar to intracellular signaling by arachidonic acid, AMPK decreases insulin signal transduction, increasing Ser307 phosphorylation of IRS-1 and a subsequent decrease in AKT phosphorylation. Overexpression of dominant-negative AMPK abolishes the effect of arachidonic acid on G6PD expression. These data suggest a role for AMPK in the inhibition of G6PD by polyunsaturated fatty acids. PMID:19646964

  13. Structural basis for inhibition of 17β-hydroxysteroid dehydrogenases by phytoestrogens: The case of fungal 17β-HSDcl.

    PubMed

    Cassetta, Alberto; Stojan, Jure; Krastanova, Ivet; Kristan, Katja; Brunskole Švegelj, Mojca; Lamba, Doriano; Lanišnik Rižner, Tea

    2017-03-01

    Phytoestrogens are plant-derived compounds that functionally and structurally mimic mammalian estrogens. Phytoestrogens have broad inhibitory activities toward several steroidogenic enzymes, such as the 17β-hydroxysteroid dehydrogenases (17β-HSDs), which modulate the biological potency of androgens and estrogens in mammals. However, to date, no crystallographic data are available to explain phytoestrogens binding to mammalian 17β-HSDs. NADP(H)-dependent 17β-HSD from the filamentous fungus Cochliobolus lunatus (17β-HSDcl) has been the subject of extensive biochemical, kinetic and quantitative structure-activity relationship studies that have shown that the flavonols are the most potent inhibitors. In the present study, we investigated the structure-activity relationships of the ternary complexes between the holo form of 17β-HSDcl and the flavonols kaempferol and 3,7-dihydroxyflavone, in comparison with the isoflavones genistein and biochanin A. Crystallographic data are accompanied by kinetic analysis of the inhibition mechanisms for six flavonols (3-hydroxyflavone, 3,7-dihydroxyflavone, kaempferol, quercetin, fisetin, myricetin), one flavanone (naringenin), one flavone (luteolin), and two isoflavones (genistein, biochanin A). The kinetics analysis shows that the degree of hydroxylation of ring B significantly influences the overall inhibitory efficacy of the flavonols. A distinct binding mode defines the interactions between 17β-HSDcl and the flavones and isoflavones. Moreover, the complex with biochanin A reveals an unusual binding mode that appears to account for its greater inhibition of 17β-HSDcl with respect to genistein. Overall, these data provide a blueprint for identification of the distinct molecular determinants that underpin 17β-HSD inhibition by phytoestrogens.

  14. Inhibition of human and rat 11beta-hydroxysteroid dehydrogenase type 1 by 18beta-glycyrrhetinic acid derivatives.

    PubMed

    Su, Xiangdong; Vicker, Nigel; Lawrence, Harshani; Smith, Andrew; Purohit, Atul; Reed, Michael J; Potter, Barry V L

    2007-05-01

    11beta-Hydroxysteroid dehydrogenase type 1 (11beta-HSD1) plays an important role in regulating the cortisol availability to bind to corticosteroid receptors within specific tissue. Recent advances in understanding the molecular mechanisms of metabolic syndrome indicate that elevation of cortisol levels within specific tissues through the action of 11beta-HSD1 could contribute to the pathogenesis of this disease. Therefore, selective inhibitors of 11beta-HSD1 have been investigated as potential treatments for metabolic diseases, such as diabetes mellitus type 2 or obesity. Here we report the discovery and synthesis of some 18beta-glycyrrhetinic acid (18beta-GA) derivatives (2-5) and their inhibitory activities against rat hepatic11beta-HSD1 and rat renal 11beta-HSD2. Once the selectivity over the rat type 2 enzyme was established, these compounds' ability to inhibit human 11beta-HSD1 was also evaluated using both radioimmunoassay (RIA) and homogeneous time resolved fluorescence (HTRF) methods. The 11-modified 18beta-GA derivatives 2 and 3 with apparent selectivity for rat 11beta-HSD1 showed a high percentage inhibition for human microsomal 11beta-HSD1 at 10 microM and exhibited IC50 values of 400 and 1100 nM, respectively. The side chain modified 18beta-GA derivatives 4 and 5, although showing selectivity for rat 11beta-HSD1 inhibited human microsomal 11beta-HSD1 with IC50 values in the low micromolar range.

  15. Pea formaldehyde-active class III alcohol dehydrogenase: common derivation of the plant and animal forms but not of the corresponding ethanol-active forms (classes I and P).

    PubMed Central

    Shafqat, J; El-Ahmad, M; Danielsson, O; Martínez, M C; Persson, B; Parés, X; Jornvall, H

    1996-01-01

    A plant class III alcohol dehydrogenase (or glutathione-dependent formaldehyde dehydrogenase) has been characterized. The enzyme is a typical class III member with enzymatic parameters and substrate specificity closely related to those of already established animal forms. Km values with the pea enzyme are 6.5 microM for NAD+, 2 microM for S-hydroxymethylglutathione, and 840 microM for octanol versus 9, 4, and 1200 microM, respectively, with the human enzyme. Structurally, the pea/human class III enzymes are closely related, exhibiting a residue identity of 69% and with only 3 of 23 residues differing among those often considered in substrate and coenzyme binding. In contrast, the corresponding ethanol-active enzymes, the long-known human liver and pea alcohol dehydrogenases, differ more (47% residue identities) and are also in functionally important active site segments, with 12 of the 23 positions exchanged, including no less than 7 at the usually much conserved coenzyme-binding segment. These differences affect functionally important residues that are often class-distinguishing, such as those at positions 48, 51, and 115, where the plant ethanol-active forms resemble class III (Thr, Tyr, and Arg, respectively) rather than the animal ethanol-active class I forms (typically Ser, His, and Asp, respectively). Calculations of phylogenetic trees support the conclusions from functional residues in subgrouping plant ethanol-active dehydrogenases and the animal ethanol-active enzymes (class I) as separate descendants from the class III line. It appears that the classical plant alcohol dehydrogenases (now called class P) have a duplicatory origin separate from that of the animal class I enzymes and therefore a paralogous relationship with functional convergence of their alcohol substrate specificity. Combined, the results establish the conserved nature of class III also in plants, and contribute to the molecular and functional understanding of alcohol dehydrogenases by

  16. Oleyl alcohol inhibits intestinal long-chain fatty acid absorption in rats.

    PubMed

    Murota, K; Kawada, T; Matsui, N; Sakakibara, M; Takahashi, N; Fushiki, T

    2000-12-01

    Long-chain fatty acids are important nutrients, but obesity is the most common nutritional disorder in humans. In this study we investigated the effect of oleyl alcohol on the intestinal long-chain fatty acid absorption in rats. We administered [14C]oleic acid and oleyl alcohol as lipid emulsion intraduodenally in unanesthetized lymph-cannulated rats and measured the lymphatic output of oleic acid. Second, we orally administered lipid emulsion with a stomach tube and measured the luminal and mucosal oleic acid residues. Furthermore, rats were fed oleyl alcohol as a dietary component for 20 days, and fecal lipid and the weight of adipose tissues were measured. In lymph-cannulated rats, triglyceride and [14C]oleic acid output in the lymph were significantly lower in the presence of oleyl alcohol when compared with the absence of oleyl alcohol in a dose-dependent manner. The radioactivity remaining in the intestinal lumen was more strongly detected in rats that had been orally administered oleyl alcohol than in the controls. The feces of rats fed an oleyl-alcohol-added diet contained much higher amounts of lipids, and the weights of their adipose tissues were significantly lower than in the control group. These results suggest that oleyl alcohol inhibits the rat gastrointestinal absorption of long-chain fatty acids in vivo.

  17. Cancer screening of upper aerodigestive tract in Japanese alcoholics with reference to drinking and smoking habits and aldehyde dehydrogenase-2 genotype.

    PubMed

    Yokoyama, A; Ohmori, T; Muramatsu, T; Higuchi, S; Yokoyama, T; Matsushita, S; Matsumoto, M; Maruyama, K; Hayashida, M; Ishii, H

    1996-11-04

    In this study, 1,000 Japanese male alcoholics were consecutively screened by upper gastrointestinal endoscopy with esophageal iodine staining. Associations among cancer-detection rates, drinking and smoking habits, and aldehyde dehydrogenase-2 (ALDH2) genotypes were evaluated. A total of 53 patients (5.3%) had histologically confirmed cancer. Esophageal cancer was diagnosed in 36, gastric cancer in 17, and oropharyngolaryngeal cancer in 9 patients: 8 of the esophageal-cancer patients were multiple-cancer patients, with additional cancer(s) in the stomach and/or oropharyngolaryngeal region. Multiple logistic regression revealed that use of stronger alcoholic beverages (whisky or shochu) in contrast with lighter beverages (sake or beer) and smoking of 50 pack-years or more increased the risks for esophageal (odds ratio 3.2 and 2.8 respectively), oropharyngolaryngeal (4.8 and 5.1 respectively) and multiple cancer (10.5 and 11.8 respectively). The inactive form of ALDH2, encoded by the gene ALDH2*1/2*2 prevalent in Orientals, exposes them to higher blood levels of acetaldehyde, a recognized animal carcinogen, after drinking. This inactive ALDH2 was detected in 19/36 (52.8%) patients with esophageal cancer, in 5/9 (55.6%) patients with oropharyngolaryngeal cancer, and in 7/8 (87.5%) patients with multiple cancer. All of these gene frequencies far exceeded that in a large alcoholic cohort (80/655, 12.2%). The triple combination of the risk factors of the inactive ALDH2, stronger alcoholic beverages and heavy smoking was more commonly associated with multiple-cancer patients than with patients with esophageal cancer alone (62.5% vs. 7.1%). These results show that the 3 risk factors are important for the development of upper-aerodigestive-tract cancer in Japanese alcoholics. For these high-risk drinkers, regimented screening appears to be indicated.

  18. Ethanol metabolism, oxidative stress, and endoplasmic reticulum stress responses in the lungs of hepatic alcohol dehydrogenase deficient deer mice after chronic ethanol feeding

    PubMed Central

    Kaphalia, Lata; Boroumand, Nahal; Ju, Hyunsu; Kaphalia, Bhupendra S.; Calhoun, William J.

    2014-01-01

    Consumption and over-consumption of alcoholic beverages are well-recognized contributors to a variety of pulmonary disorders, even in the absence of intoxication. The mechanisms by which alcohol (ethanol) may produce disease include oxidative stress and prolonged endoplasmic reticulum (ER) stress. Many aspects of these processes remain incompletely understood due to a lack of a suitable animal model. Chronic alcohol over-consumption reduces hepatic alcohol dehydrogenase (ADH), the principal canonical metabolic pathway of ethanol oxidation. We therefore modeled this situation using hepatic ADH-deficient deer mice fed 3.5% ethanol daily for 3 months. Blood ethanol concentration was 180 mg% in ethanol fed mice, compared to <0.2% in the controls. Acetaldehyde (oxidative metabolite of ethanol) was minimally, but significantly increased in ethanol-fed vs. pair-fed control mice. Total fatty acid ethyl esters (FAEEs, nonoxidative metabolites of ethanol) were 47.6 μg/g in the lungs of ethanol-fed mice as compared to 1.5 μg/g in pair-fed controls. Histological and immunohistological evaluation showed perivascular and peribronchiolar lymphocytic infiltration, and significant oxidative injury, in the lungs of ethanol-fed mice compared to pair-fed controls. Several fold increases for cytochrome P450 2E1, caspase 8 and caspase 3 found in the lungs of ethanol-fed mice as compared to pair-fed controls suggest role of oxidative stress in ethanol-induced lung injury. ER stress and unfolded protein response signaling were also significantly increased in the lungs of ethanol-fed mice. Surprisingly, no significant activation of inositol-requiring enzyme-1α and spliced XBP1 were observed indicating a lack of activation of corrective mechanisms to reinstate ER homeostasis. The data suggest that oxidative stress and prolonged ER stress, coupled with formation and accumulation of cytotoxic FAEEs may contribute to the pathogenesis of alcoholic lung disease. PMID:24625836

  19. Ethanol metabolism, oxidative stress, and endoplasmic reticulum stress responses in the lungs of hepatic alcohol dehydrogenase deficient deer mice after chronic ethanol feeding.

    PubMed

    Kaphalia, Lata; Boroumand, Nahal; Hyunsu, Ju; Kaphalia, Bhupendra S; Calhoun, William J

    2014-06-01

    Consumption and over-consumption of alcoholic beverages are well-recognized contributors to a variety of pulmonary disorders, even in the absence of intoxication. The mechanisms by which alcohol (ethanol) may produce disease include oxidative stress and prolonged endoplasmic reticulum (ER) stress. Many aspects of these processes remain incompletely understood due to a lack of a suitable animal model. Chronic alcohol over-consumption reduces hepatic alcohol dehydrogenase (ADH), the principal canonical metabolic pathway of ethanol oxidation. We therefore modeled this situation using hepatic ADH-deficient deer mice fed 3.5% ethanol daily for 3 months. Blood ethanol concentration was 180 mg% in ethanol fed mice, compared to <1.0% in the controls. Acetaldehyde (oxidative metabolite of ethanol) was minimally, but significantly increased in ethanol-fed vs. pair-fed control mice. Total fatty acid ethyl esters (FAEEs, nonoxidative metabolites of ethanol) were 47.6 μg/g in the lungs of ethanol-fed mice as compared to 1.5 μg/g in pair-fed controls. Histological and immunohistological evaluation showed perivascular and peribronchiolar lymphocytic infiltration, and significant oxidative injury, in the lungs of ethanol-fed mice compared to pair-fed controls. Several fold increases for cytochrome P450 2E1, caspase 8 and caspase 3 found in the lungs of ethanol-fed mice as compared to pair-fed controls suggest role of oxidative stress in ethanol-induced lung injury. ER stress and unfolded protein response signaling were also significantly increased in the lungs of ethanol-fed mice. Surprisingly, no significant activation of inositol-requiring enzyme-1α and spliced XBP1 was observed indicating a lack of activation of corrective mechanisms to reinstate ER homeostasis. The data suggest that oxidative stress and prolonged ER stress, coupled with formation and accumulation of cytotoxic FAEEs may contribute to the pathogenesis of alcoholic lung disease.

  20. P300 during response inhibition is associated with ad-lib alcohol consumption in social drinkers.

    PubMed

    Jones, Andrew; Field, Matt; Christiansen, Paul; Stancak, Andrej

    2013-06-01

    Reduced amplitude of the cortical P300 event-related potential (ERP) component during response inhibition is associated with vulnerability to alcohol use disorders. In the current study, we investigated the effect of an experimental manipulation of response conflict on the amplitude of the P300 component during response inhibition, and examined whether individual differences in the amplitude of this P300 component would predict voluntary ad-lib alcohol consumption, in social drinkers. Using a repeated measures design, 16 participants performed a stop-signal task after receiving instructions that either emphasised or de-emphasised response conflict while their electroencephalogram (EEG) was concurrently recorded, before their ad-lib drinking was assessed. Results revealed that task instructions had the predicted effects on behavioural indices of response inhibition and the associated P300 components. Most importantly, individual differences in the amplitude of P300 subcomponents during response inhibition were negatively correlated with ad-lib alcohol consumption. Results provide the first experimental support for theoretical models that posit that reduced amplitude of the P300 during response inhibition is associated with alcohol-seeking behaviour in humans.

  1. Purification and properties of a 3 alpha-hydroxysteroid dehydrogenase of rat liver cytosol and its inhibition by anti-inflammatory drugs.

    PubMed Central

    Penning, T M; Mukharji, I; Barrows, S; Talalay, P

    1984-01-01

    An NAD(P)-dependent 3 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.50) was purified to homogeneity from rat liver cytosol, where it is responsible for most if not all of the capacity for the oxidation of androsterone, 1-acenaphthenol and benzenedihydrodiol (trans-1,2-dihydroxycyclohexa-3,5-diene). The dehydrogenase has many properties (substrate specificity, pI, Mr, amino acid composition) in common with the dihydrodiol dehydrogenase (EC 1.3.1.20) purified from the same source [Vogel, Bentley, Platt & Oesch (1980) J. Biol. Chem. 255, 9621-9625]. Since 3 alpha-hydroxysteroids are by far the most efficient substrates, the enzyme is more appropriately designated a 3 alpha-hydroxysteroid dehydrogenase. It also promotes the NAD(P)H-dependent reductions of quinones (e.g. 9,10-phenanthrenequinone, 1,4-benzoquinone), aromatic aldehydes (4-nitrobenzaldehyde) and aromatic ketones (4-nitroacetophenone). The dehydrogenase is not inhibited by dicoumarol, disulfiram, hexobarbital or pyrazole. The mechanism of the powerful inhibition of this enzyme by both non-steroidal and steroidal anti-inflammatory drugs [Penning & Talalay (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 4504-4508] was examined with several substrates. Most non-steroidal anti-inflammatory drugs are competitive inhibitors (e.g. Ki for indomethacin, 0.20 microM for 9,10-phenanthrenequinone reduction at pH 6.0, and 0.835 microM for androsterone oxidation at pH 7.0), except for salicylates, which act non-competitively (e.g. Ki for aspirin, 650 microM for androsterone oxidation). The inhibitory potency of these agents falls sharply as the pH is increased from 6 to 9. Most anti-inflammatory steroids are likewise competitive inhibitors, except for the most potent (betamethasone and dexamethasone), which act non-competitively. The enzyme is inhibited competitively by arachidonic acid and various prostaglandins. PMID:6435601

  2. Inhibition of cereblon by fenofibrate ameliorates alcoholic liver disease by enhancing AMPK.

    PubMed

    Kim, Yong Deuk; Lee, Kwang Min; Hwang, Seung-Lark; Chang, Hyeun Wook; Kim, Keuk-Jun; Harris, Robert A; Choi, Hueng-Sik; Choi, Won-Sik; Lee, Sung-Eun; Park, Chul-Seung

    2015-12-01

    Alcohol consumption exacerbates alcoholic liver disease by attenuating the activity of AMP-activated protein kinase (AMPK). AMPK is activated by fenofibrate, a peroxisome proliferator-activated receptor α (PPARα) agonist, and inhibited by direct interaction with cereblon (CRBN), a component of an E3 ubiquitin ligase complex. Based on these preliminary findings, we investigated that CRBN would be up-regulated in the liver by alcohol consumption and that CRBN deficiency would ameliorate hepatic steatosis and pro-inflammatory responses in alcohol-fed mice by increasing AMPK activity. Wild-type, CRBN and PPARα null mice were fed an alcohol-containing liquid diet and administered with fenofibrate. Gene expression profiles and metabolic changes were measured in the liver and blood of these mice. Expression of CRBN, cytochrome P450 2E1 (CYP2E1), lipogenic genes, pro-inflammatory cytokines, serum alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were increased in the Lieber-DeCarli alcohol-challenged mice. Fenofibrate attenuated the induction of CRBN and reduced hepatic steatosis and pro-inflammatory markers in these mice. Ablation of the gene encoding CRBN produced the same effect as fenofibrate. The increase in CRBN gene expression by alcohol and the reduction of CRBN expression by fenofibrate were negated in PPARα null mice. Fenofibrate increased the recruitment of PPARα on CRBN gene promoter in WT mice but not in PPARα null mice. Silencing of AMPK prevented the beneficial effects of fenofibrate. These results demonstrate that activation of PPARα by fenofibrate alleviates alcohol-induced hepatic steatosis and inflammation by reducing the inhibition of AMPK by CRBN. CRBN is a potential therapeutic target for the alcoholic liver disease.

  3. Inhibition of alcoholic fermentation by substrate and ethanol. [Candida pseudotropicalis

    SciTech Connect

    Maulin, H.B.; Galzy, P.

    1980-11-01

    The effect of ethanol and sugars on rates of fermentation was studied. A strain of Candida pseudotropicalis was used. The specific rate of fermentation was determined by using the Warburg manometer. The effect of ethanol was formulated as an exponential function of ethanol concentration, but the empirical constant was different when glucose or lactose was used as a substrate. The effects of both ethanol and substrate were formulated. It was demonstrated that when lactose and glucose were present in the medium with a small amount of alcohol, a synergistic effect on the rate of fermentation appeared. This phenomenon considerably limits the rate of fermentation.

  4. Efficient PCR-Based Amplification of Diverse Alcohol Dehydrogenase Genes from Metagenomes for Improving Biocatalysis: Screening of Gene-Specific Amplicons from Metagenomes

    PubMed Central

    Kariya, Satomi; Kurokawa, Junji

    2014-01-01

    Screening of gene-specific amplicons from metagenomes (S-GAM) has tremendous biotechnological potential. We used this approach to isolate alcohol dehydrogenase (adh) genes from metagenomes based on the Leifsonia species adh gene (lsadh), the enzyme product of which can produce various chiral alcohols. A primer combination was synthesized by reference to homologs of lsadh, and PCR was used to amplify nearly full-length adh genes from metagenomic DNAs. All adh preparations were fused with lsadh at the terminal region and used to construct Escherichia coli plasmid libraries. Of the approximately 2,000 colonies obtained, 1,200 clones were identified as adh positive (∼60%). Finally, 40 adh genes, Hladh-001 to Hladh-040 (for homologous Leifsonia adh), were identified from 223 clones with high efficiency, which were randomly sequenced from the 1,200 clones. The Hladh genes obtained via this approach encoded a wide variety of amino acid sequences (8 to 99%). After screening, the enzymes obtained (HLADH-012 and HLADH-021) were confirmed to be superior to LSADH in some respects for the production of anti-Prelog chiral alcohols. PMID:25085492

  5. Inhibition of several enzymes by gold compounds. II. beta-Glucuronidase, acid phosphatase and L-malate dehydrogenase by sodium thiomalatoraurate (I), sodium thiosulfatoaurate (I) and thioglucosoaurate (I).

    PubMed

    Lee, M T; Ahmed, T; Haddad, R; Friedman, M E

    1989-01-01

    Bovine liver beta-D-glucuronide glucuronohydrolase, EC 3.2.1.32), wheat germ acid phosphatase (orthophosphoric monoesterphosphohydrolase, EC 3.1.3.2) and bovine liver L-malate dehydrogenase (L-malate: NAD oxidoreductase, EC 1.1.1.37) were inhibited by a series of gold (I) complexes that have been used as anti-inflammatory drugs. Both sodium thiosulfatoaurate (I) (Na AuTs) and sodium thiomalatoraurate (NaAuTM) effectively inhibited all three enzymes, while thioglucosoaurate (I) (AuTG) only inhibited L-malate dehydrogenase. The equilibrium constants (K1) ranged from nearly 4000 microM for the NaAuTM-beta-glucuronidase interaction to 24 microM for the NaAuTS-beta-glucuronidase interaction. The rate of covalent bond formation (kp) ranged from 0.00032 min-1 for NaAuTM-beta-glucuronidase formation to 1.7 min-1 for AuTG-L-malate dehydrogenase formation. The equilibrium data shows that the gold (I) drugs bind by several orders lower than the gold (III) compounds, suggesting a significantly stronger interaction between the more highly charged gold ion and the enzyme. Yet the rate of covalent bond formation depends as much on the structure of the active site as upon the lability of the gold-ligand bond. It was also observed that the more effective the gold inhibition the more toxic the compound.

  6. Inhibition of aldehyde dehydrogenase 2 activity enhances antimycin-induced rat cardiomyocytes apoptosis through activation of MAPK signaling pathway.

    PubMed

    Zhang, Peng; Xu, Danling; Wang, Shijun; Fu, Han; Wang, Keqiang; Zou, Yunzeng; Sun, Aijun; Ge, Junbo

    2011-12-01

    Aldehyde dehydrogenase 2 (ALDH2), a mitochondrial-specific enzyme, has been proved to be involved in oxidative stress-induced cell apoptosis, while little is known in cardiomyocytes. This study was aimed at investigating the role of ALDH2 in antimycin A-induced cardiomyocytes apoptosis by suppressing ALDH2 activity with a specific ALDH2 inhibitor Daidzin. Antimycin A (40μg/ml) was used to induce neonatal cardiomyocytes apoptosis. Daidzin (60μM) effectively inhibited ALDH2 activity by 50% without own effect on cell apoptosis, and significantly enhanced antimycin A-induced cardiomyocytes apoptosis from 33.5±4.4 to 56.5±6.4% (Hochest method, p<0.05), and from 57.9±1.9 to 74.0±11.9% (FACS, p<0.05). Phosphorylation of activated MAPK signaling pathway, including extracellular signal-regulated kinase (ERK1/2), c-Jun NH2-terminal kinase (JNK) and p38 was also increased in antimycin A and daidzin treated cardiomyocytes compared to the cells treated with antimycin A alone. These findings indicated that modifying mitochondrial ALDH2 activity/expression might be a potential therapeutic option on reducing oxidative insults induced cardiomyocytes apoptosis.

  7. Inhibition and biochemical characterization of methicillin-resistant Staphylococcus aureus shikimate dehydrogenase: an in silico and kinetic study.

    PubMed

    Avitia-Domínguez, Claudia; Sierra-Campos, Erick; Salas-Pacheco, José Manuel; Nájera, Hugo; Rojo-Domínguez, Arturo; Cisneros-Martínez, Jorge; Téllez-Valencia, Alfredo

    2014-04-10

    Methicillin-resistant Staphylococcus auerus (MRSA) strains are having a major impact worldwide, and due to their resistance to all β-lactams, an urgent need for new drugs is emerging. In this regard, the shikimate pathway is considered to be one of the metabolic features of bacteria and is absent in humans. Therefore enzymes involved in this route, such as shikimate dehydrogenase (SDH), are considered excellent targets for discovery of novel antibacterial drugs. In this study, the SDH from MRSA (SaSDH) was characterized. The results showed that the enzyme is a monomer with a molecular weight of 29 kDa, an optimum temperature of 65 °C, and a maximal pH range of 9-11 for its activity. Kinetic studies revealed that SDH showed Michaelis-Menten kinetics toward both substrates (shikimate and NADP+). Initial velocity analysis suggested that SaSDH catalysis followed a sequential random mechanism. Additionally, a tridimensional model of SaSDH was obtained by homology modeling and validated. Through virtual screening three inhibitors of SaSDH were found (compounds 238, 766 and 894) and their inhibition constants and mechanism were obtained. Flexible docking studies revealed that these molecules make interactions with catalytic residues. The data of this study could serve as starting point in the search of new chemotherapeutic agents against MRSA.

  8. Free fatty acid inhibition of the insulin induction of glucose-6-phosphate dehydrogenase in rat hepatocyte monolayers.

    PubMed

    Salati, L M; Adkins-Finke, B; Clarke, S D

    1988-01-01

    Rat hepatocytes in monolayer culture were utilized to determine if the decrease in glucose-6-phosphate dehydrogenase (G6PD) activity resulting from the ingestion of fat can be mimicked by the addition of fatty acids to a chemically, hormonally defined medium. G6PD activity in cultured hepatocytes was induced several-fold by insulin. Dexamethasone or T3 did not amplify the insulin induction of G6PD. Glucose alone increased G6PD activity in cultured hepatocytes from fasted donors by nearly 500%. Insulin in combination with glucose induced G6PD an additional two-fold. The increase in G6PD activity caused by glucose was greater in hepatocytes isolated from 72 hr-fasted rats as compared to fed donor rats. Such a response was reminiscent of the "overshoot" phenomenon in which G6PD activity is induced well above the normal level by fasting-refeeding rats a high glucose diet. Addition of linoleate to the medium resulted in a significant suppression of insulin's ability to induce G6PD, but linoleate had no effect on the induction of G6PD activity by glucose alone. A shift to the right in the insulin-response curve for the induction of G6PD also was detected for the induction of malic enzyme and acetyl-CoA carboxylase. Arachidonate (0.25 mM) was a significantly more effective inhibitor of the insulin action than linoleate was. Apparently rat hepatocytes in monolayer culture can be utilized as a model to investigate the molecular mechanism by which fatty acids inhibit the production of lipogenic enzymes. In part, this mechanism of fatty acid inhibition involves desensitization of hepatocytes to the lipogenic action of insulin.

  9. JWH-018 ω-OH, a shared hydroxy metabolite of the two synthetic cannabinoids JWH-018 and AM-2201, undergoes oxidation by alcohol dehydrogenase and aldehyde dehydrogenase enzymes in vitro forming the carboxylic acid metabolite.

    PubMed

    Holm, Niels Bjerre; Noble, Carolina; Linnet, Kristian

    2016-09-30

    Synthetic cannabinoids are new psychoactive substances (NPS) acting as agonists at the cannabinoid receptors. The aminoalkylindole-type synthetic cannabinoid naphthalen-1-yl-(1-pentylindol-3-yl)methanone (JWH-018) was among the first to appear on the illicit drug market and its metabolism has been extensively investigated. The N-pentyl side chain is a major site of human cytochrome P450 (CYP)-mediated oxidative metabolism, and the ω-carboxylic acid metabolite appears to be a major in vivo human urinary metabolite. This metabolite is, however, not formed to any significant extent in human liver microsomal (HLM) incubations raising the possibility that the discrepancy is due to involvement of cytosolic enzymes. Here we demonstrate in incubations with human liver cytosol (HLC), that JWH-018 ω-OH, but not the JWH-018 parent compound, is a substrate for nicotinamide adenine dinucleotide (NAD(+))-dependent alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) enzymes. The sole end-product identified in HLC was the JWH-018 ω-COOH metabolite, while trapping tests with methoxyamine proved the presence of the aldehyde intermediate. ADH/ALDH and UDP-glucuronosyl-transferases (UGT) enzymes may therefore both act on the JWH-018 ω-OH substrate. Finally, we note that for [1-(5-fluoropentyl)indol-3-yl]-naphthalen-1-yl-methanone (AM-2201), the ω-fluorinated analog of JWH-018, a high amount of JWH-018 ω-OH was formed in HLM incubated without NADPH, suggesting that the oxidative defluorination is efficiently catalyzed by non-CYP enzyme(s). The pathway presented here may therefore be especially important for N-(5-fluoropentyl) substituted synthetic cannabinoids, because the oxidative defluorination can occur even if the CYP-mediated metabolism preferentially takes place on other parts of the molecule than the N-alkyl side chain. Controlled clinical studies in humans are ultimately required to demonstrate the in vivo importance of the oxidation pathway presented here.

  10. Inhibition by alcohols of the localization of radioactive nitrosonornicotine in sites of tumor formation

    SciTech Connect

    Waddell, W.J.; Marlowe, C.

    1983-06-01

    Oral administration of ethanol, n-butanol, or t-butanol to mice 20 minutes before injection of carbon-14-labeled nitrosonornicotine inhibited the localization of radioactivity in bronchial and salivary duct epithelium and in the liver. Localization of radioactivity in the nasal epithelium and esophagus was not significantly reduced. These alcohols therefore may selectively inhibit tumor formation in three of the five sites where this carcinogen typically acts.

  11. The Genetics of a Small Autosomal Region of DROSOPHILA MELANOGASTER Containing the Structural Gene for Alcohol Dehydrogenase. I. Characterization of Deficiencies and Mapping of ADH and Visible Mutations

    PubMed Central

    Woodruff, R. C.; Ashburner, M.

    1979-01-01

    The position of the structural gene coding for alcohol dehydrogenase (ADH) in Drosophila melanogaster has been shown to be within polytene chromosome bands 35B1 and 35B3, most probably within 35B2. The genetic and cytological properties of twelve deficiencies in polytene chromosome region 34–35 have been characterized, eleven of which include Adh. Also mapped cytogenetically are seven other recessive visible mutant loci. Flies heterozygous for overlapping deficiencies that include both the Adh locus and that for the outspread mutant (osp: a recessive wing phenotype) are homozygous viable and show a complete ADH negative phenotype and strong osp phenotype. These deficiencies probably include two polytene chromosome bands, 35B2 and 35B3. PMID:115743

  12. A new regulatory element mediates ethanol repression of KlADH3, a Kluyveromyces lactis gene coding for a mitochondrial alcohol dehydrogenase.

    PubMed

    Saliola, Michele; Getuli, Claudia; Mazzoni, Cristina; Fantozzi, Ivana; Falcone, Claudio

    2007-08-01

    KlADH3 is a Kluyveromyces lactis alcohol dehydrogenase gene induced in the presence of all respiratory carbon sources except ethanol, which specifically represses this gene. Deletion analysis of the KlADH3 promoter revealed the presence of both positive and negative elements. However, by site-directed mutagenesis and gel retardation experiments, we identified a 15-bp element responsible for the transcriptional repression of this gene by ethanol. In particular, this element showed putative sites required for the sequential binding of ethanol-induced factors responsible for the repressed conditions, and the binding of additional factors relieved repression. In addition, we showed that the ethanol element was required for in vivo repression of KlAdh3 activity.

  13. Structural Studies of Cinnamoyl-CoA Reductase and Cinnamyl-Alcohol Dehydrogenase, Key Enzymes of Monolignol Biosynthesis[C][W

    PubMed Central

    Pan, Haiyun; Zhou, Rui; Louie, Gordon V.; Mühlemann, Joëlle K.; Bomati, Erin K.; Bowman, Marianne E.; Dudareva, Natalia; Dixon, Richard A.; Noel, Joseph P.; Wang, Xiaoqiang

    2014-01-01

    The enzymes cinnamoyl-CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) catalyze the two key reduction reactions in the conversion of cinnamic acid derivatives into monolignol building blocks for lignin polymers in plant cell walls. Here, we describe detailed functional and structural analyses of CCRs from Medicago truncatula and Petunia hybrida and of an atypical CAD (CAD2) from M. truncatula. These enzymes are closely related members of the short-chain dehydrogenase/reductase (SDR) superfamily. Our structural studies support a reaction mechanism involving a canonical SDR catalytic triad in both CCR and CAD2 and an important role for an auxiliary cysteine unique to CCR. Site-directed mutants of CAD2 (Phe226Ala and Tyr136Phe) that enlarge the phenolic binding site result in a 4- to 10-fold increase in activity with sinapaldehyde, which in comparison to the smaller coumaraldehyde and coniferaldehyde substrates is disfavored by wild-type CAD2. This finding demonstrates the potential exploitation of rationally engineered forms of CCR and CAD2 for the targeted modification of monolignol composition in transgenic plants. Thermal denaturation measurements and structural comparisons of various liganded and unliganded forms of CCR and CAD2 highlight substantial conformational flexibility of these SDR enzymes, which plays an important role in the establishment of catalytically productive complexes of the enzymes with their NADPH and phenolic substrates. PMID:25217505

  14. Thymoquinone Inhibition of Acquisition and Expression of Alcohol-Induced Behavioral Sensitization.

    PubMed

    Khan, Muhammad Sona; Gohar, Aneela; Abbas, Ghulam; Mahmood, Wajahat; Rauf, Khalid; Sewell, Robert D E

    2015-10-01

    Repeated low doses of alcohol have been shown to progressively enhance locomotor activity in mice, and this phenomenon is designated as behavioral sensitization. Thymoquinone, a major active component of Nigella sativa oil has been investigated in a number of studies for its neuroprotective effects against a variety of ailments. This study was conducted to explore the therapeutic potential of thymoquinone on the acquisition and expression of alcohol-induced behavioral sensitization. Mice treated with alcohol (2.2 g/kg/day) or saline for 13 days and subsequently challenged with an acute alcohol dose (2.2 g/kg) 5 days later were orally administered acute doses of thymoquinone (10, 20 and 30 mg/kg). Thymoquinone subacute treatment with all doses throughout alcohol exposure significantly inhibited both the development and expression phases of alcohol behavioral sensitization in a dose-dependent manner. However, acute treatment with thymoquinone (30 mg/kg) only reversed the expression phase of sensitization. These findings are explained in terms of the known GABA promoting action of thymoquinone in relation to the motive circuit within the limbic component of the basal ganglia. It is concluded that thymoquinone may be a potential therapeutic option for the treatment and prevention of alcohol induced behavioral sensitization.

  15. Environmental Stresses of Field Growth Allow Cinnamyl Alcohol Dehydrogenase-Deficient Nicotiana attenuata Plants to Compensate for their Structural Deficiencies1[C][W][OA

    PubMed Central

    Kaur, Harleen; Shaker, Kamel; Heinzel, Nicolas; Ralph, John; Gális, Ivan; Baldwin, Ian T.

    2012-01-01

    The organized lignocellulosic assemblies of cell walls provide the structural integrity required for the large statures of terrestrial plants. Silencing two CINNAMYL ALCOHOL DEHYDROGENASE (CAD) genes in Nicotiana attenuata produced plants (ir-CAD) with thin, red-pigmented stems, low CAD and sinapyl alcohol dehydrogenase activity, low lignin contents, and rubbery, structurally unstable stems when grown in the glasshouse (GH). However, when planted into their native desert habitat, ir-CAD plants produced robust stems that survived wind storms as well as the wild-type plants. Despite efficient silencing of NaCAD transcripts and enzymatic activity, field-grown ir-CAD plants had delayed and restricted spread of red stem pigmentation, a color change reflecting blocked lignification by CAD silencing, and attained wild-type-comparable total lignin contents. The rubbery GH phenotype was largely restored when field-grown ir-CAD plants were protected from wind, herbivore attack, and ultraviolet B exposure and grown in restricted rooting volumes; conversely, it was lost when ir-CAD plants were experimentally exposed to wind, ultraviolet B, and grown in large pots in growth chambers. Transcript and liquid chromatography-electrospray ionization-time-of-flight analysis revealed that these environmental stresses enhanced the accumulation of various phenylpropanoids in stems of field-grown plants; gas chromatography-mass spectrometry and nuclear magnetic resonance analysis revealed that the lignin of field-grown ir-CAD plants had GH-grown comparable levels of sinapaldehyde and syringaldehyde cross-linked into their lignins. Additionally, field-grown ir-CAD plants had short, thick stems with normal xylem element traits, which collectively enabled field-grown ir-CAD plants to compensate for the structural deficiencies associated with CAD silencing. Environmental stresses play an essential role in regulating lignin biosynthesis in lignin-deficient plants. PMID:22645069

  16. Characterization of the Saccharomyces cerevisiae YMR318C (ADH6) gene product as a broad specificity NADPH-dependent alcohol dehydrogenase: relevance in aldehyde reduction.

    PubMed Central

    Larroy, Carol; Fernández, M Rosario; González, Eva; Parés, Xavier; Biosca, Josep A

    2002-01-01

    YMR318C represents an open reading frame from Saccharomyces cerevisiae with unknown function. It possesses a conserved sequence motif, the zinc-containing alcohol dehydrogenase (ADH) signature, specific to the medium-chain zinc-containing ADHs. In the present study, the YMR318C gene product has been purified to homogeneity from overexpressing yeast cells, and found to be a homodimeric ADH, composed of 40 kDa subunits and with a pI of 5.0-5.4. The enzyme was strictly specific for NADPH and was active with a wide variety of substrates, including aliphatic (linear and branched-chain) and aromatic primary alcohols and aldehydes. Aldehydes were processed with a 50-fold higher catalytic efficiency than that for the corresponding alcohols. The highest k(cat)/K(m) values were found with pentanal>veratraldehyde > hexanal > 3-methylbutanal >cinnamaldehyde. Taking into consideration the substrate specificity and sequence characteristics of the YMR318C gene product, we have proposed this gene to be called ADH6. The disruption of ADH6 was not lethal for the yeast under laboratory conditions. Although S. cerevisiae is considered a non lignin-degrading organism, the catalytic activity of ADHVI can direct veratraldehyde and anisaldehyde, arising from the oxidation of lignocellulose by fungal lignin peroxidases, to the lignin biodegradation pathway. ADHVI is the only S. cerevisiae enzyme able to significantly reduce veratraldehyde in vivo, and its overexpression allowed yeast to grow under toxic concentrations of this aldehyde. The enzyme may also be involved in the synthesis of fusel alcohols. To our knowledge this is the first NADPH-dependent medium-chain ADH to be characterized in S. cerevisiae. PMID:11742541

  17. Cofactor engineering of ketol-acid reductoisomerase (IlvC) and alcohol dehydrogenase (YqhD) improves the fusel alcohol yield in algal protein anaerobic fermentation

    SciTech Connect

    Wu, Weihua; Tran-Gyamfi, Mary Bao; Jaryenneh, James Dekontee; Davis, Ryan W.

    2016-08-24

    Recently the feasibility of conversion of algal protein to mixed alcohols has been demonstrated with an engineered E.coli strain, enabling comprehensive utilization of the biomass for biofuel applications. However, the yield and titers of mixed alcohol production must be improved for market adoption. A major limiting factor for achieving the necessary yield and titer improvements is cofactor imbalance during the fermentation of algal protein. To resolve this problem, a directed evolution approach was applied to modify the cofactor specificity of two key enzymes (IlvC and YqhD) from NADPH to NADH in the mixed alcohol metabolic pathway. Using high throughput screening, more than 20 YqhD mutants were identified to show activity on NADH as a cofactor. Of these 20 mutants, the top five of YqhD mutants were selected for combination with two IlvC mutants with NADH as a cofactor for the modification of the protein conversion strain. The combination of the IlvC and YqhD mutants yielded a refined E.coli strain, subtype AY3, with increased fusel alcohol yield of ~60% compared to wild type under anaerobic fermentation on amino acid mixtures. When applied to real algal protein hydrolysates, the strain AY3 produced 100% and 38% more total mixed alcohols than the wild type strain on two different algal hydrolysates, respectively. The results indicate that cofactor engineering is a promising approach to improve the feasibility of bioconversion of algal protein into mixed alcohols as advanced biofuels.

  18. A New View of Alcohol Metabolism and Alcoholism—Role of the High-Km Class III Alcohol Dehydrogenase (ADH3)

    PubMed Central

    Haseba, Takeshi; Ohno, Youkichi

    2010-01-01

    The conventional view is that alcohol metabolism is carried out by ADH1 (Class I) in the liver. However, it has been suggested that another pathway plays an important role in alcohol metabolism, especially when the level of blood ethanol is high or when drinking is chronic. Over the past three decades, vigorous attempts to identify the enzyme responsible for the non-ADH1 pathway have focused on the microsomal ethanol oxidizing system (MEOS) and catalase, but have failed to clarify their roles in systemic alcohol metabolism. Recently, using ADH3-null mutant mice, we demonstrated that ADH3 (Class III), which has a high Km and is a ubiquitous enzyme of ancient origin, contributes to systemic alcohol metabolism in a dose-dependent manner, thereby diminishing acute alcohol intoxication. Although the activity of ADH3 toward ethanol is usually low in vitro due to its very high Km, the catalytic efficiency (kcat/Km) is markedly enhanced when the solution hydrophobicity of the reaction medium increases. Activation of ADH3 by increasing hydrophobicity should also occur in liver cells; a cytoplasmic solution of mouse liver cells was shown to be much more hydrophobic than a buffer solution when using Nile red as a hydrophobicity probe. When various doses of ethanol are administered to mice, liver ADH3 activity is dynamically regulated through induction or kinetic activation, while ADH1 activity is markedly lower at high doses (3–5 g/kg). These data suggest that ADH3 plays a dynamic role in alcohol metabolism, either collaborating with ADH1 or compensating for the reduced role of ADH1. A complex two-ADH model that ascribes total liver ADH activity to both ADH1 and ADH3 explains the dose-dependent changes in the pharmacokinetic parameters (β, CLT, AUC) of blood ethanol very well, suggesting that alcohol metabolism in mice is primarily governed by these two ADHs. In patients with alcoholic liver disease, liver ADH3 activity increases, while ADH1 activity decreases, as alcohol

  19. Engineering of the pyruvate dehydrogenase bypass in Saccharomyces cerevisiae: role of the cytosolic Mg(2+) and mitochondrial K(+) acetaldehyde dehydrogenases Ald6p and Ald4p in acetate formation during alcoholic fermentation.

    PubMed

    Remize, F; Andrieu, E; Dequin, S

    2000-08-01

    Acetic acid plays a crucial role in the organoleptic balance of many fermented products. We have investigated the factors controlling the production of acetate by Saccharomyces cerevisiae during alcoholic fermentation by metabolic engineering of the enzymatic steps involved in its formation and its utilization. The impact of reduced pyruvate decarboxylase (PDC), limited acetaldehyde dehydrogenase (ACDH), or increased acetoacetyl coenzyme A synthetase (ACS) levels in a strain derived from a wine yeast strain was studied during alcoholic fermentation. In the strain with the PDC1 gene deleted exhibiting 25% of the PDC activity of the wild type, no significant differences were observed in the acetate yield or in the amounts of secondary metabolites formed. A strain overexpressing ACS2 and displaying a four- to sevenfold increase in ACS activity did not produce reduced acetate levels. In contrast, strains with one or two disrupted copies of ALD6, encoding the cytosolic Mg(2+)-activated NADP-dependent ACDH and exhibiting 60 and 30% of wild-type ACDH activity, showed a substantial decrease in acetate yield (the acetate production was 75 and 40% of wild-type production, respectively). This decrease was associated with a rerouting of carbon flux towards the formation of glycerol, succinate, and butanediol. The deletion of ALD4, encoding the mitochondrial K(+)-activated NAD(P)-linked ACDH, had no effect on the amount of acetate formed. In contrast, a strain lacking both Ald6p and Ald4p exhibited a long delay in growth and acetate production, suggesting that Ald4p can partially replace the Ald6p isoform. Moreover, the ald6 ald4 double mutant was still able to ferment large amounts of sugar and to produce acetate, suggesting the contribution of another member(s) of the ALD family.

  20. Selective inhibition of 11 beta-hydroxysteroid dehydrogenase type 1 improves hepatic insulin sensitivity in hyperglycemic mice strains.

    PubMed

    Alberts, Pēteris; Nilsson, Cecilia; Selen, Göran; Engblom, Lars O M; Edling, Naimie H M; Norling, Solveig; Klingström, Gunnel; Larsson, Catarina; Forsgren, Margareta; Ashkzari, Mandana; Nilsson, Catrine E; Fiedler, Maj; Bergqvist, Elisabet; Ohman, Birgitta; Björkstrand, Eva; Abrahmsen, Lars B

    2003-11-01

    11 beta-Hydroxysteroid dehydrogenase type 1 (11 beta-HSD1) has been proposed as a new target for type 2 diabetes drugs. The aim of the present study was to assess the effects of inhibition of 11 beta-HSD1 on blood glucose levels, glucose tolerance, and insulin sensitivity in mouse models of type 2 diabetes. BVT.2733 is an isoform-selective inhibitor of mouse 11 beta-HSD1. Hyperglycemic and hyperinsulinemic ob/ob, db/db, KKAy, and normal C57BL/6J mice were orally administered BVT.2733 (200 mg/kg.d, twice daily). In hyperglycemic, but not in normal mice, BVT.2733 lowered circulating glucose (to 50-88% of control) and insulin (52-65%) levels. In oral glucose tolerance tests in ob/ob and KKAy mice, glucose concentrations were 65-75% of vehicle values after BVT.2733 treatment, and in KKAy mice insulin concentrations were decreased (62-74%). Euglycemic, hyperinsulinemic clamps demonstrated decreased endogenous glucose production (21-61%). Analysis of hepatic mRNA in KKAy mice showed reduced phosphoenolpyruvate carboxykinase mRNA (71%). A slight reduction in food intake was observed in ob/ob and KKAy mice. Cholesterol, triglycerides, and free fatty acid levels were decreased to 81-86% in KKAy mice after a 4-h fast. The results support previous suggestions that selective 11 beta-HSD1 inhibitors lower blood glucose levels and improve insulin sensitivity in different mouse models of type 2 diabetes.

  1. Is there sufficient evidence to consider the use of 11β-hydroxysteroid dehydrogenase type 1 inhibition in children?

    PubMed

    Fürst-Recktenwald, Sabine; Dörr, Helmuth G; Quinkler, Marcus; Dötsch, Jörg; Stewart, Paul M

    2012-08-01

    Manifestations of the metabolic syndrome [obesity, dyslipidaemia, hypertension, blood glucose derangements including prediabetes or type 2 diabetes mellitus (T2DM)] in juvenile populations are becoming increasingly prevalent throughout the world and are at the point of being a global public health concern. Derangements in cortisol regeneration seem to be involved in the pathophysiology. Treatment with selective 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) inhibitors could be a therapeutic strategy in paediatric patients with manifestations of the metabolic syndrome. Based on preclinical and clinical data regarding development of the 11β-HSD1 enzyme, it appears that maturation occurs within the first year of life. Different changes in biomarkers for assessing the efficacy and safety of 11β-HSD1 inhibitors are to be expected in paediatric patients compared to adults, reflecting differences in metabolism. The effect of 11β-HSD1 treatment in children on bone differentiation and development as well as adrenocorticotropic hormone (ACTH), circulating and local cortisol tissue concentrations, androgens and respective stress response is not yet known. Based on current literature, the concept of inhibition of 11β-HSD1 is considered a potentially effective mean to regulate local cortisol levels in the paediatric population, and 11β-HSD1 inhibitors may provide a valuable target and treatment option for the metabolic syndrome in paediatric patients. However, the uncertainty over effects on the developing skeleton combined with mild increases in adrenal androgen levels raises potential concerns regarding growth as well as onset of puberty as to their future use in children. Future clinical studies are needed to thoroughly assess the risks and benefits of this new class of drugs in the paediatric population.

  2. Low doses of alcohol potentiate GABA sub B inhibition of spontaneous activity of hippocampal CA1 neurons in vivo

    SciTech Connect

    Criado, J.R.; Thies, R. )

    1991-03-11

    Low doses of alcohol facilitate firing of hippocampal neurons. Such doses also enhance the inhibitory actions of GABA. Alcohol is known to potentiate inhibition via GABA{sub A} receptors. However, the effects of alcohol on GABA{sub B} receptor function are not understood. Spontaneous activity of single units was recorded from CA1 neurons of male rats anesthetized with 1.0% halothane. Electrical recordings and local application of drugs were done with multi-barrel pipettes. CA1 pyramidal neurons fired spontaneous bursts of action potentials. Acute alcohol decreased the interval between bursts, a mild excitatory action. Alcohol also more than doubled the period of complete inhibition produced by local application of both GABA and baclofen. These data suggest that GABA{sub B}-mediated inhibition is also potentiated by low doses of alcohol.

  3. Chronic alcohol exposure inhibits biotin uptake by pancreatic acinar cells: possible involvement of epigenetic mechanisms.

    PubMed

    Srinivasan, Padmanabhan; Kapadia, Rubina; Biswas, Arundhati; Said, Hamid M

    2014-11-01

    Chronic exposure to alcohol affects different physiological aspects of pancreatic acinar cells (PAC), but its effect on the uptake process of biotin is not known. We addressed this issue using mouse-derived pancreatic acinar 266-6 cells chronically exposed to alcohol and wild-type and transgenic mice (carrying the human SLC5A6 5'-promoter) fed alcohol chronically. First we established that biotin uptake by PAC is Na(+) dependent and carrier mediated and involves sodium-dependent multivitamin transporter (SMVT). Chronic exposure of 266-6 cells to alcohol led to a significant inhibition in biotin uptake, expression of SMVT protein, and mRNA as well as in the activity of the SLC5A6 promoter. Similarly, chronic alcohol feeding of wild-type and transgenic mice carrying the SLC5A6 promoter led to a significant inhibition in biotin uptake by PAC, as well as in the expression of SMVT protein and mRNA and the activity of the SLC5A6 promoters expressed in the transgenic mice. We also found that chronic alcohol feeding of mice is associated with a significant increase in the methylation status of CpG islands predicted to be in the mouse Slc5a6 promoters and a decrease in the level of expression of transcription factor KLF-4, which plays an important role in regulating SLC5A6 promoter activity. These results demonstrate, for the first time, that chronic alcohol exposure negatively impacts biotin uptake in PAC and that this effect is exerted (at least in part) at the level of transcription of the SLC5A6 gene and may involve epigenetic/molecular mechanisms.

  4. Gene ercA, Encoding a Putative Iron-Containing Alcohol Dehydrogenase, Is Involved in Regulation of Ethanol Utilization in Pseudomonas aeruginosa

    PubMed Central

    Hempel, Niels; Görisch, Helmut

    2013-01-01

    Several two-component regulatory systems are known to be involved in the signal transduction pathway of the ethanol oxidation system in Pseudomonas aeruginosa ATCC 17933. These sensor kinases and response regulators are organized in a hierarchical manner. In addition, a cytoplasmic putative iron-containing alcohol dehydrogenase (Fe-ADH) encoded by ercA (PA1991) has been identified to play an essential role in this regulatory network. The gene ercA (PA1991) is located next to ercS, which encodes a sensor kinase. Inactivation of ercA (PA1991) by insertion of a kanamycin resistance cassette created mutant NH1. NH1 showed poor growth on various alcohols. On ethanol, NH1 grew only with an extremely extended lag phase. During the induction period on ethanol, transcription of structural genes exa and pqqABCDEH, encoding components of initial ethanol oxidation in P. aeruginosa, was drastically reduced in NH1, which indicates the regulatory function of ercA (PA1991). However, transcription in the extremely delayed logarithmic growth phase was comparable to that in the wild type. To date, the involvement of an Fe-ADH in signal transduction processes has not been reported. PMID:23813731

  5. Degradation of Swainsonine by the NADP-Dependent Alcohol Dehydrogenase A1R6C3 in Arthrobacter sp. HW08

    PubMed Central

    Wang, Yan; Zhai, A’guan; Zhang, Yanqi; Qiu, Kai; Wang, Jianhua; Li, Qinfan

    2016-01-01

    Swainsonine is an indolizidine alkaloid that has been found in locoweeds and some fungi. Our previous study demonstrated that Arthrobacter sp. HW08 or its crude enzyme extract could degrade swainsonie efficiently. However, the mechanism of swainsonine degradation in bacteria remains unclear. In this study, we used label-free quantitative proteomics method based on liquid chromatography-electrospray ionization-tandem mass spectrometry to dissect the mechanism of swainsonine biodegradation by Arthrobacter sp. HW08. The results showed that 129 differentially expressed proteins were relevant to swainsonine degradation. These differentially expressed proteins were mostly related to the biological process of metabolism and the molecular function of catalytic activity. Among the 129 differentially expressed proteins, putative sugar phosphate isomerase/epimerase A1R5X7, Acetyl-CoA acetyltransferase A0JZ95, and nicotinamide adenine dinucleotide phosphate (NADP)-dependent alcohol dehydrogenase A1R6C3 were found to contribute to the swainsonine degradation. Notably, NADP-dependent alcohol dehyrodgenase A1R6C3 appeared to play a major role in degrading swainsonine, but not as much as Arthrobacter sp. HW08 did. Collectively, our findings here provide insights to understand the mechanism of swainsonine degradation in bacteria. PMID:27196926

  6. Gene ercA, encoding a putative iron-containing alcohol dehydrogenase, is involved in regulation of ethanol utilization in Pseudomonas aeruginosa.

    PubMed

    Hempel, Niels; Görisch, Helmut; Mern, Demissew S

    2013-09-01

    Several two-component regulatory systems are known to be involved in the signal transduction pathway of the ethanol oxidation system in Pseudomonas aeruginosa ATCC 17933. These sensor kinases and response regulators are organized in a hierarchical manner. In addition, a cytoplasmic putative iron-containing alcohol dehydrogenase (Fe-ADH) encoded by ercA (PA1991) has been identified to play an essential role in this regulatory network. The gene ercA (PA1991) is located next to ercS, which encodes a sensor kinase. Inactivation of ercA (PA1991) by insertion of a kanamycin resistance cassette created mutant NH1. NH1 showed poor growth on various alcohols. On ethanol, NH1 grew only with an extremely extended lag phase. During the induction period on ethanol, transcription of structural genes exa and pqqABCDEH, encoding components of initial ethanol oxidation in P. aeruginosa, was drastically reduced in NH1, which indicates the regulatory function of ercA (PA1991). However, transcription in the extremely delayed logarithmic growth phase was comparable to that in the wild type. To date, the involvement of an Fe-ADH in signal transduction processes has not been reported.

  7. Cofactor engineering of ketol-acid reductoisomerase (IlvC) and alcohol dehydrogenase (YqhD) improves the fusel alcohol yield in algal protein anaerobic fermentation

    DOE PAGES

    Wu, Weihua; Tran-Gyamfi, Mary Bao; Jaryenneh, James Dekontee; ...

    2016-08-24

    Recently the feasibility of conversion of algal protein to mixed alcohols has been demonstrated with an engineered E.coli strain, enabling comprehensive utilization of the biomass for biofuel applications. However, the yield and titers of mixed alcohol production must be improved for market adoption. A major limiting factor for achieving the necessary yield and titer improvements is cofactor imbalance during the fermentation of algal protein. To resolve this problem, a directed evolution approach was applied to modify the cofactor specificity of two key enzymes (IlvC and YqhD) from NADPH to NADH in the mixed alcohol metabolic pathway. Using high throughput screening,more » more than 20 YqhD mutants were identified to show activity on NADH as a cofactor. Of these 20 mutants, the top five of YqhD mutants were selected for combination with two IlvC mutants with NADH as a cofactor for the modification of the protein conversion strain. The combination of the IlvC and YqhD mutants yielded a refined E.coli strain, subtype AY3, with increased fusel alcohol yield of ~60% compared to wild type under anaerobic fermentation on amino acid mixtures. When applied to real algal protein hydrolysates, the strain AY3 produced 100% and 38% more total mixed alcohols than the wild type strain on two different algal hydrolysates, respectively. The results indicate that cofactor engineering is a promising approach to improve the feasibility of bioconversion of algal protein into mixed alcohols as advanced biofuels.« less

  8. Thiodiglycol, the Hydrolysis Product of Sulfur Mustard: Analysis of In Vitro Biotransformation by Mammalian Alcohol Dehydrogenases using Nuclear Magnetic Resonance

    DTIC Science & Technology

    2006-01-01

    secondary aliphatic and aromatic alcohols. They function as detoxifiers with the digitalis glycosides (Frey and Vallee, 1979) and permethrin (Choi et al...Command. The authors would like to thank Dr. essential to the metabolism of digitalis . Biochem. Biophys. Res. Commun. Jonghoon Choi for purifying the

  9. Alcohol

    MedlinePlus

    ... that's how many accidents occur. continue What Is Alcoholism? What can be confusing about alcohol is that ... develop a problem with it. Sometimes, that's called alcoholism (say: al-kuh-HOL - ism) or being an ...

  10. Alcohol

    MedlinePlus

    If you are like many Americans, you drink alcohol at least occasionally. For many people, moderate drinking ... risky. Heavy drinking can lead to alcoholism and alcohol abuse, as well as injuries, liver disease, heart ...

  11. Ethanol metabolism, oxidative stress, and endoplasmic reticulum stress responses in the lungs of hepatic alcohol dehydrogenase deficient deer mice after chronic ethanol feeding

    SciTech Connect

    Kaphalia, Lata; Boroumand, Nahal; Hyunsu, Ju; Kaphalia, Bhupendra S.; Calhoun, William J.

    2014-06-01

    Consumption and over-consumption of alcoholic beverages are well-recognized contributors to a variety of pulmonary disorders, even in the absence of intoxication. The mechanisms by which alcohol (ethanol) may produce disease include oxidative stress and prolonged endoplasmic reticulum (ER) stress. Many aspects of these processes remain incompletely understood due to a lack of a suitable animal model. Chronic alcohol over-consumption reduces hepatic alcohol dehydrogenase (ADH), the principal canonical metabolic pathway of ethanol oxidation. We therefore modeled this situation using hepatic ADH-deficient deer mice fed 3.5% ethanol daily for 3 months. Blood ethanol concentration was 180 mg% in ethanol fed mice, compared to < 1.0% in the controls. Acetaldehyde (oxidative metabolite of ethanol) was minimally, but significantly increased in ethanol-fed vs. pair-fed control mice. Total fatty acid ethyl esters (FAEEs, nonoxidative metabolites of ethanol) were 47.6 μg/g in the lungs of ethanol-fed mice as compared to 1.5 μg/g in pair-fed controls. Histological and immunohistological evaluation showed perivascular and peribronchiolar lymphocytic infiltration, and significant oxidative injury, in the lungs of ethanol-fed mice compared to pair-fed controls. Several fold increases for cytochrome P450 2E1, caspase 8 and caspase 3 found in the lungs of ethanol-fed mice as compared to pair-fed controls suggest role of oxidative stress in ethanol-induced lung injury. ER stress and unfolded protein response signaling were also significantly increased in the lungs of ethanol-fed mice. Surprisingly, no significant activation of inositol-requiring enzyme-1α and spliced XBP1 was observed indicating a lack of activation of corrective mechanisms to reinstate ER homeostasis. The data suggest that oxidative stress and prolonged ER stress, coupled with formation and accumulation of cytotoxic FAEEs may contribute to the pathogenesis of alcoholic lung disease. - Highlights: • Chronic

  12. Specific inhibition by synthetic analogs of pyruvate reveals that the pyruvate dehydrogenase reaction is essential for metabolism and viability of glioblastoma cells.

    PubMed

    Bunik, Victoria I; Artiukhov, Artem; Kazantsev, Alexey; Goncalves, Renata; Daloso, Danilo; Oppermann, Henry; Kulakovskaya, Elena; Lukashev, Nikolay; Fernie, Alisdair; Brand, Martin; Gaunitz, Frank

    2015-11-24

    The pyruvate dehydrogenase complex (PDHC) and its phosphorylation are considered essential for oncotransformation, but it is unclear whether cancer cells require PDHC to be functional or silenced. We used specific inhibition of PDHC by synthetic structural analogs of pyruvate to resolve this question. With isolated and intramitochondrial PDHC, acetyl phosphinate (AcPH, KiAcPH = 0.1 μM) was a much more potent competitive inhibitor than the methyl ester of acetyl phosphonate (AcPMe, KiAcPMe = 40 μM). When preincubated with the complex, AcPH also irreversibly inactivated PDHC. Pyruvate prevented, but did not reverse the inactivation. The pyruvate analogs did not significantly inhibit other 2-oxo acid dehydrogenases. Different cell lines were exposed to the inhibitors and a membrane-permeable precursor of AcPMe, dimethyl acetyl phosphonate, which did not inhibit isolated PDHC. Using an ATP-based assay, dependence of cellular viability on the concentration of the pyruvate analogs was followed. The highest toxicity of the membrane-permeable precursor suggested that the cellular action of charged AcPH and AcPMe requires monocarboxylate transporters. The relevant cell-specific transcripts extracted from Gene Expression Omnibus database indicated that cell lines with higher expression of monocarboxylate transporters and PDHC components were more sensitive to the PDHC inhibitors. Prior to a detectable antiproliferative action, AcPH significantly changed metabolic profiles of the investigated glioblastoma cell lines. We conclude that catalytic transformation of pyruvate by pyruvate dehydrogenase is essential for the metabolism and viability of glioblastoma cell lines, although metabolic heterogeneity causes different cellular sensitivities and/or abilities to cope with PDHC inhibition.

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

    PubMed

    Cheng, Shouqiang; Fan, Chenguang; Sinha, Sharmistha; Bobik, Thomas A

    2012-01-01

    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.

  14. Determination of the in vivo NAD:NADH ratio in Saccharomyces cerevisiae under anaerobic conditions, using alcohol dehydrogenase as sensor reaction.

    PubMed

    Bekers, K M; Heijnen, J J; van Gulik, W M

    2015-08-01

    With the current quantitative metabolomics techniques, only whole-cell concentrations of NAD and NADH can be quantified. These measurements cannot provide information on the in vivo redox state of the cells, which is determined by the ratio of the free forms only. In this work we quantified free NAD:NADH ratios in yeast under anaerobic conditions, using alcohol dehydrogenase (ADH) and the lumped reaction of glyceraldehyde-3-phosphate dehydrogenase and 3-phosphoglycerate kinase as sensor reactions. We showed that, with an alternative accurate acetaldehyde determination method, based on rapid sampling, instantaneous derivatization with 2,4 diaminophenol hydrazine (DNPH) and quantification with HPLC, the ADH-catalysed oxidation of ethanol to acetaldehyde can be applied as a relatively fast and simple sensor reaction to quantify the free NAD:NADH ratio under anaerobic conditions. We evaluated the applicability of ADH as a sensor reaction in the yeast Saccharomyces cerevisiae, grown in anaerobic glucose-limited chemostats under steady-state and dynamic conditions. The results found in this study showed that the cytosolic redox status (NAD:NADH ratio) of yeast is at least one order of magnitude lower, and is thus much more reduced, under anaerobic conditions compared to aerobic glucose-limited steady-state conditions. The more reduced state of the cytosol under anaerobic conditions has major implications for (central) metabolism. Accurate determination of the free NAD:NADH ratio is therefore of importance for the unravelling of in vivo enzyme kinetics and to judge accurately the thermodynamic reversibility of each redox reaction.

  15. Toxicological effects of thiomersal and ethylmercury: Inhibition of the thioredoxin system and NADP{sup +}-dependent dehydrogenases of the pentose phosphate pathway

    SciTech Connect

    Rodrigues, Juan; Branco, Vasco; Lu, Jun; Holmgren, Arne; Carvalho, Cristina

    2015-08-01

    Mercury (Hg) is a strong toxicant affecting mainly the central nervous, renal, cardiovascular and immune systems. Thiomersal (TM) is still in use in medical practice as a topical antiseptic and as a preservative in multiple dose vaccines, routinely given to young children in some developing countries, while other forms of mercury such as methylmercury represent an environmental and food hazard. The aim of the present study was to determine the effects of thiomersal (TM) and its breakdown product ethylmercury (EtHg) on the thioredoxin system and NADP{sup +}-dependent dehydrogenases of the pentose phosphate pathway. Results show that TM and EtHg inhibited the thioredoxin system enzymes in purified suspensions, being EtHg comparable to methylmercury (MeHg). Also, treatment of neuroblastoma and liver cells with TM or EtHg decreased cell viability (GI{sub 50}: 1.5 to 20 μM) and caused a significant (p < 0.05) decrease in the overall activities of thioredoxin (Trx) and thioredoxin reductase (TrxR) in a concentration- and time-dependent manner in cell lysates. Compared to control, the activities of Trx and TrxR in neuroblastoma cells after EtHg incubation were reduced up to 60% and 80% respectively, whereas in hepatoma cells the reduction was almost 100%. In addition, the activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were also significantly inhibited by all mercurials, with inhibition intensity of Hg{sup 2+} > MeHg ≈ EtHg > TM (p < 0.05). Cell incubation with sodium selenite alleviated the inhibitory effects on TrxR and glucose-6-phosphate dehydrogenase. Thus, the molecular mechanism of toxicity of TM and especially of its metabolite EtHg encompasses the blockage of the electrons from NADPH via the thioredoxin system. - Highlights: • TM and EtHg inhibit Trx and TrxR both in purified suspensions and cell lysates. • TM and EtHg also inhibit the activities of G6PDH and 6PGDH in cell lysates, • Co-exposure to selenite alleviates

  16. Alcohol

    MedlinePlus

    ... de los dientes Video: Getting an X-ray Alcohol KidsHealth > For Kids > Alcohol Print A A A What's in this article? ... What Is Alcoholism? Say No en español El alcohol Getting the Right Message "Hey, who wants a ...

  17. Pharmacological evaluation of alcohol withdrawal-induced inhibition of exploratory behaviour and supersensitivity to harmine-induced tremor.

    PubMed

    Meert, T F

    1994-01-01

    Rats given a liquid diet containing 10% (v/v) alcohol consume high quantities of alcohol. Within 8 hr after cessation of the alcohol intake, the animals will show alcohol-withdrawal reactions including a supersensitivity to harmine-induced tremor and an inhibition of exploratory behaviour in a neutral environment. Several drugs can overcome one or more of these alcohol-withdrawal reactions. A reduction of the alcohol withdrawal-induced inhibition of exploration, in terms of both the number of transits into the open field and the time spent in the open field, was obtained with chlordiazepoxide, ritanserin, mianserin and propranolol. Of these four compounds, propranolol and mianserin were also active against the supersensitivity to both 5.00 and 10.00 mg/kg harmine-induced tremor. Chlordiazepoxide and ritanserin were only active against 5.00 mg/kg harmine. Other compounds that reversed the supersensitivity to harmine-induced tremor during alcohol withdrawal included buspirone, fluoxetine, haloperidol, clonidine, flunarizine and baclofen. Very limited effects on both alcohol-withdrawal reactions were observed with ondansetron, nimodipine and MK-801. Risperidone and SCH 23390 were inactive. These results demonstrate that some alcohol withdrawal reactions can be studied in a systematic way and that various pharmacological agents can differentially interact with these alcohol withdrawal reactions.

  18. Acetaldehyde/alcohol dehydrogenase-2 (EhADH2) and clathrin are involved in internalization of human transferrin by Entamoeba histolytica.

    PubMed

    Reyes-López, Magda; Bermúdez-Cruz, Rosa María; Avila, Eva E; de la Garza, Mireya

    2011-01-01

    Transferrin (Tf) is a host glycoprotein capable of binding two ferric-iron ions to become holotransferrin (holoTf), which transports iron in to all cells. Entamoeba histolytica is a parasitic protozoan able to use holoTf as a sole iron source in vitro. The mechanism by which this parasite scavenges iron from holoTf is unknown. An E. histolytica holoTf-binding protein (EhTfbp) was purified by using an anti-human transferrin receptor (TfR) monoclonal antibody. EhTfbp was identified by MS/MS analysis and database searches as E. histolytica acetaldehyde/alcohol dehydrogenase-2 (EhADH2), an iron-dependent enzyme. Both EhTfbp and EhADH2 bound holoTf and were recognized by the anti-human TfR antibody, indicating that they correspond to the same protein. It was found that the amoebae internalized holoTf through clathrin-coated pits, suggesting that holoTf endocytosis could be important for the parasite during colonization and invasion of the intestinal mucosa and liver.

  19. Purification and characterization of a novel alcohol dehydrogenase from Leifsonia sp. strain S749: a promising biocatalyst for an asymmetric hydrogen transfer bioreduction.

    PubMed

    Inoue, Kousuke; Makino, Yoshihide; Itoh, Nobuya

    2005-07-01

    To find microorganisms that could reduce phenyl trifluoromethyl ketone (PTK) to (S)-1-phenyltrifluoroethanol [(S)-PTE], styrene-assimilating bacteria (ca. 900 strains) isolated from soil samples were screened. We found that Leifsonia sp. strain S749 was the most suitable strain for the conversion of PTK to (S)-PTE in the presence of 2-propanol as a hydrogen donor. The enzyme corresponding to the reaction was purified homogeneity, characterized and designated Leifsonia alcohol dehydrogenase (LSADH). The purified enzyme had a molecular weight of 110,000 and was composed of four identical subunits (molecular weight, 26,000). LSADH required NADH as a cofactor, showed little activity with NADPH, and reduced a wide variety of aldehydes and ketones. LSADH catalyzed the enantioselective reduction of some ketones with high enantiomeric excesses (e.e.): PTK to (S)-PTE (>99% e.e.), acetophenone to (R)-1-phenylethanol (99% e.e.), and 2-heptanone to (R)-2-heptanol (>99% e.e.) in the presence of 2-propanol without an additional NADH regeneration system. Therefore, it would be a useful biocatalyst.

  20. Lignin and lignans in plant defence: insight from expression profiling of cinnamyl alcohol dehydrogenase genes during development and following fungal infection in Populus.

    PubMed

    Bagniewska-Zadworna, Agnieszka; Barakat, Abdelali; Lakomy, Piotr; Smoliński, Dariusz J; Zadworny, Marcin

    2014-12-01

    Cinnamyl alcohol dehydrogenase (CAD) catalyses the final step in the biosynthesis of monolignol, the main component of lignin. Lignins, deposited in the secondary cell wall, play a role in plant defence against pathogens. We re-analysed the phylogeny of CAD/CAD-like genes using sequences from recently sequenced genomes, and analysed the temporal and spatial expression profiles of CAD/CAD-like genes in Populus trichocarpa healthy and infected plants. Three fungal pathogens (Rhizoctonia solani, Fusarium oxysporum, and Cytospora sp.), varying in lifestyle and pathogenicity, were used for plant infection. Phylogenetic analyses showed that CAD/CAD-like genes were distributed in classes represented by all members from angiosperm lineages including basal angiosperms and Selaginella. The analysed genes showed different expression profiles during development and demonstrated that three genes were involved in primary xylem maturation while five may function in secondary xylem formation. Expression analysis following inoculation with fungal pathogens, showed that five genes were induced in either stem or leaves. These results add further evidence that CAD/CAD-like genes have evolved specialised functions in plant development and defence against various pest and pathogens. Two genes (PoptrCAD11 and PoptrCAD15), which were induced under various stresses, could be treated as universal markers of plant defence using lignification or lignan biosynthesis.

  1. A study on the stability and enzymatic activity of yeast alcohol dehydrogenase in presence of the self-assembling block copolymer Poloxamer 407.

    PubMed

    Pucciarelli, Stefania; Bonacucina, Giulia; Bernabucci, Franco; Cespi, Marco; Mencarelli, Giovanna; De Fronzo, Giusi Serena; Natalini, Paolo; Palmieri, Giovanni Filippo

    2012-05-01

    Yeast alcohol dehydrogenase (ADH) is an enzyme widely studied for biotechnological applications due to its involvement in fermentation industry, and various attempts to improve its catalytic properties and its thermal stability have been carried out. In this paper, the influence of a block copolymer (Poloxamer 407) on ADH enzymatic activity and thermal behaviour has been studied in order to get new insights about the use of poloxamers in formulation of sustained release systems for therapeutic proteins. Poloxamer 407 has the ability to form micelles and gel due to its self-assembling and thermoresponsive properties. The effect of the copolymer towards thermal stress and pH changes, which often reduce enzymes activity it has been investigated by means of enzymatic assays and differential scanning calorimetry. Results showed that at pH 9.1 and 7.3, the Poloxamer in the form of unimeric, micellar and gel state is able to effectively preserve the enzyme from thermoinactivation. In addition by calorimetric data Poloxamer 407 has showed an effect in preserving ADH from aggregation at pH 7.3. In conclusion, Poloxamer 407 seems to be very effective in protecting ADH from stress related events, like alkaline inactivation and aggregation.

  2. Dependence of Ethanolic Fermentation, Cytoplasmic pH Regulation, and Viability on the Activity of Alcohol Dehydrogenase in Hypoxic Maize Root Tips 1

    PubMed Central

    Roberts, Justin K. M.; Chang, Keejong; Webster, Cecelia; Callis, Judy; Walbot, Virginia

    1989-01-01

    We examined the role of alcohol dehydrogenase (ADH) in the metabolism and survival of hypoxic maize (Zea mays L.) root tips. The dependence of the rate of ethanolic fermentation, cytoplasmic pH, and viability on the activity of ADH in maize root tips during extreme hypoxia was determined. Maize lines with ADH activities differing over about a 200-fold range were studied. Effects of genetic background were controlled by comparing pairs of F4 progeny of crosses between mutant (low ADH activity) and reference inbred lines. The capacity of hypoxic root tips to perform ethanolic fermentation exhibited a dependence on ADH activity only at activities found in Adh 1 nulls. The ability of maize root tips to withstand prolonged and extreme hypoxia was like-wise independent of ADH activity, except at the lowest activities. Root tips that exhibited lower tolerance of hypoxia had more acidic cytoplasm during extreme hypoxia. We conclude that the activity of ADH in normal maize root tips does not limit the capacity for energy production via fermentation, and does not determine viability under extreme hypoxia. The significance of the induction of ADH activity in plants by hypoxia is discussed. PMID:16666696

  3. Dependence of ethanolic fermentation, cytoplasmic pH regulation, and viability on the activity of alcohol dehydrogenase in hypoxic maize root tips

    SciTech Connect

    Roberts, J.; Chang, Keejong; Webster, C.; Callis, J.; Walbot, V. Stanford Univ., CA )

    1989-04-01

    We examined the role of alcohol dehydrogenase (ADH) in the metabolism and survival of hypoxic maize (Zea mays L.) root tips. The dependence of the rate of ethanolic fermentation, cytoplasmic pH, and viability on the activity of ADH in maize root tips during extreme hypoxia was determined. Maize lines with ADH activities differing over about a 200-fold range were studied. Effects of genetic background were controlled by comparing pairs of F4 progeny of crosses between mutant (low ADH activity) and reference inbred lines. The capacity of hypoxic root tips to perform ethanolic fermentation exhibited a dependence on ADH activity only at activities found in Adh 1 nulls. The ability of maize root tips to withstand prolonged and extreme hypoxia was likewise independent of ADH activity, except at the lowest activities. Root tips that exhibited lower tolerance of hypoxia had more acidic cytoplasm during extreme hypoxia. We conclude that the activity of ADH in normal maize root tips does not limit the capacity for energy production via fermentation, and does not determine viability under extreme hypoxia. The significance of the induction of ADH activity in plants by hypoxia is discussed.

  4. Manipulation of Guaiacyl and Syringyl Monomer Biosynthesis in an Arabidopsis Cinnamyl Alcohol Dehydrogenase Mutant Results in Atypical Lignin Biosynthesis and Modified Cell Wall Structure

    PubMed Central

    Anderson, Nickolas A.; Tobimatsu, Yuki; Ciesielski, Peter N.; Ximenes, Eduardo; Ralph, John; Donohoe, Bryon S.; Ladisch, Michael; Chapple, Clint

    2015-01-01

    Modifying lignin composition and structure is a key strategy to increase plant cell wall digestibility for biofuel production. Disruption of the genes encoding both cinnamyl alcohol dehydrogenases (CADs), including CADC and CADD, in Arabidopsis thaliana results in the atypical incorporation of hydroxycinnamaldehydes into lignin. Another strategy to change lignin composition is downregulation or overexpression of ferulate 5-hydroxylase (F5H), which results in lignins enriched in guaiacyl or syringyl units, respectively. Here, we combined these approaches to generate plants enriched in coniferaldehyde-derived lignin units or lignins derived primarily from sinapaldehyde. The cadc cadd and ferulic acid hydroxylase1 (fah1) cadc cadd plants are similar in growth to wild-type plants even though their lignin compositions are drastically altered. In contrast, disruption of CAD in the F5H-overexpressing background results in dwarfism. The dwarfed phenotype observed in these plants does not appear to be related to collapsed xylem, a hallmark of many other lignin-deficient dwarf mutants. cadc cadd, fah1 cadc cadd, and cadd F5H-overexpressing plants have increased enzyme-catalyzed cell wall digestibility. Given that these CAD-deficient plants have similar total lignin contents and only differ in the amounts of hydroxycinnamaldehyde monomer incorporation, these results suggest that hydroxycinnamaldehyde content is a more important determinant of digestibility than lignin content. PMID:26265762

  5. Cellulase and alcohol dehydrogenase immobilized in Langmuir and Langmuir-Blodgett films and their molecular-level effects upon contact with cellulose and ethanol.

    PubMed

    Rodrigues, Dilmer; Camilo, Fernanda Ferraz; Caseli, Luciano

    2014-02-25

    The key challenges for producing devices based on nanostructured films with control over the molecular architecture are to preserve the catalytic activity of the immobilized biomolecules and to provide a reliable method for determining the intermolecular interactions and the accommodation of molecules at very small scales. In this work, the enzymes cellulase and alcohol dehydrogenase (ADH) were coimmobilized with dipalmitoylphosphatidylcholine (DPPC) as Langmuir-Blodgett (LB) films, and their biological activities were assayed by accommodating the structure formed in contact with cellulose. For this purpose, the polysaccharide was dissolved in an ionic liquid, 1-buthyl-3-methylimidazolium chloride (BMImCl), and dropped on the top of the hybrid cellulase-ADH-DPPC LB film. The interactions between cellulose and ethanol, which are the catalytic substrates of the enzymes as well as important elements in the production of second-generation fuels, were then investigated using polarization-modulation infrared reflection-absorption spectroscopy (PM-IRRAS). Investigation of the secondary structures of the enzymes was performed using PM-IRRAS, through which the presence of ethanol and cellulose was observed to highly affect the structures of ADH and cellulase, respectively. The detection of products formed from the catalyzed reactions as well as the changes of secondary structure of the enzymes immobilization could be carried out, which opens the possibility to produce a means for producing second-generation ethanol using nanoscale arrangements.

  6. Alcohol Dehydrogenase and Pyruvate Decarboxylase Activity in Leaves and Roots of Eastern Cottonwood (Populus deltoides Bartr.) and Soybean (Glycine max L.) 1

    PubMed Central

    Kimmerer, Thomas W.

    1987-01-01

    Pyruvate decarboxylase (PDC, EC 4.1.1.1) and alcohol dehydrogenase (ADH, EC 1.1.1.1) are responsible for the anaerobic production of acetaldehyde and ethanol in higher plants. In developing soybean embryos, ADH activity increased upon imbibition and then declined exponentially with development, and was undetectable in leaves by 30 days after imbibition. PDC was not detectable in soybean leaves. In contrast, ADH activity remained high in developing cottonwood seedlings, with no decline in activity during development. ADH activity in the first fully expanded leaf of cottonwood was 230 micromoles NADH oxidized per minute per gram dry weight, and increased with leaf age. Maximal PDC activity of cottonwood leaves was 10 micromoles NADH oxidized per minute per gram dry weight. ADH activity in cottonwood roots was induced by anaerobic stress, increasing from 58 to 205 micromoles NADH oxidized per minute per gram dry weight in intact plants in 48 hours, and from 38 to 246 micromoles NADH oxidized per minute per gram dry weight in detached roots in 48 hours. Leaf ADH activity increased by 10 to 20% on exposure to anaerobic conditions. Crude leaf enzyme extracts with high ADH activity reduced little or no NADH when other aldehydes, such as trans-2-hexenal, were provided as substrate. ADH and PDC are constitutive enzyme in cottonwood leaves, but their metabolic role is not known. PMID:16665586

  7. Expression of a heat-stable NADPH-dependent alcohol dehydrogenase in Caldicellulosiruptor bescii results in furan aldehyde detoxification

    DOE PAGES

    Chung, Daehwan; Verbeke, Tobin J.; Cross, Karissa L.; ...

    2015-07-22

    Compounds such as furfural and 5-hydroxymethylfurfural (5-HMF) are generated through the dehydration of xylose and glucose, respectively, during dilute-acid pretreatment of lignocellulosic biomass and are also potent microbial growth and fermentation inhibitors. The enzymatic reduction of these furan aldehydes to their corresponding, and less toxic, alcohols is an engineering approach that has been successfully implemented in both Saccharomyces cerevisiae and ethanologenicEscherichia coli, but has not yet been investigated in thermophiles relevant to biofuel production through consolidated bioprocessing (CBP). Developing CBP-relevant biocatalysts that are either naturally resistant to such inhibitors, or are amenable to engineered resistance, is therefore, an important componentmore » in making biofuels production from lignocellulosic biomass feasible.« less

  8. Expression of a heat-stable NADPH-dependent alcohol dehydrogenase in Caldicellulosiruptor bescii results in furan aldehyde detoxification

    SciTech Connect

    Chung, Daehwan; Verbeke, Tobin J.; Cross, Karissa L.; Westpheling, Janet; Elkins, James G.

    2015-07-22

    Compounds such as furfural and 5-hydroxymethylfurfural (5-HMF) are generated through the dehydration of xylose and glucose, respectively, during dilute-acid pretreatment of lignocellulosic biomass and are also potent microbial growth and fermentation inhibitors. The enzymatic reduction of these furan aldehydes to their corresponding, and less toxic, alcohols is an engineering approach that has been successfully implemented in both Saccharomyces cerevisiae and ethanologenicEscherichia coli, but has not yet been investigated in thermophiles relevant to biofuel production through consolidated bioprocessing (CBP). Developing CBP-relevant biocatalysts that are either naturally resistant to such inhibitors, or are amenable to engineered resistance, is therefore, an important component in making biofuels production from lignocellulosic biomass feasible.

  9. Alcohol

    MedlinePlus

    ... parents and other adults use alcohol socially — having beer or wine with dinner, for example — alcohol seems ... besides just hanging out in someone's basement drinking beer all night. Plan a trip to the movies, ...

  10. The protein conformation of Cd-substituted horse liver alcohol dehydrogenase and its metal-site coordination geometry in binary and ternary inhibitor complexes.

    PubMed

    Hemmingsen, L; Bauer, R; Bjerrum, M J; Adolph, H W; Zeppezauer, M; Cedergren-Zeppezauer, E

    1996-10-15

    The coordination geometry of the metal at the active site in Cd-substituted horse liver alcohol dehydrogenase (LADH) has been investigated for the binary complexes of LADH with imidazole, isobutyramide, decanoic acid and Cl-, and for the ternary complexes of LADH with NADH and imidazole, NADH and isobutyramide, NAD+ and decanoic acid and NAD+ and Cl-, by using the method of perturbed angular correlation of gamma-rays (PAC). The spectral results are consistent with a flexible structure around the metal for the binary complexes with inhibitors. For ternary complexes, however, a rigid structure is observed. An exception is the ternary complex between LADH, NADH and imidazole, in which the metal site is still flexible. Comparing with available structures determined by X-ray crystallography, we found a correlation between open structures and flexible metal sites, and between closed structures and rigid metal sites. This indicates that the PAC technique can be applied to distinguish the two conformations in solution. The spectral parameters, omega(o) and eta, of the experiments, except for the complexes with imidazole, fall into two groups: one with low omega(o) and one with high omega(o) (eta is relatively constant in all experiments). In this work it is clarified that the low omega(o) values are connected with the presence of a negatively charged solvent ligand. Using an angular-overlap approach to interpret the results, the low omega(o) values are found to be compatible with a coordination geometry where the S-Cd-S (Cys174 and Cys46 coordinate to the metal) angle is about 110 degrees as suggested in [Hemmingsen, L., Bauer, R., Danielsen, E., Bjerrum. M. J., Zeppezauer, M., Adolph, H. W., Formicka, G. & Cedergren-Zeppezauer, E. (1995) Biochemistry 34, 7145-7153], whereas high omega(o) values are compatible with an S-Cd-S angle of 130 degrees. The presence of a negatively charged metal ligand, therefore, might trigger the movement of the sulfur of Cys174. As it is

  11. Association of Genetically Determined Aldehyde Dehydrogenase 2 Activity with Diabetic Complications in Relation to Alcohol Consumption in Japanese Patients with Type 2 Diabetes Mellitus: The Fukuoka Diabetes Registry.

    PubMed

    Idewaki, Yasuhiro; Iwase, Masanori; Fujii, Hiroki; Ohkuma, Toshiaki; Ide, Hitoshi; Kaizu, Shinako; Jodai, Tamaki; Kikuchi, Yohei; Hirano, Atsushi; Nakamura, Udai; Kubo, Michiaki; Kitazono, Takanari

    2015-01-01

    Aldehyde dehydrogenase 2 (ALDH2) detoxifies aldehyde produced during ethanol metabolism and oxidative stress. A genetic defect in this enzyme is common in East Asians and determines alcohol consumption behaviors. We investigated the impact of genetically determined ALDH2 activity on diabetic microvascular and macrovascular complications in relation to drinking habits in Japanese patients with type 2 diabetes mellitus. An ALDH2 single-nucleotide polymorphism (rs671) was genotyped in 4,400 patients. Additionally, the relationship of clinical characteristics with ALDH2 activity (ALDH2 *1/*1 active enzyme activity vs. *1/*2 or *2/*2 inactive enzyme activity) and drinking habits (lifetime abstainers vs. former or current drinkers) was investigated cross-sectionally (n = 691 in *1/*1 abstainers, n = 1,315 in abstainers with *2, n = 1,711 in *1/*1 drinkers, n = 683 in drinkers with *2). The multiple logistic regression analysis for diabetic complications was adjusted for age, sex, current smoking habits, leisure-time physical activity, depressive symptoms, diabetes duration, body mass index, hemoglobin A1c, insulin use, high-density lipoprotein cholesterol, systolic blood pressure and renin-angiotensin system inhibitors use. Albuminuria prevalence was significantly lower in the drinkers with *2 than that of other groups (odds ratio [95% confidence interval (CI)]: *1/*1 abstainers as the referent, 0.94 [0.76-1.16] in abstainers with *2, 1.00 [0.80-1.26] in *1/*1 drinkers, 0.71 [0.54-0.93] in drinkers with *2). Retinal photocoagulation prevalence was also lower in drinkers with ALDH2 *2 than that of other groups. In contrast, myocardial infarction was significantly increased in ALDH2 *2 carriers compared with that in ALDH2 *1/*1 abstainers (odds ratio [95% CI]: *1/*1 abstainers as the referent, 2.63 [1.28-6.13] in abstainers with *2, 1.89 [0.89-4.51] in *1/*1 drinkers, 2.35 [1.06-5.79] in drinkers with *2). In summary, patients with type 2 diabetes and ALDH2 *2 displayed a

  12. Fluoxetine elevates allopregnanolone in female rat brain but inhibits a steroid microsomal dehydrogenase rather than activating an aldo-keto reductase

    PubMed Central

    Fry, J P; Li, K Y; Devall, A J; Cockcroft, S; Honour, J W; Lovick, T A

    2014-01-01

    Background and Purpose Fluoxetine, a selective serotonin reuptake inhibitor, elevates brain concentrations of the neuroactive progesterone metabolite allopregnanolone, an effect suggested to underlie its use in the treatment of premenstrual dysphoria. One report showed fluoxetine to activate the aldo-keto reductase (AKR) component of 3α-hydroxysteroid dehydrogenase (3α-HSD), which catalyses production of allopregnanolone from 5α-dihydroprogesterone. However, this action was not observed by others. The present study sought to clarify the site of action for fluoxetine in elevating brain allopregnanolone. Experimental Approach Adult male rats and female rats in dioestrus were treated with fluoxetine and their brains assayed for allopregnanolone and its precursors, progesterone and 5α-dihydroprogesterone. Subcellular fractions of rat brain were also used to investigate the actions of fluoxetine on 3α-HSD activity in both the reductive direction, producing allopregnanolone from 5α-dihydroprogesterone, and the reverse oxidative direction. Fluoxetine was also tested on these recombinant enzyme activities expressed in HEK cells. Key Results Short-term treatment with fluoxetine increased brain allopregnanolone concentrations in female, but not male, rats. Enzyme assays on native rat brain fractions and on activities expressed in HEK cells showed fluoxetine did not affect the AKR producing allopregnanolone from 5α-dihydroprogesterone but did inhibit the microsomal dehydrogenase oxidizing allopregnanolone to 5α-dihydroprogesterone. Conclusions and Implications Fluoxetine elevated allopregnanolone in female rat brain by inhibiting its oxidation to 5α-dihydroprogesterone by a microsomal dehydrogenase. This is a novel site of action for fluoxetine, with implications for the development of new agents and/or dosing regimens to raise brain allopregnanolone. PMID:25161074

  13. Alcoholism.

    ERIC Educational Resources Information Center

    Caliguri, Joseph P., Ed.

    This extensive annotated bibliography provides a compilation of documents retreived from a computerized search of the ERIC, Social Science Citation Index, and Med-Line databases on the topic of alcoholism. The materials address the following areas of concern: (1) attitudes toward alcohol users and abusers; (2) characteristics of alcoholics and…

  14. Inhibitory pathways and the inhibition of luteinizing hormone-releasing hormone release by alcohol

    PubMed Central

    Lomniczi, Alejandro; Mastronardi, Claudio A.; Faletti, Alicia G.; Seilicovich, Adriana; De Laurentiis, Andrea; McCann, Samuel M.; Rettori, Valeria

    2000-01-01

    In this research we examined the mechanisms by which ethanol (EtOH) inhibits luteinizing hormone-releasing hormone (LHRH) release from incubated medial basal hypothalamic explants. EtOH (100 mM) stimulated the release of two inhibitory neurotransmitters: γ-aminobutyric acid (GABA) and β-endorphin. EtOH also inhibited NO production, indicative of a suppression of nitric oxide synthase (NOS) activity. This inhibition was reversed by naltroxone (10−8 M), a μ-opioid receptor blocker, indicating that the inhibition of NOS by EtOH is mediated by β-endorphin. EtOH also blocked N-methyl-d-aspartic acid-induced LHRH release, but the blockade could not be reversed by either the GABA receptor blocker, bicuculline (10−5 M), naltroxone (10−8 M), or both inhibitors added together. However, increasing the concentration of naltrexone (10−6 M) but not bicuculline (10−4 M) reversed the inhibition. When we lowered the concentration of EtOH (50 mM), the EtOH-induced blockade of LHRH release could be reversed by either bicuculline (10−5 M), naltroxone (10−8 M), or the combination of the two blockers. Therefore, GABA is partially responsible for the blockade of N-methyl-d-aspartic acid-induced LHRH release. The block by GABA was exerted by inhibiting the activation of cyclooxygenase by NO, because it was reversed by prostaglandin E2, the product of activation of cyclooxygenase. Because the inhibition caused by the higher concentration of EtOH could not be reduced by bicuculline (10−4 M) but was blocked by naltroxone (10−6 M), the action of alcohol can be accounted for by stimulation of β-endorphin neurons that inhibit LHRH release by inhibition of activation of NOS and stimulation of GABA release. PMID:10688896

  15. Inhibition of vertebrate squalene epoxidase by extended and truncated analogues of trisnorsqualene alcohol.

    PubMed

    Sen, S E; Wawrzeńczyk, C; Prestwich, G D

    1990-06-01

    The epoxidation of squalene to (3S)-2,3-epoxysqualene and subsequent cyclization to lanosterol are keys steps in vertebrate cholesterol biosynthesis. Trisnorsqualene alcohol (TNSA) has previously been reported as a potent inhibitor of vertebrate squalene epoxidase, with IC50 = 4 microM for pig liver (J. Am. Chem. Soc. 1989, 111, 1508-1510). Analogues with extended and truncated carbon skeletons have been prepared and tested for pig liver squalene epoxidase (SE) inhibition. Most of the structural analogues were poor inhibitors of vertebrate SE, with the exception of bisnorsqualene alcohol which had the same activity as TNSA. These results support the theory that an intact trisnorsqualene moiety is required for activity.

  16. Inosine 5'-monophosphate dehydrogenase of Escherichia coli. Purification by affinity chromatography, subunit structure and inhibition by guanosine 5'-monophosphate.

    PubMed Central

    Gilbert, H J; Lowe, C R; Drabble, W T

    1979-01-01

    Escherichia coli IMP dehydrogenase (EC 1.2.1.14) was purified by affinity chromatography on immobilized nucleotides. The enzyme binds to agarose-bound 8-(6-aminohexyl)-AMP, N6-(6-aminohexyl)-AMP and 8-(8-amino-octyl)-IMP but not to immobilized NAD+ or Cibacron Blue F3G-A. AMP proved to be an effective eluent. A large-scale purification scheme in which 8-(6-aminohexyl)-AMP-agarose was used resulted in a homogeneous preparation of IMP dehydrogenase. The enzyme was also purified by immunoprecipitation with monospecific antisera. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, N-terminal amino acid analysis and tryptic 'finger-printing' demonstrated that IMP dehydrogenase comprises identical subunits of mol.wt. 58000. Trypsin and Pronase cleave the 58000-mol.wt. subunit into peptides of mol.wts. 42000 and 14000, with a concomitant decrease in enzyme activity. These observations rationalize much of the contradictory data on the subunit composition of the enzyme found in the literature. GMP appears to be a competitive inhibitor with respect to IMP, with no evidence for regulatory behaviour being found. The two purification procedures were also used to purify inactive mutant enzymes from guaB mutant strains of E. coli. PMID:44191

  17. A novel small molecule, LAS-0811, inhibits alcohol-induced apoptosis in VL-17A cells.

    PubMed

    Kim, Tae-Hun; Venugopal, Senthil K; Zhu, Ming; Wang, Si-Si; Lau, Derick; Lam, Kit S; Clemens, Dahn L; Zern, Mark A

    2009-02-20

    One of the pathways by which alcohol induces hepatocyte apoptosis is via oxidative stress. We screened several chemically-synthesized small molecules and found LAS-0811, which inhibits oxidative stress. In this study, we elucidated its role in inhibiting alcohol-induced apoptosis in hepatocyte-like VL-17A cells. VL-17A cells were pre-incubated with LAS-0811, followed by ethanol incubation. Ethanol-induced reactive oxygen species and apoptosis were significantly inhibited in LAS-0811 pre-treated cells. VL-17A cells were transfected with a reporter (ARE/TK-GFP) plasmid containing green fluorescent protein (GFP) as a reporter gene and the anti-oxidant response element as the promoter. LAS-0811 pre-treatment significantly induced the GFP expression compared to the cells treated with ethanol alone. LAS-0811 induced the activation of nrf2 and enhanced the expression and activity of glutathione peroxidase, one of the downstream targets of nrf2. The results indicate that LAS-0811 protects VL-17A cells against ethanol-induced oxidative stress and apoptosis at least in part via nrf2 activation.

  18. Atomic resolution structures of R-specific alcohol dehydrogenase from Lactobacillus brevis provide the structural bases of its substrate and cosubstrate specificity.

    PubMed

    Schlieben, Nils Helge; Niefind, Karsten; Müller, Jörg; Riebel, Bettina; Hummel, Werner; Schomburg, Dietmar

    2005-06-17

    The R-specific alcohol dehydrogenase (RADH) from Lactobacillus brevis is an NADP-dependent, homotetrameric member of the extended enzyme family of short-chain dehydrogenases/reductases (SDR) with a high biotechnological application potential. Its preferred in vitro substrates are prochiral ketones like acetophenone with almost invariably a small methyl group as one substituent and a bulky (often aromatic) moiety as the other. On the basis of an atomic-resolution structure of wild-type RADH in complex with NADP and acetophenone, we designed the mutant RADH-G37D, which should possess an improved cosubstrate specificity profile for biotechnological purposes, namely, a preference for NAD rather than NADP. Comparative kinetic measurements with wild-type and mutant RADH showed that this aim was achieved. To characterize the successful mutant structurally, we determined several, partly atomic-resolution, crystal structures of RADH-G37D both as an apo-enzyme and as ternary complex with NAD or NADH and phenylethanol. The increased affinity of RADH-G37D for NAD(H) depends on an interaction between the adenosine ribose moiety of NAD and the inserted aspartate side-chain. A structural comparison between RADH-G37D as apo-enzyme and as a part of a ternary complex revealed significant rearrangements of Ser141, Glu144, Tyr189 and Met205 in the vicinity of the active site. This plasticity contributes to generate a small hydrophobic pocket for the methyl group typical for RADH substrates, and a hydrophobic coat for the second, more variable and often aromatic, substituent. Around Ser141 we even found alternative conformations in the backbone. A structural adaptability in this region, which we describe here for the first time for an SDR enzyme, is probably functionally important, because it concerns Ser142, a member of the highly conserved catalytic tetrad typical for SDR enzymes. Moreover, it affects an extended proton relay system that has been identified recently as a critical

  19. Selective Inhibition of Mutant Isocitrate Dehydrogenase 1 (IDH1) via Disruption of a Metal Binding Network by an Allosteric Small Molecule

    PubMed Central

    Deng, Gejing; Shen, Junqing; Yin, Ming; McManus, Jessica; Mathieu, Magali; Gee, Patricia; He, Timothy; Shi, Chaomei; Bedel, Olivier; McLean, Larry R.; Le-Strat, Frank; Zhang, Ying; Marquette, Jean-Pierre; Gao, Qiang; Zhang, Bailin; Rak, Alexey; Hoffmann, Dietmar; Rooney, Eamonn; Vassort, Aurelie; Englaro, Walter; Li, Yi; Patel, Vinod; Adrian, Francisco; Gross, Stefan; Wiederschain, Dmitri; Cheng, Hong; Licht, Stuart

    2015-01-01

    Cancer-associated point mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) confer a neomorphic enzymatic activity: the reduction of α-ketoglutarate to d-2-hydroxyglutaric acid, which is proposed to act as an oncogenic metabolite by inducing hypermethylation of histones and DNA. Although selective inhibitors of mutant IDH1 and IDH2 have been identified and are currently under investigation as potential cancer therapeutics, the mechanistic basis for their selectivity is not yet well understood. A high throughput screen for selective inhibitors of IDH1 bearing the oncogenic mutation R132H identified compound 1, a bis-imidazole phenol that inhibits d-2-hydroxyglutaric acid production in cells. We investigated the mode of inhibition of compound 1 and a previously published IDH1 mutant inhibitor with a different chemical scaffold. Steady-state kinetics and biophysical studies show that both of these compounds selectively inhibit mutant IDH1 by binding to an allosteric site and that inhibition is competitive with respect to Mg2+. A crystal structure of compound 1 complexed with R132H IDH1 indicates that the inhibitor binds at the dimer interface and makes direct contact with a residue involved in binding of the catalytically essential divalent cation. These results show that targeting a divalent cation binding residue can enable selective inhibition of mutant IDH1 and suggest that differences in magnesium binding between wild-type and mutant enzymes may contribute to the inhibitors' selectivity for the mutant enzyme. PMID:25391653

  20. Opossum alcohol dehydrogenases: Sequences, structures, phylogeny and evolution: evidence for the tandem location of ADH genes on opossum chromosome 5.

    PubMed

    Holmes, Roger S

    2009-03-16

    BLAT (BLAST-Like Alignment Tool) analyses and interrogations of the recently published opossum genome were undertaken using previously reported rat ADH amino acid sequences. Evidence is presented for six opossum ADH genes localized on chromosome 5 and organized in a comparable ADH gene cluster to that reported for human and rat ADH genes. The predicted amino acid sequences and secondary structures for the opossum ADH subunits and the intron-exon boundaries for opossum ADH genes showed a high degree of similarity with other mammalian ADHs, and four opossum ADH classes were identified, namely ADH1, ADH3, ADH6 and ADH4 (for which three genes were observed: ADH4A, ADH4B and ADH4C). Previous biochemical analyses of opossum ADHs have reported the tissue distribution and properties for these enzymes: ADH1, the major liver enzyme; ADH3, widely distributed in opossum tissues with similar kinetic properties to mammalian class 3 ADHs; and ADH4, for which several forms were localized in extrahepatic tissues, especially in the digestive system and in the eye. These ADHs are likely to perform similar functions to those reported for other mammalian ADHs in the metabolism of ingested and endogenous alcohols and aldehydes. Phylogenetic analyses examined opossum, human, rat, chicken and cod ADHs, and supported the proposed designation of opossum ADHs as class I (ADH1), class III (ADH3), class IV (ADH4A, ADH4B and ADH4C) and class VI (ADH6). Percentage substitution rates were examined for ADHs during vertebrate evolution which indicated that ADH3 is evolving at a much slower rate to that of the other ADH classes.

  1. Evidence for Lateral Transfer of Genes Encoding Ferredoxins, Nitroreductases, NADH Oxidase, and Alcohol Dehydrogenase 3 from Anaerobic Prokaryotes to Giardia lamblia and Entamoeba histolytica

    PubMed Central

    Nixon, Julie E. J.; Wang, Amy; Field, Jessica; Morrison, Hilary G.; McArthur, Andrew G.; Sogin, Mitchell L.; Loftus, Brendan J.; Samuelson, John

    2002-01-01

    Giardia lamblia and Entamoeba histolytica are amitochondriate, microaerophilic protists which use fermentation enzymes like those of bacteria to survive anaerobic conditions within the intestinal lumen. Genes encoding fermentation enzymes and related electron transport peptides (e.g., ferredoxins) in giardia organisms and amebae are hypothesized to be derived from either an ancient anaerobic eukaryote (amitochondriate fossil hypothesis), a mitochondrial endosymbiont (hydrogen hypothesis), or anaerobic bacteria (lateral transfer hypothesis). The goals here were to complete the molecular characterization of giardial and amebic fermentation enzymes and to determine the origins of the genes encoding them, when possible. A putative giardia [2Fe-2S]ferredoxin which had a hypothetical organelle-targeting sequence at its N terminus showed similarity to mitochondrial ferredoxins and the hydrogenosomal ferredoxin of Trichomonas vaginalis (another luminal protist). However, phylogenetic trees were star shaped, with weak bootstrap support, so we were unable to confirm or rule out the endosymbiotic origin of the giardia [2Fe-2S]ferredoxin gene. Putative giardial and amebic 6-kDa ferredoxins, ferredoxin-nitroreductase fusion proteins, and oxygen-insensitive nitroreductases each tentatively supported the lateral transfer hypothesis. Although there were not enough sequences to perform meaningful phylogenetic analyses, the unique common occurrence of these peptides and enzymes in giardia organisms, amebae, and the few anaerobic prokaryotes suggests the possibility of lateral transfer. In contrast, there was more robust phylogenetic evidence for the lateral transfer of G. lamblia genes encoding an NADH oxidase from a gram-positive coccus and a microbial group 3 alcohol dehydrogenase from thermoanaerobic prokaryotes. In further support of lateral transfer, the G. lamblia NADH oxidase and adh3 genes appeared to have an evolutionary history distinct from those of E. histolytica. PMID

  2. Alcohol dehydrogenase and cytochrome P450 2E1 can be induced by long-term exposure to ethanol in cultured liver HEP-G2 cells.

    PubMed

    Balusikova, Kamila; Kovar, Jan

    2013-09-01

    It has been shown in previous studies that liver HEP-G2 cells (human hepatocellular carcinoma) lose their ability to express active alcohol dehydrogenase (ADH) and cytochrome P450 2E1 (CYP2E1). Although both are ethanol-inducible enzymes, short-term exposure to ethanol does not cause any changes in expression or activity in cultured HEP-G2 cells. Therefore, we tested the effect of long-term exposure to ethanol on the expression and activity of both ADH and CYP2E1 in these cells. The expression of ADH and CYP2E1 was assessed at the mRNA and/or protein level using real-time PCR and Western blot analysis. Specific colorimetric assays were used for the measurement of ADH and CYP2E1 enzymatic activities. Caco-2 cells (active CYP2E1 and inactive ADH) were used as control cells. Significantly increased protein expression of ADH (about 2.5-fold) as well as CYP2E1 (about 1.6-fold) was found in HEP-G2 cells after long-term (12 mo) exposure to ethanol. The activity of ADH and CYP2E1 was also significantly increased from 12 ± 3 and 6 ± 1 nmol/h/mg of total protein to 191 ± 9 and 57 ± 9 nmol/h/mg of total protein, respectively. We suggest that the loss of activity of ethanol-metabolizing enzymes in cultured HEP-G2 cells is reversible and can be induced by prolonged exposure to ethanol. We are therefore able to reactivate HEP-G2 cells metabolic functions concerning ethanol oxidation just by modification of in vitro culture conditions without necessity of transfection with its side effect - enzyme overexpression.

  3. In vivo stage- and tissue-specific DNA-protein interactions at the D. melanogaster alcohol dehydrogenase distal promoter and adult enhancer.

    PubMed Central

    Jackson, J R; Benyajati, C

    1992-01-01

    We performed a high resolution analysis of the chromatin structure within the regions required for distal transcription of the Drosophila melanogaster alcohol dehydrogenase gene (Adh). Using dimethyl sulfate, DNase I, and micrococcal nuclease as structural probes, and comparing chromatin structure in tissues isolated from several developmental stages, we have identified several sites of stage- and tissue-specific DNA-protein interactions that correlate with distal transcription initiation. Most were within previously identified cis-acting elements and/or in vitro protein binding sites of the adult enhancer (AAE) and distal promoter, including the TATA box. We also detected a novel stage-specific DNA-protein interaction at the Adf-2a binding site where a non-histone protein was bound to the DNA on the surface of a positioned nucleosome previously identified between the distal promoter and adult enhancer. In addition to footprints, we have also revealed stage- and tissue-specific DNA helix deformations between many of the non-histone protein binding sites. These helix distortions suggest there are interactions among the adjacently bound proteins that result in bending or kinking of the intervening DNA. The distal promoter and AAE have an accessible chromatin conformation in fat body prior to the third larval instar and many of the regulatory proteins that bind in these regions are also available before distal transcription begins. Nevertheless, the timing of DNA-protein interactions in the distal promoter and AAE suggest these proteins do not bind individually or assemble progressively as they and their binding sites become available. Instead, there appears to be a coordinated assembly of a large cooperative complex of proteins interacting with the distal promoter, the positioned nucleosome, the enhancer of the distal promoter (the AAE), and each other. Images PMID:1437559

  4. Screening, Molecular Cloning, and Biochemical Characterization of an Alcohol Dehydrogenase from Pichia pastoris Useful for the Kinetic Resolution of a Racemic β-Hydroxy-β-trifluoromethyl Ketone.

    PubMed

    Bulut, Dalia; Duangdee, Nongnaphat; Gröger, Harald; Berkessel, Albrecht; Hummel, Werner

    2016-07-15

    The stereoselective synthesis of chiral 1,3-diols with the aid of biocatalysts is an attractive tool in organic chemistry. Besides the reduction of diketones, an alternative approach consists of the stereoselective reduction of β-hydroxy ketones (aldols). Thus, we screened for an alcohol dehydrogenase (ADH) that would selectively reduce a β-hydroxy-β-trifluoromethyl ketone. One potential starting material for this process is readily available by aldol addition of acetone to 2,2,2-trifluoroacetophenone. Over 200 strains were screened, and only a few yeast strains showed stereoselective reduction activities. The enzyme responsible for the reduction of the β-hydroxy-β-trifluoromethyl ketone was identified after purification and subsequent MALDI-TOF mass spectrometric analysis. As a result, a new NADP(+) -dependent ADH from Pichia pastoris (PPADH) was identified and confirmed to be capable of stereospecific and diastereoselective reduction of the β-hydroxy-β-trifluoromethyl ketone to its corresponding 1,3-diol. The gene encoding PPADH was cloned and heterologously expressed in Escherichia coli BL21(DE3). To determine the influence of an N- or C-terminal His-tag fusion, three different recombinant plasmids were constructed. Interestingly, the variant with the N-terminal His-tag showed the highest activity; consequently, this variant was purified and characterized. Kinetic parameters and the dependency of activity on pH and temperature were determined. PPADH shows a substrate preference for the reduction of linear and branched aliphatic aldehydes. Surprisingly, the enzyme shows no comparable activity towards ketones other than the β-hydroxy-β-trifluoromethyl ketone.

  5. Molecular cloning, nucleotide sequencing, and expression of genes encoding alcohol dehydrogenases from the thermophile Thermoanaerobacter brockii and the mesophile Clostridium beijerinckii.

    PubMed

    Peretz, M; Bogin, O; Tel-Or, S; Cohen, A; Li, G; Chen, J S; Burstein, Y

    1997-08-01

    Proteins play a pivotal role in thermophily. Comparing the molecular properties of homologous proteins from thermophilic and mesophilic bacteria is important for understanding the mechanisms of microbial adaptation to extreme environments. The thermophile Thermoanaerobacter (Thermoanaerobium) brockii and the mesophile Clostridium beijerinckii contain an NADP(H)-linked, zinc-containing secondary alcohol dehydrogenase (TBADH and CBADH) showing a similarly broad substrate range. The structural genes encoding the TBADH and the CBADH were cloned, sequenced, and highly expressed in Escherichia coli. The coding sequences of the TB adh and the CB adh genes are, respectively, 1056 and 1053 nucleotides long. The TB adh gene encoded an amino acid sequence identical to that of the purified TBADH. Alignment of the deduced amino acid sequences of the TB and CB adh genes showed a 76% identity and a 86% similarity, and the two genes had a similar preference for codons with A or T in the third position. Multiple sequence alignment of ADHs from different sources revealed that two (Cys-46 and His-67) of the three ligands for the catalytic Zn atom of the horse-liver ADH are preserved in TBADH and CBADH. Both the TBADH and CBADH were homotetramers. The substrate specificities and thermostabilities of the TBADH and CBADH expressed inE. coli were identical to those of the enzymes isolated from T. brockii and C. beijerinckii, respectively. A comparison of the amino acid composition of the two ADHs suggests that the presence of eight additional proline residues in TBADH than in CBADH and the exchange of hydrophilic and large hydrophobic residues in CBADH for the small hydrophobic amino acids Pro, Ala, and Val in TBADH might contribute to the higher thermostability of the T. brockii enzyme.

  6. Cinnamyl alcohol dehydrogenases in the mesocarp of ripening fruit of Prunus persica genotypes with different flesh characteristics: changes in activity and protein and transcript levels.

    PubMed

    Gabotti, Damiano; Negrini, Noemi; Morgutti, Silvia; Nocito, Fabio F; Cocucci, Maurizio

    2015-07-01

    Development of fruit flesh texture quality traits may involve the metabolism of phenolic compounds. This study presents molecular and biochemical results on the possible role played by cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.195) during ripening [S3, S4 I (pre-climacteric) and S4 III (climacteric) stages] of peach [Prunus persica (L.) Batsch] fruit with different flesh firmness [non-melting flesh (NMF) 'Oro A'/melting flesh (MF) 'Springcrest' and 'Sanguinella'] and color (blood-flesh Sanguinella). A total of 24 putative full-length PRUPE_CAD genes were identified (in silico analysis) in the peach genome. The most abundant CAD isoforms, encoded by genes located on scaffolds 8 and 6, were probed by specifically developed anti-PRUPE_CAD sc8 and by anti-FaCAD (PRUPE_CAD sc6) polyclonal antibodies, respectively. PRUPE_CAD sc8 proteins (SDS-PAGE and native-PAGE/western blot) appeared responsible for the CAD activity (in vitro/in-gel assays) that increased with ripening (parallel to PRUPE_ACO1 transcripts accumulation and ethylene evolution) only in the mesocarp of Oro A and blood-flesh Sanguinella. Accumulation of PRUPE_CAD sc8 transcripts (semi-quantitative RT-PCR) occurred in all three cultivars, but in Oro A and Springcrest it was not always accompanied by that of the related proteins, suggesting possible post-transcriptional regulation. Flesh firmness, as well as levels of lignin, total phenolics and, where present (Sanguinella), anthocyanins, declined with ripening, suggesting that, at least in the studied peach cultivars, CAD activity is related to neither lignification nor differences in flesh firmness (NMF/MF). Further studies are necessary to clarify whether the high levels of CAD activity/expression in Sanguinella play a role in determining the characteristics of this blood-flesh fruit.

  7. Picosecond-Resolved Fluorescence Studies of Substrate and Cofactor-Binding Domain Mutants in a Thermophilic Alcohol Dehydrogenase Uncover an Extended Network of Communication

    PubMed Central

    2015-01-01

    Time-resolved fluorescence dynamics are investigated in two mutants of a thermophilic alcohol dehydrogenase (ht-ADH): Y25A (at the dimer interface) and V260A (at the cofactor-binding domain). These residues, ca. 32 Å apart, are shown to exhibit opposing low-temperature effects on the hydride tunneling step. Using single-tryptophan constructs at the active site (Trp87) and a remote, surface-exposed site (Trp167), time-dependent Stokes shifts and collisional quenching data allow an analysis of intra-protein dynamical communication. A double mutant, Y25A:V260A, was also inserted into each single-Trp construct and analyzed accordingly. None of the mutations affect fluorescence lifetimes, Stokes shift relaxation rates, and quenching data for the surface-exposed Trp167 to an appreciable extent. By contrast, fluorescent probes of the active-site tryptophan 87 reveal distinctive forms of dynamical communication. Stokes shifts show that the distal Y25A increases active-site flexibility, V260A introduces a temperature-dependent equilibration process not previously reported by such measurements, and the double mutant (Y25A:V260A) eliminates the temperature-dependent transition sensed by the active-site tryptophan in the presence of V260A. Collisional quenching data at Trp87 further show a structural change in the active-site environment/solvation for V260A. In the aggregate, the temperature dependencies of the fluorescence data are distinct from the breaks in behavior previously reported for catalysis and hydrogen/deuterium exchange, attributed to time scales for the interconversion of protein conformational substates that are slower and more global than the local motions monitored within. An extended network of dynamical communication between the protein dimer surface and substrate- and cofactor-binding domains emerges from the flourescent data. PMID:25314615

  8. Disrupting the cinnamyl alcohol dehydrogenase 1 gene (BdCAD1) leads to altered lignification and improved saccharification in Brachypodium distachyon.

    PubMed

    Bouvier d'Yvoire, Madeleine; Bouchabke-Coussa, Oumaya; Voorend, Wannes; Antelme, Sébastien; Cézard, Laurent; Legée, Frédéric; Lebris, Philippe; Legay, Sylvain; Whitehead, Caragh; McQueen-Mason, Simon J; Gomez, Leonardo D; Jouanin, Lise; Lapierre, Catherine; Sibout, Richard

    2013-02-01

    Brachypodium distachyon (Brachypodium) has been proposed as a model for grasses, but there is limited knowledge regarding its lignins and no data on lignin-related mutants. The cinnamyl alcohol dehydrogenase (CAD) genes involved in lignification are promising targets to improve the cellulose-to-ethanol conversion process. Down-regulation of CAD often induces a reddish coloration of lignified tissues. Based on this observation, we screened a chemically induced population of Brachypodium mutants (Bd21-3 background) for red culm coloration. We identified two mutants (Bd4179 and Bd7591), with mutations in the BdCAD1 gene. The mature stems of these mutants displayed reduced CAD activity and lower lignin content. Their lignins were enriched in 8-O-4- and 4-O-5-coupled sinapaldehyde units, as well as resistant inter-unit bonds and free phenolic groups. By contrast, there was no increase in coniferaldehyde end groups. Moreover, the amount of sinapic acid ester-linked to cell walls was measured for the first time in a lignin-related CAD grass mutant. Functional complementation of the Bd4179 mutant with the wild-type BdCAD1 allele restored the wild-type phenotype and lignification. Saccharification assays revealed that Bd4179 and Bd7591 lines were more susceptible to enzymatic hydrolysis than wild-type plants. Here, we have demonstrated that BdCAD1 is involved in lignification of Brachypodium. We have shown that a single nucleotide change in BdCAD1 reduces the lignin level and increases the degree of branching of lignins through incorporation of sinapaldehyde. These changes make saccharification of cells walls pre-treated with alkaline easier without compromising plant growth.

  9. Rice alcohol dehydrogenase 1 promotes survival and has a major impact on carbohydrate metabolism in the embryo and endosperm when seeds are germinated in partially oxygenated water

    PubMed Central

    Takahashi, Hirokazu; Greenway, Hank; Matsumura, Hideo; Tsutsumi, Nobuhiro; Nakazono, Mikio

    2014-01-01

    Background and Aims Rice (Oryza sativa) has the rare ability to germinate and elongate a coleoptile under oxygen-deficient conditions, which include both hypoxia and anoxia. It has previously been shown that ALCOHOL DEHYDROGENASE 1 (ADH1) is required for cell division and cell elongation in the coleoptile of submerged rice seedlings by means of studies using a rice ADH1-deficient mutant, reduced adh activity (rad). The aim of this study was to understand how low ADH1 in rice affects carbohydrate metabolism in the embryo and endosperm, and lactate and alanine synthesis in the embryo during germination and subsequent coleoptile growth in submerged seedlings. Methods Wild-type and rad mutant rice seeds were germinated and grown under complete submergence. At 1, 3, 5 and 7 d after imbibition, the embryo and endosperm were separated and several of their metabolites were measured and compared. Key results In the rad embryo, the rate of ethanol fermentation was halved, while lactate and alanine concentrations were 2·4- and 5·7- fold higher in the mutant than in the wild type. Glucose and fructose concentrations in the embryos increased with time in the wild type, but not in the rad mutant. The rad mutant endosperm had lower amounts of the α-amylases RAMY1A and RAMY3D, resulting in less starch degradation and lower glucose concentrations. Conclusions These results suggest that ADH1 is essential for sugar metabolism via glycolysis to ethanol fermentation in both the embryo and endosperm. In the endosperm, energy is presumably needed for synthesis of the amylases and for sucrose synthesis in the endosperm, as well as for sugar transport to the embryo. PMID:24431339

  10. Cloning and overexpression of an NADH-dependent alcohol dehydrogenase gene from Candida maris involved in (R)-selective reduction of 5-acetylfuro[2,3-c]pyridine.

    PubMed

    Kawano, Shigeru; Yano, Miho; Hasegawa, Junzo; Yasohara, Yoshihiko

    2011-01-01

    5-((R)-1-Hydroxyethyl)-furo[2,3-c]pyridine ((R)-FPH) is a useful chiral building block in the synthesis of pharmaceuticals. An NADH-dependent alcohol dehydrogenase (AFPDH) isolated from Candida maris catalyzed the reduction of 5-acetylfuro[2,3-c]pyridine (AFP) to (R)-FPH with 100% enantiomeric excess. The gene encoding AFPDH was cloned and sequenced. The AFPDH gene comprises 762 bp and encodes a polypeptide of 27,230 Da. The deduced amino acid sequence showed a high degree of similarity to those of other members of the short-chain alcohol dehydrogenase superfamily. The AFPDH gene was overexpressed in Escherichia coli under the control of the lac promoter. One L of the cultured broth of an E. coli transformant coexpressing AFPDH and the glucose dehydrogenase (GDH) gene reduced 250 g of AFP to (R)-FPH in an organic solvent two-phase system. Under coupling with NADH regeneration using 2-propanol, 1 L of the cultured broth of an E. coli transformant expressing the AFPDH gene reduced 150 g of AFP to (R)-FPH. The optical purity of the (R)-FPH formed was 100% enantiomeric excess under both reaction conditions.

  11. Selective inhibition of 11beta-hydroxysteroid dehydrogenase 1 by 18alpha-glycyrrhetinic acid but not 18beta-glycyrrhetinic acid.

    PubMed

    Classen-Houben, Dirk; Schuster, Daniela; Da Cunha, Thierry; Odermatt, Alex; Wolber, Gerhard; Jordis, Ulrich; Kueenburg, Bernhard

    2009-02-01

    Elevated cortisol concentrations have been associated with metabolic diseases such as diabetes type 2 and obesity. 11beta-hydroxysteroid dehydrogenase (11beta-HSD) type 1, catalyzing the conversion of inactive 11-ketoglucocorticoids into their active 11beta-hydroxy forms, plays an important role in the regulation of cortisol levels within specific tissues. The selective inhibition of 11beta-HSD1 is currently considered as promising therapeutic strategy for the treatment of metabolic diseases. In recent years, natural compound-derived drug design has gained considerable interest. 18beta-glycyrrhetinic acid (GA), a metabolite of the natural product glycyrrhizin, is not selective and inhibits both 11beta-HSD1 and 11beta-HSD2. Here, we compare the biological activity of 18beta-GA and its diastereomer 18alpha-GA against the two enzymes in lysates of transfected HEK-293 cells and show that 18alpha-GA selectively inhibits 11beta-HSD1 but not 11beta-HSD2. This is in contrast to 18beta-GA, which preferentially inhibits 11beta-HSD2. Using a pharmacophore model based on the crystal structure of the GA-derivative carbenoxolone in complex with human 11beta-HSD1, we provide an explanation for the differences in the activities of 18alpha-GA and 18beta-GA. This model will be used to design novel selective derivatives of GA.

  12. Trypanosoma cruzi: inhibition of alpha-hydroxyacid dehydrogenase isozyme II by N-allyl and N-propyl oxamates and their effects on intact epimastigotes.

    PubMed

    Chena, Miguel A; Elizondo-Jiménez, Silvia; Rodríguez-Páez, Lorena; Nogueda-Torres, Benjamín; Baeza-Ramírez, Isabel; Wong-Ramírez, Carlos

    2004-12-01

    N-allyl (NAOx) and N-propyl (NPOx) oxamates were designed as inhibitors of alpha-hydroxyacid dehydrogenase (HADH) isozyme II from Trypanosoma cruzi. The kinetic studies showed that NAOx and NPOx were competitive inhibitors of HADH-isozyme II (Ki = 72 microM, IC50 = 0.33 mM and 70 microM, IC50 = 0.32 mM, respectively). The attachment of the allylic and propylic chains to nitrogen of the competitive inhibitor oxamate (Ki = 0.91 mM, IC50 = 4.25 mM), increased 12.6 and 13-folds respectively, the affinity for T. cruzi HADH-isozyme II. NAOx and NPOx were selective inhibitors of HADH-isozyme II, because other T. cruzi dehydrogenases were not inhibited by these substances. Since HADH-isozyme II participates in the energy metabolism of T. cruzi, a trypanocidal effect can be expected with these inhibitors. However, we were not able to detect any trypanocidal activity with these oxamates. When the corresponding ethyl esters of N-allyl (Et-NAOx) and N-propyl (Et-NPOx) oxamates were tested as a possible trypanocidal prodrugs, in comparison with nifurtimox and benznidazole, the expected trypanocidal effects were obtained.

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

    PubMed

    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.

  14. Inhibition and deactivation effects in catalytic wet oxidation of high-strength alcohol-distillery liquors

    SciTech Connect

    Belkacemi, K.; Larachi, F.; Hamoudi, S.; Turcotte, G.; Sayari, A.

    1999-06-01

    The removal efficiency of total organic carbon (TOC) from raw high-strength alcohol-distillery waste liquors was evaluated using three different treatments: thermolysis (T), noncatalytic wet oxidation (WO), and solid-catalyzed wet oxidation (CWO). The distillery liquors (TOC = 22,500 mg/l, sugars = 18,000 mg/l, and proteins = 13,500 mg/l) were produced by alcoholic fermentation of enzymatic hydrolyzates from steam-exploded timothy grass. TOC-abatement studies were conducted batchwise in a stirred autoclave to evaluate the influence of the catalyst (7:3, MnO{sub 2}/CeO{sub 2} mixed oxide), oxygen partial pressure (0.5--2.5 MPa), and temperature (453--523 K) on T, WO, and CWO processes. Although CWO outperformed T and WO, TOC conversions did not exceed {approximately}60% at the highest temperature used. Experiments provided prima facie evidence for a gradual fouling of the catalyst and a developing inhibition in the liquors which impaired deep TOC removals. Occurrence of catalyst deactivation by carbonaceous deposits was proven experimentally through quantitative and qualitative experiments such as elemental analysis and X-ray photoelectron spectroscopy. Inhibition toward further degradation of the liquors was ascribed to the occurrence of highly stable antioxidant intermediates via the Maillard reactions between dissolved sugars and proteins. A lumping kinetic model involving both reaction inhibition by dissolved intermediates and catalyst deactivation by carbonaceous deposits was proposed to account for the distribution of carbon in the liquid, solid, and the vapor phases.

  15. High-Affinity Inhibitors of Human NAD+-Dependent 15-Hydroxyprostaglandin Dehydrogenase: Mechanisms of Inhibition and Structure-Activity Relationships

    PubMed Central

    Niesen, Frank H.; Schultz, Lena; Jadhav, Ajit; Bhatia, Chitra; Guo, Kunde; Maloney, David J.; Pilka, Ewa S.; Wang, Minghua; Oppermann, Udo; Heightman, Tom D.; Simeonov, Anton

    2010-01-01

    Background 15-hydroxyprostaglandin dehydrogenase (15-PGDH, EC 1.1.1.141) is the key enzyme for the inactivation of prostaglandins, regulating processes such as inflammation or proliferation. The anabolic pathways of prostaglandins, especially with respect to regulation of the cyclooxygenase (COX) enzymes have been studied in detail; however, little is known about downstream events including functional interaction of prostaglandin-processing and -metabolizing enzymes. High-affinity probes for 15-PGDH will, therefore, represent important tools for further studies. Principal Findings To identify novel high-affinity inhibitors of 15-PGDH we performed a quantitative high-throughput screen (qHTS) by testing >160 thousand compounds in a concentration-response format and identified compounds that act as noncompetitive inhibitors as well as a competitive inhibitor, with nanomolar affinity. Both types of inhibitors caused strong thermal stabilization of the enzyme, with cofactor dependencies correlating with their mechanism of action. We solved the structure of human 15-PGDH and explored the binding modes of the inhibitors to the enzyme in silico. We found binding modes that are consistent with the observed mechanisms of action. Conclusions Low cross-reactivity in screens of over 320 targets, including three other human dehydrogenases/reductases, suggest selectivity of the present inhibitors for 15-PGDH. The high potencies and different mechanisms of action of these chemotypes make them a useful set of complementary chemical probes for functional studies of prostaglandin-signaling pathways. Enhanced version This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S2. PMID:21072165

  16. Feedback inhibition of chorismate mutase/prephenate dehydrogenase (TyrA) of Escherichia coli: generation and characterization of tyrosine-insensitive mutants.

    PubMed

    Lütke-Eversloh, Tina; Stephanopoulos, Gregory

    2005-11-01

    In order to get insights into the feedback regulation by tyrosine of the Escherichia coli chorismate mutase/prephenate dehydrogenase (CM/PDH), which is encoded by the tyrA gene, feedback-inhibition-resistant (fbr) mutants were generated by error-prone PCR. The tyrA(fbr) mutants were selected by virtue of their resistance toward m-fluoro-D,L-tyrosine, and seven representatives were characterized on the biochemical as well as on the molecular level. The PDH activities of the purified His6-tagged TyrA proteins exhibited up to 35% of the enzyme activity of TyrA(WT), but tyrosine did not inhibit the mutant PDH activities. On the other hand, CM activities of the TyrA(fbr) mutants were similar to those of the TyrA(WT) protein. Analyses of the DNA sequences of the tyrA genes revealed that tyrA(fbr) contained amino acid substitutions either at Tyr263 or at residues 354 to 357, indicating that these two sites are involved in the feedback inhibition by tyrosine.

  17. Selective oxidation of glycerol to 1,3-dihydroxyacetone by covalently immobilized glycerol dehydrogenases with higher stability and lower product inhibition.

    PubMed

    Rocha-Martin, Javier; Acosta, Andreína; Berenguer, Jose; Guisan, Jose M; Lopez-Gallego, Fernando

    2014-10-01

    Glycerol dehydrogenase (GlyDH) catalyzes the regioselective oxidation of glycerol to yield 1,3-dihydroxyacetone (DHA); an important building block in chemical industry. Three recombinant GlyDHs from Geobacillus stearothermophilus, from Citrobacter braakii and from Cellulomonas sp. were stabilized by covalent immobilization. The highest activity recoveries (40-50%) of the insoluble preparations were obtained by immobilizing these enzymes in presence of polyethylene glycol (PEG). Noteworthy, these immobilized preparations were more stable and less inhibited by DHA than their soluble counterparts. In particular, GlyDH from G.stearothermophilus immobilized on agarose activated with both amine and glyoxyl groups and crosslinked with dextran aldehyde was 3.7-fold less inhibited by DHA than its soluble form and retained 100% of its initial activity after 18h of incubation at 65°C and pH 7. This is one of the few examples where the same immobilization protocol has minimized enzyme product inhibition and maximized thermal stability.

  18. Influence of human serum albumin on the bile acid-mediated inhibition of liver microsomal type 1 11β-hydroxysteroid dehydrogenase.

    PubMed

    Maeda, Yorio; Funagayama, Mayumi; Shinohara, Akio; Koshimoto, Chihiro; Furusawa, Hidemi; Nakahara, Hiroshi; Yamaguchi, Yukiko; Saitoh, Tomokazu; Yamamoto, Takashi; Komaki, Kansei

    2014-09-01

    The influence of human serum albumin (HSA) on the bile acid-mediated inhibition of liver microsomal type 1 11β-hydroxysteroid dehydrogenase (11β-HSD1) was studied in vitro. A rat liver microsomal fraction was prepared, and the 11β-HSD1 enzyme activity in the presence of various concentrations of bile acids and HSA was determined using hydrocortisone as the substrate. The products of the reaction were extracted and analyzed using high-performance liquid chromatography. The magnitude of the inhibition decreased with the addition of HSA in a dose-dependent manner. Four percent human albumin decreased the inhibitory effects of 100 μM chenodeoxycholic acid and lithocholic acid from 89.9 ± 5.6 to 54.5 ± 6.1% and from 83.8 ± 4.8 to 20.8 ± 4.2%, respectively. In contrast, ursodeoxycholic acid and deoxycholic acid showed no inhibitory effect on the enzyme activity in the presence of 4% human serum albumin, and the addition of 1% γ-globulin to the assay mixture in the presence of bile acids did not affect the enzyme activity. Our in vitro study showed that the addition of HSA ameliorated the inhibition of 11β-HSD1 and that the magnitude of the change is dependent on the species of bile acid, presumably based on the numbers of hydroxyl groups. These results suggest that HSA seems to protect the bile acid-mediated inhibition of 11β-HSD1 in the healthy subject. On the other hand, in the patients with obstructive biliary diseases, not only elevated serum bile acid but also the accompanying hypoalbuminemia is important to evaluate the pathophysiology of the bile acid-mediated inhibition of 11β-HSD1 of the disease.

  19. Alcohol

    MedlinePlus

    ... created when grains, fruits, or vegetables are fermented . Fermentation is a process that uses yeast or bacteria ... change the sugars in the food into alcohol. Fermentation is used to produce many necessary items — everything ...

  20. Alcohol.

    ERIC Educational Resources Information Center

    Schibeci, Renato

    1996-01-01

    Describes the manufacturing of ethanol, the effects of ethanol on the body, the composition of alcoholic drinks, and some properties of ethanol. Presents some classroom experiments using ethanol. (JRH)

  1. Activation of Ice Recrystallization Inhibition Activity of Poly(vinyl alcohol) using a Supramolecular Trigger†

    PubMed Central

    Phillips, Daniel J.; Congdon, Thomas R.; Gibson, Matthew I.

    2016-01-01

    Antifreeze (glyco)proteins (AF(G)Ps) have potent ice recrystallisation inhibition (IRI) activity – a desirable phenomenon in applications such as cryopreservation, frozen food and more. In Nature AF(G)P activity is regulated by protein expression levels in response to an environmental stimulus; temperature. However, this level of regulation is not possible in synthetic systems. Here, a synthetic macromolecular mimic is introduced, using supramolecular assembly to regulate activity. Catechol-terminated poly(vinyl alcohol) was synthesised by RAFT polymerization. Upon addition of Fe3+, larger supramolecular star polymers form by assembly with two or three catechols. This increase in molecular weight effectively ‘switches on’ the IRI activity and is the first example of external control over the function of AFP mimetics. This provides a simple but elegant solution to the challenge of external control of AFP-mimetic function. PMID:28003855

  2. Activation of Group II Metabotropic Glutamate Receptors Inhibits the Discriminative Stimulus Effects of Alcohol via Selective Activity Within the Amygdala

    PubMed Central

    Cannady, Reginald; Grondin, Julie JM; Fisher, Kristen R; Hodge, Clyde W; Besheer, Joyce

    2011-01-01

    Metabotropic glutamate receptor subtypes (mGlu2/3) regulate a variety of alcohol-associated behaviors, including alcohol reinforcement, and relapse-like behavior. To date, the role of mGlu2/3 receptors in modulating the discriminative stimulus effects of alcohol has not been examined. Given that the discriminative stimulus effects of drugs are determinants of abuse liability and can influence drug seeking, we examined the contributions of mGlu2/3 receptors in modulating the discriminative stimulus effects of alcohol. In male Long-Evans rats trained to discriminate between alcohol (1 g/kg, IG) and water, the mGlu2/3 agonist LY379268 (0.3–10 mg/kg) did not produce alcohol-like stimulus effects. However, pretreatment with LY379268 (1 and 3 mg/kg; in combination with alcohol) inhibited the stimulus effects of alcohol (1 g/kg). Systemic LY379268 (3 mg/kg, i.p.) was associated with increases in neuronal activity within the amygdala, but not the nucleus accumbens, as assessed by c-Fos immunoreactivity. Intra-amygdala activation of mGlu2/3 receptors by LY379268 (6 μg) inhibited the discriminative stimulus effects of alcohol, without altering response rate. In contrast, intra-accumbens LY379268 (3 μg) profoundly reduced response rate; however, at lower LY379268 doses (0.3, 1 μg), the discriminative stimulus effects of alcohol and response rate were not altered. These data suggest that amygdala mGlu2/3 receptors have a functional role in modulating the discriminative stimulus properties of alcohol and demonstrate differential motor sensitivity to activation of mGlu2/3 receptors in the amygdala and the accumbens. Understanding the neuronal mechanisms that underlie the discriminative stimulus effects of alcohol may prove to be important for future development of pharmacotherapies for treating alcoholism. PMID:21734651

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

    PubMed

    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.

  4. Amyloid beta protein inhibits cellular MTT reduction not by suppression of mitochondrial succinate dehydrogenase but by acceleration of MTT formazan exocytosis in cultured rat cortical astrocytes.

    PubMed

    Abe, K; Saito, H

    1998-08-01

    Alzheimer's disease amyloid beta protein (Abeta) inhibits cellular reduction of the dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Kaneko et al. have previously hypothesized that Abeta works by suppressing mitochondrial succinate dehydrogenase (SDH), but Liu and Schubert have recently demonstrated that Abeta decreases cellular MTT reduction by accelerating the exocytosis of MTT formazan in neuronal cells. To ask which is the case in astrocytes, we compared the effects of Abeta and 3-nitropropionic acid (3-NP), a specific SDH inhibitor, on MTT reduction in cultured rat cortical astrocytes. Treatment with 3-NP (10 mM) decreased cellular activity of MTT reduction, regardless of the time of incubation with MTT. On the other hand. Abeta-induced inhibition of cellular MTT reduction was dependent on the time of incubation with MTT. The cells treated with Abeta (0.1-1000 nM) exhibited normal capacity for MTT reduction at an early stage of incubation ( < 30 min), but ceased to reduce MTT at the late stage (> 1 h). Microscopic examination revealed that Abeta treatment accelerated the appearance of needle-like MTT formazan crystals at the cell surface. These observations support that Abeta accelerates the exocytosis of MTT formazan in astrocytes. In addition to inhibition of MTT reduction, Abeta is known to induce morphological changes in astrocytes. Following addition of Abeta (20 microM), polygonal astrocytes changed into process-bearing stellate cells. To explore a possible linkage between these two effects of Abeta, we tested if astrocyte stellation is induced by agents that mimic the effect of Abeta on MTT reduction. Cholesterol (5 5000 nM) and lysophosphatidic acid (0.2-20 microg/ml) were found to accelerate the exocytosis of MTT formazan in a similar manner to Abeta, but failed to induce astrocyte stellation. Therefore, Abeta-induced inhibition of MTT reduction is unlikely to be directly linked to its effect on astrocyte morphology.

  5. miR-339-5p inhibits alcohol-induced brain inflammation through regulating NF-κB pathway.

    PubMed

    Zhang, Yu; Wei, Guangkuan; Di, Zhiyong; Zhao, Qingjie

    2014-09-26

    Alcohol-induced neuroinflammation is mediated by the innate immunesystem. Pro-inflammatory responses to alcohol are modulated by miRNAs. The miRNA miR-339-5p has previously been found to be upregulated in alcohol-induced neuroinflammation. However, little has been elucidated on the regulatory functions of this miRNA in alcohol-induced neuroinflammation. We investigated the function of miR-339-5p in alcohol exposed brain tissue and isolated microglial cells using ex vivo and in vitro techniques. Our results show that alcohol induces transcription of miR 339-5p, IL-6, IL-1β and TNF-α in mouse brain tissue and isolated microglial cells by activating NF-κB. Alcohol activation of NF-κB allows for nuclear translocation of the NF-κB subunit p65 and expression of pro-inflammatory mediators. miR-339-5p inhibited expression of these pro-inflammatory factors through the NF-κB pathway by abolishing IKK-β and IKK-ε activity.

  6. Molecular Characterization and Transcriptional Analysis of adhE2, the Gene Encoding the NADH-Dependent Aldehyde/Alcohol Dehydrogenase Responsible for Butanol Production in Alcohologenic Cultures of Clostridium acetobutylicum ATCC 824

    PubMed Central

    Fontaine, Lisa; Meynial-Salles, Isabelle; Girbal, Laurence; Yang, Xinghong; Croux, Christian; Soucaille, Philippe

    2002-01-01

    The adhE2 gene of Clostridium acetobutylicum ATCC 824, coding for an aldehyde/alcohol dehydrogenase (AADH), was characterized from molecular and biochemical points of view. The 2,577-bp adhE2 codes for a 94.4-kDa protein. adhE2 is expressed, as a monocistronic operon, in alcohologenic cultures and not in solventogenic cultures. Primer extension analysis identified two transcriptional start sites 160 and 215 bp upstream of the adhE2 start codon. The expression of adhE2 from a plasmid in the DG1 mutant of C. acetobutylicum, a mutant cured of the pSOL1 megaplasmid, restored butanol production and provided elevated activities of NADH-dependent butyraldehyde and butanol dehydrogenases. The recombinant AdhE2 protein expressed in E. coli as a Strep-tag fusion protein and purified to homogeneity also demonstrated NADH-dependent butyraldehyde and butanol dehydrogenase activities. This is the second AADH identified in C. acetobutylicum ATCC 824, and to our knowledge this is the first example of a bacterium with two AADHs. It is noteworthy that the two corresponding genes, adhE and adhE2, are carried by the pSOL1 megaplasmid of C. acetobutylicum ATCC 824. PMID:11790753

  7. miR-339-5p inhibits alcohol-induced brain inflammation through regulating NF-κB pathway

    SciTech Connect

    Zhang, Yu; Wei, Guangkuan; Di, Zhiyong; Zhao, Qingjie

    2014-09-26

    Graphical abstract: - Highlights: • Alcohol upregulates miR-339-5p expression. • miR-339-5p inhibits the NF-kB pathway. • miR-339-5p interacts with and blocks activity of IKK-beat and IKK-epsilon. • miR-339-5p modulates IL-1β, IL-6 and TNF-α. - Abstract: Alcohol-induced neuroinflammation is mediated by the innate immunesystem. Pro-inflammatory responses to alcohol are modulated by miRNAs. The miRNA miR-339-5p has previously been found to be upregulated in alcohol-induced neuroinflammation. However, little has been elucidated on the regulatory functions of this miRNA in alcohol-induced neuroinflammation. We investigated the function of miR-339-5p in alcohol exposed brain tissue and isolated microglial cells using ex vivo and in vitro techniques. Our results show that alcohol induces transcription of miR 339-5p, IL-6, IL-1β and TNF-α in mouse brain tissue and isolated microglial cells by activating NF-κB. Alcohol activation of NF-κB allows for nuclear translocation of the NF-κB subunit p65 and expression of pro-inflammatory mediators. miR-339-5p inhibited expression of these pro-inflammatory factors through the NF-κB pathway by abolishing IKK-β and IKK-ε activity.

  8. Inhibition of telomerase activity preferentially targets aldehyde dehydrogenase-positive cancer stem-like cells in lung cancer

    PubMed Central

    2011-01-01

    Background Mortality rates for advanced lung cancer have not declined for decades, even with the implementation of novel chemotherapeutic regimens or the use of tyrosine kinase inhibitors. Cancer Stem Cells (CSCs) are thought to be responsible for resistance to chemo/radiotherapy. Therefore, targeting CSCs with novel compounds may be an effective approach to reduce lung tumor growth and metastasis. We have isolated and characterized CSCs from non-small cell lung cancer (NSCLC) cell lines and measured their telomerase activity, telomere length, and sensitivity to the novel telomerase inhibitor MST312. Results The aldehyde dehydrogenase (ALDH) positive lung cancer cell fraction is enriched in markers of stemness and endowed with stem cell properties. ALDH+ CSCs display longer telomeres than the non-CSC population. Interestingly, MST312 has a strong antiproliferative effect on lung CSCs and induces p21, p27 and apoptosis in the whole tumor population. MST312 acts through activation of the ATM/pH2AX DNA damage pathway (short-term effect) and through decrease in telomere length (long-term effect). Administration of this telomerase inhibitor (40 mg/kg) in the H460 xenograft model results in significant tumor shrinkage (70% reduction, compared to controls). Combination therapy consisting of irradiation (10Gy) plus administration of MST312 did not improve the therapeutic efficacy of the telomerase inhibitor alone. Treatment with MST312 reduces significantly the number of ALDH+ CSCs and their telomeric length in vivo. Conclusions We conclude that antitelomeric therapy using MST312 mainly targets lung CSCs and may represent a novel approach for effective treatment of lung cancer. PMID:21827695

  9. Active site-specific reconstituted copper(II) horse liver alcohol dehydrogenase: a biological model for type 1 Cu2+ and its changes upon ligand binding and conformational transitions.

    PubMed

    Maret, W; Dietrich, H; Ruf, H H; Zeppezauer, M

    1980-06-01

    Insertion of Cu2+ ions into horse liver alcohol dehydrogenase depleted of its catalytic Zn2+ ions creates an artificial blue copper center similar to that of plastocyanin and similar copper proteins. The esr spectrum of a frozen solution and the optical spectra at 296 and 77 K are reported, together with the corresponding data for binary and ternary complexes with NAD+ and pyrazole. The binary complex of the cupric enzyme with pyrazole establishes a novel type of copper proteins having the optical characteristics of Type 1 and the esr parameters of Type 2 Cu2+. Ternary complex formation with NAD+ converts the Cu2+ ion to a Type 1 center. By an intramolecular redox reaction the cuprous enzyme is formed from the cupric enzyme. Whereas the activity of the cupric alcohol dehydrogenase is difficult to assess (0.5%-1% that of the native enzyme), the cuprous enzyme is distinctly active (8% of the native enzyme). The implications of these findings are discussed in view of the coordination of the metal in native copper proteins.

  10. Short-term inhibition of 11β-hydroxysteroid dehydrogenase type 1 reversibly improves spatial memory but persistently impairs contextual fear memory in aged mice

    PubMed Central

    Wheelan, Nicola; Webster, Scott P.; Kenyon, Christopher J.; Caughey, Sarah; Walker, Brian R.; Holmes, Megan C.; Seckl, Jonathan R.; Yau, Joyce L.W.

    2015-01-01

    High glucocorticoid levels induced by stress enhance the memory of f