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Sample records for alcohol dehydrogenase cad

  1. Molecular cloning, functional analysis of three cinnamyl alcohol dehydrogenase (CAD) genes in the leaves of tea plant, Camellia sinensis.

    PubMed

    Deng, Wei-Wei; Zhang, Ming; Wu, Jian-Qiang; Jiang, Zheng-Zhong; Tang, Lei; Li, Ye-Yun; Wei, Chao-Ling; Jiang, Chang-Jun; Wan, Xiao-Chun

    2013-02-15

    Cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.195) is considered to be a key enzyme in lignin biosynthesis, but little was known about CADs in tea plants (Camellia sinensis). A full-length cDNA sequence (CsCAD2) was isolated by suppressive subtractive hybridization (SSH) in Ectropis oblique feeding-induced tea plants, and another two full-length cDNA sequences (CsCAD1 and CsCAD3) were obtained from a transcriptome obtained by deep sequencing. However, they showed only 20-54% identities. Phylogenetic analysis revealed that they belonged to three different families. DNA gel blotting analysis revealed that two copies of CsCAD1 and CsCAD2 genes existed in tea genome, but CsCAD3 likely had only one copy. Recombinant proteins of these CsCADs were produced in Escherichia coli. The activity of purified recombinant CsCAD2 protein was up to 0.43 μmol min(-1) mg(-1). However, the other two recombinant proteins had lower activities, probably due to incomplete refolding. qRT-PCR analysis indicated that while CsCAD3 was strongly up-regulated in tea plants after E. oblique attack and mechanical damage, CsCAD1 and CsCAD2 showed only moderate or no changes in transcript levels. Treatment of defence-related hormones methyl jasmonate (MeJA) and salicylic acid (SA) elevated the expression of CsCAD1 and CsCAD2, but decreased the transcript abundance of CsCAD3. The transcript levels of CsCAD2 did not change after applying abscisic acid (ABA), whereas CsCAD1 and CsCAD3 were induced. These results suggested that these three CsCAD genes in tea plants may play a role in defense against insects and pathogens and adaptation to abiotic stresses and these genes likely have divergant functions.

  2. 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

  3. [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. PMID:25739284

  4. 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

  5. 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

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

    PubMed

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

    2012-10-01

    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 49 kDa. 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

  7. Unusual NADPH conformation in the crystal structure of a cinnamyl alcohol dehydrogenase from Helicobacter pylori in complex with NADP(H) and substrate docking analysis.

    PubMed

    Seo, Kyung Hye; Zhuang, Ningning; Chen, Cong; Song, Jae-Young; Kang, Hyung-Lyun; Rhee, Kwang-Ho; Lee, Kon Ho

    2012-02-17

    Cinnamyl alcohol dehydrogenase is a zinc- and NADPH-dependent dehydrogenase catalyzing the reversible conversion of p-hydroxycinnamaldehydes to their corresponding hydroxycinnamyl alcohols. A CAD homolog from Helicobacter pylori (HpCAD) possesses broad substrate specificities like the plant CADs and additionally a dismutation activity converting benzaldehyde to benzyl alcohol and benzoic acid. We have determined the crystal structure of HpCAD complexed with NADP(H) at 2.18Å resolution to get a better understanding of this class of CAD outside of plants. The structure of HpCAD is highly homologous to the sinapyl alcohol dehydrogenase and the plant CAD with well-conserved residues involved in catalysis and zinc binding. However, the NADP(H) binding mode of the HpCAD has been found to be significantly different from those of plant CADs.

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

    PubMed

    Ma, Qing-Hu

    2010-06-01

    Cinnamyl alcohol dehydrogenase (CAD) catalyses the final step in the biosynthesis of monolignols. In the present study, a cDNA encoding a CAD was isolated from wheat, designated as TaCAD1. A genome-wide data mining in the wheat EST database revealed another 10 CAD-like homologues, namely TaCAD2 to TaCAD11. A phylogenetic analysis showed that TaCAD1 belonged to the bona fide CAD group involved in lignin synthesis. Two other putative CADs from the wheat genome (TaCAD2 and TaCAD4) also belonged to this group and were very close to TaCAD1, but lacked C-terminal domain, suggesting that they are pseudogenes. DNA gel blot analysis for the wheat genome showed two to three copies of CAD related to TaCAD1, but RNA gel blot analysis revealed only single band for TaCAD1, which was highly expressed in stem, with quite low expression in leaf and undetectable expression in root. The predicted three-dimension structure of TaCAD1 resembled that of AtCAD5, but two amino acid substitutions were identified in the substrate binding region. Recombinant TaCAD1 protein used coniferyl aldehyde as the most favoured substrate, also showed high efficiencies toward sinapyl and p-coumaryl aldehydes. TaCAD1 was an enzyme being pH-dependent and temperature-sensitive, and showing a typical random catalysing mechanism. At the milky stage of wheat, TaCAD1 mRNA abundance, protein level and enzyme activity in stem tissues were higher in a lodging-resistant cultivar (H4546) than in lodging-sensitive cultivar (C6001). These properties were correlated to the lignin contents and lodging indices of the two cultivars. These data suggest that TaCAD1 is the predominant CAD in wheat stem for lignin biosynthesis and is critical for lodging resistance.

  9. 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.

  10. The physiological role of liver alcohol dehydrogenase.

    PubMed

    Krebs, H A; Perkins, J R

    1970-07-01

    1. Yeast alcohol dehydrogenase was used to determine ethanol in the portal and hepatic veins and in the contents of the alimentary canal of rats given a diet free from ethanol. Measurable amounts of a substance behaving like ethanol were found. Its rate of interaction with yeast alcohol dehydrogenase and its volatility indicate that the substance measured was in fact ethanol. 2. The mean alcohol concentration in the portal blood of normal rats was 0.045mm. In the hepatic vein, inferior vena cava and aorta it was about 15 times lower. 3. The contents of all sections of the alimentary canal contained measurable amounts of ethanol. The highest values (average 3.7mm) were found in the stomach. 4. Infusion of pyrazole (an inhibitor of alcohol dehydrogenase) raised the alcohol concentration in the portal vein 10-fold and almost removed the difference between portal and hepatic venous blood. 5. Addition of antibiotics to the food diminished the ethanol concentration of the portal blood to less than one-quarter and that of the stomach contents to less than one-fortieth. 6. The concentration of alcohol in the alimentary canal and in the portal blood of germ-free rats was much decreased, to less than one-tenth in the alimentary canal and to one-third in the portal blood, but detectable quantities remained. These are likely to arise from acetaldehyde formed by the normal pathways of degradation of threonine, deoxyribose phosphate and beta-alanine. 7. The results indicate that significant amounts of alcohol are normally formed in the gastro-intestinal tract. The alcohol is absorbed into the circulation and almost quantitatively removed by the liver. Thus the function, or a major function, of liver alcohol dehydrogenase is the detoxication of ethanol normally present. 8. The alcohol concentration in the stomach of alloxan-diabetic rats was increased about 8-fold. 9. The activity of liver alcohol dehydrogenase is generally lower in carnivores than in herbivores and omnivores

  11. 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.

  12. 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...

  13. 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. PMID:24064207

  14. Syringyl Lignin Is Unaltered by Severe Sinapyl Alcohol Dehydrogenase Suppression in Tobacco[W

    PubMed Central

    Barakate, Abdellah; Stephens, Jennifer; Goldie, Alison; Hunter, William N.; Marshall, David; Hancock, Robert D.; Lapierre, Catherine; Morreel, Kris; Boerjan, Wout; Halpin, Claire

    2011-01-01

    The manipulation of lignin could, in principle, facilitate efficient biofuel production from plant biomass. Despite intensive study of the lignin pathway, uncertainty exists about the enzyme catalyzing the last step in syringyl (S) monolignol biosynthesis, the reduction of sinapaldehyde to sinapyl alcohol. Traditional schemes of the pathway suggested that both guaiacyl (G) and S monolignols are produced by a single substrate-versatile enzyme, cinnamyl alcohol dehydrogenase (CAD). This was challenged by the discovery of a novel sinapyl alcohol dehydrogenase (SAD) that preferentially uses sinapaldehyde as a substrate and that was claimed to regulate S lignin biosynthesis in angiosperms. Consequently, most pathway schemes now show SAD (or SAD and CAD) at the sinapaldehyde reduction step, although functional evidence is lacking. We cloned SAD from tobacco (Nicotiana tabacum) and suppressed it in transgenic plants using RNA interference–inducing vectors. Characterization of lignin in the woody stems shows no change to content, composition, or structure, and S lignin is normal. By contrast, plants additionally suppressed in CAD have changes to lignin structure and S:G ratio and have increased sinapaldehyde in lignin, similar to plants suppressed in CAD alone. These data demonstrate that CAD, not SAD, is the enzyme responsible for S lignin biosynthesis in woody angiosperm xylem. PMID:22158465

  15. Effects of herbal infusions, tea and carbonated beverages on alcohol dehydrogenase and aldehyde dehydrogenase activity.

    PubMed

    Li, Sha; Gan, Li-Qin; Li, Shu-Ke; Zheng, Jie-Cong; Xu, Dong-Ping; Li, Hua-Bin

    2014-01-01

    Various alcoholic beverages containing different concentrations of ethanol are widely consumed, and excessive alcohol consumption may result in serious health problems. The consumption of alcoholic beverages is often accompanied by non-alcoholic beverages, such as herbal infusions, tea and carbonated beverages to relieve drunk symptoms. The aim of this study was to supply new information on the effects of these beverages on alcohol metabolism for nutritionists and the general public, in order to reduce problems associated with excessive alcohol consumption. The effects of 57 kinds of herbal infusions, tea and carbonated beverages on alcohol dehydrogenase and aldehyde dehydrogenase activity were evaluated. Generally, the effects of these beverages on alcohol dehydrogenase and aldehyde dehydrogenase activity are very different. The results suggested that some beverages should not be drank after excessive alcohol consumption, and several beverages may be potential dietary supplements for the prevention and treatment of problems related to excessive alcohol consumption.

  16. 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

  17. Interactions between heparinoids and alcohol dehydrogenase.

    PubMed

    Paulíková, H; Valusová, E; Antalík, M

    1997-07-01

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

  18. Fast internal dynamics in alcohol dehydrogenase.

    PubMed

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

    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 D2O solvent. It is tempting to associate the fast internal process with solvent exposed amino acid residues with dangling side chains. PMID:26298156

  19. 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.

  20. Partial Similarities Between Yeast and Liver Alcohol Dehydrogenases

    PubMed Central

    Jörnvall, Hans

    1973-01-01

    The primary structure of about half of the protein chain of yeast alcohol dehydrogenase has been determined and compared with the amino-acid sequences of other dehydrogenases. The enzyme is found to be distantly related to horse-liver alcohol dehydrogenase, although these two proteins have different quaternary structures and subunit sizes. Some regions show no significant similarities, but long segments within the N-terminal parts of the molecules are homologous, suggesting a common and important function for these segments. Ancestral connections between some different dehydrogenases can be concluded and the degree of evolutionary changes may be estimated. PMID:4599620

  1. Elusive transition state of alcohol dehydrogenase unveiled

    PubMed Central

    Roston, Daniel; Kohen, Amnon

    2010-01-01

    For several decades the hydride transfer catalyzed by alcohol dehydrogenase has been difficult to understand. Here we add to the large corpus of anomalous and paradoxical data collected for this reaction by measuring a normal (> 1) 2° kinetic isotope effect (KIE) for the reduction of benzaldehyde. Because the relevant equilibrium effect is inverse (< 1), this KIE eludes the traditional interpretation of 2° KIEs. It does, however, enable the development of a comprehensive model for the “tunneling ready state” (TRS) of the reaction that fits into the general scheme of Marcus-like models of hydrogen tunneling. The TRS is the ensemble of states along the intricate reorganization coordinate, where H tunneling between the donor and acceptor occurs (the crossing point in Marcus theory). It is comparable to the effective transition state implied by ensemble-averaged variational transition state theory. Properties of the TRS are approximated as an average of the individual properties of the donor and acceptor states. The model is consistent with experimental findings that previously appeared contradictory; specifically, it resolves the long-standing ambiguity regarding the location of the TRS (aldehyde-like vs. alcohol-like). The new picture of the TRS for this reaction identifies the principal components of the collective reaction coordinate and the average structure of the saddle point along that coordinate. PMID:20457944

  2. 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

  3. 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

  4. Optimization of adsorptive immobilization of alcohol dehydrogenases.

    PubMed

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

    2005-04-01

    In this work, a systematic examination of various parameters of adsorptive immobilization of alcohol dehydrogenases (ADHs) on solid support is performed and the impact of these parameters on immobilization efficiency is studied. Depending on the source of the enzymes, these parameters differently influence the immobilization efficiency, expressed in terms of residual activity and protein loading. Residual activity of 79% was achieved with ADH from bakers' yeast (YADH) after optimizing the immobilization parameters. A step-wise drying process has been found to be more effective than one-step drying. A hypothesis of deactivation through bubble nucleation during drying of the enzyme/glass bead suspension at low drying pressure (<45 kPa) is experimentally verified. In the case of ADH from Lactobacillus brevis (LBADH), >300% residual activity was found after drying. Hyperactivation of the enzyme is probably caused by structural changes in the enzyme molecule during the drying process. ADH from Thermoanaerobacter species (ADH T) is found to be stable under drying conditions (>15 kPa) in contrast to LBADH and YADH.

  5. 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

  6. 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

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

    PubMed

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

    2015-09-01

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

  8. Multiple retinoid dehydrogenases in testes cytosol from alcohol dehydrogenase negative or positive deermice.

    PubMed

    Posch, K C; Napoli, J L

    1992-05-28

    Retinoic acid syntheses from retinol by cytosol from testes of alcohol dehydrogenase negative or positive deermice were similar in specific activity and in their insensitivity to 1 M ethanol or 100 mM 4-methylpyrazole. Anion-exchange followed by size-exclusion chromatography revealed multiple and similarly migrating peaks in each cytosol that had both retinol and retinal dehydrogenase activities. Thus, the effects of ethanol on testes cannot be caused by direct inhibition of cytosolic retinoic acid synthesis because retinoid dehydrogenases distinct from mouse class A2 alcohol dehydrogenases, which corresponds to human class I, occurred in testes and they were not inhibited by ethanol. These data also demonstrate the occurrence of multiple cytosolic retinoic acid synthesis activities and indicate that the two reactions of cytosolic retinoic acid synthesis, retinol and retinal dehydrogenation, may be catalyzed by enzymes that occur as complexes. PMID:1599517

  9. 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

  10. 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. PMID:24532464

  11. Marked reduction of alcohol dehydrogenase in keratoconus corneal fibroblasts

    PubMed Central

    Kanoff, J.M.; Shankardas, J.; Dimitrijevich, S.

    2009-01-01

    Purpose To identify differentially expressed genes in keratoconus (KC) corneal fibroblasts. Methods Stromal keratocytes (having a fibroblast morphology) from KC keratoplasty specimens and eye bank donor corneas were isolated and expanded using a serum containing medium. RNA was isolated from three KC fibroblast cultures and five eye bank donor cornea fibroblast cultures. The targets from the cultured fibroblasts were hybridized to the Affymetrix U133 Plus 2.0 microarrays. Western blot analyses of cell lysates were performed to examine protein levels of interest in the two groups. Protein levels of select differentially expressed genes were further examined by immunohistochemistry. Keratocyte staining of archived KC keratoplasty specimens were graded using a 0 to 3+ scale and compared to five archived whole globes having normal corneas as well as to 10 Fuchs’ dystrophy keratoplasty specimens. Results Microarray analysis revealed up to a 212 fold reduction in the mRNA levels of alcohol dehydrogenase (class 1) beta polypeptide (ADH1B) in KC fibroblasts (p=0.04). Decreased alcohol dehydrogenase in KC fibroblasts was confirmed by western blot analysis of early passage primary keratocyte cell lysates. Immunohistochemistry using a monoclonal mouse immunoglobulin G (IgG) against human liver alcohol dehydrogenase revealed a dramatic difference in protein staining in the keratocytes of the KC group compared to the normal cornea group. Immunohistochemistry also showed decreased immunostaining against alcohol dehydrogenase in the KC stromal sections compared to those obtained from Fuchs’ endothelial corneal dystrophy samples. Conclusions Decreased alcohol dehydrogenase in KC corneal fibroblasts represents a strong marker and possible mediator of keratoconus. PMID:19365573

  12. 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.

  13. 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. PMID:16954110

  14. 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.

  15. Histidine 51 facilitates proton transfer in alcohol dehydrogenase

    SciTech Connect

    Gould, R.M.; Plapp, B.V.

    1987-05-01

    Operating through a proton relay system, His-51 has been proposed to serve as a base during ethanol oxidation by alcohol dehydrogenase. This residue is highly conserved in alcohol dehydrogenases. They have used mutamer directed mutagenesis to change this residue to Gln-51. Diethyl pyrocarbonate treatment decreases the activity of the wild type enzyme 60-fold, whereas the Gln-51 enzyme is inactivated by only 5-fold. The rate of inactivation is also much slower with the mutant enzyme. They conclude that His-51 is the reactive residue in yeast alcohol dehydrogenase. The mutation also alters the Km for acetaldehyde and the pH dependence of several kinetic constants. At pH 7.0 the Km for acetaldehyde is 18-fold higher in the Gln-51 enzyme, whereas Vmax for acetaldehyde reduction is the same as with the wild type enzyme. For ethanol oxidation the pH dependence of the log of Vmax and V/K shows a linear dependence with a slope of 0.5 and no discernible pK. They propose a mechanism that can explain these data. For the Gln-51 enzyme, after the ternary complex has formed in an Ordered Bi mechanism, a random component for proton release and hydride transfer occurs. With histidine at position 51, serving as a base, a more rapid proton release from the enzyme-NAD-ethanol complex precedes product formation.

  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. Cloning and sequencing of the gene encoding the 72-kilodalton dehydrogenase subunit of alcohol dehydrogenase from Acetobacter aceti.

    PubMed

    Inoue, T; Sunagawa, M; Mori, A; Imai, C; Fukuda, M; Takagi, M; Yano, K

    1989-06-01

    A genomic library of Acetobacter aceti DNA was constructed by using a broad-host-range cosmid vector. Complementation of a spontaneous alcohol dehydrogenase-deficient mutant resulted in the isolation of a plasmid designated pAA701. Subcloning and deletion analysis of pAA701 limited the region that complemented the deficiency in alcohol dehydrogenase activity of the mutant. The nucleotide sequence of this region was determined and showed that this region contained the full structural gene for the 72-kilodalton dehydrogenase subunit of the alcohol dehydrogenase enzyme complex. The predicted amino acid sequence of the gene showed homology with sequences of methanol dehydrogenase structural genes of Paracoccus denitrificans and Methylobacterium organophilum.

  18. Direct Observation of Correlated Interdomain Motion in Alcohol Dehydrogenase

    SciTech Connect

    Biehl, Ralf; Monkenbusch, Michael; Richter, Dieter; Hoffmann, Bernd; Merkel, Rudolf; Falus, Peter; Preost, Sylvain

    2008-09-26

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

  19. 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.

  20. 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. PMID:19444390

  1. Kinetic models for synthesis by a thermophilic alcohol dehydrogenase

    SciTech Connect

    Ford, J.B.; Askins, K.J.; Taylor, K.B. )

    1993-07-01

    Alcohol dehydrogenase from Thermoanearobium brockii at 25[degree] C and at 65[degree]C is more active with secondary than primary alcohols. The enzyme utilizes NADP and NADPH as cosubstrates better than NAD and NADH. The maximum velocities (V[sub m]) for secondary alcohols at 65[degree] C are 10 to 100 times higher than those at 25[degree] C, whereas the K[sub m] values are more comparable. At both 25[degree] C and 65[degree] C the substrate analogue 1,1,1,3,3,3-hexafluoro-2-propanol inhibited the oxidation of alcohol competitively with respect to cyclopentanol, and uncompetitively with respect to NADP. Dimethylsulfoxide inhibited the reduction of cyclopentanone competitively with respect to cyclopentanone, and uncompetitively with respect to NADPH. As a product inhibitor, NADP was competitive with respect to NADPH. These results demonstrate that the enzyme binds the nucleotide and then the alcohol or ketone to form a ternary complex which is converted to a product ternary complex that releases product and nucleotide in that order. At 25[degree] C, all aldehydes and ketones examined inhibited the enzyme at concentrations above their Michaelis constants. The substrate inhibition by cyclopentanone was incomplete, and it was uncompetitive with respect to NADPH. Furthermore, cyclopentanone as a product inhibitor showed intercept-linear, slope-parabolic inhibition with respect to cyclopentanol. These results indicate that cyclopentanone binds to the enzyme-NADP complex at high concentrations. The resulting ternary complex slowly dissociates NADP and cyclopentanone. At 65[degree] C, all of the secondary alcohols, with the exception of cyclohexanol, show substrate activation at high concentration.

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

    PubMed

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

    1999-08-01

    The alcohol dehydrogenase (ADH) gene family encodes enzymes that metabolize a wide variety of substrates, including ethanol, retinol, other aliphatic alcohols, hydroxysteroids, and lipid peroxidation products. Studies on 19 vertebrate animals have identified ADH orthologs across several species, and this has now led to questions of how best to name ADH proteins and genes. Seven distinct classes of vertebrate ADH encoded by non-orthologous genes have been defined based upon sequence homology as well as unique catalytic properties or gene expression patterns. Each class of vertebrate ADH shares <70% sequence identity with other classes of ADH in the same species. Classes may be further divided into multiple closely related isoenzymes sharing >80% sequence identity such as the case for class I ADH where humans have three class I ADH genes, horses have two, and mice have only one. Presented here is a nomenclature that uses the widely accepted vertebrate ADH class system as its basis. It follows the guidelines of human and mouse gene nomenclature committees, which recommend coordinating names across species boundaries and eliminating Roman numerals and Greek symbols. We recommend that enzyme subunits be referred to by the symbol "ADH" (alcohol dehydrogenase) followed by an Arabic number denoting the class; i.e. ADH1 for class I ADH. For genes we recommend the italicized root symbol "ADH" for human and "Adh" for mouse, followed by the appropriate Arabic number for the class; i.e. ADH1 or Adh1 for class I ADH genes. For organisms where multiple species-specific isoenzymes exist within a class, we recommend adding a capital letter after the Arabic number; i.e. ADH1A, ADH1B, and ADH1C for human alpha, beta, and gamma class I ADHs, respectively. This nomenclature will accommodate newly discovered members of the vertebrate ADH family, and will facilitate functional and evolutionary studies. PMID:10424757

  3. The structure of the quinoprotein alcohol dehydrogenase of Acetobacter aceti modelled on that of methanol dehydrogenase from Methylobacterium extorquens.

    PubMed

    Cozier, G E; Giles, I G; Anthony, C

    1995-06-01

    The 1.94 A structure of methanol dehydrogenase has been used to provide a model structure for part of a membrane quinohaemoprotein alcohol dehydrogenase. The basic superbarrel structure and the active-site region are retained, indicating essentially similar mechanisms of action, but there are considerable differences in the external loops, particularly those involved in formation of the shallow funnel leading to the active site.

  4. [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. PMID:27266256

  5. Physicochemical Characterization of a Thermostable Alcohol Dehydrogenase from Pyrobaculum aerophilum

    PubMed Central

    Vitale, Annalisa; Thorne, Natasha; Lovell, Scott; Battaile, Kevin P.; Hu, Xin; Shen, Min; D'Auria, Sabato; Auld, Douglas S.

    2013-01-01

    In this work we characterize an alcohol dehydrogenase (ADH) from the hyperthermophilic archaeon Pyrobaculum aerophilum (PyAeADHII). We have previously found that PyAeADHII has no activity when standard ADH substrates are used but is active when α-tetralone is used as substrate. Here, to gain insights into enzyme function, we screened several chemical libraries for enzymatic modulators using an assay employing α-tetralone. The results indicate that PyAeADHII activity in the presence of α-tetralone was inhibited by compounds such as flunarizine. We also examined metal coordination of the enzyme in solution by performing metal substitution of the enzyme-bound zinc (Zn2+) with cobalt. The solution-based absorption spectra for cobalt substituted PyAeADHII supports substitution at the structural Zn2+ site. To gain structural insight, we obtained the crystal structure of both wild-type and cobalt-substituted PyAeADHII at 1.75 Å and 2.20 Å resolution, respectively. The X-ray data confirmed one metal ion per monomer present only at the structural site with otherwise close conservation to other ADH enzymes. We next determined the co-crystal structure of the NADPH-bound form of the enzyme at 2.35 Å resolution to help define the active site region of the enzyme and this data shows close structural conservation with horse ADH, despite the lack of a catalytic Zn2+ ion in PyAeADHII. Modeling of α-tetralone into the NADPH bound structure suggests an arginine as a possible catalytic residue. The data presented here can yield a better understanding of alcohol dehydrogenases lacking the catalytic zinc as well as the structural features inherent to thermostable enzymes. PMID:23755111

  6. 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.

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

    PubMed Central

    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 Vmax 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.—Rivera-Meza, M., Quintanilla, M. E., Tampier, L., Mura, C. V., Sapag, A., Israel, Y. Mechanism of protection against alcoholism by an alcohol dehydrogenase polymorphism: development of an animal model. PMID:19710201

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

    PubMed

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

    2014-12-01

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

  9. 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

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

    PubMed

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

    2015-06-01

    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 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.

  11. Structural determinants of stereospecificity in yeast alcohol dehydrogenase.

    PubMed Central

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

    1991-01-01

    Replacing Leu-182 by Ala in yeast alcohol dehydrogenase (YADH; alcohol:NAD+ oxidoreductase, EC 1.1.1.1) yields a mutant that retains 34% of its kcat value and makes one stereochemical "mistake" every 850,000 turnovers (instead of approximately 1 error every 7,000,000,000 turnovers in native YADH) in its selection of the 4-Re hydrogen of NADH. Half of the decrease in stereochemical fidelity comes from an increase in the rate of transfer of the 4-Si hydrogen of NADH. The mutant also accepts 5-methylnicotinamide adenine dinucleotide, a cofactor analog not accepted by native YADH. The stereospecificity of the mutant is lower still with analogs of NADH where the carboxamide group of the nicotinamide ring is replaced by groups with weaker hydrogen bonding potential. For example, with thio-NADH, the mutant enzyme makes 1 stereochemical "mistake" every 450 turnovers. Finally, the double mutant T157S/L182A, in which Thr-157 is replaced by Ser and Leu-182 is replaced by Ala, also shows decreased stereochemical fidelity. These results suggest that Si transfer in the mutant enzymes arises from NADH bound in a syn conformation in the active site and that this binding is not obstructed in native YADH by side chains essential for catalysis. PMID:1924300

  12. Structural determinants of stereospecificity in yeast alcohol dehydrogenase.

    PubMed

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

    1991-10-01

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

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

    PubMed Central

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

    2013-01-01

    Class I alcohol dehydrogenases (ADH1s) are the rate-limiting enzymes for ethanol and vitamin A (retinol) metabolism in the liver. Because previous studies have shown that human ADH1 enzymes may participate in bile acid metabolism, we investigated whether the bile acid-activated nuclear receptor farnesoid X receptor (FXR) regulates ADH1 genes. In human hepatocytes, both the endogenous FXR ligand chenodeoxycholic acid and synthetic FXR-specific agonist GW4064 increased ADH1 mRNA, protein, and activity. Moreover, overexpression of a constitutively active form of FXR induced ADH1A and ADH1B expression, whereas silencing of FXR abolished the effects of FXR agonists on ADH1 expression and activity. Transient transfection studies and electrophoretic mobility shift assays revealed functional FXR response elements in the ADH1A and ADH1B proximal promoters, thus indicating that both genes are direct targets of FXR. These findings provide the first evidence for direct connection of bile acid signaling and alcohol metabolism. PMID:23772048

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

    PubMed

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

    2013-09-01

    Class I alcohol dehydrogenases (ADH1s) are the rate-limiting enzymes for ethanol and vitamin A (retinol) metabolism in the liver. Because previous studies have shown that human ADH1 enzymes may participate in bile acid metabolism, we investigated whether the bile acid-activated nuclear receptor farnesoid X receptor (FXR) regulates ADH1 genes. In human hepatocytes, both the endogenous FXR ligand chenodeoxycholic acid and synthetic FXR-specific agonist GW4064 increased ADH1 mRNA, protein, and activity. Moreover, overexpression of a constitutively active form of FXR induced ADH1A and ADH1B expression, whereas silencing of FXR abolished the effects of FXR agonists on ADH1 expression and activity. Transient transfection studies and electrophoretic mobility shift assays revealed functional FXR response elements in the ADH1A and ADH1B proximal promoters, thus indicating that both genes are direct targets of FXR. These findings provide the first evidence for direct connection of bile acid signaling and alcohol metabolism.

  15. 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

  16. Origins of the high catalytic activity of human alcohol dehydrogenase 4 studied with horse liver A317C alcohol dehydrogenase.

    PubMed

    Herdendorf, Timothy J; Plapp, Bryce V

    2011-05-30

    The turnover numbers and other kinetic constants for human alcohol dehydrogenase (ADH) 4 ("stomach" isoenzyme) are substantially larger (10-100-fold) than those for human class I and horse liver alcohol dehydrogenases. Comparison of the primary amino acid sequences (69% identity) and tertiary structures of these enzymes led to the suggestion that residue 317, which makes a hydrogen bond with the nicotinamide amide nitrogen of the coenzyme, may account for these differences. Ala-317 in the class I enzymes is substituted with Cys in human ADH4, and locally different conformations of the peptide backbones could affect coenzyme binding. This hypothesis was tested by making the A317C substitution in horse liver ADH1E and comparisons to the wild-type ADH1E. The steady-state kinetic constants for the oxidation of benzyl alcohol and the reduction of benzaldehyde catalyzed by the A317C enzyme were very similar (up to about 2-fold differences) to those for the wild-type enzyme. Transient kinetics showed that the rate constants for binding of NAD(+) and NADH were also similar. Transient reaction data were fitted to the full Ordered Bi Bi mechanism and showed that the rate constants for hydride transfer decreased by about 2.8-fold with the A317C substitution. The structure of A317C ADH1E complexed with NAD(+) and 2,3,4,5,6-pentafluorobenzyl alcohol at 1.2 Å resolution is essentially identical to the structure of the wild-type enzyme, except near residue 317 where the additional sulfhydryl group displaces a water molecule that is present in the wild-type enzyme. ADH is adaptable and can tolerate internal substitutions, but the protein dynamics apparently are affected, as reflected in rates of hydride transfer. The A317C substitution is not solely responsible for the larger kinetic constants in human ADH4; thus, the differences in catalytic activity must arise from one or more of the other hundred substitutions in the enzyme.

  17. Salivary alcohol dehydrogenase in non-smoking and smoking alcohol-dependent persons.

    PubMed

    Waszkiewicz, Napoleon; Jelski, Wojciech; Zalewska, Anna; Szulc, Agata; Szmitkowski, Maciej; Zwierz, Krzysztof; Szajda, Sławomir Dariusz

    2014-09-01

    Increasing attention to the importance of saliva testing is not surprising because smoking and alcohol drinking act synergistically on oral tissues, and their metabolite levels, e.g., acetaldehyde, are much higher in saliva than in blood. The activity of salivary alcohol dehydrogenase (ADH) comes from oral microbiota, mucosa, and salivary glands. The purpose of this study was to investigate the involvement of ADH in the oral health pathology of smoking (AS) and non-smoking (ANS) alcohol-dependent males. The results indicated that the AS group had a more significant and longer duration (until the 30th day of alcohol abstinence) decrease in ADH activity and output than the ANS group (until the 15th day of alcohol abstinence) compared to controls (social drinkers; C). The decreased salivary flow (SF) in alcoholics was observed longer in the ANS group (until the 30th day of alcohol abstinence), whereas in the AS group SF normalized at the 15th day, probably due to the irritating effect of tobacco smoke on the oral mucosa. Because saliva was centrifuged to remove cells and debris (including microbial cells), the detected salivary ADH activity was derived from salivary glands and/or oral mucosa. A more profound and longer decrease in ADH activity/output in smoking than non-smoking alcoholics was likely due to the damaged salivary glands and/or oral mucosa, caused by the synergistic effect of alcohol drinking and smoking. The lower values of salivary ADH in smoking than non-smoking alcoholics might also be partly due to the reversed/inhibited ADH reaction by high levels of accumulated acetaldehyde.

  18. [Spectral study of the conformational change of yeast alcohol dehydrogenase induced by alcohol].

    PubMed

    Zhang, Q; Yuan, J

    1998-02-01

    The conformational change of Yeast Alcohol Dehydrogenase (YADH) at the different concentration of ethanol, n-propanol and ethylene glycol was studied by means of ultraviolet spectrum, fluorescence spectrum and circular dichroism spectrum. The results showed that the ultraviolet absorbance at 220nm and 280nm as well as the relative fluorescence intensity at 336nm of YADH increased with increasing alcohol concentration. The negative peakes at 208nm and 220nm of YADH in circular dichroism spectrum with the solvent of ethanol, ethylene glycol were obviously intensified, but the 220nm peak of YADH was increased in the presence of n-propanol while the 208nm peak was decreased and red-shifted in position as to completely lost. According to the data above, it indicates that the conformation of YADH was changed with losing activity at the various concentration of alcohol.

  19. 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.

  20. 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. PMID:25805841

  1. Drosophila melanogaster alcohol dehydrogenase: mechanism of aldehyde oxidation and dismutation.

    PubMed

    Winberg, J O; McKinley-McKee, J S

    1998-02-01

    Drosophila alcohol dehydrogenase (Adh) catalyses the oxidation of both alcohols and aldehydes. In the latter case, the oxidation is followed by a reduction of the aldehyde, i.e. a dismutation reaction. At high pH, dismutation is accompanied by a small release of NADH, which is not observed at neutral pH. Previously it has been emphasized that kinetic coefficients obtained by measuring the increase in A340, i.e. the release of NADH at high pH is not a direct measure of the aldehyde oxidation reaction and these values cannot be compared with those for alcohol dehydrogenation. In this article we demonstrate that this is not entirely true, and that the coefficients phiB and phiAB, where B is the aldehyde and A is NAD+, are the same for a dismutation reaction and a simple aldehyde dehydrogenase reaction. Thus the substrate specificity of the aldehyde oxidation reaction can be determined by simply measuring the NADH release. The coefficients for oxidation and dehydrogenation reactions (phi0d and phiAd respectively) are complex and involve the constants for the dismutation reaction. However, dead-end inhibitors can be used to determine the quantitative contribution of the kinetic constants for the aldehyde oxidation and reduction pathways to the phi0d and phiAd coefficients. The combination of dead-end and product inhibitors can be used to determine the reaction mechanism for the aldehyde oxidation pathway. Previously, we showed that with Drosophila Adh, the interconversion between alcohols and aldehydes followed a strictly compulsory ordered pathway, although aldehydes and ketones formed binary complexes with the enzyme. This raised the question regarding the reaction mechanism for the oxidation of aldehydes, i.e. whether a random ordered pathway was followed. In the present work, the mechanism for the oxidation of different aldehydes and the accompanying dismutation reaction with the slow alleloenzyme (AdhS) from Drosophila melanogaster has been studied. To obtain

  2. Drosophila melanogaster alcohol dehydrogenase: mechanism of aldehyde oxidation and dismutation.

    PubMed Central

    Winberg, J O; McKinley-McKee, J S

    1998-01-01

    Drosophila alcohol dehydrogenase (Adh) catalyses the oxidation of both alcohols and aldehydes. In the latter case, the oxidation is followed by a reduction of the aldehyde, i.e. a dismutation reaction. At high pH, dismutation is accompanied by a small release of NADH, which is not observed at neutral pH. Previously it has been emphasized that kinetic coefficients obtained by measuring the increase in A340, i.e. the release of NADH at high pH is not a direct measure of the aldehyde oxidation reaction and these values cannot be compared with those for alcohol dehydrogenation. In this article we demonstrate that this is not entirely true, and that the coefficients phiB and phiAB, where B is the aldehyde and A is NAD+, are the same for a dismutation reaction and a simple aldehyde dehydrogenase reaction. Thus the substrate specificity of the aldehyde oxidation reaction can be determined by simply measuring the NADH release. The coefficients for oxidation and dehydrogenation reactions (phi0d and phiAd respectively) are complex and involve the constants for the dismutation reaction. However, dead-end inhibitors can be used to determine the quantitative contribution of the kinetic constants for the aldehyde oxidation and reduction pathways to the phi0d and phiAd coefficients. The combination of dead-end and product inhibitors can be used to determine the reaction mechanism for the aldehyde oxidation pathway. Previously, we showed that with Drosophila Adh, the interconversion between alcohols and aldehydes followed a strictly compulsory ordered pathway, although aldehydes and ketones formed binary complexes with the enzyme. This raised the question regarding the reaction mechanism for the oxidation of aldehydes, i.e. whether a random ordered pathway was followed. In the present work, the mechanism for the oxidation of different aldehydes and the accompanying dismutation reaction with the slow alleloenzyme (AdhS) from Drosophila melanogaster has been studied. To obtain

  3. 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.

  4. A model system for QTL analysis: Effects of alcohol dehydrogenase genotype on alcohol pharmacokinetics

    SciTech Connect

    Martin, N.G.; Nightingale, B.; Whitfield, J.B.

    1994-09-01

    There is much interest in the detection of quantitative trait loci (QTL) - major genes which affect quantitative phenotypes. The relationship of polymorphism at known alcohol metabolizing enzyme loci to alcohol pharmacokinetics is a good model system. The three class I alcohol dehydrogenase genes are clustered on chromosome 4 and protein electrophoresis has revealed polymorphisms at the ADH2 and ADH3 loci. While different activities of the isozymes have been demonstrated in vitro, little work has been done in trying to relate ADH polymorphism to variation in ethanol metabolism in vivo. We previously measured ethanol metabolism and psychomotor reactivity in 206 twin pairs and demonstrated that most of the repeatable variation was genetic. We have now recontacted the twins to obtain DNA samples and used PCR with allele specific primers to type the ADH2 and ADH3 polymorphisms in 337 individual twins. FISHER has been used to estimate fixed effects of typed polymorphisms simultaneously with remaining linked and unlinked genetic variance. The ADH2*1-2 genotypes metabolize ethanol faster and attain a lower peak blood alcohol concentration than the more common ADH2*1-1 genotypes, although less than 3% of the variance is accounted for. There is no effect of ADH3 genotype. However, sib-pair linkage analysis suggests that there is a linked polymorphism which has a much greater effect on alcohol metabolism that those typed here.

  5. 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.

  6. 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.

  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. 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. PMID:25280628

  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. 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.

  11. Bifunctional aldehyde/alcohol dehydrogenase (ADHE) in chlorophyte algal mitochondria.

    PubMed

    Atteia, Ariane; van Lis, Robert; Mendoza-Hernández, Guillermo; Henze, Katrin; Martin, William; Riveros-Rosas, Hector; González-Halphen, Diego

    2003-09-01

    Protein profiles of mitochondria isolated from the heterotrophic chlorophyte Polytomella sp. grown on ethanol at pH 6.0 and pH 3.7 were analyzed by Blue Native and denaturing polyacrylamide gel electrophoresis. Steady-state levels of oxidative phosphorylation complexes were influenced by external pH. Levels of an abundant, soluble, mitochondrial protein of 85 kDa and its corresponding mRNA increased at pH 6.0 relative to pH 3.7. N-terminal and internal sequencing of the 85 kDa mitochondrial protein together with the corresponding cDNA identified it as a bifunctional aldehyde/alcohol dehydrogenase (ADHE) with strong similarity to homologues from eubacteria and amitochondriate protists. A mitochondrial targeting sequence of 27 amino acids precedes the N-terminus of the mature mitochondrial protein. A gene encoding an ADHE homologue was also identified in the genome of Chlamydomonas reinhardtii, a photosynthetic relative of Polytomella. ADHE reveals a complex picture of sequence similarity among homologues. The lack of ADHE from archaebacteria indicates a eubacterial origin for the eukaryotic enzyme. Among eukaryotes, ADHE has hitherto been characteristic of anaerobes since it is essential to cytosolic energy metabolism of amitochondriate protists such as Giardia intestinalis and Entamoeba histolytica. Its abundance and expression pattern suggest an important role for ADHE in mitochondrial metabolism of Polytomella under the conditions studied. The current data are compatible with the view that Polytomella ADHE could be involved either in ethanol production or assimilation, or both, depending upon environmental conditions. Presence of ADHE in an oxygen-respiring algal mitochondrion and co-expression at ambient oxygen levels with respiratory chain components is unexpected with respect to the view that eukaryotes acquired ADHE genes specifically as an adaptation to an anaerobic lifestyle.

  12. Bifunctional aldehyde/alcohol dehydrogenase (ADHE) in chlorophyte algal mitochondria.

    PubMed

    Atteia, Ariane; van Lis, Robert; Mendoza-Hernández, Guillermo; Henze, Katrin; Martin, William; Riveros-Rosas, Hector; González-Halphen, Diego

    2003-09-01

    Protein profiles of mitochondria isolated from the heterotrophic chlorophyte Polytomella sp. grown on ethanol at pH 6.0 and pH 3.7 were analyzed by Blue Native and denaturing polyacrylamide gel electrophoresis. Steady-state levels of oxidative phosphorylation complexes were influenced by external pH. Levels of an abundant, soluble, mitochondrial protein of 85 kDa and its corresponding mRNA increased at pH 6.0 relative to pH 3.7. N-terminal and internal sequencing of the 85 kDa mitochondrial protein together with the corresponding cDNA identified it as a bifunctional aldehyde/alcohol dehydrogenase (ADHE) with strong similarity to homologues from eubacteria and amitochondriate protists. A mitochondrial targeting sequence of 27 amino acids precedes the N-terminus of the mature mitochondrial protein. A gene encoding an ADHE homologue was also identified in the genome of Chlamydomonas reinhardtii, a photosynthetic relative of Polytomella. ADHE reveals a complex picture of sequence similarity among homologues. The lack of ADHE from archaebacteria indicates a eubacterial origin for the eukaryotic enzyme. Among eukaryotes, ADHE has hitherto been characteristic of anaerobes since it is essential to cytosolic energy metabolism of amitochondriate protists such as Giardia intestinalis and Entamoeba histolytica. Its abundance and expression pattern suggest an important role for ADHE in mitochondrial metabolism of Polytomella under the conditions studied. The current data are compatible with the view that Polytomella ADHE could be involved either in ethanol production or assimilation, or both, depending upon environmental conditions. Presence of ADHE in an oxygen-respiring algal mitochondrion and co-expression at ambient oxygen levels with respiratory chain components is unexpected with respect to the view that eukaryotes acquired ADHE genes specifically as an adaptation to an anaerobic lifestyle. PMID:14756315

  13. 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

    2016-07-01

    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.

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

    PubMed

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

    2013-09-01

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

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

    PubMed Central

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

    2013-01-01

    Alcohol dehydrogenases (ADHs) are a group of dehydrogenase enzymes that facilitate the interconversion between alcohols and aldehydes or ketones with the reduction of NAD+ to NADH. In bacteria, some alcohol dehydrogenases catalyze the opposite reaction as part of fermentation to ensure a constant supply of NAD+. The adh gene from Kangiella koreensis was cloned and the protein (KkADH) was expressed, purified and crystallized. A KkADH crystal diffracted to 2.5 Å resolution and belonged to the monoclinic space group P21, with unit-cell parameters a = 94.1, b = 80.9, c = 115.6 Å, β = 111.9°. Four monomers were present in the asymmetric unit, with a corresponding V M of 2.55 Å3 Da−1 and a solvent content of 51.8%. PMID:23989158

  16. Crystal structure of cod liver class I alcohol dehydrogenase: substrate pocket and structurally variable segments.

    PubMed Central

    Ramaswamy, S.; el Ahmad, M.; Danielsson, O.; Jörnvall, H.; Eklund, H.

    1996-01-01

    The structural framework of cod liver alcohol dehydrogenase is similar to that of horse and human alcohol dehydrogenases. In contrast, the substrate pocket differs significantly, and main differences are located in three loops. Nevertheless, the substrate pocket is hydrophobic like that of the mammalian class I enzymes and has a similar topography in spite of many main-chain and side-chain differences. The structural framework of alcohol dehydrogenase is also present in a number of related enzymes like glucose dehydrogenase and quinone oxidoreductase. These enzymes have completely different substrate specificity, but also for these enzymes, the corresponding loops of the substrate pocket have significantly different structures. The domains of the two subunits in the crystals of the cod enzyme further differ by a rotation of the catalytic domains by about 6 degrees. In one subunit, they close around the coenzyme similarly as in coenzyme complexes of the horse enzyme, but form a more open cleft in the other subunit, similar to the situation in coenzyme-free structures of the horse enzyme. The proton relay system differs from the mammalian class I alcohol dehydrogenases. His 51, which has been implicated in mammalian enzymes to be important for proton transfer from the buried active site to the surface is not present in the cod enzyme. A tyrosine in the corresponding position is turned into the substrate pocket and a water molecule occupies the same position in space as the His side chain, forming a shorter proton relay system. PMID:8845755

  17. Characterization of alcohol dehydrogenase 1 and 3 from Neurospora crassa FGSC2489.

    PubMed

    Park, Yong-Cheol; San, Ka-Yiu; Bennett, George N

    2007-08-01

    Alcohol dehydrogenase (ADH) is a key enzyme in the production and utilization of alcohols. Some also catalyze the formation of carboxylate esters from alcohols and aldehydes. The ADH1 and ADH3 genes of Neurospora crassa FGSC2489 were cloned and expressed in recombinant Escherichia coli to investigate their alcohol dehydrogenation and carboxylate ester formation abilities. Homology analysis and sequence alignment of amino acid sequence indicated that ADH1 and ADH3 of N. crassa contained a zinc-binding consensus sequence and a NAD(+)-binding motif and showed 54-75% identity with fungi ADHs. N. crassa ADH1 was expressed in E. coli to give a specific activity of 289 +/- 9 mU/mg using ethanol and NAD(+) as substrate and cofactor, respectively. Corresponding experiments on the expression and activity of ADH3 gave 4 mU/mg of specific activity. N. crassa ADH1 preferred primary alcohols containing C3-C8 carbons to secondary alcohols such as 2-propanol and 2-butanol. N. crassa ADH1 possessed 5.3 mU/mg of specific carboxylate ester-forming activity accumulating 0.4 mM of ethyl acetate in 18 h. Substrate specificity of various linear alcohols and aldehydes indicated that short chain-length alcohols and aldehydes were good substrates for carboxylate ester production. N. crassa ADH1 was a primary alcohol dehydrogenase using cofactor NAD(+) preferably and possessed carboxylate ester-forming activity with short chain alcohols and aldehydes. PMID:17516063

  18. Dehydrogenation of 3-phenoxybenzyl alcohol in isolated perfused rabbit skin, skin homogenate and purified dehydrogenases.

    PubMed

    Bast, G E; Kampffmeyer, H G

    1998-01-01

    The formation of 3-phenoxybenzoic acid from 3-phenoxybenzyl alcohol was determined in (a) rabbit ears, single-pass perfused with a protein-free buffer, pH 7.4; (b) the microsomal fraction and its supernatant from homogenized rabbit skin; and (c) purified alcohol dehydrogenase from horse liver and baker's yeast. The inhibition of product formation in (a) was about 60% by various 4-methylpyrazole concentrations, but metyrapone had no effect. Following ultracentrifugation, only the supernatant of homogenized skin showed product formation (apparent Vmay: 32 pmol/min per cm2 skin; apparent Km: 64 microM). 3-Phenoxybenzyl alcohol and ethanol dehydrogenation was similar by alcohol dehydrogenase from horse liver (apparent Km: 0.7 vs. 0.4 mM; apparent Vmax: 0.3 vs. 0.2 U/ microg protein). In baker's yeast, the apparent Km of 3-phenoxybenzoic acid formation was several times larger than that for ethanol dehydrogenation. The KI of 4-methylpyrazole for alcohol dehydrogenase from horse liver was 0.6 (3-phenoxybenzyl alcohol) vs. 0.04 microM (ethanol). The KI for ethanol in baker's yeast was 470 microM. In conclusion dehydrogenation is an important metabolic pathway in the skin for xenobiotics with an aliphatic alcohol at a side chain. PMID:9885409

  19. 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

  20. Transcriptome-wide identification and characterization of CAD isoforms specific for podophyllotoxin biosynthesis from Podophyllum hexandrum.

    PubMed

    Bhattacharyya, Dipto; Hazra, Saptarshi; Banerjee, Anindyajit; Datta, Riddhi; Kumar, Deepak; Chakrabarti, Saikat; Chattopadhyay, Sharmila

    2016-09-01

    Podophyllotoxin (ptox) is a therapeutically important lignan derived from Podophyllum hexandrum and is used as a precursor for the synthesis of anticancer drugs etoposide, teniposide and etopophose. In spite of its enormous economic significance, genomic information on this endangered medicinal herb is scarce. We have performed de novo transcriptome analysis of methyl jasmonate (MeJA)-treated P. hexandrum cell cultures exhibiting enhanced ptox accumulation. The results revealed the maximum up-regulation of several isoforms of cinnamyl alcohol dehydrogenase (CAD). CAD catalyzes the synthesis of coniferyl alcohol and sinapyl alcohol from coniferaldehyde (CAld) and sinapaldehyde respectively. Coniferyl alcohol can produce both lignin and lignan while sinapyl alcohol produces only lignin. To isolate the CAD isoforms favoring ptox, we deduced full length cDNA sequences of four CAD isoforms: PhCAD1, PhCAD2, PhCAD3 and PhCAD4 from the contigs of the transcriptome data. In vitro enzyme assays indicated a higher affinity for CAld over sinapaldehyde for each isoform. In silico molecular docking analyses also suggested that PhCAD3 has a higher binding preference with CAld over sinapaldehyde, followed by PhCAD4, PhCAD2, and PhCAD1, respectively. The transgenic cell cultures overexpressing these isoforms independently revealed that PhCAD3 favored the maximum accumulation of ptox as compared to lignin followed by PhCAD4 and PhCAD2, whereas, PhCAD1 favored both equally. Together, our study reveals transcriptome-wide identification and characterization of ptox specific CAD isoforms from P. hexandrum. It provides a useful resource for future research not only on the ptox biosynthetic pathway but on overall P. hexandrum, an endangered medicinal herb with immense therapeutic importance.

  1. Optical spectroscopy of nicotinoprotein alcohol dehydrogenase from Amycolatopsis methanolica: a comparison with horse liver alcohol dehydrogenase and UDP-galactose epimerase.

    PubMed

    Piersma, S R; Visser, A J; de Vries, S; Duine, J A

    1998-03-01

    The NADH absorbance spectrum of nicotinoprotein (NADH-containing) alcohol dehydrogenase from Amycolatopsis methanolica has a maximum at 326 nm. Reduced enzyme-bound pyridine dinucleotide could be reversibly oxidized by acetaldehyde. The fluorescence excitation spectrum for NADH bound to the enzyme has a maximum at 325 nm. Upon excitation at 290 nm, energy transfer from tryptophan to enzyme-bound NADH was negligible. The fluorescence emission spectrum (excitation at 325 nm) for NADH bound to the enzyme has a maximum at 422 nm. The fluorescence intensity is enhanced by a factor of 3 upon binding of isobutyramide (Kd = 59 microM). Isobutyramide acts as competitive inhibitor (Ki = 46 microM) with respect to the electron acceptor NDMA (N,N-dimethyl-p-nitrosoaniline), which binds to the enzyme containing the reduced cofactor. The nonreactive substrate analogue trifluoroethanol acts as a competitive inhibitor with respect to the substrate ethanol (Ki = 1.6 microM), which binds to the enzyme containing the oxidized cofactor. Far-UV circular dichroism spectra of the enzyme containing NADH and the enzyme containing NAD+ were identical, indicating that no major conformational changes occur upon oxidation or reduction of the cofactor. Near-UV circular dichroism spectra of NADH bound to the enzyme have a minimum at 323 nm (Deltaepsilon = -8.6 M-1 cm-1). The fluorescence anisotropy decay of enzyme-bound NADH showed no rotational freedom of the NADH cofactor. This implies a rigid environment as well as lack of motion of the fluorophore. The average fluorescence lifetime of NADH bound to the enzyme is 0.29 ns at 20 degreesC and could be resolved into at least three components (in the range 0.13-0.96 ns). Upon binding of isobutyramide to the enzyme-containing NADH, the average excited-state lifetime increased to 1.02 ns and could be resolved into two components (0.37 and 1.11 ns). The optical spectra of NADH bound to nicotinoprotein alcohol dehydrogenase have blue-shifted maxima

  2. 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…

  3. Rapid Microscale Isolation and Purification of Yeast Alcohol Dehydrogenase Using Cibacron Blue Affinity Chromatography

    NASA Astrophysics Data System (ADS)

    Morgan, Chad; Moir, Neil

    1996-11-01

    A rapid microscale procedure has been developed for the isolation and purification of yeast alcohol dehydrogenase. Glass beads are used for cytolysis, PEG precipitation for partial purification and a cibacron blue affinity column for the final step. A 27.5 fold purification can be achieved in 2 - 3 hours.

  4. 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…

  5. [Molecular evidences of non-ADH pathway in alcohol metabolism and Class III alcohol dehydrogenase (ADH3)].

    PubMed

    Haseba, Takeshi

    2014-06-01

    Class I alcohol dehydrogenase (ADH1), a key enzyme of alcohol metabolism, contributes around 70% to the systemic alcohol metabolism and also to the acceleration of the metabolism due to chronic alcohol consumption by increasing its liver content, if the liver damage or disease is not apparent. However, the contribution of ADH1 to alcohol metabolism decreases in case of acute alcohol poisoning or chronic alcohol consumption inducing liver damage or disease. On the contrary, non-ADH pathway, which is independent of ADH1, increases the contribution to alcohol metabolism in these cases, by complementing the reduced role of ADH1. The molecular substantiality of non-ADH pathway has been still unknown in spite of the long and hot controversy between two candidates of microsomal ethanol oxidizing system (MEOS) and catalase. This research history suggests the existence of other candidates. Among ADH isozymes, Class III (ADH3) has the highest Km for ethanol and the highest resistance to pyrazole reagents of specific ADH inhibitors. This ADH3 was demonstrated to increase the contribution to alcohol metabolism in vivo dose-dependently, therefore, is a potent candidate of non-ADH pathway. Moreover, ADH3 is considered to increase the contribution to alcohol metabolism in case of alcoholic liver diseases, because the enzyme content increases in damaged tissues with increased hydrophobicity or the activity of the liver correlates with the accumulated alcohol consumptions of patients with alcoholic liver diseases. Such adaptation of ADH3 to alcohol metabolism in these pathological conditions makes patients possible to keep drinking a lot in spite of decrease of ADH1 activity and develops alcoholism seriously.

  6. 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. PMID:26743658

  7. 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

  8. 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.

  9. 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

    Anderson, Nickolas A; Tobimatsu, Yuki; Ciesielski, Peter N; Ximenes, Eduardo; Ralph, John; Donohoe, Bryon S; Ladisch, Michael; Chapple, Clint

    2015-08-01

    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

  10. 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

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

    PubMed

    Dallet, S; Legoy, M D

    1996-05-01

    A comparison between the pressure effects on the catalysis of Thermoanaerobium brockii alcohol dehydrogenase (TBADH: a thermostable tetrameric enzyme) and yeast alcohol dehydrogenase (YADH: a mesostable tetrameric enzyme) revealed a different behaviour. YADH activity is continuously inhibited by an increase of pressure, whereas YADH affinity seems less sensitive to pressure. TBADH activity is enhanced by pressure up to 100 MPa. TBADH affinity for alcoholic substrates increases if pressure increases, was TBADH affinity for NADP decreases when pressure increases. Hypothesis has been raised concerning the dissociation of oligomeric enzymes under high hydrostatic pressure ( < 200 MPa) [1]. But in the case of these two enzymes, unless the oligomers reassociate very quickly (< 1 min), the activity inhibition of YADH at all pressures and TBADH for pressures above 100 MPa is not correlated to subunit dissociation. Hence we suggest that enzymes under pressure encounter a molecular rearrangement which can either have a positive or a negative effect on activity. Finally, we have observed that the catalytic behaviour of alcohol dehydrogenases under pressure is connected to their thermostability.

  12. 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.

  13. Purification and characterization of benzyl alcohol- and benzaldehyde- dehydrogenase from Pseudomonas putida CSV86.

    PubMed

    Shrivastava, Rahul; Basu, Aditya; Phale, Prashant S

    2011-08-01

    Pseudomonas putida CSV86 utilizes benzyl alcohol via catechol and methylnaphthalenes through detoxification pathway via hydroxymethylnaphthalenes and naphthaldehydes. Based on metabolic studies, benzyl alcohol dehydrogenase (BADH) and benzaldehyde dehydrogenase (BZDH) were hypothesized to be involved in the detoxification pathway. BADH and BZDH were purified to apparent homogeneity and were (1) homodimers with subunit molecular mass of 38 and 57 kDa, respectively, (2) NAD(+) dependent, (3) broad substrate specific accepting mono- and di-aromatic alcohols and aldehydes but not aliphatic compounds, and (4) BADH contained iron and magnesium, while BZDH contained magnesium. BADH in the forward reaction converted alcohol to aldehyde and required NAD(+), while in the reverse reaction it reduced aldehyde to alcohol in NADH-dependent manner. BZDH showed low K (m) value for benzaldehyde as compared to BADH reverse reaction. Chemical cross-linking studies revealed that BADH and BZDH do not form multi-enzyme complex. Thus, the conversion of aromatic alcohol to acid is due to low K (m) and high catalytic efficiency of BZDH. Phylogenetic analysis revealed that BADH is a novel enzyme and diverged during the evolution to gain the ability to utilize mono- and di-aromatic compounds. The wide substrate specificity of these enzymes enables strain to detoxify methylnaphthalenes to naphthoic acids efficiently.

  14. 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.

  15. 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

    2015-01-01

    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.

  16. 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. PMID:21416338

  17. 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.

  18. 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.

  19. Loss of function of cinnamyl alcohol dehydrogenase 1 leads to unconventional lignin and a temperature-sensitive growth defect in Medicago truncatula

    PubMed Central

    Zhao, Qiao; Tobimatsu, Yuki; Zhou, Rui; Pattathil, Sivakumar; Gallego-Giraldo, Lina; Fu, Chunxiang; Jackson, Lisa A.; Hahn, Michael G.; Kim, Hoon; Chen, Fang; Ralph, John; Dixon, Richard A.

    2013-01-01

    There is considerable debate over the capacity of the cell wall polymer lignin to incorporate unnatural monomer units. We have identified Tnt1 retrotransposon insertion mutants of barrel medic (Medicago truncatula) that show reduced lignin autofluorescence under UV microscopy and red coloration in interfascicular fibers. The phenotype is caused by insertion of retrotransposons into a gene annotated as encoding cinnamyl alcohol dehydrogenase, here designated M. truncatula CAD1. NMR analysis indicated that the lignin is derived almost exclusively from coniferaldehyde and sinapaldehyde and is therefore strikingly different from classical lignins, which are derived mainly from coniferyl and sinapyl alcohols. Despite such a major alteration in lignin structure, the plants appear normal under standard conditions in the greenhouse or growth chamber. However, the plants are dwarfed when grown at 30 °C. Glycome profiling revealed an increased extractability of some xylan and pectin epitopes from the cell walls of the cad1-1 mutant but decreased extractability of others, suggesting that aldehyde-dominant lignin significantly alters cell wall structure. PMID:23901113

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

    PubMed

    Paulíková, H; Molnárová, M; Podhradský, D

    1998-12-01

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

  1. Taraxerone enhances alcohol oxidation via increases of alcohol dehyderogenase (ADH) and acetaldehyde dehydrogenase (ALDH) activities and gene expressions.

    PubMed

    Sung, Chang-Keun; Kim, Seung-Mi; Oh, Chang-Jin; Yang, Sun-A; Han, Byung-Hee; Mo, Eun-Kyoung

    2012-07-01

    The present study, taraxerone (d-friedoolean-14-en-3-one) was isolated from Sedum sarmentosum with purity 96.383%, and its enhancing effects on alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) activities were determined: EC(50) values were 512.42 ± 3.12 and 500.16 ± 3.23 μM for ADH and ALDH, respectively. In order to obtain more information on taraxerone related with the alcohol metabolism, 40% ethanol (5 mL/kg body weight) with 0.5-1mM of taraxerone were administered to mice. The plasma alcohol and acetaldehyde concentrations of taraxerone-treated groups were significantly lowered than those of the control group (p<0.01): approximately 20-67% and 7-57% lowered for plasma alcohol and acetaldehyde, respectively. Compare to the control group, the ADH and ALDH expressions in the liver tissues were abruptly increased in the taraxerone-treated groups after ethanol exposure. In addition, taraxerone prevented catalase, superoxide dismutase, and reduced glutathione concentrations from the decrease induced by ethanol administration with the concentration dependent manner.

  2. New studies of the alcohol dehydrogenase cline in D. melanogaster from Mexico.

    PubMed

    Pipkin, S B; Franklin-Springer, E; Law, S; Lubega, S

    1976-01-01

    An altitudinal cline of frequencies of alcohol dehydrogenase alleles occurs in D. melanogaster populations of southeastern Mexico. A similar cline of two aldehyde oxidase alleles is present, but frequencies of esterase-6 alleles are not distributed clinically. Collections were made from small dispersed populations. Some gene flow occurred throughout the lowlands according to the distribution of two moderately endemic autosomal inversions and five previously described inversions. The clines are believed dependent on a limited gene flow between temperature races of D. melanogaster.

  3. Cupriavidus necator JMP134 rapidly reduces furfural with a Zn-dependent alcohol dehydrogenase.

    PubMed

    Li, Qunrui; Metthew Lam, L K; Xun, Luying

    2011-11-01

    Ethanol is a renewable biofuel, and it can be produced from lignocellulosic biomass. The biomass is usually converted to hydrolysates that consist of sugar and sugar derivatives, such as furfural. Yeast ferments sugar to ethanol, but furfural higher than 3 mM is inhibitory. It can take several days for yeast cells to reduce furfural to non-inhibitory furfuryl alcohol before producing ethanol. Bioreduction of furfural to furfuryl alcohol before fermentation may relieve yeast from furfural toxicity. We observed that Cupriavidus necator JMP134, a strict aerobe, rapidly reduced 17 mM furfural to less than 3 mM within 14 min with cell turbidity of 1.0 at 600 nm at 50°C. The rapid reduction consumed ethanol. The "furfural reductase" (FurX) was purified, and it oxidized ethanol to acetaldehyde and reduced furfural to furfuryl alcohol with NAD(+) as the cofactor. The protein was identified with mass spectrometry fingerprinting to be a hypothetical protein belonging to Zn-dependent alcohol dehydrogenase family. The furX-inactivation mutant of C. necator JMP134 lost the ability to rapidly reduce furfural, and Escherichia coli producing recombinant FurX gained the ability. Thus, an alcohol dehydrogenase enabled bacteria to rapidly reduce furfural with ethanol as the reducing power.

  4. The first step in polyethylene glycol degradation by sphingomonads proceeds via a flavoprotein alcohol dehydrogenase containing flavin adenine dinucleotide.

    PubMed

    Sugimoto, M; Tanabe, M; Hataya, M; Enokibara, S; Duine, J A; Kawai, F

    2001-11-01

    Several Sphingomonas spp. utilize polyethylene glycols (PEGs) as a sole carbon and energy source, oxidative PEG degradation being initiated by a dye-linked dehydrogenase (PEG-DH) that oxidizes the terminal alcohol groups of the polymer chain. Purification and characterization of PEG-DH from Sphingomonas terrae revealed that the enzyme is membrane bound. The gene encoding this enzyme (pegA) was cloned, sequenced, and expressed in Escherichia coli. The purified recombinant enzyme was vulnerable to aggregation and inactivation, but this could be prevented by addition of detergent. It is as a homodimeric protein with a subunit molecular mass of 58.8 kDa, each subunit containing 1 noncovalently bound flavin adenine dinucleotide but not Fe or Zn. PEG-DH recognizes a broad variety of primary aliphatic and aromatic alcohols as substrates. Comparison with known sequences revealed that PEG-DH belongs to the group of glucose-methanol-choline (GMC) flavoprotein oxidoreductases and that it is a novel type of flavoprotein alcohol dehydrogenase related (percent identical amino acids) to other, so far uncharacterized bacterial, membrane-bound, dye-linked dehydrogenases: alcohol dehydrogenase from Pseudomonas oleovorans (46%); choline dehydrogenase from E. coli (40%); L-sorbose dehydrogenase from Gluconobacter oxydans (38%); and 4-nitrobenzyl alcohol dehydrogenase from a Pseudomonas species (35%). PMID:11673442

  5. Geometric specificity of alcohol dehydrogenases and its potential for separation of trans and cis isomers of unsaturated aldehydes.

    PubMed Central

    Klibanov, A M; Giannousis, P P

    1982-01-01

    The geometric specificity of three different alcohol dehydrogenases (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) (from yeast, from horse liver, and from Leuconostoc mesenteroides) in the reduction of trans- and cis-cinnamaldehydes has been investigated. All three enzymes display a remarkable trans specificity: they react with the trans isomer 7 to 647 times faster than with its cis counterpart. Experiments with the enzymatic reduction of 3-phenylpropionaldehyde, a saturated analog of cinnamaldehyde, have revealed that whereas trans-cinnamaldehyde possesses the "right" configuration for the active centers of the alcohol dehydrogenases, the cis isomer apparently does not fit the active centers well. All three alcohol dehydrogenases studied also exhibit a marked trans specificity in the reaction with alpha-methylcinnamaldehyde. The geometric specificity of alcohol dehydrogenases can be used for the production of otherwise hard to synthesize cis isomers of unsaturated aldehydes from their readily available trans counterparts: trans-cinnamaldehyde was irradiated with ultraviolet light (which converted it to a mixture of trans and cis isomers) then treated with NADH and yeast alcohol dehydrogenase (which selectively reduces only trans aldehyde into the alcohol), and finally the mixture of cis-cinnamaldehyde and trans-cinnamyl alcohol was separated easily by preparative column chromatography. PMID:7048306

  6. Human liver alcohol dehydrogenase. 1. The primary structure of the beta 1 beta 1 isoenzyme.

    PubMed

    Hempel, J; Bühler, R; Kaiser, R; Holmquist, B; de Zalenski, C; von Wartburg, J P; Vallee, B; Jörnvall, H

    1984-12-17

    Determination of the amino acid sequence of the beta 1 subunit from the class I (pyrazole-sensitive) human liver alcohol dehydrogenase isoenzyme beta 1 beta 1 revealed a 373-residue structure differing at 48 positions (including a gap) from that of the subunit of the well studied horse liver alcohol dehydrogenase EE isoenzyme. The structure deduced is compatible with known differences in composition, ultraviolet absorbance, electrophoretic mobility and catalytic properties between the horse and human enzymes. All zinc-liganding residues of the horse E subunit are strictly conserved in the human beta 1 subunit, despite an earlier report of a mutation involving Cys-46. This residue therefore remains conserved in all known alcohol dehydrogenase structures. However, the total cysteine content of the beta 1 structure is raised from 14 in the subunit of the horse enzyme to 15 by a Tyr----Cys exchange. Most exchanges are on the surface of the molecule and of a well conserved nature. Substitutions close to the catalytic centre are of interest to explain the altered substrate specificity and different catalytic activity of the beta 1 homodimer. Functionally, a Ser----Thr exchange at position 48 appears to be of special importance, since Thr-48 in beta 1 instead of Ser-48 in the horse enzyme can restrict available space. Four other substitutions also line the active-site pocket, and appear to constitute partly compensated exchanges. PMID:6391920

  7. 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.

  8. Catalytic and Molecular Properties of the Quinohemoprotein Tetrahydrofurfuryl Alcohol Dehydrogenase from Ralstonia eutropha Strain Bo

    PubMed Central

    Zarnt, Grit; Schräder, Thomas; Andreesen, Jan R.

    2001-01-01

    The quinohemoprotein tetrahydrofurfuryl alcohol dehydrogenase (THFA-DH) from Ralstonia eutropha strain Bo was investigated for its catalytic properties. The apparent kcat/Km and Ki values for several substrates were determined using ferricyanide as an artificial electron acceptor. The highest catalytic efficiency was obtained with n-pentanol exhibiting a kcat/Km value of 788 × 104 M−1 s−1. The enzyme showed substrate inhibition kinetics for most of the alcohols and aldehydes investigated. A stereoselective oxidation of chiral alcohols with a varying enantiomeric preference was observed. Initial rate studies using ethanol and acetaldehyde as substrates revealed that a ping-pong mechanism can be assumed for in vitro catalysis of THFA-DH. The gene encoding THFA-DH from R. eutropha strain Bo (tfaA) has been cloned and sequenced. The derived amino acid sequence showed an identity of up to 67% to the sequence of various quinoprotein and quinohemoprotein dehydrogenases. A comparison of the deduced sequence with the N-terminal amino acid sequence previously determined by Edman degradation analysis suggested the presence of a signal sequence of 27 residues. The primary structure of TfaA indicated that the protein has a tertiary structure quite similar to those of other quinoprotein dehydrogenases. PMID:11222593

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

    PubMed

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

    2013-10-01

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

  10. 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

  11. Alcohol and polyol dehydrogenases are both divided into two protein types, and structural properties cross-relate the different enzyme activities within each type.

    PubMed Central

    Jörnvall, H; Persson, M; Jeffery, J

    1981-01-01

    Sorbitol dehydrogenase from sheep liver shows similarities to mammalian and yeast alcohol dehydrogenases. Comparisons based on peptides from segments of sorbitol dehydrogenase reveal that homologous regions with 38% identity include two ligands to the active site zinc atom in liver alcohol dehydrogenase, as well as further important residues. Similarities in in other regions are less extensive, exactly as they are between different alcohol dehydrogenases. In all aspects, sorbitol dehydrogenase appears as a typical member of the alcohol dehydrogenase family. On the other hand, alcohol dehydrogenase from Drosophila, which has a shorter subunit, is not closely related to either of these enzymes, except for a region that probably corresponds to the first part of the coenzyme binding domain in many dehydrogenases. Instead, Drosophila alcohol dehydrogenase in its supposed catalytic region shows similarities toward Klebsiella ribitol dehydrogenase, which also has a small subunit. It may be concluded that both alcohol and polyol dehydrogenases show two types of protein subunit, reflecting an early subdivision of polypeptide types into "long" and "short" subunits rather than into different enzymatic specificities or quaternary structures. The relationships explain known properties of all these enzymes and provide insight into functional mechanisms and evolutionary interpretations. PMID:7027257

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

    PubMed

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

    2000-12-20

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

  13. Substrate Specificity of the Purified Primary Alcohol Dehydrogenases from Methanol-Oxidizing Bacteria

    PubMed Central

    Sperl, George T.; Forrest, Hugh S.; Gibson, David T.

    1974-01-01

    Hyphomicrobium strain WC, Pseudomonas strain TP-1, and Pseudomonas strain W1 are capable of growth on methanol as the sole source of carbon and energy. Methanol-grown cells of each organism contain a primary alcohol dehydrogenase that has been purified to homogeneity. Each enzyme has a molecular weight of 120,000 and shows an in vitro requirement for phenazine methosulfate and ammonium ions for enzymatic activity. Normal aliphatic alcohols are oxidized rapidly by each enzyme. The presence of a methyl group on the carbon atom adjacent to the primary alcohol group lowers the enzymatic activity. This effect is reduced as the methyl substituent is moved further away from the hydroxyl group. The effect of other substituents on enzymatic activity is reported. Methanol, formaldehyde, and to a limited extent acetaldehyde are oxidized by the primary alcohol dehydrogenases. Higher aldehydes are not oxidized. A possible explanation for this specificity, with regard to aldehydes, is presented in terms of degree of hydration of the aldehyde. Images PMID:4828309

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

    PubMed

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

    1984-11-01

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

  15. 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.

  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. Ethanol-Induced Alcohol Dehydrogenase E (AdhE) Potentiates Pneumolysin in Streptococcus pneumoniae

    PubMed Central

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

    2014-01-01

    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. PMID:25312953

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

    PubMed

    Kittridge, K J; Willson, R L

    1984-05-01

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

  20. Structure of Liver Alcohol Dehydrogenase at 2.9-Å Resolution

    PubMed Central

    Brändén, Carl-Ivar; Eklund, Hans; Nordström, Bo; Boiwe, Torne; Söderlund, Gustaf; Zeppezauer, Eila; Ohlsson, Ingrid; Åkeson, Åke

    1973-01-01

    The conformation of the polypeptide chain in horse liver alcohol dehydrogenase (EC 1.1.1.1), as well as the binding sites for some inhibitor molecules, have been determined from x-ray crystallographic data to a resolution of 2.9 Å. Each subunit of the dimeric molecule is organized into two parts unequal in size and separated by a wide and deep active-site cleft. The adenosine moiety of the coenzyme is bound within the smaller region. Interactions between these coenzyme-binding substructures define the subunit contact area of the molecule. The “catalytic” zinc atoms are bound at the bottom of the clefts about 20 Å from the surface of the molecule. The coenzyme binding region has a main-chain conformation very similar to a corresponding region in lactate and malate dehydrogenase. It is suggested that this substructure is a general one for binding of nucleotides and, in particular, the coenzyme NAD+. PMID:4365379

  1. 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.

  2. Human liver alcohol dehydrogenase. 2. The primary structure of the gamma 1 protein chain.

    PubMed

    Bühler, R; Hempel, J; Kaiser, R; de Zalenski, C; von Wartburg, J P; Jörnvall, H

    1984-12-17

    The primary structure of the gamma 1 subunit of human liver alcohol dehydrogenase isoenzyme gamma 1 gamma 1 was deduced by characterization of 36 tryptic and 2 CNBr peptides. The polypeptide chain is composed of 373 amino acid residues. gamma 1 differs from the beta 1 subunit of human liver alcohol dehydrogenase at 21 positions, and from the E subunit of horse liver alcohol dehydrogenase at 43 positions including a gap at position 128 as in the beta 1 subunit. All zinc-liganding residues from the E subunit of the horse protein and the beta 1 subunit of the human enzyme are conserved, but like beta 1, gamma 1 also has an additional cysteine residue at position 286 (in the positional numbering system of the horse enzyme) due to a Tyr----Cys exchange. Most amino acid exchanges preserve the properties of the residues affected and are largely located on the surface of the molecules, away from the active site and the coenzyme binding region. However, eight positions with charge differences in relation to the E subunit of the horse enzyme are noticed. These result in a net positive charge increase of one in gamma 1 versus E, explaining the electrophoretic mobilities on starch gels. Of functional significance is the conservation of Ser-48 in gamma 1 relative to E. The residue is close to the active site but different (Thr-48) in the beta 1 subunit of the human enzyme. Thus, the closer structural relationship between human gamma 1 and horse E enzyme subunit than between beta 1 and E is also reflected in functionally important residues, explaining a greater similarity between gamma 1 gamma 1 and EE than between beta 1 beta 1 and EE. PMID:6391921

  3. A kinetic locking-on strategy for bioaffinity purification: further studies with alcohol dehydrogenase.

    PubMed

    O'flaherty, M; McMahon, M; Mulcahy, P

    1999-02-01

    The kinetic locking-on strategy improves the selectivity of protein purification procedures based on immobilized cofactor derivatives through use of enzyme-specific substrate analogues in irrigants to promote biospecific adsorption. This paper describes the development and application of this strategy to the one-chromatographic step affinity purification of NAD(P)+-dependent alcohol dehydrogenases using 8'-azo-linked immobilized NAD(P)+, S6-linked and N6-linked immobilized NAD+, and N6-linked immobilized NADP+ derivatives. These studies were carried out using alcohol dehydrogenases from Saccharomyces cerevisiae (YADH, EC 1.1.1.1), equine liver (HLADH, EC 1.1.1.1), and Thermoanaerobium brockii (TBADH, EC 1.1.1.2). The results reveal that the factors which require careful consideration before development of a truly biospecific system based on the locking-on strategy include: (i) the stability of the immobilized cofactor derivative; (ii) the spacer-arm composition of the affinity derivative; (iii) the accessible immobilized cofactor concentration; (iv) the soluble locking-on ligand concentration; (v) the dissociation constant of locking-on ligand, and (vi) the identification and elimination of nonbiospecific interference. The S6-linked immobilized NAD+ derivative (synthesized with a hydrophilic spacer arm) proved to be the most suitable of the affinity adsorbents investigated in the present study for use with the locking-on strategy. This conclusion was based primarily on the observations that this affinity adsorbent was stable, retained cofactor activity with the "test" enzymes under study, and was not prone to nonbiospecific interactions. Using this immobilized derivative in conjunction with the locking-on strategy, alcohol dehydrogenase from Saccharomyces cerevisiae was purified to electrophoretic homogeneity in a single affinity chromatographic step.

  4. The protective effects of osmolytes on yeast alcohol dehydrogenase conformational stability and aggregation.

    PubMed

    Han, Hong-Yan; Yao, Zhi-Gang; Gong, Cheng-Liang; Xu, Wei-An

    2010-08-01

    The protective effects of four osmolytes (trehalose, dimethysulfoxide, glycine and proline) on the conformational stability and aggregation of guanidine-denatured yeast alcohol dehydrogenase (YADH) have been investigated in this paper. The results show that the four osmolytes protect YADH against unfolding and inactivation by reducing ki (inactivation rate constants), increasing DeltaDeltaGi (transition free energy changes at 25 degrees C), increasing Cm (value for the midpoint of denaturation) and decreasing its ANS-binding fluorescence intensity. Furthermore, these osmolytes can prevent YADH aggregation in a concentration-dependent manner during YADH refolding.

  5. Fusion of pyruvate decarboxylase and alcohol dehydrogenase increases ethanol production in Escherichia coli.

    PubMed

    Lewicka, Aleksandra J; Lyczakowski, Jan J; Blackhurst, Gavin; Pashkuleva, Christiana; Rothschild-Mancinelli, Kyle; Tautvaišas, Dainius; Thornton, Harry; Villanueva, Hugo; Xiao, Weike; Slikas, Justinas; Horsfall, Louise; Elfick, Alistair; French, Christopher

    2014-12-19

    Ethanol is an important biofuel. Heterologous expression of Zymomonas mobilis pyruvate decarboxylase (Pdc) and alcohol dehydrogenase (AdhB) increases ethanol production in Escherichia coli. A fusion of PDC and ADH was generated and expressed in E. coli. The fusion enzyme was demonstrated to possess both activities. AdhB activity was significantly lower when fused to PDC than when the two enzymes were expressed separately. However, cells expressing the fusion protein generated ethanol more rapidly and to higher levels than cells coexpressing Pdc and AdhB, suggesting a specific rate enhancement due to the fusion of the two enzymes.

  6. Effects of Macromolecular Crowding on Alcohol Dehydrogenase Activity Are Substrate-Dependent.

    PubMed

    Wilcox, A E; LoConte, Micaela A; Slade, Kristin M

    2016-06-28

    Enzymes operate in a densely packed cellular environment that rarely matches the dilute conditions under which they are studied. To better understand the ramifications of this crowding, the Michaelis-Menten kinetics of yeast alcohol dehydrogenase (YADH) were monitored spectrophotometrically in the presence of high concentrations of dextran. Crowding decreased the maximal rate of the reaction by 40% for assays with ethanol, the primary substrate of YADH. This observation was attributed to slowed release of the reduced β-nicotinamide adenine dinucleotide product, which is rate-limiting. In contrast, when larger alcohols were used as the YADH substrate, the rate-limiting step becomes hydride transfer and crowding instead increased the maximal rate of the reaction by 20-40%. This work reveals the importance of considering enzyme mechanism when evaluating the ways in which crowding can alter kinetics.

  7. Influence of Tris(hydroxymethyl)aminomethane on kinetic mechanism of yeast alcohol dehydrogenase.

    PubMed

    Trivić, S; Leskovac, V; Zeremski, J; Stancić, B; Anderson, B M

    1998-02-01

    Acetaldehyde, propionaldehyde, glyceraldehyde-3-P and 4-dimethylaminocinnamaldehyde form Schiff bases in Tris. HCl buffers; the rates of formation and dissociation of Schiff bases, and equilibrium constants for their formation are very similar for the first three aldehydes. The steady-state kinetic constants for the yeast alcohol dehydrogenase-catalyzed reaction, propan-1-ol + NAD+ reversible propionaldehyde + NADH + H+, have been determined in several Tris. HCl buffers of increasing concentration at pH 8.1. In the forward direction, oxidation of alcohol, most kinetic constants are increased by increasing concentrations of Tris. In the reverse direction, reduction of aldehyde, substrate, NADH, Tris and Schiff base were equilibrated before enzyme reaction was started. It was found that Schiff base, rather than Tris, binds to free enzyme competitively with respect to NADH. Tris and Schiff base do not influence the binding of aldehyde to enzyme in any way. PMID:9879514

  8. Investigation of asymmetric alcohol dehydrogenase (ADH) reduction of acetophenone derivatives: effect of charge density.

    PubMed

    Naik, Hemantkumar G; Yeniad, Bahar; Koning, Cor E; Heise, Andreas

    2012-07-01

    In an effort to study the effect of substituent groups of the substrate on the alcohol dehydrogenase (ADH) reductions of aryl-alkyl ketones, several derivatives of acetophenone have been evaluated against ADHs from Lactobacillus brevis (LB) and Thermoanaerobacter sp. (T). Interestingly, ketones with non-demanding (neutral) para-substituents were reduced to secondary alcohols by these enzymes in enantiomerically pure form whereas those with demanding (ionizable) substituents could not be reduced. The effect of substrate size, their solubility in the reaction medium, electron donating and withdrawing properties of the ligand and also the electronic charge density distribution on the substrate molecules have been studied and discussed in detail. From the results, it is observed that the electronic charge distribution in the substrate molecules is influencing the orientation of the substrate in the active site of the enzyme and hence the ability to reduce the substrate.

  9. 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

  10. 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.

  11. NAD(P)-Dependent Aldehyde Dehydrogenases Induced during Growth of Ralstonia eutropha Strain Bo on Tetrahydrofurfuryl Alcohol

    PubMed Central

    Schräder, Thomas; Zarnt, Grit; Andreesen, Jan R.

    2001-01-01

    Different aldehyde dehydrogenases (AlDHs) were formed during growth of Ralstonia eutropha Bo on tetrahydrofurfuryl alcohol (THFA). One of these enzymes, AlDH 4, was purified and characterized as a homodimer containing no prosthetic groups, showing a strong substrate inhibition, and having an N-terminal sequence similar to those of various NAD(P)-dependent AlDHs. The conversion rate of THFA by the quinohemoprotein THFA dehydrogenase was increased by AlDH 4. PMID:11717302

  12. Biochemical characterization of ethanol-dependent reduction of furfural by alcohol dehydrogenases.

    PubMed

    Li, Qunrui; Metthew Lam, L K; Xun, Luying

    2011-11-01

    Lignocellulosic biomass is usually converted to hydrolysates, which consist of sugars and sugar derivatives, such as furfural. Before yeast ferments sugars to ethanol, it reduces toxic furfural to non-inhibitory furfuryl alcohol in a prolonged lag phase. Bioreduction of furfural may shorten the lag phase. Cupriavidus necator JMP134 rapidly reduces furfural with a Zn-dependent alcohol dehydrogenase (FurX) at the expense of ethanol (Li et al. 2011). The mechanism of the ethanol-dependent reduction of furfural by FurX and three homologous alcohol dehydrogenases was investigated. The reduction consisted of two individual reactions: ethanol-dependent reduction of NAD(+) to NADH and then NADH-dependent reduction of furfural to furfuryl alcohol. The kinetic parameters of the coupled reaction and the individual reactions were determined for the four enzymes. The data indicated that limited NADH was released in the coupled reaction. The enzymes had high affinities for NADH (e.g., K ( d ) of 0.043 μM for the FurX-NADH complex) and relatively low affinities for NAD(+) (e.g., K ( d ) of 87 μM for FurX-NAD(+)). The kinetic data suggest that the four enzymes are efficient "furfural reductases" with either ethanol or NADH as the reducing power. The standard free energy change (ΔG°') for ethanol-dependent reduction of furfural was determined to be -1.1 kJ mol(-1). The physiological benefit for ethanol-dependent reduction of furfural is likely to replace toxic and recalcitrant furfural with less toxic and more biodegradable acetaldehyde.

  13. Genic Heterogeneity at Two Alcohol Dehydrogenase Loci in DROSOPHILA PSEUDOOBSCURA and DROSOPHILA PERSIMILIS

    PubMed Central

    Coyne, Jerry A.; Felton, Alexander A.

    1977-01-01

    A sequential electrophoretic survey of the second chromosome loci, alcohol dehydrogenase-6 (Adh-6) and octanol dehydrogenase ( Odh), was performed on 147 isochromosomal lines of Drosophila pseudoobscura and 60 lines of its sibling species, D. persimilis. Gels run with a variety of acrylamide concentrations and buffer pH's revealed the presence of 18 alleles of Adh-6 in the two species, where only eight had been previously detected by conventional electrophoretic methods. Only two alleles were added with our techniques to the previous total of nine in both species at the largely monomorphic Odh locus. Both enzymes show a predominance of one allele, with the other variants being fairly rare. There was no evidence of increased genetic divergence between the two species, but we found a striking increase in differentiation of Adh-6 alleles between the main body of D. pseudoobscura populations and the conspecific isolate from Bogotá, Colombia. These results are compared with our previous surveys of xanthine dehydrogenase in these species and discussed in reference to theories of genic polymorphism. PMID:17248763

  14. Importance of the structural zinc atom for the stability of yeast alcohol dehydrogenase.

    PubMed Central

    Magonet, E; Hayen, P; Delforge, D; Delaive, E; Remacle, J

    1992-01-01

    Yeast alcohol dehydrogenase is a tetrameric enzyme containing zinc. Initially we confirmed the presence of two zinc atoms per subunit. Incubation of the enzyme with increasing concentrations of dithiothreitol, a method for partial chelation, allowed first the reduction of four disulphide bridges per enzyme, but eventually was sufficient to chelate the structural zinc atom without having any effect on the zinc located in the active site. The enzyme activity was not affected but the enzyme became very sensitive to heat denaturation. Chelation by EDTA was also performed. Given its location at an external position in the globular protein, protected in each subunit by one disulphide bridge, the results establish that the second zinc atom present on each enzymic subunit plays a prominent conformational role, probably by stabilizing the tertiary structure of yeast alcohol dehydrogenase. Recovery experiments were performed by incubation of the native enzyme, or the dithiothreitol-treated enzyme, with a small amount of Zn2+. A stabilization effect was found when the structural zinc was re-incorporated after its removal by dithiothreitol. In all cases a large increase in activity was also observed, which was much greater than that expected based on the amount of re-incorporated zinc atom, suggesting the re-activation of some inactive commercial enzyme which had lost some of its original catalytic zinc atoms. PMID:1445195

  15. The conserved Glu-60 residue in Thermoanaerobacter brockii alcohol dehydrogenase is not essential for catalysis

    PubMed Central

    Kleifeld, Oded; Shi, Shu Ping; Zarivach, Raz; Eisenstein, Miriam; Sagi, Irit

    2003-01-01

    Glu-60 of the zinc-dependent Thermoanaerobacter brockii alcohol dehydrogenase (TbADH) is a strictly conserved residue in all members of the alcohol dehydrogenase (ADH) family. Unlike most other ADHs, the crystal structures of TbADH and its analogs, ADH from Clostridium beijerinckii (CbADH), exhibit a unique zinc coordination environment in which this conserved residue is directly coordinated to the catalytic zinc ion in the native form of the enzymes. To explore the role of Glu-60 in TbADH catalysis, we have replaced it by alanine (E60A-TbADH) and aspartate (E60D-TbADH). Steady-state kinetic measurements show that the catalytic efficiency of these mutants is only four- and eightfold, respectively, lower than that of wild-type TbADH. We applied X-ray absorption fine-structure (EXAFS) and near-UV circular dichroism to characterize the local environment around the catalytic zinc ion in the variant enzymes in their native, cofactor-bound, and inhibited forms. We show that the catalytic zinc site in the studied complexes of the variant enzymes exhibits minor changes relative to the analogous complexes of wild-type TbADH. These moderate changes in the kinetic parameters and in the zinc ion environment imply that the Glu-60 in TbADH does not remain bound to the catalytic zinc ion during catalysis. Furthermore, our results suggest that a water molecule replaces this residue during substrate turnover. PMID:12592017

  16. Molecular analysis of mutant and wild type alcohol dehydrogenase alleles from Drosophila

    SciTech Connect

    Batzer, M.A.

    1988-01-01

    Wild type alcohol dehydrogenase polypeptides (ADH) from Drosophila melanogaster transformants were examined using western blots and polyclonal antiserum specific for Drosophila melanogaster ADH. Mutants induced in Drosophila spermatozoa at the alcohol dehydrogenase (Adh) locus using X-rays, 1-ethyl-1-nitrosourea (ENU) or ethyl methanesulfonate (EMS) were characterized using genetic complementation tests, western blots, Southern blots, northern blots and enzymatic amplification of the Adh locus. Genetic complementation tests showed that 22/30 X-ray-induced mutants, and 3/13 ENU and EMS induced mutants were multi-locus deficiencies. Western blot analysis of the intragenic mutations showed that 4/7 X-ray-induced mutants produced detectable polypeptides, one of which was normal in molecular weight and charge. In contrast 8/10 intragenic ENU and EMS induced mutants produced normal polypeptides. Southern blot analysis showed that 5/7 intragenic X-ray induced mutants and all 10 of the intragenic ENU and EMS induced mutants were normal with respect to the alleles they were derived from.

  17. 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.

  18. 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.

  19. 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.

  20. Switchgrass PviCAD1: Understanding residues important for substrate preferences and activity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lignin is a major component of plant cell walls and is a complex aromatic heteropolymer. Reducing lignin content improves conversion efficiency into liquid fuels, and enzymes involved in lignin biosynthesis are attractive targets for bioengineering. Cinnamyl alcohol dehydrogenase (CAD) catalyzes t...

  1. Adaptation of methods for glutamate dehydrogenase and alcohol dehydrogenase activities to a centrifugal analyser: assessment of their clinical use in anoxic states of the liver.

    PubMed Central

    Shephard, M D; Penberthy, L A; Berry, M N

    1987-01-01

    Sensitive, precise, and rapid methods for the measurement of alcohol dehydrogenase (ADH) and glutamate dehydrogenase (GDH) were developed on the Cobas Bio centrifugal analyser. The optimal pH for ADH in caucasians was 9.8. Non-linearity of ADH enzyme activity was observed when samples were diluted in saline; linearity was restored when inactivated serum was used as diluent. ADH was shown to be a sensitive index of liver anoxia due to cardiorespiratory disturbance (clinical sensitivity 90%) and generalised anoxia. GDH exhibited sensitivity equal to that of alanine aminotransferase (ALT) but was inferior to gamma-glutamyltransferase (GGT) in the detection of specific liver disease. Both ADH and GDH were sensitive indicators of alcoholic liver disease. PMID:2890662

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

    PubMed

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

    2013-07-01

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

  3. 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.

  4. The binding of an aminoazo dye carcinogen to a specific methionine residue in rat liver alcohol dehydrogenase in vivo.

    PubMed

    Coles, B; Beale, D; Miller, D; Lay, J; Kadlubar, F; Aitken, A; Ketterer, B

    1987-01-01

    On the administration of 3'-methyl-N,N-dimethyl-4-aminoazobenzene to rats pure aminoazo dye-bound alcohol dehydrogenase accounting for 45% of the total soluble protein bound aminoazo dye is isolated from the liver soluble supernatant. Tryptic digestion of that purified aminoazo dye-bound enzyme yields an aminoazo dye-bound nonapeptide which has a sequence identical to amino acids 301-309 in the known sequence of alcohol dehydrogenase (H. Jornvall and O. Markovic, Eur. J. Biochem., 29 (1972) 167-174) with the exception of methionine 306 which is replaced by an aminoazo dye modified amino acid. The nature of the aminoazo dye adduct was determined by studying the structure of the related tetrapeptide obtained by Pronase B digestion and shown by proton NMR spectroscopy and fast atom bombardment mass spectroscopy to have the structure 3-(Val. Asn. Pro. Homocystein-S-yl)-4-methylamino-3'-methylazobenzene. This carcinogen-protein adduct is assumed to arise from attack of the ultimate carcinogenic metabolite, N-sulphonyloxy-4-methylamino-3'-methylazobenzene (FF. Kadlubar, J.A. Miller and E.C. Miller, Cancer Res., 36 (1976) 2350-2359) at the sulphur of methionine 306 followed by spontaneous S-demethylation. This highly specific reaction of carcinogen with alcohol dehydrogenase lowers its Vmax and increases its Km with cyclohexanone thereby reducing its catalytic efficiency for this substrate. This highly specific reaction of the carcinogen with alcohol dehydrogenase may be regarded as a major detoxication reaction.

  5. Comparison of inactivation and conformational changes of native and apo yeast alcohol dehydrogenase during thermal denaturation.

    PubMed

    Yang, Y; Chen, R; Zhou, H M

    1998-07-01

    The conformational changes of native and apo yeast alcohol dehydrogenase during thermal denaturation have been followed by fluorescence emission and circular dichroism spectra. A comparison of inactivation and conformational changes during thermal denaturation shows that for the native enzyme and for the apo-I YADH which has the conformational zinc removed, the extent of inactivation was larger than the extent of conformational changes at the same temperature. This result supported the suggestion by Tsou (Trends Biochem. Sci. 1986, 11, 427-429: Science 1993, 262, 380-381) that the enzyme active site is more flexible. The results also show that apo-I YADH without the conformational zinc was more easily inactivated with increasing incubation temperature, indicating that the stability of the apo-I YADH decreased. Kinetic analysis suggest that the substrate does not provide any protective effect during thermal inactivation of native and apo-I YADH.

  6. Mechanism of thermal aggregation of yeast alcohol dehydrogenase I: role of intramolecular chaperone.

    PubMed

    Markossian, Kira A; Golub, Nikolay V; Khanova, Helen A; Levitsky, Dmitrii I; Poliansky, Nikolay B; Muranov, Konstantin O; Kurganov, Boris I

    2008-09-01

    Kinetics of thermal aggregation of yeast alcohol dehydrogenase I (yADH) have been studied using dynamic light scattering at a fixed temperature (56 degrees C) and under the conditions where the temperature was elevated at a constant rate (1 K/min). The initial parts of the dependences of the hydrodynamic radius on time (or temperature) follow the exponential law. At rather high values of time splitting of the population of aggregates into two components occurs. It is assumed that such peculiarities of the kinetics of thermal aggregation of yADH are due to the presence of a sequence -YSGVCHTDLHAWHGDWPLPVK- in the polypeptide chain possessing chaperone-like activity. Thermodynamic parameters for thermal denaturation of yADH have been calculated from the differential scanning calorimetry data.

  7. Activity and stability of yeast alcohol dehydrogenase (YADH) entrapped in aerosol OT reverse micelles.

    PubMed

    Sarcar, S; Jain, T K; Maitra, A

    1992-02-20

    The activity and stability of yeast alcohol dehydrogenase (YADH) entrapped in aerosol OT reverse micellar droplets have been investigated spectrophotometrically. Various physical parameters, e.g., water pool size, w(0), pH, and temperature, were optimized for YADH in water/AOT/isooctane reverse micelles. It was found that the enzyme exhibits maximum activity at w(0) = 28 and pH 8.1. It was more active in reverse micelles than in aqueous buffers at a particular temperature and was denatured at about 307 degrees C in both the systems. At a particular temperature YADH entrapped in reverse micelles was less stable than when it was dissolved in aqueous buffer.

  8. Kinetics of irreversible inhibition of yeast alcohol dehydrogenase during modification by o-phthaldehyde.

    PubMed

    Le, W P; Yan, S X; Huang, M Q; Zhang, Y X; Zhou, H M

    The kinetic theory of the substrate reaction during irreversible inhibition of enzyme activity described previously has been applied to a study on the kinetics of the course of inactivation of yeast alcohol dehydrogenase (YADH) by o-phthaldehyde (OPTA). The microscopic constants for the reaction of the inactivators with the free enzyme and with the enzyme-substrate complexes were determined. The inactivation is a monophasic pseudo-first-order reaction with OPTA. The apparent rate constant A is independent of the OPTA concentration, indicating that the inactivation is a noncomplexing inhibition. The marked protective effect of substrates on the inactivation of YADH by OPTA has been observed. This result suggests that the modification of the enzyme by OPTA may occur at the active site.

  9. Kinetics of irreversible inhibition of yeast alcohol dehydrogenase during modification by 4,4'-dithiodipyridine.

    PubMed

    Zheng, S Y; Xu, D; Wang, H R; Li, J; Zhou, H M

    1997-07-01

    The course of inactivation of yeast alcohol dehydrogenase (YADH) using 4,4'-dithiodipyridine (DSDP) has been studied in this paper. The results show that the reaction mechanism between DSDP and YADH is a competitive, complexing inhibition. The microscopic constants for the inactivation of the free enzyme and the enzyme-substrate complex were determined. The presence of the substrate NAD+ offers strong protection for this enzyme against inactivation by DSDP. The above results suggest that two Cys residues are essential for activity and are situated at the active site. These essential Cys residues should be Cys-46 and Cys-174 which are ligands to the catalytic zinc ion. Another Cys residue, which can be modified by DSDP, is non-essential for activity of the enzyme.

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

    PubMed

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

    2011-08-16

    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.

  11. 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.

  12. 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.

  13. Zeta-crystallin, a novel protein from the guinea pig lens is related to alcohol dehydrogenases.

    PubMed

    Rodokanaki, A; Holmes, R K; Borrás, T

    1989-05-30

    zeta-Crystallin is a major component of the water-soluble proteins of the guinea pig lens. We have constructed a lens cDNA library from one- to seven-day-old guinea pigs in the plasmid Bluescript KS+ and used the 16 amino acid (aa) sequence of a CNBr peptide to design an oligodeoxyribonucleotide probe. Analysis of two positive clones and direct sequence of the 5' end of the RNA resulted in the completion of a most probably full-length mRNA comprising 1842 nucleotides (nt). The ATG start codon occurs 83 nt downstream from the 5' end. The open reading frame, ending with a stop codon at nt position 1070, predicts a protein of 328 aa with a calculated Mr of 35,071. Comparison of the amino acid sequence with the National Biomedical Research Foundation protein data base reveals a significant similarity of zeta-crystallin with the enzyme of the alcohol dehydrogenase family.

  14. The partial purification and characterization of cytosol alcohol dehydrogenase from Astasia

    PubMed Central

    Morosoli, Rolf; Bégin-Heick, Nicole

    1974-01-01

    1. The cytosol alcohol dehydrogenase (alcohol–NAD oxidoreductase, EC 1.1.1.1) of Astasia longa was partially purified and characterized from cells grown in the presence of air+CO2 (95:5) or of O2+CO2 (95:5). 2. Under both these growth conditions, the cells contained a fraction, ADHII, which was characterized by its electrophoretic properties, by a high degree of resistance to heat inactivation, by a sharp pH optimum at 8.2 and by its kinetic properties. The estimated molecular weight of this fraction was approx. 150000, which is similar to that of yeast alcohol dehydrogenase. 3. Cells grown in air+CO2 (95:5) contain another fraction, ADHI, which can be further separated into two subfractions by polyacrylamide-gel electrophoresis and by DEAE-cellulose chromatography. This was termed fraction `ADHI-air'. 4. In addition to fraction ADHII, cells grown in the presence of O2 have a twofold increase in fraction ADHI-air activity as well as two new fractions that could not be demonstrated in air-grown cells. These new fractions which we have called fraction `ADHI-O2', account for about 10% of the total activity. 5. The ADHI fractions (air) and (O2) have similar broad pH–activity curves and similar kinetic properties, both having a lower Km for ethanol and NAD than fraction ADHII. However, they differ from each other with respect to their activity with various substrates. The estimated molecular weight of these two ADHI fractions and their chromatographic behaviour on hydroxyapatite and on DEAE-cellulose also distinguish them. ImagesPLATE 1Fig. 1. PMID:4455216

  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. 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.

  17. Optical isopropanol biosensor using NADH-dependent secondary alcohol dehydrogenase (S-ADH).

    PubMed

    Chien, Po-Jen; Ye, Ming; Suzuki, Takuma; Toma, Koji; Arakawa, Takahiro; Iwasaki, Yasuhiko; Mitsubayashi, Kohji

    2016-10-01

    Isopropanol (IPA) is an important solvent used in industrial activity often found in hospitals as antiseptic alcohol rub. Also, IPA may have the potential to be a biomarker of diabetic ketoacidosis. In this study, an optical biosensor using NADH-dependent secondary alcohol dehydrogenase (S-ADH) for IPA measurement was constructed and evaluated. An ultraviolet light emitting diode (UV-LED, λ=340nm) was employed as the excitation light to excite nicotinamide adenine dinucleotide (NADH). A photomultiplier tube (PMT) was connected to a two-way branch optical fiber for measuring the fluorescence emitted from the NADH. S-ADH was immobilized on the membrane to catalyze IPA to acetone and reduce NAD(+) to be NADH. This IPA biosensor shows highly sensitivity and selectivity, the calibration range is from 500 nmol L(-1) to 1mmolL(-1). The optimization of buffer pH, temperature, and the enzyme-immobilized method were also evaluated. The detection of IPA in nail related cosmetic using our IPA biosensor was also carried out. The results showed that large amounts of IPA were used in these kinds of cosmetics. This IPA biosensor comes with the advantages of rapid reaction, good reproducibility, and wide dynamic range, and is also expected to use for clinical IPA detections in serum or other medical and health related applications.

  18. Optical isopropanol biosensor using NADH-dependent secondary alcohol dehydrogenase (S-ADH).

    PubMed

    Chien, Po-Jen; Ye, Ming; Suzuki, Takuma; Toma, Koji; Arakawa, Takahiro; Iwasaki, Yasuhiko; Mitsubayashi, Kohji

    2016-10-01

    Isopropanol (IPA) is an important solvent used in industrial activity often found in hospitals as antiseptic alcohol rub. Also, IPA may have the potential to be a biomarker of diabetic ketoacidosis. In this study, an optical biosensor using NADH-dependent secondary alcohol dehydrogenase (S-ADH) for IPA measurement was constructed and evaluated. An ultraviolet light emitting diode (UV-LED, λ=340nm) was employed as the excitation light to excite nicotinamide adenine dinucleotide (NADH). A photomultiplier tube (PMT) was connected to a two-way branch optical fiber for measuring the fluorescence emitted from the NADH. S-ADH was immobilized on the membrane to catalyze IPA to acetone and reduce NAD(+) to be NADH. This IPA biosensor shows highly sensitivity and selectivity, the calibration range is from 500 nmol L(-1) to 1mmolL(-1). The optimization of buffer pH, temperature, and the enzyme-immobilized method were also evaluated. The detection of IPA in nail related cosmetic using our IPA biosensor was also carried out. The results showed that large amounts of IPA were used in these kinds of cosmetics. This IPA biosensor comes with the advantages of rapid reaction, good reproducibility, and wide dynamic range, and is also expected to use for clinical IPA detections in serum or other medical and health related applications. PMID:27474326

  19. Human class II (pi) alcohol dehydrogenase has a redox-specific function in norepinephrine metabolism.

    PubMed Central

    Mårdh, G; Dingley, A L; Auld, D S; Vallee, B L

    1986-01-01

    Studies of the function of human alcohol dehydrogenase (ADH) have revealed substrates that are virtually unique for class II ADH (pi ADH). It catalyzes the formation of the intermediary glycols of norepinephrine metabolism, 3,4-dihydroxyphenylglycol and 4-hydroxy-3-methoxyphenylglycol, from the corresponding aldehydes 3,4-dihydroxymandelaldehyde and 4-hydroxy-3-methoxymandelaldehyde with Km values of 55 and 120 microM and kcat/Km ratios of 14,000 and 17,000 mM-1 X min-1; these are from 60- to 210-fold higher than those obtained with class I ADH isozymes. The catalytic preference of class II ADH also extends to benzaldehydes. The kcat/Km values for the reduction of benzaldehyde, 3,4-dihydroxybenzaldehyde and 4-hydroxy-3-methoxybenzaldehyde by pi ADH are from 9- to 29-fold higher than those for a class I isozyme, beta 1 gamma 2 ADH. Furthermore, the norepinephrine aldehydes are potent inhibitors of alcohol (ethanol) oxidation by pi ADH. The high catalytic activity of pi ADH-catalyzed reduction of the aldehydes in combination with a possible regulatory function of the aldehydes in the oxidative direction leads to essentially "unidirectional" catalysis by pi ADH. These features and the presence of pi ADH in human liver imply a physiological role for pi ADH in the degradation of circulating epinephrine and norepinephrine. PMID:3466164

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

    PubMed

    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.

  1. 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.

  2. Association between common alcohol dehydrogenase gene (ADH) variants and schizophrenia and autism

    PubMed Central

    Wang, Kesheng; Zhang, Xiang-Yang; Pan, Xinghua; Wang, Guilin; Tan, Yunlong; Zhong, Chunlong; Krystal, John H.; State, Matthew; Zhang, Heping

    2013-01-01

    Humans express at least seven alcohol dehydrogenase (ADH) isoforms that are encoded by ADH gene cluster (ADH7–ADH1C–ADH1B–ADH1A–ADH6–ADH4–ADH5) at chromosome 4. ADHs are key catabolic enzymes for retinol and ethanol. The functional ADH variants (mostly rare) have been implicated in alcoholism risk. In addition to catalyzing the oxidation of retinol and ethanol, ADHs may be involved in the metabolic pathways of several neurotransmitters that are implicated in the neurobiology of neuropsychiatric disorders. In the present study, we comprehensively examined the associations between common ADH variants [minor allele frequency (MAF) >0.05] and 11 neuropsychiatric and neurological disorders. A total of 50,063 subjects in 25 independent cohorts were analyzed. The entire ADH gene cluster was imputed across these 25 cohorts using the same reference panels. Association analyses were conducted, adjusting for multiple comparisons. We found 28 and 15 single nucleotide polymorphisms (SNPs), respectively, that were significantly associated with schizophrenia in African-Americans and autism in European-Americans after correction by false discovery rate (FDR) (q <0.05); and 19 and 6 SNPs, respectively, that were significantly associated with these two disorders after region-wide correction by SNPSpD (8.9 × 10−5 ≤ p ≤ 0.0003 and 2.4 × 10−5 ≤ p ≤ 0.0003, respectively). No variants were significantly associated with the other nine neuropsychiatric disorders, including alcohol dependence. We concluded that common ADH variants conferred risk for both schizophrenia in African-Americans and autism in European-Americans. PMID:23468174

  3. Association between common alcohol dehydrogenase gene (ADH) variants and schizophrenia and autism.

    PubMed

    Zuo, Lingjun; Wang, Kesheng; Zhang, Xiang-Yang; Pan, Xinghua; Wang, Guilin; Tan, Yunlong; Zhong, Chunlong; Krystal, John H; State, Matthew; Zhang, Heping; Luo, Xingguang

    2013-07-01

    Humans express at least seven alcohol dehydrogenase (ADH) isoforms that are encoded by ADH gene cluster (ADH7-ADH1C-ADH1B-ADH1A-ADH6-ADH4-ADH5) at chromosome 4. ADHs are key catabolic enzymes for retinol and ethanol. The functional ADH variants (mostly rare) have been implicated in alcoholism risk. In addition to catalyzing the oxidation of retinol and ethanol, ADHs may be involved in the metabolic pathways of several neurotransmitters that are implicated in the neurobiology of neuropsychiatric disorders. In the present study, we comprehensively examined the associations between common ADH variants [minor allele frequency (MAF) >0.05] and 11 neuropsychiatric and neurological disorders. A total of 50,063 subjects in 25 independent cohorts were analyzed. The entire ADH gene cluster was imputed across these 25 cohorts using the same reference panels. Association analyses were conducted, adjusting for multiple comparisons. We found 28 and 15 single nucleotide polymorphisms (SNPs), respectively, that were significantly associated with schizophrenia in African-Americans and autism in European-Americans after correction by false discovery rate (FDR) (q < 0.05); and 19 and 6 SNPs, respectively, that were significantly associated with these two disorders after region-wide correction by SNPSpD (8.9 × 10(-5) ≤ p ≤ 0.0003 and 2.4 × 10(-5) ≤ p ≤ 0.0003, respectively). No variants were significantly associated with the other nine neuropsychiatric disorders, including alcohol dependence. We concluded that common ADH variants conferred risk for both schizophrenia in African-Americans and autism in European-Americans.

  4. Effect of pressure on a heavy-atom isotope effect of yeast alcohol dehydrogenase.

    PubMed

    Park, Hyun; Girdaukas, Gary G; Northrop, Dexter B

    2006-02-15

    Hydrostatic pressure causes a monophasic decrease in the (13)C primary isotope effect expressed on the oxidation of benzyl alcohol by yeast alcohol dehydrogenase. The primary isotope effect was measured by the competitive method, using whole-molecule mass spectrometry. The effect is, therefore, an expression of isotopic discrimination on the kinetic parameter V/K, which measures substrate capture. Moderate pressure increases capture by activating hydride transfer, the transition state of which must therefore have a smaller volume than the free alcohol plus the capturing form of enzyme [Cho, Y.-K.; Northrop, D. B. Biochemistry 1999, 38, 7470-7475]. The decrease in the (13)C isotope effect with increasing pressure means that the transition state for hydride transfer from the heavy atom must have an even smaller volume, measured here to be 13 mL.mol(-1). The pressure data factor the kinetic isotope effect into a semiclassical reactant-state component, with a null value of k(12)/k(13) = 1, and a transition-state component of Q(12)/Q(13) = 1.028 (borrowing Bell's nomenclature for hydrogen tunneling corrections). A similar experiment involving a deuterium isotope effect previously returned the same volume and null value, plus a pressure-sensitive isotope effect [Northrop, D. B.; Cho, Y.-K. Biochemistry 2000, 39, 2406-2412]. Consistent with precedence in the chemical literature, the latter suggested a possibility of hydrogen tunneling; however, it is unlikely that carbon can engage in significant tunneling at ambient temperature. The fact that the decrease in activation volumes for hydride transfer is equivalent when one mass unit is added to the carbon end of a scissile C-H bond and when one mass unit is added to the hydrogen end is significant and suggests a common origin.

  5. Microbial production of methylketones: properties of purified yeast secondary alcohol dehydrogenase

    SciTech Connect

    Patel, R.N.; Hou, C.T.; Laskin, A.I.; Derelanko, P.

    1981-06-01

    Secondary alcohol dehydrogenase (SADH) was purified from extracts of a methanol-grown yeast, Pichia sp. The purified enzyme was homogeneous as judged by ultracentrifugation and by polyacrylamide gel electrophoresis. The purified SADH has a molecular weight of 98,000 as determined by gel filtration and 102,000 as determined by sedimentation equilibrium analysis. The sedimentation constant s/sub 20,w/ was 6.0. The subunit size of the SADH was 48,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that it consists of two subunits. The purified SADH contained two atoms of zinc per mole of enzyme protein. SADH catalyzed the oxidation of secondary alcohols. Primary alcohols (C/sub 1/ to C/sub 8/ tested) were not oxidized. The purified SADH and extracts of various yeasts and bacteria also catalyzed the reduction of methylketones to the corresponding secondary alcohols in the presence of reduced NAD/sup +/ as an electron donor. Both reactions (oxidation of secondary alcohols in the presence of NAD/sup +/ and reduction of methylketones in the presence of reduced NAD/sup +/) catalyzed by the purified SADH were inhibited by metal-chelating agents, thio reagent, and by antisera prepared against the purified enzyme. The apparent K/sub m/ values for NAD/sup +/, reduced NAD/sup +/, reduced NAD/sup +/, 2-butanol, and 2-butanone are 0.05, 0.1, 0.4, and 1 mM, respectively. The purified enzyme preferentially oxidized (-)-2-butanol and (-)-2-octanol, the rate of oxidation of (+)-2-butanol and (+)-2-octanol was 36% and 13% of that of 100% with (-)-2-butanol and (-)-2-octanol, respectively. The K/sub m/ values for (-)-2-butanol and (+)-2-butanol were 3.0 and 0.75 mM, respectively. Antisera prepared against purified Pichia SADH cross-reacted with the SADH derived from bacteria. This suggests difference in immunological properties between yeast and bacterial SADH.

  6. Aldehyde dehydrogenase 2 deficiency ameliorates alcoholic fatty liver but worsens liver inflammation and fibrosis in mice

    PubMed Central

    Kwon, Hyo-Jung; Won, Young-Suk; Park, Ogyi; Chang, Binxia; Duryee, Michael J.; Thiele, Geoffrey E.; Matsumoto, Akiko; Singh, Surendra; Abdelmegeed, Mohamed A.; Song, Byoung-Joon; Kawamoto, Toshihiro; Vasiliou, Vasilis; Thiele, Geoffrey M.; Gao, Bin

    2014-01-01

    Aldehyde dehydrogenase 2 (ALDH2) is the major enzyme that metabolizes acetaldehyde produced from alcohol metabolism. Approximately 40~50% of East Asians carry an inactive ALDH2 gene and exhibit acetaldehyde accumulation after alcohol consumption. However, the role of ALDH2 deficiency in the pathogenesis of alcoholic liver injury remains obscure. In the present study, wild-type and ALDH2−/− mice were subjected to ethanol feeding and/or carbon tetrachloride (CCl4) treatment, and liver injury was assessed. Compared with wild-type mice, ethanol-fed ALDH2−/− mice had higher levels of malondialdehyde-acetaldehyde (MAA) adduct and greater hepatic inflammation, with higher hepatic IL-6 expression but surprisingly lower levels of steatosis and serum ALT. Higher IL-6 levels were also detected in ethanol-treated precision-cut-liver-slices from ALDH2−/− mice and in Kupffer cells isolated from ethanol-fed ALDH2−/− mice than those levels in wild-type mice. In vitro incubation with MAA enhanced the LPS-mediated stimulation of IL-6 production in Kupffer cells. In agreement with these findings, hepatic activation of the major IL-6 downstream signaling molecule signal transducer and activator of transcription 3 (STAT3) was higher in ethanol-fed ALDH2−/− mice than in wild-type mice. An additional deletion of hepatic STAT3 increased steatosis and hepatocellular damage in ALDH2−/− mice. Finally, ethanol-fed ALDH2−/− mice were more prone to CCl4-induced liver inflammation and fibrosis than ethanol-fed wild-type mice. Conclusions: ALDH2−/− mice are resistant to ethanol-induced steatosis but prone to inflammation and fibrosis via MAA-mediated paracrine activation of IL-6 in Kupffer cells. These findings suggest that alcohol, via acetaldehyde and its associated adducts, stimulates hepatic inflammation and fibrosis independent from causing hepatocyte death, and that ALDH2-deficient individuals may be resistant to steatosis and blood ALT elevation, but are

  7. A comparison of two novel alcohol dehydrogenase enzymes (ADH1 and ADH2) from the extreme halophile Haloferax volcanii.

    PubMed

    Timpson, Leanne M; Liliensiek, Ann-Kathrin; Alsafadi, Diya; Cassidy, Jennifer; Sharkey, Michael A; Liddell, Susan; Allers, Thorsten; Paradisi, Francesca

    2013-01-01

    Haloarchaeal alcohol dehydrogenases are exciting biocatalysts with potential industrial applications. In this study, two alcohol dehydrogenase enzymes from the extremely halophilic archaeon Haloferax volcanii (HvADH1 and HvADH2) were homologously expressed and subsequently purified by immobilized metal-affinity chromatography. The proteins appeared to copurify with endogenous alcohol dehydrogenases, and a double Δadh2 Δadh1 gene deletion strain was constructed to prevent this occurrence. Purified HvADH1 and HvADH2 were compared in terms of stability and enzymatic activity over a range of pH values, salt concentrations, and temperatures. Both enzymes were haloalkaliphilic and thermoactive for the oxidative reaction and catalyzed the reductive reaction at a slightly acidic pH. While the NAD(+)-dependent HvADH1 showed a preference for short-chain alcohols and was inherently unstable, HvADH2 exhibited dual cofactor specificity, accepted a broad range of substrates, and, with respect to HvADH1, was remarkably stable. Furthermore, HvADH2 exhibited tolerance to organic solvents. HvADH2 therefore displays much greater potential as an industrially useful biocatalyst than HvADH1.

  8. Display of Bombyx mori Alcohol Dehydrogenases on the Bacillus subtilis Spore Surface to Enhance Enzymatic Activity under Adverse Conditions

    PubMed Central

    Wang, Nan; Chang, Cheng; Yao, Qin; Li, Guohui; Qin, Lvgao; Chen, Liang; Chen, Keping

    2011-01-01

    Alcohol dehydrogenases (ADHs) are oxidoreductases catalyzing the reversible oxidation of alcohols to corresponding aldehydes or ketones accompanied by nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP) as coenzyme. ADHs attract major scientific and industrial interest for the evolutionary perspectives, afforded by their wide occurrence in nature, and for their use in industrial synthesis. However, the low activity of ADHs under extremes of pH and temperature often limits their application. To obtain ADH with high activity, in this study, we used Bombyx mori alcohol dehydrogenases (BmADH) as foreign gene and constructed a recombinant integrative plasmid pJS700-BmADH. This pJS700-BmADH was transformed into Bacillus subtilis by double cross-over and produced an amylase inactivated mutant. The fusion protein containing BmADH was expressed on the spore surface and recognized by BmADH-specific antibody. We also assayed the alcohol dehydrogenase activity of the fusion protein together with the native BmADH at different pH and temperature levels, which indicated the recombinant enzyme exhibits activity over wider ranges of temperature and pH than its native form, perhaps due to the resistance properties of B. subtilis spores against adverse conditions. PMID:21738670

  9. 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.

  10. Optimization of enzyme assisted extraction of Fructus Mori polysaccharides and its activities on antioxidant and alcohol dehydrogenase.

    PubMed

    Deng, Qingfang; Zhou, Xin; Chen, Huaguo

    2014-10-13

    In the present study, enzyme assisted extraction of Fructus Mori polysaccharides (FMPS) from F. mori using four kinds of enzymes and three compound enzymes were examined. Research found that glucose oxidase offered a better performance in enhancement of the extraction yields of FMPS, antioxidant and activate alcohol dehydrogenase activities. The glucose oxidase assisted extraction process was further optimized by using response surface method (RSM) to obtain maximum yield of crude FMPS. The results showed that optimized extraction conditions were ratio of enzyme amount 0.40%, enzyme treated time 38 min, treated temperature 58 °C and liquid-solid radio 11.0. Under these conditions, the mean experimental value of extraction yield (16.16 ± 0.14%) corresponded well with the predicted values and increased 160% than none enzyme treated ones. Pharmacological verification tests showed that F. mori crude polysaccharides had good antioxidant and activate alcohol dehydrogenase activities in vitro. PMID:25037415

  11. Optimization of enzyme assisted extraction of Fructus Mori polysaccharides and its activities on antioxidant and alcohol dehydrogenase.

    PubMed

    Deng, Qingfang; Zhou, Xin; Chen, Huaguo

    2014-10-13

    In the present study, enzyme assisted extraction of Fructus Mori polysaccharides (FMPS) from F. mori using four kinds of enzymes and three compound enzymes were examined. Research found that glucose oxidase offered a better performance in enhancement of the extraction yields of FMPS, antioxidant and activate alcohol dehydrogenase activities. The glucose oxidase assisted extraction process was further optimized by using response surface method (RSM) to obtain maximum yield of crude FMPS. The results showed that optimized extraction conditions were ratio of enzyme amount 0.40%, enzyme treated time 38 min, treated temperature 58 °C and liquid-solid radio 11.0. Under these conditions, the mean experimental value of extraction yield (16.16 ± 0.14%) corresponded well with the predicted values and increased 160% than none enzyme treated ones. Pharmacological verification tests showed that F. mori crude polysaccharides had good antioxidant and activate alcohol dehydrogenase activities in vitro.

  12. Effects of high pressure on solvent isotope effects of yeast alcohol dehydrogenase.

    PubMed Central

    Northrop, D B; Cho, Y K

    2000-01-01

    The effect of pressure on the capture of a substrate alcohol by yeast alcohol dehydrogenase is biphasic. Solvent isotope effects accompany both phases and are expressed differently at different pressures. These differences allow the extraction of an inverse intrinsic kinetic solvent isotope effect of 1.1 (i.e., (D(2(O)))V/K = 0.9) accompanying hydride transfer and an inverse equilibrium solvent isotope effect of 2.6 (i.e., (D(2(O)))K(s) = 0.4) accompanying the binding of nucleotide, NAD(+). The value of the kinetic effect is consistent with a reactant-state E-NAD(+)-Zn-OH(2) having a fractionation factor of phi approximately 0.5 for the zinc-bound water in conjunction with a transition-state proton exiting a low-barrier hydrogen bond with a fractionation factor between 0.6 and 0.9. The value of the equilibrium effect is consistent with restrictions of torsional motions of multiple hydrogens of the enzyme protein during the conformational change that accompanies the binding of NAD(+). The absence of significant commitments to catalysis accompanying the kinetic solvent isotope effect means that this portion of the proton transfer occurs in the same reactive step as hydride transfer in a concerted chemical mechanism. The success of this analysis suggests that future measurements of solvent isotope effects as a function of pressure, in the presence of moderate commitments to catalysis, may yield precise estimates of intrinsic solvent isotope effects that are not fully expressed on capture at atmospheric pressure. PMID:10969022

  13. 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.

  14. The effects of alcohol and aldehyde dehydrogenases on disorders of hematopoiesis.

    PubMed

    Smith, Clay; Gasparetto, Maura; Jordan, Craig; Pollyea, Daniel A; Vasiliou, Vasilis

    2015-01-01

    Hematopoiesis involves the orderly production of millions of blood cells per second from a small number of essential bone marrow cells termed hematopoietic stem cells (HSCs). Ethanol suppresses normal hematopoiesis resulting in leukopenia, anemia, and thrombocytopenia and may also predispose to the development of diseases such as myelodysplasia (MDS) and acute leukemia. Currently the exact mechanisms by which ethanol perturbs hematopoiesis are unclear. The aldehyde dehydrogenase (ALDH) gene family plays a major role in the metabolism of reactive aldehydes derived from ethanol in the liver and other organs. At least one of the ALDH isoforms, ALDH1A1, is expressed at high levels in HSCs in humans, mice, and other organisms. Recent data indicate that ALDH1A1 and possibly other ALDH isoforms may metabolize reactive aldehydes in HSCs and other hematopoietic cells as they do in the liver and elsewhere. In addition, loss of these ALDHs leads to perturbation of a variety of cell processes that may predispose HSCs to disorders in growth and leukemic transformation. From these findings, we suggest a hypothesis that the cytopenias and possible increased risk of MDS and acute leukemia in heavy alcohol users is due to polymorphisms in genes responsible for metabolism of alcohol derived reactive aldehydes and repair of their DNA adducts in HSCs and other hematopoietic cells. In the article, we will summarize the biological properties of hematopoietic cells and diseases related to ethanol consumption, discuss molecular characteristics of ethanol metabolism, and describe a model to explain how ethanol derived reactive aldehydes may promote HSC damage. PMID:25427917

  15. Complete amino acid sequence and characterization of the reaction mechanism of a glucosamine-induced novel alcohol dehydrogenase from Agrobacterium radiobacter (tumefaciens).

    PubMed

    Iwamoto, Ryoko; Kubota, Humie; Hosoki, Tomoko; Ikehara, Kenji; Tanaka, Mieko

    2002-02-15

    A glucosamine-induced novel alcohol dehydrogenase has been isolated from Agrobacterium radiobacter (tumefaciens) and its fundamental properties have been characterized. The enzyme catalyzes NAD-dependent dehydrogenation of aliphatic alcohols and amino alcohols. In this work, the complete amino acid sequence of the alcohol dehydrogenase was determined by PCR method using genomic DNA of A. radiobacter as template. The enzyme comprises 336 amino acids and has a molecular mass of 36 kDa. The primary structure of the enzyme demonstrates a high homology to structures of alcohol dehydrogenases from Shinorhizobium meliloti (83% identity, 90% positive) and Pseudomonas aeruginosa (65% identity, 76% positive). The two Zn(2+) ion binding sites, both the active site and another site that contributed to stabilization of the enzyme, are conserved in those enzymes. Sequences analysis of the NAD-dependent dehydrogenase family using a hypothetical phylogenetic tree indicates that these three enzymes form a new group distinct from other members of the Zn-containing long-chain alcohol dehydrogenase family. The physicochemical properties of alcohol dehydrogenase from A. radiobacter were characterized as follows. (1) Stereospecificity of the hydride transfer from ethanol to NADH was categorized as pro-R type by NMR spectra of NADH formed in the enzymatic reaction using ethanol-D(6) was used as substrate. (2) Optimal pH for all alcohols with no amino group examined was pH 8.5 (of the C(2)-C(6) alcohols, n-amyl alcohol demonstrated the highest activity). Conversely, glucosaminitol was optimally dehydrogenated at pH 10.0. (3) The rate-determining step of the dehydrogenase for ethanol is deprotonation of the enzyme-NAD-Zn-OHCH(2)CH(3) complex to enzyme-NAD-Zn-O(-)CH(2)CH(3) complex and that for glucosaminitol is H(2)O addition to enzyme-Zn-NADH complex. PMID:11831851

  16. Complete amino acid sequence and characterization of the reaction mechanism of a glucosamine-induced novel alcohol dehydrogenase from Agrobacterium radiobacter (tumefaciens).

    PubMed

    Iwamoto, Ryoko; Kubota, Humie; Hosoki, Tomoko; Ikehara, Kenji; Tanaka, Mieko

    2002-02-15

    A glucosamine-induced novel alcohol dehydrogenase has been isolated from Agrobacterium radiobacter (tumefaciens) and its fundamental properties have been characterized. The enzyme catalyzes NAD-dependent dehydrogenation of aliphatic alcohols and amino alcohols. In this work, the complete amino acid sequence of the alcohol dehydrogenase was determined by PCR method using genomic DNA of A. radiobacter as template. The enzyme comprises 336 amino acids and has a molecular mass of 36 kDa. The primary structure of the enzyme demonstrates a high homology to structures of alcohol dehydrogenases from Shinorhizobium meliloti (83% identity, 90% positive) and Pseudomonas aeruginosa (65% identity, 76% positive). The two Zn(2+) ion binding sites, both the active site and another site that contributed to stabilization of the enzyme, are conserved in those enzymes. Sequences analysis of the NAD-dependent dehydrogenase family using a hypothetical phylogenetic tree indicates that these three enzymes form a new group distinct from other members of the Zn-containing long-chain alcohol dehydrogenase family. The physicochemical properties of alcohol dehydrogenase from A. radiobacter were characterized as follows. (1) Stereospecificity of the hydride transfer from ethanol to NADH was categorized as pro-R type by NMR spectra of NADH formed in the enzymatic reaction using ethanol-D(6) was used as substrate. (2) Optimal pH for all alcohols with no amino group examined was pH 8.5 (of the C(2)-C(6) alcohols, n-amyl alcohol demonstrated the highest activity). Conversely, glucosaminitol was optimally dehydrogenated at pH 10.0. (3) The rate-determining step of the dehydrogenase for ethanol is deprotonation of the enzyme-NAD-Zn-OHCH(2)CH(3) complex to enzyme-NAD-Zn-O(-)CH(2)CH(3) complex and that for glucosaminitol is H(2)O addition to enzyme-Zn-NADH complex.

  17. Alcohol dehydrogenases from Scheffersomyces stipitis involved in the detoxification of aldehyde inhibitors derived from lignocellulosic biomass conversion.

    PubMed

    Ma, Menggen; Wang, Xu; Zhang, Xiaoping; Zhao, Xianxian

    2013-09-01

    Aldehyde inhibitors such as furfural and 5-hydroxymethylfurfural (HMF) are generated from biomass pretreatment. Scheffersomyces stipitis is able to reduce furfural and HMF to less toxic furanmethanol and furan-2,5-dimethanol; however, the enzymes involved in the reductive reaction still remain unknown. In this study, transcription responses of two known and five putative alcohol dehydrogenase genes from S. stipitis were analyzed under furfural and HMF stress conditions. All the seven alcohol dehydrogenase genes were also cloned and overexpressed for their activity analyses. Our results indicate that transcriptions of SsADH4 and SsADH6 were highly induced under furfural and HMF stress conditions, and the proteins encoded by them exhibited NADH- and/or NADPH-dependent activities for furfural and HMF reduction, respectively. For furfural reduction, NADH-dependent activity was also observed in SsAdh1p and NAD(P)H-dependent activities were also observed in SsAdh5p and SsAdh7p. For HMF reduction, NADPH-dependent activities were also observed in SsAdh5p and SsAdh7p. SsAdh4p displayed the highest NADPH-dependent specific activity and catalytic efficiency for reduction of both furfural and HMF among the seven alcohol dehydrogenases. Enzyme activities of all SsADH proteins were more stable under acidic condition. For most SsADH proteins, the optimum temperature for enzyme activities was 30 °C and more than 50 % enzyme activities remained at 60 °C. Reduction activities of formaldehyde, acetaldehyde, isovaleraldehyde, benzaldehyde, and phenylacetaldehyde were also observed in some SsADH proteins. Our results indicate that multiple alcohol dehydrogenases in S. stipitis are involved in the detoxification of aldehyde inhibitors derived from lignocellulosic biomass conversion. PMID:23912116

  18. Origin and evolution of a new gene descended from alcohol dehydrogenase in Drosophila.

    PubMed

    Begun, D J

    1997-02-01

    Drosophila alcohol dehydrogenase (Adh) is highly conserved in size, organization, and amino acid sequence. Adh-psi was hypothesized to be a pseudogene derived from an Adh duplication in the repleta group of Drosophila; however, several results from molecular analyses of this gene conflict with currently held notions of molecular evolution. Perhaps the most difficult observations to reconcile with the pseudogene hypothesis are that the hypothetical replacement sites of Adh-psi evolve only slightly more quickly than replacement sites of closely related, functional Adh genes, and that the replacement sites of the pseudogenes evolve considerably more slowly than neighboring silent sites. The data have been presented as a paradox that challenges our understanding of the mechanisms underlying DNA sequence divergence. Here I show that Adh-psi is actually a new, functional gene recently descended from an Adh duplication. This descendant recruited approximately 60 new N-terminal amino acids, is considerably more basic than ADH, and is evolving at a faster rate than Adh. Furthermore, though the descendant is clearly functional, as inferred from molecular evolution and population genetic data, it retains no obvious ADH activity. This probably reflects functional divergence from its Adh ancestor.

  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. Determination of Kinetic Isotope Effects in Yeast Alcohol Dehydrogenase Using Transition Path Sampling

    NASA Astrophysics Data System (ADS)

    Varga, Matthew; Schwartz, Steven

    2015-03-01

    The experimental determination of kinetic isotope effects in enzymatic systems can be a difficult, time-consuming, and expensive process. In this study, we use the Chandler-Bolhius method for the determination of reaction rates within transition path sampling (rTPS) to determine the primary kinetic isotope effect in yeast alcohol dehydrogenase (YADH). In this study, normal mode centroid molecular dynamics (CMD) was applied to the transferring hydride/deuteride in order to correctly incorporate quantum effects into the molecular simulations. Though previous studies have used rTPS to calculate reaction rate constants in various model and real systems, it has not been applied to a system as large as YADH. Due to the fact that particle transfer is not wholly indicative of the chemical step, this method cannot be used to determine reaction rate constants in YADH. However, it is possible to determine the transition rate constant of the particle transfer, and the kinetic isotope effect of that step. This method provides a set of tools to determine kinetic isotope effects with the atomistic detail of molecular simulations.

  1. Study on immobilization of yeast alcohol dehydrogenase on nanocrystalline Ni-Co ferrites as magnetic support.

    PubMed

    Shakir, Mohammad; Nasir, Zeba; Khan, Mohd Shoeb; Lutfullah; Alam, Md Fazle; Younus, Hina; Al-Resayes, Saud Ibrahim

    2015-01-01

    The covalent binding of yeast alcohol dehydrogenase (YADH) enzyme complex in a series of magnetic crystalline Ni-Co nanoferrites, synthesized via sol-gel auto combustion technique was investigated. The structural analysis, morphology and magnetic properties of Ni-Co nanoferrites were determined by X-ray diffraction (XRD), scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDS), vibrating-sample magnetometer (VSM), high resolution transmission electron microscopy (HRTEM) and Fourier transform infrared spectroscopy (FTIR). The comparative analysis of the HRTEM micrographs of bare magnetic nanoferrite particles and particles immobilized with enzyme revealed an uniform distribution of the particles in both the cases without undergoing change in the size which was found to be in the range 20-30 nm. The binding of YADH to Ni-Co nanoferrites and the possible binding mechanism have been suggested by comparing the FTIR results. The binding properties of the immobilized YADH enzyme were also studied by kinetic parameters, optimum operational pH, temperature, thermal stability and reusability. The immobilized YADH exhibits enhanced thermal stability as compared to the free enzyme over a wide range of temperature and pH, and showed good durability after recovery by magnetic separation for repeated use.

  2. Activity and Conformation of Yeast Alcohol Dehydrogenase (YADH) Entrapped in Reverse Micelles.

    PubMed

    Das; Mozumdar; Maitra

    2000-10-15

    Yeast alcohol dehydrogenase (YADH) solubilized in reverse micelles of aerosol OT (i.e., AOT or sodium bis (2-ethyl hexyl) sulfosuccinate) in isooctane has been shown to be catalytically more active than that in aqueous buffer under optimum conditions of pH, temperature, and water content in reverse micelles. Studies of the secondary structure conformational changes of the enzyme in reverse micelles have been made from circular dichroism spectroscopy. It has been seen that the conformation of YADH in reverse micelles is extremely sensitive to pH, temperature, and water content. A comparison has been made between the catalytic activity of the enzyme and the alpha-helix content in the conformation and it has been observed that the enzyme is most active at the maximum alpha-helix content. While the beta-sheet content in the conformation of the entrapped enzyme was found to be dependent on the enzyme-micelle interface interaction, the alpha-helix and random coil conformations are governed by the degree of entrapment and the extent of rigidity provided by the micelle core to the enzyme structure. Copyright 2000 Academic Press.

  3. The three zinc-containing alcohol dehydrogenases from baker's yeast, Saccharomyces cerevisiae.

    PubMed

    Leskovac, Vladimir; Trivić, Svetlana; Pericin, Draginja

    2002-12-01

    This review is a summary of our current knowledge of the structure, function and mechanism of action of the three zinc-containing alcohol dehydrogenases, YADH-1, YADH-2 and YADH-3, in baker's yeast, Saccharomyces cerevisiae. The opening section deals with the substrate specificity of the enzymes, covering the steady-state kinetic data for its most known substrates. In the following sections, the kinetic mechanism for this enzyme is reported, along with the values of all rate constants in the mechanism. The complete primary structures of the three isoenzymes of YADH are given, and the model of the 3D structure of the active site is presented. All known artificial mutations in the primary structure of the YADH are covered in full and described in detail. Further, the chemical mechanism of action for YADH is presented along with the complement of steady-state and ligand-binding data supporting this mechanism. Finally, the bio-organic chemistry of the hydride-transfer reactions catalyzed by the enzyme is covered: this chemistry explains the narrow substrate specificity and the enantioselectivity of the yeast enzyme.

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

    PubMed

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

    2007-01-01

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

  5. Inactivation of yeast alcohol dehydrogenase by alkylperoxyl radicals. Characteristics and influence of nicotinamide-adenine dinucleotides.

    PubMed

    Videla, L A; Salim-Hanna, M; Lissi, E A

    1992-10-01

    The study of the interaction of alkylperoxyl radicals generated by the aerobic thermolysis of 2,2'-azobis(2-amidinopropane) (AAP) with yeast alcohol dehydrogenase (YADH) revealed a high reactivity of the enzyme, with an average of about 20 radicals per added YADH tetramer being needed to elicit its total inactivation. NAD+ enhanced YADH inactivation at NAD+/YADH molar ratios from 0.25 to 1, decreasing the rate of the process when added in excess to the enzyme concentration. At NADH/YADH molar ratios greater than 1, NADH exhibited a protective effect characterized by a poorly defined induction time and lower inactivation rates, which progressively increased during the reaction period. These changes occurred concomitantly with the oxidation of NADH into NAD+, which might counteract the protective effect of NADH. Under similar conditions, NADP+ did not modify AAP-induced YADH inactivation, while NADPH exhibited a modest protection at NADPH/YADH molar ratios greater than 1. It is concluded that YADH inactivation by alkylperoxyl radicals is strongly dependent on the redox state of the NADH-NAD+ couple, as the rates of the process at different time intervals inversely correlate with the respective NADH/NAD+ ratios.

  6. Suitability of the hydrocarbon-hydroxylating molybdenum-enzyme ethylbenzene dehydrogenase for industrial chiral alcohol production.

    PubMed

    Tataruch, M; Heider, J; Bryjak, J; Nowak, P; Knack, D; Czerniak, A; Liesiene, J; Szaleniec, M

    2014-12-20

    The molybdenum/iron-sulfur/heme protein ethylbenzene dehydrogenase (EbDH) was successfully applied to catalyze enantiospecific hydroxylation of alkylaromatic and alkylheterocyclic compounds. The optimization of the synthetic procedure involves use of the enzyme in a crude purification state that saves significant preparation effort and is more stable than purified EbDH without exhibiting unwanted side reactions. Moreover, immobilization of the enzyme on a crystalline cellulose support and changes in reaction conditions were introduced in order to increase the amounts of product formed (anaerobic atmosphere, electrochemical electron acceptor recycling or utilization of ferricyanide as alternative electron acceptor in high concentrations). We report here on an extension of effective enzyme activity from 4h to more than 10 days and final product yields of up to 0.4-0.5g/l, which represent a decent starting point for further optimization. Therefore, we expect that the hydrocarbon-hydroxylation capabilities of EbDH may be developed into a new process of industrial production of chiral alcohols.

  7. 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.

  8. Selection variability for Arg48His in alcohol dehydrogenase ADH1B among Asian populations.

    PubMed

    Evsyukov, Alexey; Ivanov, Denis

    2013-08-01

    The variant His at codon 48 of the alcohol dehydrogenase gene (ADH1B) results in more efficient ethanol metabolism than with the "typical" codon 48Arg. In this study we introduced selection properties of Arg48His genotypes of ADH1B and estimated fitness in four ethnic-geographical clusters in Asia. Population genetics models were employed that derive observed gene frequencies from fitness relationships among genotypes, to infer the selection pattern of polymorphisms in an indirect manner. The data were analyzed using the model of "complete stationary distribution" by Wright that takes into account random genetic drift, pressure of migrations, mutations, and selection as influential factors of gene frequency. We found that the different population groups showed some variation in the types of selection for Arg48His. Han Chinese from eastern and southeastern China and the Japanese and Korean populations showed stabilizing selection, while the groups from Central Asian and Indochina showed divergent selection. However, all the groups demonstrated a strong positive selection for Arg48His. PMID:25019189

  9. Developmentally Regulated RNA Transcripts Coding for Alcohol Dehydrogenase in DROSOPHILA AFFINIDISJUNCTA

    PubMed Central

    Rowan, Robert G.; Brennan, Mark D.; Dickinson, W. J.

    1986-01-01

    The organization of the gene coding for alcohol dehydrogenase (Adh) in Drosophila affinidisjuncta has been determined by physically mapping Adh RNA transcripts to cloned genomic DNA. Two distinct transcript types accumulate with developmental specificity. Because only a single genomic Adh locus is detected in D. affinidisjuncta , and since all Adh transcripts appear to be identical except at their termini, the two Adh RNA types are products of the same gene. One type of transcript, abundant in adults, contains a small 5' terminal exon that is completely lacking in the other type of transcript, which accumulates in larvae. This 5' end difference suggests that the D. affinidisjuncta Adh gene, like the homologous gene from the distantly related species D. melanogaster, is expressed from two promoters. According to the transcription map, these D. affinidisjuncta promoters are separated by approximately 560 base pairs of genomic DNA sequence. D. affinidisjuncta Adh transcripts also resemble D. melanogaster Adh transcripts in both their overall organization and their developmental distribution. Multiple 3' ends are responsible for the size heterogeneity of both types of D. affinidisjuncta Adh RNA, and some of these also appear with stage specificity. PMID:2429897

  10. Subcellular localization of the NAD+-dependent alcohol dehydrogenase in Entamoeba histolytica trophozoites.

    PubMed

    Avila, Eva E; Martínez-Alcaraz, Edith R; Barbosa-Sabanero, Gloria; Rivera-Baron, Elda I; Arias-Negrete, Sergio; Zazueta-Sandoval, Roberto

    2002-04-01

    The protozoan parasite Entamoeba histolytica is an ancient eukaryotic cell that shows morphologically atypical organelles and differs metabolically from higher eukaryotic cells. The aim of this study was to determine the subcellular localization of ameba NAD+-dependent alcohol dehydrogenase (ADH2). The enzyme activity was present in soluble and mainly in particulate material whose density was 1.105 in a sucrose gradient. By differential centrifugation, most of the ADH activity sedimented at 160,000 g (160,000-g pellet), similar to the Escherichia coli polymeric ADHE. In the Coomassie staining of the 160,000-g pellet analyzed by electrophoresis, a 96-kDa protein was more prominent than in other fractions; this band was recognized by antibodies against Lactococcus lactis ADHE. By gold labeling, the antibodies recognized the granular material that mainly constitutes the 160,000-g pellet and a material that sedimented along with the internal membrane vesicles. By negative staining, the 160,000-g fraction showed helical rodlike structures with an average length of 103 nm; almost no membrane vesicles were observed in this pellet. In internal membrane fractions, no rodlike structures were found, but protomerlike round structures were observed. These results indicate that the main amebic NAD+-dependent ADH2 activity is naturally organized as rodlike helical particles, similar to bacterial ADHE. Detection of ADH2 in membrane fractions might be explained by cosedimentation of the multimeric ADH during membrane purification.

  11. Characterization of thermotolerant Acetobacter pasteurianus strains and their quinoprotein alcohol dehydrogenases.

    PubMed

    Kanchanarach, Watchara; Theeragool, Gunjana; Yakushi, Toshiharu; Toyama, Hirohide; Adachi, Osao; Matsushita, Kazunobu

    2010-01-01

    We isolated several thermotolerant Acetobacter species of which MSU10 strain, identified as Acetobacter pasteurianus, could grow well on agar plates at 41 degrees C, tolerate to 1.5% acetic acid or 4% ethanol at 39 degrees C, similarly seen with A. pasteurianus SKU1108 previously isolated. The MSU10 strain showed higher acetic acid productivity in a medium containing 6% ethanol at 37 degrees C than SKU1108 while SKU1108 strain could accumulate more acetic acid in a medium supplemented with 4-5% ethanol at the same temperature. The fermentation ability at 37 degrees C of these thermotolerant strains was superior to that of mesophilic A. pasteurianus IFO3191 strain having weak growth and very delayed acetic acid production at 37 degrees C even at 4% ethanol. Alcohol dehydrogenases (ADHs) were purified from MSU10, SKU1108, and IFO3191 strains, and their properties were compared related to the thermotolerance. ADH of the thermotolerant strains had a little higher optimal temperature and heat stability than that of mesophilic IFO3191. More critically, ADHs from MSU10 and SKU1108 strains exhibited a higher resistance to ethanol and acetic acid than IFO3191 enzyme at elevated temperature. Furthermore, in this study, the ADH genes were cloned, and the amino acid sequences of ADH subunit I, subunit II, and subunit III were compared. The difference in the amino acid residues could be seen, seemingly related to the thermotolerance, between MSU10 or SKU1108 ADH and IFO 3191 ADH.

  12. Furfural reduction mechanism of a zinc-dependent alcohol dehydrogenase from Cupriavidus necator JMP134

    PubMed Central

    Kang, ChulHee; Hayes, Robert; Sanchez, Emiliano J.; Webb, Brian N.; Li, Qunrui; Hooper, Travis; Nissen, Mark S.; Xun, Luying

    2012-01-01

    Summary FurX is a tetrameric Zn-dependent alcohol dehydrogenase (ADH) from Cupriavidus necator JMP134. The enzyme rapidly reduces furfural with NADH as the reducing power. For the first time among characterized ADHs, the high-resolution structures of all reaction steps were obtained in a time-resolved manner, thereby illustrating the complete catalytic events of NADH-dependent reduction of furfural and the dynamic Zn2+ coordination among Glu66, water, substrate and product. In the fully closed conformation of the NADH complex, the catalytic turnover proved faster than observed for the partially closed conformation due to an effective proton transfer network. The domain motion triggered by NAD(H) association/dissociation appeared to facilitate dynamic interchanges in Zn2+ coordination with substrate and product molecules, ultimately increasing the enzymatic turnover rate. NAD+ dissociation appeared to be a slow process, involving multiple steps in concert with a domain opening and reconfiguration of Glu66. This agrees with the report that the cofactor is not dissociated from FurX during ethanol-dependent reduction of furfural, in which ethanol reduces NAD+ to NADH that is subsequently used for furfural reduction. PMID:22081946

  13. Substitution of arginine for histidine-47 in the coenzyme binding site of yeast alcohol dehydrogenase I

    SciTech Connect

    Gould, R.M.; Plapp, B.V. )

    1990-06-12

    Molecular modeling of alcohol dehydrogenases suggests that His-47 in the yeast enzyme (His-44 in the protein sequence, corresponding to Arg-47 in the horse liver enzyme) binds the pyrophosphate of the NAD coenzyme. His-47 in the Saccharomyces cerevisiae isoenzyme I was substituted with an arginine by a directed mutation. Steady-state kinetic results at pH 7.3 and 30{degree}C of the mutant and wild-type enzymes were consistent with an ordered Bi-Bi mechanism. The substitution decreased dissociation constants by 4-fold for NAD{sup +} and 2-fold for NADH while turnover numbers were decreased by 4-fold for ethanol oxidation and 6-fold for acetaldehyde reduction. The magnitudes of these effects are smaller than those found for the same mutation in the human liver {beta} enzyme, suggesting that other amino acid residues in the active site modulate the effects of the substitution. The pH dependencies of dissociation constants and other kinetic constants were similar in the two yeast enzymes. Thus, it appears that His-47 is not solely responsible for a pK value near 7 that controls activity and coenzyme binding rates in the wild-type enzyme. The small substrate deuterium isotope effect above pH 7 and the single exponential phase of NADH production during the transient oxidation of ethanol by the Arg-47 enzyme suggest that the mutation makes an isomerization of the enzyme-NAD{sup +} complex limiting for turnover with ethanol.

  14. 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.

  15. 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.

  16. The activity of liver alcohol dehydrogenase with nicotinamide–adenine dinucleotide phosphate as coenzyme

    PubMed Central

    Dalziel, K.; Dickinson, F. M.

    1965-01-01

    1. The separation of nucleotide impurities from commercial NADP preparations by chromatography is described. All the preparations studied contained 0·1–0·2% of NAD. 2. The activity of pure crystalline liver alcohol dehydrogenase with NADP as coenzyme has been confirmed. Initial-rate data are reported for the reaction at pH 6·0 and 7·0 with ethanol and acetaldehyde as substrates. With NADP and NADPH2 of high purity, the maximal specific rates were similar to those obtained with NAD and NADH2, but the Michaelis constants for the former coenzymes were much greater than those for the latter. 3. The oxidation of ethanol by NADP is greatly inhibited by NADH2, and this accounts for low values of certain initial-rate parameters obtained with commercial NADP preparations containing NAD. The kinetics of the inhibition are consistent with competitive inhibition in a compulsory-order mechanism. 4. Initial-rate data with NAD and NADPH2 do not conform to the requirements of the mechanism proposed by Theorell & Chance (1951), in contrast with results previously obtained with NAD and NADH2. The possibility that the deviations are due to competing nucleotide impurity in the oxidized coenzyme cannot be excluded. The data show that the enzyme reacts more slowly with, and has a smaller affinity for, NADP and NADPH2 than NAD and NADH2. 5. Phosphate behaves as a competitive inhibitor towards NADP. PMID:14340079

  17. 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.

  18. 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.

  19. 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.

  20. 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

  1. Molecular phylogeny and evolution of alcohol dehydrogenase (Adh) genes in legumes

    PubMed Central

    Fukuda, Tatsuya; Yokoyama, Jun; Nakamura, Toru; Song, In-Ja; Ito, Takuro; Ochiai, Toshinori; Kanno, Akira; Kameya, Toshiaki; Maki, Masayuki

    2005-01-01

    Background Nuclear genes determine the vast range of phenotypes that are responsible for the adaptive abilities of organisms in nature. Nevertheless, the evolutionary processes that generate the structures and functions of nuclear genes are only now be coming understood. The aim of our study is to isolate the alcohol dehydrogenase (Adh) genes in two distantly related legumes, and use these sequences to examine the molecular evolutionary history of this nuclear gene. Results We isolated the expressed Adh genes from two species of legumes, Sophora flavescens Ait. and Wisteria floribunda DC., by a RT-PCR based approach and found a new Adh locus in addition to homologues of the Adh genes found previously in legumes. To examine the evolution of these genes, we compared the species and gene trees and found gene duplication of the Adh loci in the legumes occurred as an ancient event. Conclusion This is the first report revealing that some legume species have at least two Adh gene loci belonging to separate clades. Phylogenetic analyses suggest that these genes resulted from relatively ancient duplication events. PMID:15836788

  2. Mechanisms of mutagenesis: Analysis through the use of alcohol dehydrogenase in Drosophila: Final report

    SciTech Connect

    Sofer, W.H.

    1986-12-01

    Our original objective was to understand the mechanism of mutagenesis of several important mutagens in higher organisms. Our approach was to try to deduce this mechanism by working backwards from its final effects. The strategy that we used in an effort to carry out our studies was to make mutations in the alcohol dehydrogenase gene of Drosophila melanogaster and sequence the modified genes. Most of our work was focused on an array of mutants that we had induced with formaldehyde, a potent mutagen in Drosophila, and with ethyl methane sulfonate. Over the course of the project period we cloned and sequenced the ADH gene from four formalde-induced mutants and from one EMS mutant. We showed that the four formaldehyde-induced mutants contained small deletions within the protein-coding region of their ADH genes ranging in size from between 6 and 34 bp. The one EMS-induced mutant was shown by DNA sequencing to bear an AT to GC sequence change at a tryptophan codon near the c-terminal coding portion of the gene. These results have significantly increased our understanding of the mechanism(s) of mutagenesis in higher organisms. 20 refs., 1 fig.

  3. 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.

  4. Study on immobilization of yeast alcohol dehydrogenase on nanocrystalline Ni-Co ferrites as magnetic support.

    PubMed

    Shakir, Mohammad; Nasir, Zeba; Khan, Mohd Shoeb; Lutfullah; Alam, Md Fazle; Younus, Hina; Al-Resayes, Saud Ibrahim

    2015-01-01

    The covalent binding of yeast alcohol dehydrogenase (YADH) enzyme complex in a series of magnetic crystalline Ni-Co nanoferrites, synthesized via sol-gel auto combustion technique was investigated. The structural analysis, morphology and magnetic properties of Ni-Co nanoferrites were determined by X-ray diffraction (XRD), scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDS), vibrating-sample magnetometer (VSM), high resolution transmission electron microscopy (HRTEM) and Fourier transform infrared spectroscopy (FTIR). The comparative analysis of the HRTEM micrographs of bare magnetic nanoferrite particles and particles immobilized with enzyme revealed an uniform distribution of the particles in both the cases without undergoing change in the size which was found to be in the range 20-30 nm. The binding of YADH to Ni-Co nanoferrites and the possible binding mechanism have been suggested by comparing the FTIR results. The binding properties of the immobilized YADH enzyme were also studied by kinetic parameters, optimum operational pH, temperature, thermal stability and reusability. The immobilized YADH exhibits enhanced thermal stability as compared to the free enzyme over a wide range of temperature and pH, and showed good durability after recovery by magnetic separation for repeated use. PMID:25450541

  5. Suitability of the hydrocarbon-hydroxylating molybdenum-enzyme ethylbenzene dehydrogenase for industrial chiral alcohol production.

    PubMed

    Tataruch, M; Heider, J; Bryjak, J; Nowak, P; Knack, D; Czerniak, A; Liesiene, J; Szaleniec, M

    2014-12-20

    The molybdenum/iron-sulfur/heme protein ethylbenzene dehydrogenase (EbDH) was successfully applied to catalyze enantiospecific hydroxylation of alkylaromatic and alkylheterocyclic compounds. The optimization of the synthetic procedure involves use of the enzyme in a crude purification state that saves significant preparation effort and is more stable than purified EbDH without exhibiting unwanted side reactions. Moreover, immobilization of the enzyme on a crystalline cellulose support and changes in reaction conditions were introduced in order to increase the amounts of product formed (anaerobic atmosphere, electrochemical electron acceptor recycling or utilization of ferricyanide as alternative electron acceptor in high concentrations). We report here on an extension of effective enzyme activity from 4h to more than 10 days and final product yields of up to 0.4-0.5g/l, which represent a decent starting point for further optimization. Therefore, we expect that the hydrocarbon-hydroxylation capabilities of EbDH may be developed into a new process of industrial production of chiral alcohols. PMID:24998764

  6. Unexpected properties of NADP-dependent secondary alcohol dehydrogenase (ADH-1) in Trichomonas vaginalis and other microaerophilic parasites.

    PubMed

    Leitsch, David; Williams, Catrin F; Lloyd, David; Duchêne, Michael

    2013-07-01

    Our previous observation that NADP-dependent secondary alcohol dehydrogenase (ADH-1) is down-regulated in metronidazole-resistant Trichomonas vaginalis isolates prompted us to further characterise the enzyme. In addition to its canonical enzyme activity as a secondary alcohol dehydrogenase, a pronounced, so far unknown, background NADPH-oxidising activity in absence of any added substrate was observed when the recombinant enzyme or T. vaginalis extract were used. This activity was strongly enhanced at low oxygen concentrations. Unexpectedly, all functions of ADH-1 were efficiently inhibited by coenzyme A which is a cofactor of a number of key enzymes in T. vaginalis metabolism, i.e. pyruvate:ferredoxin oxidoreductase (PFOR). These observations could be extended to Entamoeba histolytica and Tritrichomonas foetus, both of which have a homologue of ADH-1, but not to Giardia lamblia which lacks an NADP-dependent secondary alcohol dehydrogenase. Although we could not identify the substrate of the observed background activity, we propose that ADH-1 functions as a major sink for NADPH in microaerophilic parasites at low oxygen tension.

  7. Unexpected properties of NADP-dependent secondary alcohol dehydrogenase (ADH-1) in Trichomonas vaginalis and other microaerophilic parasites.

    PubMed

    Leitsch, David; Williams, Catrin F; Lloyd, David; Duchêne, Michael

    2013-07-01

    Our previous observation that NADP-dependent secondary alcohol dehydrogenase (ADH-1) is down-regulated in metronidazole-resistant Trichomonas vaginalis isolates prompted us to further characterise the enzyme. In addition to its canonical enzyme activity as a secondary alcohol dehydrogenase, a pronounced, so far unknown, background NADPH-oxidising activity in absence of any added substrate was observed when the recombinant enzyme or T. vaginalis extract were used. This activity was strongly enhanced at low oxygen concentrations. Unexpectedly, all functions of ADH-1 were efficiently inhibited by coenzyme A which is a cofactor of a number of key enzymes in T. vaginalis metabolism, i.e. pyruvate:ferredoxin oxidoreductase (PFOR). These observations could be extended to Entamoeba histolytica and Tritrichomonas foetus, both of which have a homologue of ADH-1, but not to Giardia lamblia which lacks an NADP-dependent secondary alcohol dehydrogenase. Although we could not identify the substrate of the observed background activity, we propose that ADH-1 functions as a major sink for NADPH in microaerophilic parasites at low oxygen tension. PMID:23578856

  8. Unexpected properties of NADP-dependent secondary alcohol dehydrogenase (ADH-1) in Trichomonas vaginalis and other microaerophilic parasites

    PubMed Central

    Leitsch, David; Williams, Catrin F.; Lloyd, David; Duchêne, Michael

    2013-01-01

    Our previous observation that NADP-dependent secondary alcohol dehydrogenase (ADH-1) is down-regulated in metronidazole-resistant Trichomonas vaginalis isolates prompted us to further characterise the enzyme. In addition to its canonical enzyme activity as a secondary alcohol dehydrogenase, a pronounced, so far unknown, background NADPH-oxidising activity in absence of any added substrate was observed when the recombinant enzyme or T. vaginalis extract were used. This activity was strongly enhanced at low oxygen concentrations. Unexpectedly, all functions of ADH-1 were efficiently inhibited by coenzyme A which is a cofactor of a number of key enzymes in T. vaginalis metabolism, i.e. pyruvate:ferredoxin oxidoreductase (PFOR). These observations could be extended to Entamoeba histolytica and Tritrichomonas foetus, both of which have a homologue of ADH-1, but not to Giardia lamblia which lacks an NADP-dependent secondary alcohol dehydrogenase. Although we could not identify the substrate of the observed background activity, we propose that ADH-1 functions as a major sink for NADPH in microaerophilic parasites at low oxygen tension. PMID:23578856

  9. Suppression of cellulase and polygalacturonase and induction of alcohol dehydrogenase isoenzymes in avocado fruit mesocarp subjected to low oxygen stress.

    PubMed

    Kanellis, A K; Solomos, T; Roubelakis-Angelakis, K A

    1991-05-01

    Expression of polygalacturonase and cellulase, two hydrolytic enzymes of avocado (Persea americana, cv Hass) fruit which are synthesized de novo during ripening, and alcohol dehydrogenase, a known anaerobic protein, were studied under different O(2) regimes. Low O(2) concentrations (2.5-5.5%) diminished the accumulation of polygalacturonase and cellulase proteins and the expression of their isoenzymes. This pattern of change in cellulase protein was also reflected in the steady-state amount of its mRNA. In contrast, 7.5 and 10% O(2) did not alter the changes observed in fruits ripened in air. On the other hand, alcohol dehydrogenase was induced in 2.5, 3.5, and 5.5% O(2) but not in 7.5 or 10% O(2). The recovery from the hypoxic stress upon returning the fruits back to air for 24 hours, was also a function of O(2) tensions under which the fruits were kept. Thus, the synthesis of polygalacturonase and cellulase was directly related to O(2) levels, while the activity of the isoenzymes of alcohol dehydrogenase was inversely related to O(2) levels. The results indicate that hypoxia exerts both negative and positive effects on the expression of certain genes and that these effects are initiated at the same levels of O(2).

  10. 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.

  11. Biophysical and mutagenic analysis of Thermoanaerobacter ethanolicus secondary-alcohol dehydrogenase activity and specificity.

    PubMed Central

    Burdette, D S; Secundo, F; Phillips, R S; Dong, J; Scott, R A; Zeikus, J G

    1997-01-01

    The Thermoanaerobacter ethanolicus 39E adhB gene encoding the secondary-alcohol dehydrogenase (secondary ADH) was overexpressed in Escherichia coli at more than 10% of total protein. The recombinant enzyme was purified in high yield (67%) by heat-treatment at 85 degrees C and (NH4)2SO4 precipitation. Site-directed mutants (C37S, H59N, D150N, D150Eand D150C were analysed to test the peptide sequence comparison-based predictions of amino acids responsible for putative catalytic Zn binding. X-ray absorption spectroscopy confirmed the presence of a protein-bound Zn atom with ZnS1(imid)1(N,O)3 co-ordination sphere. Inductively coupled plasma atomic emission spectrometry measured 0.48 Zn atoms per wild-type secondary ADH subunit. The C37S, H59N and D150N mutant enzymes bound only 0.11, 0.13 and 0.33 Zn per subunit respectively,suggesting that these residues are involved in Zn liganding. The D150E and D150C mutants retained 0.47 and 1.2 Zn atoms per subunit, indicating that an anionic side-chain moiety at this position preserves the bound Zn. All five mutant enzymes had

  12. 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.

  13. Tryptophan in Alcoholism Treatment I:  Kynurenine Metabolites Inhibit the Rat Liver Mitochondrial Low Km Aldehyde Dehydrogenase Activity, Elevate Blood Acetaldehyde Concentration and Induce Aversion to Alcohol

    PubMed Central

    Badawy, Abdulla A.-B.; Bano, Samina; Steptoe, Alex

    2011-01-01

    Aims: The aims were to provide proofs of mechanism and principle by establishing the ability of kynurenine metabolites to inhibit the liver mitochondrial low Km aldehyde dehydrogenase (ALDH) activity after administration and in vivo, and to induce aversion to alcohol. Methods: Kynurenic acid (KA), 3-hydroxykynurenine (3-HK) and 3-hydroxyanthranilic acid (3-HAA) were administered to normal male Wistar rats and ALDH activity was determined both in vitro in liver homogenates and in vivo (by measuring blood acetaldehyde following ethanol administration). Alcohol consumption was studied in an aversion model in rats and in alcohol-preferring C57 mice. Results: ALDH activity was significantly inhibited by all three metabolites by doses as small as 1 mg/kg body wt. Blood acetaldehyde accumulation after ethanol administration was strongly elevated by KA and 3-HK and to a lesser extent by 3-HAA. All three metabolites induced aversion to alcohol in rats and decreased alcohol preference in mice. Conclusions: The above kynurenine metabolites of tryptophan induce aversion to alcohol by inhibiting ALDH activity. An intellectual property covering the use of 3-HK and 3-HAA and derivatives thereof in the treatment of alcoholism by aversion awaits further development. PMID:21896552

  14. Phytophenols in whisky lower blood acetaldehyde level by depressing alcohol metabolism through inhibition of alcohol dehydrogenase 1 (class I) in mice.

    PubMed

    Haseba, Takeshi; Sugimoto, Junichi; Sato, Shigeo; Abe, Yuko; Ohno, Youkichi

    2008-12-01

    We recently reported that the maturation of whisky prolongs the exposure of the body to a given dose of alcohol by reducing the rate of alcohol metabolism and thus lowers the blood acetaldehyde level (Alcohol Clin Exp Res. 2007;31:77s-82s). In this study, administration of the nonvolatile fraction of whisky was found to lower the concentration of acetaldehyde in the blood of mice by depressing alcohol metabolism through the inhibition of liver alcohol dehydrogenase (ADH). Four of the 12 phenolic compounds detected in the nonvolatile fraction (caffeic acid, vanillin, syringaldehyde, ellagic acid), the amounts of which increase during the maturation of whisky, were found to strongly inhibit mouse ADH 1 (class I). Their inhibition constant values for ADH 1 were 0.08, 7.9, 15.6, and 22.0 mumol/L, respectively, whereas that for pyrazole, a well-known ADH inhibitor, was 5.1 mumol/L. The 2 phenolic aldehydes and ellagic acid exhibited a mixed type of inhibition, whereas caffeic acid showed the competitive type. When individually administered to mice together with ethanol, each of these phytophenols depressed the elimination of ethanol, thereby lowering the acetaldehyde concentration of blood. Thus, it was demonstrated that the enhanced inhibition of liver ADH 1 due to the increased amounts of these phytophenols in mature whisky caused the depression of alcohol metabolism and a consequent lowering of blood acetaldehyde level. These substances are commonly found in various food plants and act as antioxidants and/or anticarcinogens. Therefore, the intake of foods rich in them together with alcohol may not only diminish the metabolic toxicity of alcohol by reducing both the blood acetaldehyde level and oxidative stress, but also help limit the amount of alcohol a person drinks by depressing alcohol metabolism.

  15. Effect of zinc ions on conformational stability of yeast alcohol dehydrogenase.

    PubMed

    Yang, Y; Zhou, H M

    2001-01-01

    Yeast alcohol dehydrogenase preparations were prepared with the conformational zinc ion removed (Apo-I YADH) and with both the conformational and catalytic zinc ions removed (Apo-II YADH). The unfolding of Apo-I YADH and Apo-II YADH during denaturation in urea solutions was then followed by fluorescence emission, circular dichroism, and second-derivative optical spectroscopies. Compared with the native enzyme, Apo-I YADH incurred some slight unfolding, and its stability against urea was markedly decreased, while Apo-II YADH incurred marked unfolding but contained residual ordered structure even at high urea concentrations. The results show that native YADH is more conformationally stable against urea denaturation than Apo-I YADH, indicating that the conformational Zn(2+) plays an important role in stabilizing the conformation of the YADH molecule. However, unfolding of the region around the conformational zinc ion is shown not to be the rate limited step in the unfolding of the molecule by the fact that the unfolding and inactivation rate constants of native and Apo-I YADH are the same. It is suggested that the catalytic zinc ion is more important in maintaining the structure of YADH. YADH lost its cooperative unfolding ability after the zinc ions were removed. The shape of the transition curves of Apo-I YADH suggests the existence of an unfolding intermediate. For both native and Apo-I YADH, inactivation occurs at much lower urea concentrations than that needed to produce significant conformational changes of the enzyme molecule. At urea concentration above 4 M, the inactivation rate constants are much higher than those of the fast phase of the reaction of unfolding. These results support the suggestion of flexibility at the active site of the enzyme (C. L. Tsou (1986) Trends Biochem. Sci., 11, 427-429; (1993) Science, 262, 308-381).

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

    PubMed

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

    2007-01-01

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

  17. 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

  18. Non-alcohol dehydrogenase-mediated metabolism of methylazoxymethanol in the deer mouse, Peromyscus maniculatus

    SciTech Connect

    Fiala, E.S.; Caswell, N.; Sohn, O.S.; Felder, M.R.; McCoy, G.D.; Weisburger, J.H.

    1984-07-01

    The concept that alcohol dehydrogenase (ADH) is involved in the metabolism of methylazoxymethanol (MAM) was examined in a model consisting of two strains of the deer mouse, Peromyscus maniculatus, one of which has a normal complement of the enzyme (ADH(+)), and the other, which completely lacks it (ADH(-)). Both the ADH(+) and the ADH(-) strains rapidly metabolized (/sup 14/C)MAM, administered in the form of the acetic acid ester, (/sup 14/C) MAMOAc, to /sup 14/CO2, and the rates and extents of metabolism were virtually identical. Determination of O6-methylguanine and 7-methylguanine in liver DNA 6 and 24 hr after MAMOAc (25 mg/kg) administration showed that the levels of DNA methylation induced by the carcinogen were not significantly different in the two strains, indicating that both are capable of the metabolic activation of MAM to methylating species. Pyrazole, a potent inhibitor of ADH, inhibited MAM metabolism as well as liver DNA methylation in the ADH(+) strain; however similar inhibition of these processes also occurred in the ADH(-) strain. 3-Methylpyrazole, a weak or noninhibitor of ADH, also decreased the levels of MAM metabolism in both the ADH(+) and the ADH(-) strains. From these results, the authors conclude that ADH is not obligatory either in the metabolism or in the metabolic activation of MAM. As a possible alternative to ADH, liver microsomes were examined for their ability to metabolize MAM. In the presence of a NADPH-generating system, liver microsomes from both strains converted (/sup 14/C)MAM to /sup 14/CH3OH and /sup 14/CH2O, although liver microsomes from the ADH(-) strain were more active in this respect. The microsomal metabolism was sensitive to inhibition by CO as well as to inhibition by pyrazole and 3-methylpyrazole.

  19. Some properties of an alcohol dehydrogenase partially purified from baker's yeast grown without added zinc.

    PubMed Central

    Dickenson, C J; Dickinson, F M

    1976-01-01

    Alcohol dehydrogenase was partially purified from yeast (Saccharomyces cerevisiae) grown in the presence of 20 muM-MnSO4 without added Zn2+ and from yeast grown in the presence of 1.8 muM-MnSO4. The enzyme from yeast grown with added Zn2+ has the same properties as the crystalline enzyme from commercial supplies of baker's yeast. The enzyme from yeast grown without added An2+ has quite different properties. It has a mol.wt. in the region of 72000 and an S 20 w of 5.8S. The values can be compared with a mol.wt. of 141000 and an S 20 w of 7.6S for the crystalline enzyme. ADP-ribose, a common impurity in commercial samples of NAD+, is a potent competitive inhibitor of the new enzyme (K1 = 0.5 muM), but is not so for the crystalline enzyme. The observed maximum rate of ethanol oxidation at pH 7.05 and 25 degrees C was decreased 12-fold by the presence of 0.06 mol of inhibitor/mol of NAD+ when using the enzyme from Zn2+-deficient yeast, but with crystalline enzyme the maximum rate was essentially unchanged by this concentration of inhibitor. The kinetic characteristics for the two enzymes with ethanol, butan-1-ol, acetaldehyde and butyraldehyde as substrates are markedly different. These kinetic differences are discussed in relation to the mechanism of catalysis for the enzyme from Zn2+-deficient yeast. PMID:179534

  20. An experimental test for lineage-specific position effects on alcohol dehydrogenase (Adh) genes in Drosophila

    PubMed Central

    Siegal, Mark L.; Hartl, Daniel L.

    1998-01-01

    Independent transgene insertions differ in expression based on their location in the genome; these position effects are of interest because they reflect the influence of genome organization on gene regulation. Position effects also represent potentially insurmountable obstacles to the rigorous functional comparison of homologous genes from different species because (i) quantitative variation in expression of each gene across genomic positions (generalized position effects, or GPEs) may overwhelm differences between the genes of interest, or (ii) divergent genes may be differentially sensitive to position effects, reflecting unique interactions between each gene and its genomic milieu (lineage-specific position effects, or LSPEs). We have investigated both types of position-effect variation by applying our method of transgene coplacement, which allows comparisons of transgenes in the same position in the genome of Drosophila melanogaster. Here we report an experimental test for LSPE in Drosophila. The alcohol dehydrogenase (Adh) genes of D. melanogaster and Drosophila affinidisjuncta differ in both tissue distribution and amounts of ADH activity. Despite this striking regulatory divergence, we found a very high correlation in overall ADH activity between the genes of the two species when placed in the same genomic position as assayed in otherwise Adh-null adults and larvae. These results argue against the influence of LSPE for these sequences, although the effects of GPE are significant. Our new findings validate the coplacement approach and show that it greatly magnifies the power to detect differences in expression between transgenes. Transgene coplacement thus dramatically extends the range of functional and evolutionary questions that can be addressed by transgenic technology. PMID:9861000

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

    PubMed

    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

  2. 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.

  3. General base catalysis in a glutamine for histidine mutant at position 51 of human liver alcohol dehydrogenase.

    PubMed

    Ehrig, T; Hurley, T D; Edenberg, H J; Bosron, W F

    1991-01-29

    On the basis of the three-dimensional structure of horse liver alcohol dehydrogenase determined by X-ray crystallography, His 51 has been proposed to act as a general base during catalysis by abstracting a proton from the alcohol substrate. A hydrogen-bonding system (proton relay system) connecting the alcohol substrate and His 51 has been proposed to mediate proton transfer. We have mutated His 51 to Gln in the homologous human liver beta 1 beta 1 alcohol dehydrogenase isoenzyme which is expected to have a similar proton relay system. The mutation resulted in an about 6-fold drop in V/Kb (Vmax for ethanol oxidation divided by Km for ethanol) at pH 7.0 and a 12-fold drop at pH 6.5. V/Kb could be restored completely or partially by the presence of high concentrations of glycylglycine, glycine, and phosphate buffers. A Brønsted plot of the effect on V/Kb versus the pKa of these bases plus H2O and OH- was linear. Only secondary or tertiary amine buffers differed from linearity, presumably due to steric hindrance. These results suggest that His 51 acts as a general base catalyst during alcohol oxidation in the wild-type enzyme and can be functionally replaced in the mutant enzyme by general base catalysts present in the solvent. Steady-state kinetic constants for NAD+ and the trifluoroethanol inhibition patterns were similar between the wild-type and the mutant enzyme. Differences in the inhibition constants (Ki) of caprate and trifluoroethanol below pH 7.8 and in the pH dependence of Ki can be explained by the substitution of neutral Gln for positively charged His. PMID:1989677

  4. Mössbauer characterization of the Fe-S center in the catalytic metal binding site of alcohol dehydrogenase

    NASA Astrophysics Data System (ADS)

    Haas, C.; Dietrich, H.; Maret, W.; Zeppezauer, M.; Montiel-Montoya, R.; Bill, E.; Trautwein, A. X.

    1986-02-01

    Iron in its divalent and trivalent form can be substituted in the catalytic zinc site of alcohol dehydrogenase from horse liver (HLADH). Fe-HLADH in either oxidation state does not show enzymatic activity in the oxidation of ethanol. Nevertheless, Mössbauer studies of this material are of considerable interest, because they elucidate the effect of pH and coenzyme-induced conformational changes of the protein on the metal ion. It is of importance to note that zinc itself due to the lack of suitable chromophoric and magnetic properties does not provide any comparable information.

  5. 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.

  6. 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

  7. Liposomal encapsulation of yeast alcohol dehydrogenase with cofactor for stabilization of the enzyme structure and activity.

    PubMed

    Yoshimoto, Makoto; Sato, Mami; Yoshimoto, Noriko; Nakao, Katsumi

    2008-01-01

    Yeast alcohol dehydrogenase (YADH) with its cofactor nicotinamide adenine dinucleotide (NAD+) could be stably encapsulated in liposomes composed of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine). The YADH- and NAD+-containing liposomes (YADH-NADL) were 100 nm in mean diameter. The liposomal YADH and NAD+ concentrations were 2.3 mg/mL and 3.9 mM, respectively. A synergistic effect of the liposomal encapsulation and the presence of NAD+ was examined on the thermal stability of YADH at 45 and 50 degrees C. The enzyme stability of the YADH-NADL was compared to the stabilities of the liposomal YADH (YADHL) containing 3.3 mg/mL YADH without NAD+ as well as the free YADH with and without NAD+. Free YADH was increasingly deactivated during its incubation at 45 degrees C for 2 h with decrease of the enzyme concentration from 3.3 to 0.01 mg/mL because of the dissociation of tetrameric YADH into its subunits. At that temperature, the coexistence of free NAD+ at 3.9 mM improved the stability of free YADH at 2.3 mg/mL through forming their thermostable complex, although the stabilization effect of NAD+ was lowered at 50 degrees C. The turbidity measurements for the above free YADH solution with and without NAD+ revealed that the change in the enzyme tertiary structure was much more pronounced at 50 degrees C than at 45 degrees C even in the presence of NAD+. This suggests that YADH was readily deactivated in free solution due to a decrease in the inherent affinity of YADH with NAD+. On the other hand, both liposomal enzyme systems, YADH-NADL and YADHL, showed stabilities at both 45 and 50 degrees C much higher than those of the above free enzyme systems, YADH/NAD+ and YADH. These results imply that the liposome membranes stabilized the enzyme tertiary and thus quaternary structures. Furthermore, the enzyme activity of the YADH-NADL showed a stability higher than that of the YADHL with a more remarkable effect of NAD+ at 50 degrees C than at 45 degrees C. This was

  8. 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

  9. 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

  10. Hydroperoxidic inhibitor of horse liver alcohol dehydrogenase activity, tightly bound to the enzyme-NAD+ complex, characteristically degrades the coenzyme.

    PubMed

    Skurský, L; Rezác, M; Khan, A N; Zídek, L; Rocek, J

    1992-01-01

    The strong inhibition of horse liver alcohol dehydrogenase (HLAD) by p-methylbenzyl hydroperoxide (XyHP) is only transient, XyHP behaves also as a pseudo-substrate of the enzyme and in the presence of NAD+, is degraded by HLAD to (as yet unidentified) non-inhibiting products while the NAD+ is converted to a derivative similar to the "NADX", originally observed in an analogous reaction of HLAD with hydrogen peroxide. The apparent KM for XyHP is approximately 10(4) times smaller than that for H2O2. The catalytic constant kcat for HLAD degradation of XyHP is two orders of magnitude less than that for ethanol dehydrogenation. XyHP inhibits both directions of the alcohol-aldehyde interconversion with equal potency. The first step of the inhibition mechanism is a tight binding of XyHP to the binary HLAD-NAD+ complex. PMID:1284958

  11. Three alcohol dehydrogenase genes and one acetyl-CoA synthetase gene are responsible for ethanol utilization in Yarrowia lipolytica.

    PubMed

    Gatter, Michael; Ottlik, Stephanie; Kövesi, Zsolt; Bauer, Benjamin; Matthäus, Falk; Barth, Gerold

    2016-10-01

    The non-conventional yeast Yarrowia lipolytica is able to utilize a wide range of different substrates like glucose, glycerol, ethanol, acetate, proteins and various hydrophobic molecules. Although most metabolic pathways for the utilization of these substrates have been clarified by now, it was not clear whether ethanol is oxidized by alcohol dehydrogenases or by an alternative oxidation system inside the cell. In order to detect the genes that are required for ethanol utilization in Y. lipolytica, eight alcohol dehydrogenase (ADH) genes and one alcohol oxidase gene (FAO1) have been identified and respective deletion strains were tested for their ability to metabolize ethanol. As a result of this, we found that the availability of ADH1, ADH2 or ADH3 is required for ethanol utilization in Y. lipolytica. A strain with deletions in all three genes is lacking the ability to utilize ethanol as sole carbon source. Although Adh2p showed by far the highest enzyme activity in an in vitro assay, the availability of any of the three genes was sufficient to enable a decent growth. In addition to ADH1, ADH2 and ADH3, an acetyl-CoA synthetase encoding gene (ACS1) was found to be essential for ethanol utilization. As Y. lipolytica is a non-fermenting yeast, it is neither able to grow under anaerobic conditions nor to produce ethanol. To investigate whether Y. lipolytica may produce ethanol, the key genes of alcoholic fermentation in S. cerevisiae, ScADH1 and ScPDC1, were overexpressed in an ADH and an ACS1 deletion strain. However, instead of producing ethanol, the respective strains regained the ability to use ethanol as single carbon source and were still not able to grow under anaerobic conditions. PMID:27486067

  12. Three alcohol dehydrogenase genes and one acetyl-CoA synthetase gene are responsible for ethanol utilization in Yarrowia lipolytica.

    PubMed

    Gatter, Michael; Ottlik, Stephanie; Kövesi, Zsolt; Bauer, Benjamin; Matthäus, Falk; Barth, Gerold

    2016-10-01

    The non-conventional yeast Yarrowia lipolytica is able to utilize a wide range of different substrates like glucose, glycerol, ethanol, acetate, proteins and various hydrophobic molecules. Although most metabolic pathways for the utilization of these substrates have been clarified by now, it was not clear whether ethanol is oxidized by alcohol dehydrogenases or by an alternative oxidation system inside the cell. In order to detect the genes that are required for ethanol utilization in Y. lipolytica, eight alcohol dehydrogenase (ADH) genes and one alcohol oxidase gene (FAO1) have been identified and respective deletion strains were tested for their ability to metabolize ethanol. As a result of this, we found that the availability of ADH1, ADH2 or ADH3 is required for ethanol utilization in Y. lipolytica. A strain with deletions in all three genes is lacking the ability to utilize ethanol as sole carbon source. Although Adh2p showed by far the highest enzyme activity in an in vitro assay, the availability of any of the three genes was sufficient to enable a decent growth. In addition to ADH1, ADH2 and ADH3, an acetyl-CoA synthetase encoding gene (ACS1) was found to be essential for ethanol utilization. As Y. lipolytica is a non-fermenting yeast, it is neither able to grow under anaerobic conditions nor to produce ethanol. To investigate whether Y. lipolytica may produce ethanol, the key genes of alcoholic fermentation in S. cerevisiae, ScADH1 and ScPDC1, were overexpressed in an ADH and an ACS1 deletion strain. However, instead of producing ethanol, the respective strains regained the ability to use ethanol as single carbon source and were still not able to grow under anaerobic conditions.

  13. Retinoic acid response element in the human alcohol dehydrogenase gene ADH3: implications for regulation of retinoic acid synthesis.

    PubMed Central

    Duester, G; Shean, M L; McBride, M S; Stewart, M J

    1991-01-01

    Retinoic acid regulation of one member of the human class I alcohol dehydrogenase (ADH) gene family was demonstrated, suggesting that the retinol dehydrogenase function of ADH may play a regulatory role in the biosynthetic pathway for retinoic acid. Promoter activity of human ADH3, but not ADH1 or ADH2, was shown to be activated by retinoic acid in transient transfection assays of Hep3B human hepatoma cells. Deletion mapping experiments identified a region in the ADH3 promoter located between -328 and -272 bp which confers retinoic acid activation. This region was also demonstrated to confer retinoic acid responsiveness on the ADH1 and ADH2 genes in heterologous promoter fusions. Within a 34-bp stretch, the ADH3 retinoic acid response element (RARE) contains two TGACC motifs and one TGAAC motif, both of which exist in RAREs controlling other genes. A block mutation of the TGACC sequence located at -289 to -285 bp eliminated the retinoic acid response. As assayed by gel shift DNA binding studies, the RARE region (-328 to -272 bp) of ADH3 bound the human retinoic acid receptor beta (RAR beta) and was competed for by DNA containing a RARE present in the gene encoding RAR beta. Since ADH catalyzes the conversion of retinol to retinal, which can be further converted to retinoic acid by aldehyde dehydrogenase, these results suggest that retinoic acid activation of ADH3 constitutes a positive feedback loop regulating retinoic acid synthesis. Images PMID:1996113

  14. Determination of the effects of alcohol dehydrogenase (ADH) 1B and ADH1C polymorphisms on alcohol dependence in Turkey.

    PubMed

    Aktas, Ekin Ozgur; Kocak, Aytaç; Senol, Ender; Celik, Handan Ak; Coskunol, Hakan; Berdeli, Afig; Aydin, Hikmet Hakan

    2012-03-01

    Alcoholism is a complex genetically influenced disorder which refers to alcohol abuse and alcohol dependence. There are controversial results on the role of gene polymorphisms in alcohol dependence in the literature. Differences in population groups and selective inclusion criteria for alcohol dependence may affect results. In this study, we investigated the role of ADH1B Arg48His (rs1229984) and, ADH1C Ile350Val (rs698) gene polymorphisms in Turkish population. 100 healthy volunteers and 75 patients who were admitted to Ege University Alcohol Dependence Unit enrolled in the study. We found significant increase both in ADH1B (Arg48His) polymorphism Arg allele and Arg/Arg genotype frequency in patients. No profound connection between alcohol dependence and ADH1C Ile350Val gene polymorphism was detected. Alcohol dependence is an important health problem that depends on many genetic and environmental factors but we think that it is possible to interpret genetic risk for developing early diagnostic methods and treatment strategies by comprehensive linkage and association studies.

  15. Structure-Guided Engineering of Lactococcus lactis Alcohol Dehydrogenase LlAdhA for Improved Conversion of Isobutyraldehyde to Isobutanol

    PubMed Central

    Liu, Xiang; Bastian, Sabine; Snow, Christopher D.; Brustad, Eric M.; Saleski, Tatyana E.; Xu, Jian-He; Meinhold, Peter; Arnold, Frances H.

    2012-01-01

    We have determined the X-ray crystal structures of the NADH-dependent alcohol dehydrogenase LlAdhA from Lactococcus lactis and its laboratory-evolved variant LlAdhARE1 at 1.9 Å and 2.5 Å resolution, respectively. LlAdhARE1, which contains three amino acid mutations (Y50F, I212T, and L264V), was engineered to increase the microbial production of isobutanol (2-methylpropan-1-ol) from isobutyraldehyde (2-methylpropanal). Structural comparison of LlAdhA and LlAdhARE1 indicates that the enhanced activity on isobutyraldehyde stems from increases in the protein’s active site size, hydrophobicity, and substrate access. Further structure-guided mutagenesis generated a quadruple mutant (Y50F/N110S/I212T/L264V), whose KM for isobutyraldehyde is ~17-fold lower and catalytic efficiency (kcat/KM) is ~160-fold higher than wild-type LlAdhA. Combining detailed structural information and directed evolution, we have achieved significant improvements in non-native alcohol dehydrogenase activity that will facilitate the production of next-generation fuels such as isobutanol from renewable resources. PMID:22974724

  16. 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.

  17. 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

  18. Protein engineering of alcohol dehydrogenase--1. Effects of two amino acid changes in the active site of yeast ADH-1.

    PubMed

    Murali, C; Creaser, E H

    1986-01-01

    One of the promises held out by protein engineering is the ability to alter predictably the properties of an enzyme to enable it to find new substrates or catalyse existing substrates more efficiently, such manipulations being of interest both enzymologically and, potentially, industrially. It has been postulated that in yeast alcohol dehydrogenase (YADH-1) certain amino acids such as Trp 93 and Thr 48 constrict the active site due to their bulky side chains and thus impede catalysis of molecules larger than ethanol. To study effects of enlarging the active site we have made two changes into YADH-1, replacing Trp 93 with Phe and Thr 48 with Ser. Kinetic experiments showed that this enzyme had marked increases in reaction velocity for the n-alcohols propanol, butanol, pentanol, hexanol, heptanol, octanol and cinnamyl alcohol compared to the parent, agreeing with the prediction that expanding the active site should facilitate the oxidation of larger alcohols. The substrate affinities were slightly reduced in the altered enzyme, possibly due to its having reduced hydrophobicity at Phe 93.

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

    PubMed

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

    2012-01-20

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

  20. 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

  1. 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.

  2. Disruption of seven hypothetical aryl alcohol dehydrogenase genes from Saccharomyces cerevisiae and construction of a multiple knock-out strain.

    PubMed

    Delneri, D; Gardner, D C; Bruschi, C V; Oliver, S G

    1999-11-01

    By in silicio analysis, we have discovered that there are seven open reading frames (ORFs) in Saccharomyces cerevisiae whose protein products show a high degree of amino acid sequence similarity to the aryl alcohol dehydrogenase (AAD) of the lignin-degrading fungus Phanerochaete chrysosporium. Yeast cultures grown to stationary phase display a significant aryl alcohol dehydrogenase activity by degrading aromatic aldehydes to the corresponding alcohols. To study the biochemical and the biological role of each of the AAD genes, a series of mutant strains carrying deletion of one or more of the AAD-coding sequences was constructed by PCR-mediated gene replacement, using the readily selectable marker kanMX. The correct targeting of the PCR-generated disruption cassette into the genomic locus was verified by analytical PCR and by pulse-field gel electrophoresis (PFGE) followed by Southern blot analysis. Double, triple and quadruple mutant strains were obtained by classical genetic methods, while the construction of the quintuple, sextuple and septuple mutants was achieved by using the marker URA3 from Kluyveromyces lactis, HIS3 from Schizosaccharomyces pombe and TRP1 from S. cerevisiae. None of the knock-out strains revealed any mutant phenotype when tested for the degradation of aromatic aldehydes using both spectrophotometry and high performance liquid chromatography (HPLC). Specific tests for changes in the ergosterol and phospholipids profiles did not reveal any mutant phenotype and mating and sporulation efficiencies were not affected in the septuple deletant. Compared to the wild-type strain, the septuple deletant showed an increased resistance to the anisaldehyde, but there is a possibility that the nutritional markers used for gene replacement are causing this effect.

  3. 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.

  4. Interaction of alcohol dehydrogenase with tert-butylhydroperoxide: stimulation of the horse liver and inhibition of the yeast enzymes.

    PubMed

    Tkachenko, A G; Winston, G W

    2000-08-01

    Preincubation of horse liver alcohol dehydrogenase (HLADH) with the oxidative agent, tert-butyl hydroperoxide (tBOOH) results in a twofold stimulation of the ethanol dehydrogenase activity of this enzyme. This stimulation was dependent on tBOOH concentration up to 100 mM; above this concentration tBOOH did not further stimulate ethanol oxidation by HLADH. Active-site-directed reagents and classical ADH binary complexes were used to probe the possible mechanism of this activating effect. The rate and extent of stimulation by tBOOH is strongly reduced by binary complexes with NAD(+) or NADH, whose pyrophosphate groups bind to Arg-47 and Arg-369. In contrast stimulation by tBOOH was not prevented by AMP or the sulfhydryl reagents dithiothreitol and glutathione, suggesting, respectively, a lack of role for Lys-228 and sulfhydryl group oxidation in the stimulation by tBOOH. In contrast to the liver enzyme, treatment of yeast ADH (YADH) with tBOOH irreversibly inhibited its ethanol dehydrogenase activity. Inhibition of YADH by tBOOH approximated first-order rate kinetics with respect to enzyme at fixed concentrations of tBOOH between 0.5 to 300 mM. Four -SH groups per molecule of YADH were modified by tBOOH, whereas only two -SH groups were modified in HLADH. The stimulation of HLADH by tBOOH is suggested to be due to destabilization of the catalytic Zn-coordination sphere and amino acids associated with coenzyme binding in the active site, while inactivation of YADH appears to be associated with -SH group oxidation by the peroxide.

  5. Polyphosphazenes as tunable and recyclable supports to immobilize alcohol dehydrogenases and lipases: synthesis, catalytic activity, and recycling efficiency.

    PubMed

    Cuetos, Aníbal; Valenzuela, María L; Lavandera, Iván; Gotor, Vicente; Carriedo, Gabino A

    2010-05-10

    The polyphosphazene {NP[O(2)C(12)H(7.5)(NH(2))(0.5)]}(n), prepared by reacting {NP[O(2)C(12)H(7.5)(NO(2))(0.5)]} with the Lalancette's reagent, was used for attaching enzymes such as alcohol dehydrogenase (ADH-A) and lipase (CAL-B). The resulting new biocatalysts exhibited great potential as tunable supports for enzymatic reactions in both aqueous and organic media. The material with immobilized ADH-A was as efficient as the commercial enzyme to perform stereoselective bioreductions of ketones in aqueous solutions and could be used for the reduction of various aliphatic and aromatic ketones up to 60 degrees C and recycled several times without significant loss of activity even after three months of storage. The biocatalyst obtained with CAL-B was more efficient than the free enzyme for kinetic resolutions in organic solvents and exhibited a moderately good capability of reutilization. PMID:20359182

  6. Comparison of fluorescence properties of wild type and the W15F mutant of horse liver alcohol dehydrogenase

    NASA Astrophysics Data System (ADS)

    Eftink, Maurice R.; Wong, Cing-Yuen; Park, Doo-Hong; Shearer, Gretchen L.; Plapp, Bryce V.

    1994-08-01

    Horse liver alcohol dehydrogenase is a homodimeric protein; each subunit has two tryptophan residues that are in distinctly different microenvironments. Trp-15 is located on the surface and Trp-314 is buried at the intersubunit interface. Steady-state and time-resolved fluorescence and phosphorescence studies have enabled the assignment of parameters, e.g., quantum yield, emission maximum, decay times, to the individual tryptophan residues of the protein. We have prepared, by site-directed mutagenesis, the mutated W15F protein and have characterized its fluorescence properties. We show that the Trp-314 of the mutant experiences an apolar microenvironment, but that the fluorescence decay and exposure to solute quenchers of the mutant are somewhat different than was expected from the assignments for the wild type.

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

    PubMed

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

    2013-01-16

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

  8. Cloning and sequencing of the gene cluster encoding two subunits of membrane-bound alcohol dehydrogenase from Acetobacter polyoxogenes.

    PubMed

    Tamaki, T; Fukaya, M; Takemura, H; Tayama, K; Okumura, H; Kawamura, Y; Nishiyama, M; Horinouchi, S; Beppu, T

    1991-02-16

    The membrane-bound alcohol dehydrogenase (ADH) from Acetobacter polyoxogenes NBI1028 is composed of a 72 kDa subunit and a 44 kDa cytochrome c subunit. The amino acid sequences of the two regions of the 72 kDa subunit were determined to prepare oligonucleotides for the purpose of amplification of a DNA fragment corresponding to the intermediate region by the polymerase chain reaction. A 0.5 kb DNA fragment thus amplified was used as the probe to clone a 7.0 kb PstI fragment coding for the whole 72 kDa subunit. Nucleotide sequencing and immunoblot analysis revealed that the cloned fragment contained the full structural genes for the 72 kDa and the 44 kDa subunits and they were clustered with the same transcription polarity. The predicted amino acid sequence of the gene for the 72 kDa subunit showed homology with that of the 72 kDa subunit from ADH of A. aceti and those of methanol dehydrogenase from methylotrophic bacteria. The 72 and 44 kDa subunits contained one and three typical haem binding sequences, respectively.

  9. 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.

  10. 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

  11. Proteomic comparison of Entamoeba histolytica and Entamoeba dispar and the role of E. histolytica alcohol dehydrogenase 3 in virulence.

    PubMed

    Davis, Paul H; Chen, Minghe; Zhang, Xiaochun; Clark, C Graham; Townsend, R Reid; Stanley, Samuel L

    2009-01-01

    The protozoan intestinal parasite Entamoeba histolytica infects millions of people worldwide and is capable of causing amebic dysentery and amebic liver abscess. The closely related species Entamoeba dispar colonizes many more individuals, but this organism does not induce disease. To identify molecular differences between these two organisms that may account for their differential ability to cause disease in humans, we used two-dimensional gel-based (DIGE) proteomic analysis to compare whole cell lysates of E. histolytica and E. dispar. We observed 141 spots expressed at a substantially (>5-fold) higher level in E. histolytica HM-1:IMSS than E. dispar and 189 spots showing the opposite pattern. Strikingly, 3 of 4 proteins consistently identified as different at a greater than 5-fold level between E. histolytica HM-1:IMSS and E. dispar were identical to proteins recently identified as differentially expressed between E. histolytica HM-1:IMSS and the reduced virulence strain E. histolytica Rahman. One of these was E. histolytica alcohol dehydrogenase 3 (EhADH3). We found that E. histolytica possesses a higher level of NADP-dependent alcohol dehydrogenase activity than E. dispar and that some EhADH3 can be localized to the surface of E. histolytica. Episomal overexpression of EhADH3 in E. histolytica trophozoites resulted in only subtle phenotypic differences in E. histolytica virulence in animal models of amebic colitis and amebic liver abscess, making it difficult to directly link EhADH3 levels to virulence differences between E. histolytica and less-pathogenic Entamoeba.

  12. Fourier transform infrared spectroscopic studies of proton transfer processes and the dissociation of Zn2+-bound water in alcohol dehydrogenases.

    PubMed

    Nadolny, C; Zundel, G

    1997-08-01

    The following complexes were investigated by Fourier transform difference spectroscopy: binary complexes of alcohol dehydrogenases from yeast (YADH) and horse liver (LADH) with nicotinamide adenine dinucleotide (NAD+) and adenosine (5')-diphospho(5)-beta-D-ribose (ADP-Rib); the binary complex of Zn2+-free YADH with NAD+, the ternary complex of LADH with NAD+ and 2,2,2-trifluoroethanol. After addition of NAD+ to YADH and LADH, protonation of the N1 atom of the adenine ring of NAD+ is observed. It is shown that this proton arises from the dissociation of the Zn2+-bound water. The interaction of the Zn2+ ion with water is very strong, since this interaction is not just an electrostatic interaction. If the Zn2+ ions are in a tetrahedral environment, a large covalent contribution also occurs. If ADP-Rib is present instead of NAD+, no protonation of the N1 atom of the adenine ring of ADP-Rib is found, which demonstrates that the positively charged nicotinamide ring favors the conduction of the positive charge. All these results confirm the mechanism of Brändén et al. (1975): the Zn2+-bound water is split and the arising (OH)- deprotonates the alcohol. In the case of the ternary complex of LADH with NAD+ and 2,2,2-trifluoroethanol, we demonstrate that the alcohol is deprotonated and the alcoholate ion is bound directly to the Zn2+ ion. The conduction of the proton from the active site to the N1 atom of adenine occurs via a hydrogen-bonded chain with large proton polarizability due to collective proton motion. The nature and mechanism of this pathway are discussed on the basis of data from previous studies.

  13. Influence of gender on ethanol-induced ventricular myocyte contractile depression in transgenic mice with cardiac overexpression of alcohol dehydrogenase.

    PubMed

    Duan, Jinhong; Esberg, Lucy B; Ye, Gang; Borgerding, Anthony J; Ren, Bonnie H; Aberle, Nicholas S; Epstein, Paul N; Ren, Jun

    2003-03-01

    Acute ethanol exposure depresses ventricular contractility and contributes to alcoholic cardiomyopathy in both men and women chronically consuming ethanol. However, a gender-related difference in the severity of myopathy exists with female being more sensitive to ethanol-induced tissue damage. Acetaldehyde (ACA), the major oxidized product of ethanol, has been implicated to play a role in the pathogenesis and gender-related difference of alcoholic cardiomyopathy, possibly due to its direct cardiac effect and interaction with estrogen. This study was designed to compare the effects of cardiac overexpression of alcohol dehydrogenase (ADH), which converts ethanol into ACA, on the cardiac contractile response to ethanol in ventricular myocytes isolated from age-matched adult male and female transgenic (ADH) and wild-type (FVB) mice. Mechanical properties were measured with an IonOptix SoftEdge system. ACA production was assessed by gas chromatography. The ADH myocytes from both genders exhibited similar mechanical properties but a higher efficacy to produce ACA compared to FVB myocytes. Exposure to ethanol (80-640 mg/dl) for 60 min elicited concentration-dependent decrease of cell shortening in both FVB and ADH groups. The ethanol-induced depression on cell shortening was significantly augmented in female but not male ADH group. ADH transgene did not exacerbate the ethanol-induced inhibition of maximal velocity of shortening/relengthening in either gender. In addition, neither ethanol nor ADH transgene affect the duration of shortening and relengthening in male or female mice. These data suggest that females may be more sensitive to ACA-induced cardiac contractile depression than male, which may attribute to the gender-related difference of alcoholic cardiomyopathy.

  14. 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

  15. 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.

  16. 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.

  17. The Bifunctional Alcohol and Aldehyde Dehydrogenase Gene, adhE, Is Necessary for Ethanol Production in Clostridium thermocellum and Thermoanaerobacterium saccharolyticum

    PubMed Central

    Lo, Jonathan; Zheng, Tianyong; Hon, Shuen; Olson, Daniel G.

    2015-01-01

    ABSTRACT Thermoanaerobacterium saccharolyticum and Clostridium thermocellum are anaerobic thermophilic bacteria being investigated for their ability to produce biofuels from plant biomass. The bifunctional alcohol and aldehyde dehydrogenase gene, adhE, is present in these bacteria and has been known to be important for ethanol formation in other anaerobic alcohol producers. This study explores the inactivation of the adhE gene in C. thermocellum and T. saccharolyticum. Deletion of adhE reduced ethanol production by >95% in both T. saccharolyticum and C. thermocellum, confirming that adhE is necessary for ethanol formation in both organisms. In both adhE deletion strains, fermentation products shifted from ethanol to lactate production and resulted in lower cell density and longer time to reach maximal cell density. In T. saccharolyticum, the adhE deletion strain lost >85% of alcohol dehydrogenase (ADH) activity. Aldehyde dehydrogenase (ALDH) activity did not appear to be affected, although ALDH activity was low in cell extracts. Adding ubiquinone-0 to the ALDH assay increased activity in the T. saccharolyticum parent strain but did not increase activity in the adhE deletion strain, suggesting that ALDH activity was inhibited. In C. thermocellum, the adhE deletion strain lost >90% of ALDH and ADH activity in cell extracts. The C. thermocellum adhE deletion strain contained a point mutation in the lactate dehydrogenase gene, which appears to deregulate its activation by fructose 1,6-bisphosphate, leading to constitutive activation of lactate dehydrogenase. IMPORTANCE Thermoanaerobacterium saccharolyticum and Clostridium thermocellum are bacteria that have been investigated for their ability to produce biofuels from plant biomass. They have been engineered to produce higher yields of ethanol, yet questions remain about the enzymes responsible for ethanol formation in these bacteria. The genomes of these bacteria encode multiple predicted aldehyde and alcohol

  18. Elevated glutathione level does not protect against chronic alcohol mediated apoptosis in recombinant human hepatoma cell line VL-17A over-expressing alcohol metabolizing enzymes--alcohol dehydrogenase and Cytochrome P450 2E1.

    PubMed

    Chandrasekaran, Karthikeyan; Swaminathan, Kavitha; Kumar, S Mathan; Chatterjee, Suvro; Clemens, Dahn L; Dey, Aparajita

    2011-06-01

    Chronic consumption of alcohol leads to liver injury. Ethanol-inducible Cytochrome P450 2E1 (CYP2E1) plays a critical role in alcohol mediated oxidative stress due to its ability to metabolize ethanol. In the present study, using the recombinant human hepatoma cell line VL-17A that over-expresses the alcohol metabolizing enzymes-alcohol dehydrogenase (ADH) and CYP2E1; and control HepG2 cells, the mechanism and mode of cell death due to chronic ethanol exposure were studied. Untreated VL-17A cells exhibited apoptosis and oxidative stress when compared with untreated HepG2 cells. Chronic alcohol exposure, i.e., 100 mM ethanol treatment for 72 h caused a significant decrease in viability (47%) in VL-17A cells but not in HepG2 cells. Chronic ethanol mediated cell death in VL-17A cells was predominantly apoptotic, with increased oxidative stress as the underlying mechanism. Chronic ethanol exposure of VL-17A cells resulted in 1.1- to 2.5-fold increased levels of ADH and CYP2E1. Interestingly, the level of the antioxidant GSH was found to be 3-fold upregulated in VL-17A cells treated with ethanol, which may be a metabolic adaptation to the persistent and overwhelming oxidative stress. In conclusion, the increased GSH level may not be sufficient enough to protect VL-17A cells from chronic alcohol mediated oxidative stress and resultant apoptosis. PMID:21414402

  19. A unique enzyme of acetic acid bacteria, PQQ-dependent alcohol dehydrogenase, is also present in Frateuria aurantia.

    PubMed

    Trček, Janja; Matsushita, Kazunobu

    2013-08-01

    A membrane-bound, pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenase (ADH) was purified from Frateuria aurantia LMG 1558(T). Although F. aurantia belongs to a group of γ-Proteobacteria, the characteristics of its PQQ-ADH were similar to the enzyme characteristics of the typical high-acetic acid-resistant bacterium Gluconacetobacter europaeus from the group of α-Proteobacteria. The PQQ-dependent ADH was solubilized from the membranes and purified after anionic, cationic, and affinity chromatography with specific activity of 117 U/mg. The purified enzyme was estimated to be composed of two subunits of ca. 72 and 45 kDa, as judged by SDS-polyacrylamide gel electrophoresis. The purified enzyme had maximum activity at pH 4.5 and showed the highest substrate specificity to ethanol, isoamyl alcohol, 1-butanol, and 1-propanol. The deduced sequences of cloned genes adhA and adhB encoding subunits I and II of PQQ-ADH showed 80 % amino acid (AA) identity to AdhA and 68 % AA identity to AdhB of Ga. europaeus V3 (LMG 18494). Because of the high similarity between genes encoding subunits I and II of PQQ-ADH and its homologous genes found in a distantly related taxonomic group of acetic acid bacteria, the results suggest the possibility of horizontal gene transfer between these two groups of genera.

  20. 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.

  1. 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. PMID:26329849

  2. 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.

  3. 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.

  4. Uniform expression of alcohol dehydrogenase 3 in epithelia regenerated with cultured normal, immortalised and malignant human oral keratinocytes.

    PubMed

    Hedberg, J J; Hansson, A; Nilsson, J A; Höög, J O; Grafström, R C

    2001-01-01

    The human oral epithelium is a target for damage from the inhalation of formaldehyde. However, most experimental studies on this chemical have relied on laboratory animals that are obligatory nose breathers, including rats and mice. Therefore, in vitro model systems that mimic the structure of the human oral epithelium and which retain normal tissue-specific metabolic competence are desirable. Based on the established role of alcohol dehydrogenase 3 (ADH3), also known as glutathione-dependent formaldehyde dehydrogenase, as the primary enzyme catalysing the detoxification of formaldehyde, the aim of this study was to investigate the expression of ADH3 in organotypic epithelia regenerated with normal (NOK), immortalised (SVpgC2a) and malignant (SqCC/Y1) human oral keratinocytes. Organotypic epithelia, usually consisting of 5-10 cell layers, were produced at the air-liquid interface of collagen gels containing human oral fibroblasts, after culture for 10 days in a standardised serum-free medium. Immunochemical staining demonstrated uniform expression of ADH3 in these organotypic epithelia, as well as in the epithelial cells of oral tissue. The specificity of the ADH3 antiserum was ascertained from the complete neutralisation of the immunochemical reaction with purified ADH3 protein. Assessment of the staining intensities indicated that the expression levels were similar among the regenerated epithelia. Furthermore, the regenerated epithelia showed similar ADH3 expression to the epithelium in oral tissue. Therefore, a tissue-like expression pattern for ADH3 can be generated from the culture of various oral keratinocyte lines in an organotypic state. Similar expression levels among the various cell lines indicate the preservation of ADH3 during malignant transformation, and therefore that NOK, SVpgC2a and SqCC/Y1 represent functional models for in vitro studies of formaldehyde metabolism in human oral mucosa.

  5. Cloning and expression of a putative alcohol dehydrogenase gene of Entamoeba histolytica and its application to immunological examination.

    PubMed Central

    Kimura, A; Hara, Y; Kimoto, T; Okuno, Y; Minekawa, Y; Nakabayashi, T

    1996-01-01

    To clone and express the genes encoding major antigens of Entamoeba histolytica, we constructed a lambda gt11 cDNA library for E. histolytica HM1:IMSS and screened it with pooled sera from patients with amoebiasis. A 1,223-bp cDNA was cloned (clone 1223), and its nucleotide sequence was determined. The amino acid sequence predicted to be encoded by the open reading frame of clone 1223 consisted of 396 residues and showed 32.5 and 32.3% homology to the NADH-dependent butanol dehydrogenases I and II (bdhA and bdhB) of Clostridium acetobutylicum, respectively. In addition, 29 of the 34 consensus positions of bdhA and bdhB were also well conserved in clone 1223. The recombinant protein expressed from clone 1223 had an estimated molecular mass of 43.5 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The antigenicity and specificity of the recombinant protein were evaluated by an enzyme-linked immunosorbent assay using sera obtained from two clinical groups of patients with amoebiasis and a group of healthy controls. The recombinant protein had potent and specific antigenicity. In all, 53 serum samples (88.3%) from 60 patients with amoebiasis were positive for immunoglobulin G antibody against the recombinant protein, with a mean optical density value of 0.42. In contrast, 53 of 54 healthy control serum samples were negative, with only 1 positive serum sample showing the lower optical density value. These results suggested that clone 1223 is promising in terms of providing a useful antigen for the accurate serodiagnosis of amoebiasis and that the gene encodes a putative alcohol dehydrogenase of E. histolytica. PMID:8705667

  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. Impact of multiple Alcohol Dehydrogenase gene polymorphisms on risk of laryngeal, esophageal, gastric and colorectal cancers in Chinese Han population

    PubMed Central

    An, Jiaze; Zhao, Junsheng; Zhang, Xiyang; Ding, Rui; Geng, Tingting; Feng, Tian; Jin, Tianbo

    2015-01-01

    Alcohol intake is positively associated with the risk of upper aerodigestive tract (UADT) cancers; but its effect on gastric or colorectal cancer is controversial. Previous study had identified several single nucleotide polymorphisms (SNPs) of Alcohol Dehydrogenase (ADH) genes associated with UADT cancers in European and Japanese populations. We sought to determine if these SNPs associated with laryngeal, esophageal, gastric or colorectal cancer in Chinese population. We conducted a case-control study among 1577 cases and 1013 healthy controls from northwest China. Five SNPs associated with UADT cancers risk were selected from previous genome-wide association studies and genotyped using Sequenom Mass-ARRAY technology. Odds ratios and 95% confidence intervals (CIs) were calculated by unconditional logistic regression adjusting for age and gender. We identified that the minor alleles of rs1789924 and rs971074 were associated with decreased risk of laryngeal cancer (OR = 0.311; 95% CI = 0.161-0.602; P < 0.001) and esophagus cancer (OR = 0.711; 95% CI = 0.526-0.962; P = 0.027) in allelic model analysis, respectively. In the genetic model analysis, we found the “C/T” genotype of rs1789924 was associated with decreased laryngeal cancer risk in codominant model (P = 0.046) and overdominant model (P = 0.013); the “C/T-T/T” genotype of rs1789924 was associated with reduced risk of laryngeal cancer under dominant model (P = 0.013). Additionally, none of the SNPs was associated with gastric or colorectal cancer in our study. Our data shed new light on the association between ADH SNPs and respiratory and digestive tract cancers susceptibility in the Han Chinese population. PMID:26396927

  8. Effects of water activity and aqueous solvent ordering on thermal stability of lysozyme, alpha-chymotrypsinogen A, and alcohol dehydrogenase.

    PubMed

    Matsue, S; Fujii, T; Miyawaki, O

    2001-06-12

    Effects of water activity (aW) and solvent ordering were separately analyzed on the thermal unfolding of lysozyme and alpha-chymotrypsinogen A, and also on the thermal deactivation of yeast alcohol dehydrogenase (YADH) in aqueous solutions with various additives. With the coexistence of additives, water activity was the determinant of the extent of the change in the thermal stability of proteins while solvent ordering was the determinant of the direction of the change. The parameter alpha, determined from the activity coefficient of water, representing the deviation of aW from that of the ideal solution, was useful as a quantitative index of the solvent ordering showing good correlations with the unfolding temperature and enthalpy of lysozyme and alpha-chymotrypsinogen A and also with the thermal deactivation rate constant of YADH at a constant aW. Solvent ordering seemed to affect the thermal stability of proteins mainly through its effect on the intramolecular hydrophobic interaction among amino acid residues in a protein molecule but the contribution of the electrostatic interaction including hydrogen bonding through the change in permittivity of solution was also suggested.

  9. Pyrene excimer fluorescence of yeast alcohol dehydrogenase: a sensitive probe to investigate ligand binding and unfolding pathway of the enzyme.

    PubMed

    Santra, Manas Kumar; Dasgupta, Debjani; Panda, Dulal

    2006-01-01

    The cysteine residues of yeast alcohol dehydrogenase (YADH) were covalently modified by N-(1-pyrenyl) maleimide (PM). A maximum of 3.4 cysteines per YADH monomer could be modified by PM. The secondary structure of PM-YADH was found to be similar to that of the native YADH using far-UV circular dichroism. The covalent modification of YADH by PM inhibited the enzymatic activity indicating that the active site of the enzyme was altered. PM-YADH displayed maximum excimer fluorescence at an incorporation ratio of 2.6 mol of PM per monomeric subunit of YADH. Nucleotide adenine dinucleotide (NAD) divalent zinc and ethanol reduced the excimer fluorescence of PM-YADH indicating that these agents induce conformational changes in the enzyme. Guanidinium hydrochloride (GdnHCl)-induced unfolding of YADH was analyzed using tryptophan fluorescence, pyrene excimer fluorescence and enzymatic activity. The unfolding of YADH was found to occur in a stepwise manner. The loss of enzymatic activity preceded the global unfolding of the protein. Further, changes in tryptophan fluorescence with increasing GdnHCl suggested that YADH was completely unfolded by 2.5 M GdnHCl. Interestingly, residual structures of YADH were detected even in the presence of 5 M GdnHCl using the excimer fluorescence of PM-YADH.

  10. 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.

  11. Alkaline unfolding and salt-induced folding of yeast alcohol dehydrogenase under high pH conditions.

    PubMed

    Le, W P; Yan, S X; Li, S; Zhong, H N; Zhou, H M

    1996-06-01

    The conformational changes of yeast alcohol dehydrogenase during unfolding at alkaline pH have been followed by fluorescence emission and circular dichroism spectra. A result of comparison of inactivation and conformational changes shows that much lower values of alkaline pH are required to bring about inactivation than significant conformational change of the enzyme molecule. At pH 9.5, although the enzyme has been completely inactivated, no marked conformational changes can be observed. Even at pH 12, the apparently fully unfolded enzyme retains some ordered secondary structure. After removal of Zn2+ from the enzyme molecule, the conformational stability decreased. At pH 12 by adding the salt, the relatively unfolded state of denatured enzyme changes into a compact conformational state by hydrophobic collapsing. Folded states induced by salt bound ANS strongly, indicating the existence of increased hydrophobic surface. More extensive studies showed that although apo-YADH and holo-YADH exhibited similar behavior, the folding cooperative ability of apo-enzyme was lower than that of holo-enzyme. The above results suggest that the zinc ion plays an important role in helping the folding of YADH and in stabilizing its native conformation.

  12. 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.

  13. 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.

  14. Fast quantification of ethanol in whole blood specimens by the enzymatic alcohol dehydrogenase method. Optimization by experimental design.

    PubMed

    Kristoffersen, Lena; Skuterud, Bjørn; Larssen, Bente R; Skurtveit, Svetlana; Smith-Kielland, Anne

    2005-01-01

    A sensitive, fast, simple, and high-throughput enzymatic method for the quantification of ethanol in whole blood (blood) on Hitachi 917 is presented. Alcohol dehydrogenase (ADH) oxidizes ethanol to acetaldehyde using the coenzyme nicotinamide adenine dinucleotide (NAD), which is concurrently reduced to form NADH. Method development was performed with the aid of factorial design, varying pH, and concentrations of NAD+ and ADH. The linear range increased and reaction end point decreased with increasing NAD+ concentration and pH. The method was linear in the concentration range 0.0024-0.4220 g/dL. The limits of detection and quantification were 0.0007 g/dL and 0.0024 g/dL, respectively. Relative standard deviations for the repeatability and within-laboratory reproducibility were in the ranges 0.7-5.7% and 1.6-8.9%, respectively. The correlation coefficient when compared with headspace gas chromatography-flame ionization detection methods was 0.9903. Analysis of authentic positive blood specimens gave results that were slightly lower than those of the reference method.

  15. Promoter Strength Comparisons of Maize Shrunken 1 and Alcohol Dehydrogenase 1 and 2 Promoters in Mono- and Dicotyledonous Species 1

    PubMed Central

    Hauptmann, R. M.; Ashraf, M.; Vasil, V.; Hannah, L. C.; Vasil, I. K.; Ferl, R.

    1988-01-01

    Promoter strengths of two maize alcohol dehydrogenase genes, Adh1 and Adh2, and the maize shrunken-1 gene, Sh1, were evaluated by transient expression in cultured protoplasts of Panicum maximum, Triticum monococcum, and Daucus carota. Promoter elements were ligated in correct and opposite orientations as transcriptional gene fusions to the chloramphenicol acetyl transferase gene containing the nopaline synthase 3′ polyadenylation signal. The relative levels of gene expression were compared to the cauliflower mosaic virus 35S promoter. The full length Adh1 promoter (−1100 to +15) functioned in all species, but at a reduced level in D. carota. An Adh1 promoter deletion from −304 to −1100 did not express at detectable levels in any species nor did the Sh1 promoter construction. The Adh2 promoter (−860 to +90) only expressed in D. carota. The full length Adh1 promoter gave the highest level of CAT expression in the monocot cells but at levels which were approximately 30% compared to the CaMV 35S promoter. This was reduced further in D. carota to approximately 4%. These data suggest that at least some of the regulatory factors responsible for promoter function are somewhat species specific and that these differences should be considered in gene expression studies. Images Fig. 2 PMID:16666422

  16. Surface functionalization of chitosan-coated magnetic nanoparticles for covalent immobilization of yeast alcohol dehydrogenase from Saccharomyces cerevisiae

    NASA Astrophysics Data System (ADS)

    Li, Gui-yin; Zhou, Zhi-de; Li, Yuan-jian; Huang, Ke-long; Zhong, Ming

    2010-12-01

    A novel and efficient immobilization of yeast alcohol dehydrogenase (YADH, EC1.1.1.1) from Saccharomyces cerevisiae has been developed by using the surface functionalization of chitosan-coated magnetic nanoparticles (Fe 3O 4/KCTS) as support. The magnetic Fe 3O 4/KCTS nanoparticles were prepared by binding chitosan alpha-ketoglutaric acid (KCTS) onto the surface of magnetic Fe 3O 4 nanoparticles. Later, covalent immobilization of YADH was attempted onto the Fe 3O 4/KCTS nanoparticles. The effect of various preparation conditions on the immobilized YADH process such as immobilization time, enzyme concentration and pH was investigated. The influence of pH and temperature on the activity of the free and immobilized YADH using phenylglyoxylic acid as substrate has also been studied. The optimum reaction temperature and pH value for the enzymatic conversion catalyzed by the immobilized YADH were 30 °C and 7.4, respectively. Compared to the free enzyme, the immobilized YADH retained 65% of its original activity and exhibited significant thermal stability and good durability.

  17. 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

  18. Genetic and Biochemical Basis of Enzyme Activity Variation in Natural Populations. I. Alcohol Dehydrogenase in DROSOPHILA MELANOGASTER

    PubMed Central

    McDonald, John F.; Ayala, Francisco J.

    1978-01-01

    Recent studies by various authors suggest that variation in gene regulation may be common in nature, and might be of great evolutionary consequence; but the ascertainment of variation in gene regulation has proven to be a difficult problem. In this study, we explore this problem by measuring alcohol dehydrogenase (ADH) activity in Drosophila melanogaster strains homozygous for various combinations of given second and third chromosomes sampled from a natural population. The structural locus (Adh) coding for ADH is on the second chromosome. The results show that: (1) there are genes, other than Adh, that affect the levels of ADH activity; (2) at least some of these "regulatory" genes are located on the third chromosome, and thus are not adjacent to the Adh locus; (3) variation exists in natural populations for such regulatory genes; (4) the effect of these regulatory genes varies as they interact with different second chromosomes; (5) third chromosomes with high-activity genes are either partially or completely dominant over chromosomes with low-activity genes; (6) the effects of the regulatory genes are pervasive throughout development; and (7) the third chromosome genes regulate the levels of ADH activity by affecting the number of ADH molecules in the flies. The results are consistent with the view that the evolution of regulatory genes may play an important role in adaptation. PMID:97168

  19. 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.

  20. The alcohol dehydrogenase gene adh1 is induced in Aspergillus flavus grown on medium conducive to aflatoxin biosynthesis.

    PubMed Central

    Woloshuk, C P; Payne, G A

    1994-01-01

    An Aspergillus flavus cDNA library was screened by differential hybridization to isolate clones corresponding to genes that are actively transcribed under culture conditions conducive to aflatoxin biosynthesis. One clone with a 1.28-kb insert was isolated, and its nucleotide sequence was determined. The nucleotide sequence of this clone had 75% DNA identity to those of the alcohol dehydrogenase genes from Aspergillus nidulans, and the putative polypeptide translated from the cDNA sequence had 82% similarity with the amino acid sequences of the A. nidulans proteins. Thus, this gene has been designated adh1. Southern hybridization analysis of genomic DNA from A. flavus indicated that there was one copy of the adh1 gene. Northern (RNA) hybridization analysis indicated that the adh1 transcript accumulated in culture medium conducive to aflatoxin production and the timing of accumulation of adh1 transcripts was similar to that for aflatoxin. Fusion of the promoter region of adh1 to a beta-glucuronidase reporter gene indicated that accumulation of the adh1 transcript was the result of transcriptional activation. These molecular data support previous physiological evidence that showed the importance of carbohydrate metabolism during aflatoxin biosynthesis. Images PMID:8135521

  1. 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.

  2. 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.

  3. 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

  4. 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

  5. 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

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

    PubMed

    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

  7. The kinetics behavior of the reduction of formaldehyde catalyzed by Alcohol Dehydrogenase (ADH) and partial uncompetitive substrate inhibition by NADH.

    PubMed

    Wen, Nuan; Liu, Wenfang; Hou, Yanhui; Zhao, Zhiping

    2013-05-01

    Alcohol dehydrogenase (ADH) catalyzes the final step in the biosynthesis of methanol from CO2. Here, we report the steady-state kinetics for ADH, using a homogeneous enzyme preparation with formaldehyde as the substrate and nicotinamide adenine dinucleotide (NADH) as the cofactor. When changing NADH concentrations with the fixed concentrations of HCHO (more or less than NADH), kinetic studies revealed a particular zigzag phenomenon for the first time. Increasing formaldehyde concentration can weaken substrate inhibition and improve catalytic efficiency. The kinetic mechanism of ADH was analyzed using the secondary fitting method. The double reciprocal plots (1/v∼1/[HCHO] and 1/[NADH]) strongly demonstrated that the substrate inhibition by NADH was uncompetitive versus formaldehyde and partial. In the direction of formaldehyde reduction, ADH has an ordered kinetic mechanism with formaldehyde adding to enzyme first and product methanol released last. The second reactant NADH can combine with the enzyme-methanol complex and then methanol dissociates from it at a slower rate than from enzyme-methanol. The reaction velocity depends on the relative rates of the alternative pathways. The addition of NADH also accelerates the releasing of methanol. As a result, substrate inhibition and activation occurred intermittently, and the zigzag double reciprocal plot (1/v∼1/[NADH]) was obtained.

  8. 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. PMID:26333028

  9. Performance and value of CAD-deficient pine- Final Report

    SciTech Connect

    Bailian Li; Houmin Chang; Hasan Jameel

    2007-02-28

    The southern US produces 58% of the nation's timber, much of it grown in intensively managed plantations of genetically improved loblolly pine. One of the fastest-growing loblolly pine selections made by the NCSU-Industry Cooperative Tree Improvement Program, whose progeny are widely planted, is also the only known natural carrier of a rare gene, cadn1. This allele codes for deficiency in an enzyme, cinnamyl alcohol dehydrogenase, which catalyzes the last step in the biosynthesis of lignin precursors. This study is to characterize this candidate gene for marker-assisted selection and deployment in the breeding program. This research will enhance the sustainability of forest production in the South, where land-use pressures will limit the total area available in the future for intensively managed plantations. Furthermore, this research will provide information to establish higher-value plantation forests with more desirable wood/fiber quality traits. A rare mutant allele (cad-n1) of the cad gene in loblolly pine (Pinus taeda L.) causes a deficiency in the production of cinnamyl alcohol dehydrogenase (CAD). The effects of this allele were examined by comparing wood density and growth traits of cad-n1 heterozygous trees with those of wild-type trees in a 10-year-old open-pollinated family trial growing under two levels of fertilization in Scotland County, North Carolina. In all, 200 trees were sampled with 100 trees for each treatment. Wood density measurements were collected from wood cores at breast height using x-ray densitometry. We found that the substitution of cad-n1 for a wild-type allele (Cad) was associated with a significant effect on wood density. The cad-n1 heterozygotes had a significantly higher wood density (+2.6%) compared to wild-type trees. The higher density was apparently due to the higher percentage of latewood in the heterozygotes. The fertilization effect was highly significant for both growth and wood density traits. While no cad genotype x

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

    PubMed Central

    Conway, T; Ingram, L O

    1989-01-01

    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' 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. Images PMID:2661535

  11. 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.

  12. 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.

  13. 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

  14. 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

    2015-01-01

    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

  15. 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.

  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. Phosphorescence maxima and triplet state lifetimes of NAD+ and epsilon-NAD+ in ternary complexes with horse liver alcohol dehydrogenase.

    PubMed

    Rousslang, K; Allen, L; Ross, J B

    1989-02-01

    This paper describes the phosphorescence emission and decay times of NAD+ and its fluorescent etheno derivative, epsilon-NAD+, in the pyrazole ternary complex with horse liver alcohol dehydrogenase (ADH). We show that the epsilon-NAD+ triplet state, as well as the tryptophan triplet state, can be utilized to monitor the coenzyme-enzyme interaction. The decays of NAD+ and AMP are single exponential, and the lifetimes are the same within experimental error. The phosphorescence lifetimes, evaluated as single exponentials, are slightly shorter in epsilon-NAD+ than they are in epsilon-AMP. Whereas the decay of epsilon-AMP was adequately fit by a single exponential with a time constant of very close to 0.5 s, it was necessary to fit the decay of epsilon-NAD+ to a double exponential. Ternary complexes with NAD+ excited at 297 nm exhibit decay kinetics nearly identical to those of ADH by itself. On the other hand, when excitation of the epsilon-NAD+ ternary complex is provided at 313 nm, where there is very little absorption by either tryptophan residue, the decay law of the ternary complex is similar to that of epsilon-NAD+ in solution. Our results demonstrate that NAD+ and epsilon-NAD+ quench tryptophan phosphorescence in ADH. Normalizing the phosphorescence intensity to the 0-0 vibronic band assigned to Trp-15 (blue-edge), we calculate a 21% decrease in the phosphorescence associated with Trp-314 at stoichiometric saturation of the coenzyme binding sites with NAD+ in the ternary complex. When the active sites are saturated with epsilon-NAD+, the relative phosphorescence due to Trp-314 decreases by 63%.(ABSTRACT TRUNCATED AT 250 WORDS)

  18. Elemental sulfur: toxicity in vivo and in vitro to bacterial luciferase, in vitro yeast alcohol dehydrogenase, and bovine liver catalase.

    PubMed

    Cetkauskaite, Anolda; Pessala, Piia; Södergren, Anders

    2004-08-01

    The aim of this research was to analyze the effects and the modes of action of elemental sulfur (S(0)) in bioluminescence and respiration of Vibrio fischeri cells and the enzymes crude luciferase, pure catalase, and alcohol dehydrogenase (ADH). Metallic copper removed sulfur and reduced the toxicity of acetone extracts of sediment samples analyzed in the bioluminescence test. The sulfur inhibition of cell bioluminescence was noncompetitive with decanal, the luciferase substrate; reversible, with maximum toxicity after 15 min (EC(50) = 11.8 microg/L); and almost totally recovered after 2 h. In vitro preincubation of crude luciferase extract with sulfur (0.28 ppm) weakly inhibited bioluminescence at 5 min, but at 30 min the inhibition reached 60%. Increasing the concentration of sulfur in the parts per million concentration range in vitro decreased bioluminescence, which was not constant, but depended on exposure time, and no dead-end/total inhibition was observed. The redox state of enzymes in the in vitro system significantly affected inhibition. Hydrogen peroxide restored fully and the reducing agent dithiothreitol, itself toxic, restored only partially luciferase activity in the presence of sulfur. Sulfur (5.5 ppm) slightly inhibited ADH and catalase, and dithiothreitol enhanced sulfur inhibition. High sulfur concentrations (2.2 ppm) inhibited the bioluminescence and enhanced the respiration rate of V. fischeri cells. Elemental sulfur data were interpreted to show that sulfur acted on at least a few V. fischeri cell sites: reversibly modifying luciferase at sites sensitive to/protected by oxidative and reducing agents and by affecting electron transport processes, resulting in enhanced oxygen consumption. Sulfur together with an enzyme reducing agent inhibited the oxidoreductive enzymes ADH and catalase, which have --SH groups, metal ion cofactors, or heme, respectively, in their active centers. PMID:15269910

  19. The Natural History of Class I Primate Alcohol Dehydrogenases Includes Gene Duplication, Gene Loss, and Gene Conversion

    PubMed Central

    Carrigan, Matthew A.; Uryasev, Oleg; Davis, Ross P.; Zhai, LanMin; Hurley, Thomas D.; Benner, Steven A.

    2012-01-01

    Background Gene duplication is a source of molecular innovation throughout evolution. However, even with massive amounts of genome sequence data, correlating gene duplication with speciation and other events in natural history can be difficult. This is especially true in its most interesting cases, where rapid and multiple duplications are likely to reflect adaptation to rapidly changing environments and life styles. This may be so for Class I of alcohol dehydrogenases (ADH1s), where multiple duplications occurred in primate lineages in Old and New World monkeys (OWMs and NWMs) and hominoids. Methodology/Principal Findings To build a preferred model for the natural history of ADH1s, we determined the sequences of nine new ADH1 genes, finding for the first time multiple paralogs in various prosimians (lemurs, strepsirhines). Database mining then identified novel ADH1 paralogs in both macaque (an OWM) and marmoset (a NWM). These were used with the previously identified human paralogs to resolve controversies relating to dates of duplication and gene conversion in the ADH1 family. Central to these controversies are differences in the topologies of trees generated from exonic (coding) sequences and intronic sequences. Conclusions/Significance We provide evidence that gene conversions are the primary source of difference, using molecular clock dating of duplications and analyses of microinsertions and deletions (micro-indels). The tree topology inferred from intron sequences appear to more correctly represent the natural history of ADH1s, with the ADH1 paralogs in platyrrhines (NWMs) and catarrhines (OWMs and hominoids) having arisen by duplications shortly predating the divergence of OWMs and NWMs. We also conclude that paralogs in lemurs arose independently. Finally, we identify errors in database interpretation as the source of controversies concerning gene conversion. These analyses provide a model for the natural history of ADH1s that posits four ADH1 paralogs in

  20. Immersive CAD

    SciTech Connect

    Ames, A.L.

    1999-02-01

    This paper documents development of a capability for performing shape-changing editing operations on solid model representations in an immersive environment. The capability includes part- and assembly-level operations, with part modeling supporting topology-invariant and topology-changing modifications. A discussion of various design considerations in developing an immersive capability is included, along with discussion of a prototype implementation we have developed and explored. The project investigated approaches to providing both topology-invariant and topology-changing editing. A prototype environment was developed to test the approaches and determine the usefulness of immersive editing. The prototype showed exciting potential in redefining the CAD interface. It is fun to use. Editing is much faster and friendlier than traditional feature-based CAD software. The prototype algorithms did not reliably provide a sufficient frame rate for complex geometries, but has provided the necessary roadmap for development of a production capability.

  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. The multifunctional isopropyl alcohol dehydrogenase of Phytomonas sp. could be the result of a horizontal gene transfer from a bacterium to the trypanosomatid lineage.

    PubMed

    Molinas, Sara M; Altabe, Silvia G; Opperdoes, Fred R; Rider, Mark H; Michels, Paul A M; Uttaro, Antonio D

    2003-09-19

    Isopropyl alcohol dehydrogenase (iPDH) is a dimeric mitochondrial alcohol dehydrogenase (ADH), so far detected within the Trypanosomatidae only in the genus Phytomonas. The cloning, sequencing, and heterologous expression of the two gene alleles of the enzyme revealed that it is a zinc-dependent medium-chain ADH. Both polypeptides have 361 amino acids. A mitochondrial targeting sequence was identified. The mature proteins each have 348 amino acids and a calculated molecular mass of 37 kDa. They differ only in one amino acid, which can explain the three isoenzymes and their respective isoelectric points previously found. A phylogenetic analysis locates iPDH within a cluster with fermentative ADHs from bacteria, sharing 74% similarity and 60% identity with Ralstonia eutropha ADH. The characterization of the two bacterially expressed Phytomonas enzymes and the comparison of their kinetic properties with those of the wild-type iPDH and of the R. eutropha ADH strongly support the idea of a horizontal gene transfer event from a bacterium to a trypanosomatid to explain the origin of the iPDH in Phytomonas. Phytomonas iPDH and R. eutropha ADH are able to use a wide range of substrates with similar Km values such as primary and secondary alcohols, diols, and aldehydes, as well as ketones such as acetone, diacetyl, and acetoin. We speculate that, as for R. eutropha ADH, Phytomonas iPDH acts as a safety valve for the release of excess reducing power. PMID:12853449

  3. Impact of chronic low to moderate alcohol consumption on blood lipid and heart energy profile in acetaldehyde dehydrogenase 2-deficient mice

    PubMed Central

    Fan, Fan; Cao, Quan; Wang, Cong; Ma, Xin; Shen, Cheng; Liu, Xiang-wei; Bu, Li-ping; Zou, Yun-zeng; Hu, Kai; Sun, Ai-jun; Ge, Jun-bo

    2014-01-01

    Aim: To investigate the roles of acetaldehyde dehydrogenase 2 (ALDH2), the key enzyme of ethanol metabolism, in chronic low to moderate alcohol consumption-induced heart protective effects in mice. Methods: Twenty-one male wild-type (WT) or ALDH2-knockout (KO) mice were used in this study. In each genotype, 14 animals received alcohol (2.5%, 5% and 10% in week 1–3, respectively, and 18% in week 4–7), and 7 received water for 7 weeks. After the treatments, survival rate and general characteristics of the animals were evaluated. Serum ethanol and acetaldehyde levels and blood lipids were measured. Metabolomics was used to characterize the heart and serum metabolism profiles. Results: Chronic alcohol intake decreased the survival rate of KO mice by 50%, and significantly decreased their body weight, but did not affect those of WT mice. Chronic alcohol intake significantly increased the serum ethanol levels in both WT and KO mice, but KO mice had significantly higher serum acetaldehyde levels than WT mice. Chronic alcohol intake significantly increased the serum HDL cholesterol levels in WT mice, and did not change the serum HDL cholesterol levels in KO mice. After chronic alcohol intake, WT and KO mice showed differential heart and serum metabolism profiles, including the 3 main energy substrate types (lipids, glucose and amino acids) and three carboxylic acid cycles. Conclusion: Low to moderate alcohol consumption increases HDL cholesterol levels and improves heart energy metabolism profile in WT mice but not in ALDH2-KO mice. Thus, preserved ALDH2 function is essential for the protective effect of low to moderate alcohol on the cardiovascular system. PMID:24998256

  4. In vitro characterization of an enzymatic redox cascade composed of an alcohol dehydrogenase, an enoate reductases and a Baeyer–Villiger monooxygenase

    PubMed Central

    Oberleitner, Nikolin; Peters, Christin; Rudroff, Florian; Bornscheuer, Uwe T.; Mihovilovic, Marko D.

    2014-01-01

    An artificial enzyme cascade composed of an alcohol dehydrogenase, an enoate reductase and a Baeyer–Villiger monooxygenase was investigated in vitro to gain deeper mechanistic insights and understand the assets and drawbacks of this multi-step biocatalysis. Several substrates composed of different structural motifs were examined and provided access to functionalized chiral compounds in high yields (up to >99%) and optical purities (up to >99%). Hence, the applicability of the presented enzymatic cascade was exploited for the synthesis of biorenewable polyesters. PMID:24746588

  5. 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.

  6. Sulfur-rich zinc chemistry: new tris(thioimidazolyl)hydroborate ligands and their zinc complex chemistry related to the structure and function of alcohol dehydrogenase.

    PubMed

    Tesmer, M; Shu, M; Vahrenkamp, H

    2001-07-30

    The 1-substituted tris(2-thioimidazolyl)hydroborate ligands Tt(R) were prepared as the potassium salts from KBH(4) and the corresponding 1-R-2-thioimidazole for R = t-Bu and C(6)H(4)-p-CH(CH(3))(2) (Cum). Their reactions with zinc salts yielded the tetrahedral complexes Tt(R)Zn-X with X = F, Cl, ONO(2) and (Tt(t)()(-)(Bu))(2)Zn. With zinc perchlorate the labile perchlorate complexes Tt(R)Zn-OClO(3) were obtained. They served as starting materials for the incorporation of substrates which are relevant for the chemistry of horse liver alcohol dehydrogenase: Ethanol led to [Tt(t)()(-Bu)Zn.EtOH] ClO(4).EtOH, p-nitrophenol (NitOH) yielded Tt(Cum)Zn-ONit. Pyridine-2-carbaldehyde and salicylic aldehyde were incorporated as N(pyridine) and O(phenolate) coligands with possible additional O(aldehyde) coordination. Substituted pyridyl methanols (R-PyCH(2)OH) yielded the trinuclear complexes [(Tt(t)()(-Bu))(2)Zn(3)(R-PyCH(2)O)(2)] (ClO(4))(2) with bridging Tt and pyridylmethoxide ligands. Preliminary experiments on the functional modeling of alcohol dehydrogenase have shown that TtZn complexes promote both the dehydrogenation of 2-propanol and the hydrogenation of pentafluorobenzaldehyde. PMID:11466063

  7. 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

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

    PubMed Central

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

    1991-01-01

    Zymomonas mobilis genes for pyruvate decarboxylase (pdc) and alcohol dehydrogenase II (adhB) were integrated into the Escherichia coli chromosome within or near the pyruvate formate-lyase gene (pfl). Integration improved the stability of the Z. mobilis genes in E. coli, but further selection was required to increase expression. Spontaneous mutants were selected for resistance to high level of chloramphenicol that also expressed high levels of the Z. mobilis genes. Analogous mutants were 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). PMID:2059047

  9. 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. PMID:25772736

  10. 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.

  11. CAD: Designs on Business.

    ERIC Educational Resources Information Center

    Milburn, Ken

    1988-01-01

    Provides a general review of the field of Computer-Aided Design Software including specific reviews of "Autosketch,""Generic CADD,""Drafix 1 Plus,""FastCAD," and "Autocad Release 9." Brief articles include "Blueprint for Generation,""CAD for Every Department,""Ideas Sketched in Glass,""CAD on the MAC," and "A CAD Package Sampler." (CW)

  12. 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

  13. 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.

  14. 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

  15. 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.

  16. Alcohol

    MedlinePlus

    ... How Can I Help a Friend Who Cuts? Alcohol KidsHealth > For Teens > Alcohol Print A A A ... you can make an educated choice. What Is Alcohol? Alcohol is created when grains, fruits, or vegetables ...

  17. Cloning, sequencing, and characterization of the gene encoding the smallest subunit of the three-component membrane-bound alcohol dehydrogenase from Acetobacter pasteurianus.

    PubMed

    Kondo, K; Beppu, T; Horinouchi, S

    1995-09-01

    The membrane-bound alcohol dehydrogenase (ADH) of Acetobacter pasteurianus NCI1452 consists of three different subunits, a 78-kDa dehydrogenase subunit, a 48-kDa cytochrome c subunit, and a 20-kDa subunit of unknown function. For elucidation of the function of the smallest subunit, this gene was cloned from this strain by the oligonucleotide-probing method, and its nucleotide sequence was determined. Comparison of the deduced amino acid sequence and the NH2-terminal sequence determined for the purified protein indicated that the smallest subunit contained a typical signal peptide of 28 amino acids, as did the larger two subunits. This gene complemented the ADH activity of a mutant strain which had lost the smallest subunit. Disruption of this gene on the chromosome resulted in loss of ADH activity in Acetobacter aceti, indicating that the smallest subunit was essential for ADH activity. Immunoblot analyses of cell lysates prepared from various ADH mutants suggested that the smallest subunit was concerned with the stability of the 78-kDa subunit and functioned as a molecular coupler of the 78-kDa subunit to the 48-kDa subunit on the cytoplasmic membrane.

  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. 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.

  20. I86A/C295A mutant secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus has broadened substrate specificity for aryl ketones.

    PubMed

    Nealon, Christopher M; Welsh, Travis P; Kim, Chang Sup; Phillips, Robert S

    2016-09-15

    Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase (SADH) reduces aliphatic ketones according to Prelog's Rule, with binding pockets for small and large substituents. It was shown previously that the I86A mutant SADH reduces acetophenone, which is not a substrate of wild-type SADH, to give the anti-Prelog R-product (Musa, M. M.; Lott, N.; Laivenieks, M.; Watanabe, L.; Vieille, C.; Phillips, R. S. ChemCatChem2009, 1, 89-93.). However, I86A SADH did not reduce aryl ketones with substituents larger than fluorine. We have now expanded the small pocket of the active site of I86A SADH by mutation of Cys-295 to alanine to allow reaction of substituted acetophenones. As predicted, the double mutant I86A/C295A SADH has broadened substrate specificity for meta-substituted, but not para-substituted, acetophenones. However, the increase of the substrate specificity of I86A/C295A SADH is accompanied by a decrease in the kcat/Km values of acetophenones, possibly due to the substrates fitting loosely inside the more open active site. Nevertheless, I86A/C295A SADH gives high conversions and very high enantiomeric excess of the anti-Prelog R-alcohols from the tested substrates. PMID:27495738

  1. 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

  2. Teaching for CAD Expertise

    ERIC Educational Resources Information Center

    Chester, Ivan

    2007-01-01

    CAD (Computer Aided Design) has now become an integral part of Technology Education. The recent introduction of highly sophisticated, low-cost CAD software and CAM hardware capable of running on desktop computers has accelerated this trend. There is now quite widespread introduction of solid modeling CAD software into secondary schools but how…

  3. 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

  4. Alcohol

    MedlinePlus

    ... Text Size: A A A Listen En Español Alcohol Wondering if alcohol is off limits with diabetes? Most people with diabetes can have a moderate amount of alcohol. Research has shown that there can be some ...

  5. 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 ...

  6. Sorbitol dehydrogenase is a zinc enzyme.

    PubMed Central

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

    1984-01-01

    Evidence is given that tetrameric sorbitol dehydrogenase from sheep liver contains one zinc atom per subunit, most probably located at the active site, and no other specifically bound zinc or iron atom. In alcohol dehydrogenases that are structurally related to sorbitol dehydrogenase, more than one zinc atom per subunit can complicate investigations of zinc atom function. Therefore, sorbitol dehydrogenase will be particularly valuable for defining the precise roles of zinc in alcohol and polyol dehydrogenases, and for establishing correlations of structure and function with other important zinc-containing proteins. PMID:6370679

  7. Interdependence of coenzyme-induced conformational work and binding potential in yeast alcohol and porcine heart lactate dehydrogenases: a hydrogen-deuterium exchange study.

    PubMed

    De Weck, Z; Pande, J; Kägi, J H

    1987-07-28

    Binding of NAD coenzymes to yeast alcohol dehydrogenase (YADH) and porcine heart lactate dehydrogenase (PHLDH) was studied by hydrogen-deuterium exchange with the infrared technique. Conformational changes in the enzymes specific to the coenzymes and their fragments were observed, and the pH dependence of the exchange reaction shows that it conforms to the EX-2 scheme. In both YADH and PHLDH the magnitude of the conformational change of measured by exchange retardation is considerably larger for NAD+ than for NADH. Studies with coenzyme fragments like ADP-ribose, ADP, and AMP also highlight the lack of rigorous correlation between structural features such as charge and size and their influence on exchange behavior. Ternary complexes such as YADH-NAD+-pyrazole, PHLDH-NAD+-oxalate, and PHLDH-NADH-oxamate, which mimic the transition state, have a significantly more pronounced effect on exchange rates than the corresponding binary complexes. The outstanding feature of this study is the demonstration that in the binary enzyme-coenzyme complexes the more loosely bound NAD+ is more effective in retarding exchange than the more firmly bound NADH. These differences are attributed to the unequal structural constraints exerted by the two coenzymes upon the enzymes, which translate to unequal expenditure of transconformational work in the formation of the two complexes. The opposing variation in the free energy of binding and the transconformational work expended can be viewed as an unequal partitioning of the net free energy gain resulting from the protein-ligand interaction into a binding term and that required for conformational change.

  8. A tightly bound quinone functions in the ubiquinone reaction sites of quinoprotein alcohol dehydrogenase of an acetic acid bacterium, Gluconobacter suboxydans.

    PubMed

    Matsushita, Kazunobu; Kobayashi, Yoshiki; Mizuguchi, Mitsuhiro; Toyama, Hirohide; Adachi, Osao; Sakamoto, Kimitoshi; Miyoshi, Hideto

    2008-10-01

    Quinoprotein alcohol dehydrogenase (ADH) of acetic acid bacteria is a membrane-bound enzyme that functions as the primary dehydrogenase in the ethanol oxidase respiratory chain. It consists of three subunits and has a pyrroloquinoline quinone (PQQ) in the active site and four heme c moieties as electron transfer mediators. Of these, three heme c sites and a further site have been found to be involved in ubiquinone (Q) reduction and ubiquinol (QH2) oxidation respectively (Matsushita et al., Biochim. Biophys. Acta, 1409, 154-164 (1999)). In this study, it was found that ADH solubilized and purified with dodecyl maltoside, but not with Triton X-100, had a tightly bound Q, and thus two different ADHs, one having the tightly bound Q (Q-bound ADH) and Q-free ADH, could be obtained. The Q-binding sites of both the ADHs were characterized using specific inhibitors, a substituted phenol PC16 (a Q analog inhibitor) and antimycin A. Based on the inhibition kinetics of Q2 reductase and ubiquinol-2 (Q2H2) oxidase activities, it was suggested that there are one and two PC16-binding sites in Q-bound ADH and Q-free ADH respectively. On the other hand, with antimycin A, only one binding site was found for Q2 reductase and Q2H2 oxidase activities, irrespective of the presence of bound Q. These results suggest that ADH has a high-affinity Q binding site (QH) besides low-affinity Q reduction and QH2 oxidation sites, and that the bound Q in the QH site is involved in the electron transfer between heme c moieties and bulk Q or QH2 in the low-affinity sites.

  9. 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.

  10. Pyruvate:ferredoxin oxidoreductase and bifunctional aldehyde-alcohol dehydrogenase are essential for energy metabolism under oxidative stress in Entamoeba histolytica.

    PubMed

    Pineda, Erika; Encalada, Rusely; Rodríguez-Zavala, José S; Olivos-García, Alfonso; Moreno-Sánchez, Rafael; Saavedra, Emma

    2010-08-01

    The in vitro Entamoeba histolytica pyruvate:ferredoxin oxidoreductase (EhPFOR) kinetic properties and the effect of oxidative stress on glycolytic pathway enzymes and fluxes in live trophozoites were evaluated. EhPFOR showed a strong preference for pyruvate as substrate over other oxoacids. The enzyme was irreversibly inactivated by a long period of saturating O(2) exposure (IC(50) 0.034 mm), whereas short-term exposure (< 30 min) leading to > 90% inhibition allowed for partial restoration by addition of Fe(2+). CoA and acetyl-CoA prevented, whereas pyruvate exacerbated, inactivation induced by short-term saturating O(2) exposure. Superoxide dismutase was more effective than catalase in preventing the inactivation, indicating that reactive oxygen species (ROS) were involved. Hydrogen peroxide caused inactivation in an Fe(2+)-reversible fashion that was not prevented by the coenzymes, suggesting different mechanisms of enzyme inactivation by ROS. Structural analysis on an EhPFOR 3D model suggested that the protection against ROS provided by coenzymes could be attributable to their proximity to the Fe-S clusters. After O(2) exposure, live parasites displayed decreased enzyme activities only for PFOR (90%) and aldehyde dehydrogenase (ALDH; 68%) of the bifunctional aldehyde-alcohol dehydrogenase (EhADH2), whereas acetyl-CoA synthetase remained unchanged, explaining the increased acetate and lowered ethanol fluxes. Remarkably, PFOR and ALDH activities were restored after return of the parasites to normoxic conditions, which correlated with higher ethanol and lower acetate fluxes. These results identified amebal PFOR and ALDH of EhADH2 activities as markers of oxidative stress, and outlined their relevance as significant controlling steps of energy metabolism in parasites subjected to oxidative stress. PMID:20629749

  11. Molecular cloning and characterization of two inducible NAD⁺-adh genes encoding NAD⁺-dependent alcohol dehydrogenases from Acetobacter pasteurianus SKU1108.

    PubMed

    Masud, Uraiwan; Matsushita, Kazunobu; Theeragool, Gunjana

    2011-11-01

    The cytosolic NAD⁺-dependent alcohol dehydrogenases (NAD⁺-ADHs) are induced in the quinoprotein ADH-(PQQ-ADH) defective Acetobacter pasteurianus SKU1108 mutant during growth in an ethanol medium. The adhI and adhII genes, which encode NAD⁺-ADH I and ADH II, respectively, of this strain have been cloned and characterized. Sequence analyses have revealed that the adhI gene consists of 1029 bp coding for 342 amino acids, which share 99.71% identity with the same protein from A. pasteurianus IFO 3283. Conversely, the adhII gene is composed of 762 bp encoding for a polypeptide of 253 amino acids, which exhibit 99.60% identity with the A. pasteurianus IFO 3283 protein. ADH I is a member of the group I Zn-dependent long-chain ADHs, while the ADH II belongs to the group II short-chain dehydrogenase/reductase NAD⁺-ADHs. The NAD⁺-adh gene disruptants exhibited a growth reduction when grown in an ethanol medium. In Escherichia coli, ethanol induced adhI and adhII promoter activities by approximately 1.5 and 2.0 times, respectively, and the promoter activity of the adhII gene exceeded that of the adhI gene by approximately 3.5 times. The possible promoter regions of the adhI and adhII genes are located at approximately 81-105 bp and 74-92 bp, respectively, from their respective ATG start codons. Their repressor regions might be located in proximity to these promoters and may repress gene expression in the wild-type, where the membrane-bound ADH effectively functions.

  12. The PduQ Enzyme Is an Alcohol Dehydrogenase Used to Recycle NAD+ Internally within the Pdu Microcompartment of Salmonella enterica

    PubMed Central

    Cheng, Shouqiang; Fan, Chenguang; Sinha, Sharmistha; Bobik, Thomas A.

    2012-01-01

    Salmonella enterica uses a bacterial microcompartment (MCP) for coenzyme B12-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. PMID:23077559

  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. Alcohol dehydrogenase of class IV (sigma sigma-ADH) from human stomach. cDNA sequence and structure/function relationships.

    PubMed

    Farrés, J; Moreno, A; Crosas, B; Peralba, J M; Allali-Hassani, A; Hjelmqvist, L; Jörnvall, H; Parés, X

    1994-09-01

    Human stomach mucosa contains a characteristic alcohol dehydrogenase (ADH) enzyme, sigma sigma-ADH. Its cDNA has been cloned from a human stomach library and sequenced. The deduced amino acid sequence shows 59-70% identities with the other human ADH classes, demonstrating that the stomach enzyme represents a distinct structure, constituting class IV, coded by a separate gene, ADH7. The amino acid identity with the rat stomach class IV ADH is 88%, which is intermediate between constant and variable dehydrogenases. This value reflects higher conservation than for the classical liver enzymes of class I, compatible with a separate functional significance of the class IV enzyme. Its enzymic features can be correlated with its structural characteristics. The residues lining the substrate-binding cleft are bulky and hydrophobic, similar to those of the class I enzyme; this explains the similar specificity of both classes, compatible with the origin of class IV from class I. Position 47 has Arg, in contrast to Gly in the rat class IV enzyme, but this Arg is still associated with an extremely high activity (kcat = 1510 min-1) and weak coenzyme binding (KiaNAD+ = 1.6 mM). Thus, the strong interaction with coenzyme imposed by Arg47 in class I is probably compensated for in class IV by changes that may negatively affect coenzyme binding: Glu230, His271, Asn260, Asn261, Asn363. The still higher activity and weaker coenzyme binding of rat class IV (kcat = 2600 min-1, KiaNAD = 4 mM) can be correlated to the exchanges to Gly47, Gln230 and Tyr363. An important change at position 294, with Val in human and Ala in rat class IV, is probably responsible for the dramatic difference in Km values for ethanol between human (37 mM) and rat (2.4 M) class IV enzymes.

  15. Application of nicotin amide-adenine dinucleotide analogs for clinical enzymology: alcohol dehydrogenase activity in liver injury.

    PubMed

    Fujisawa, K; Kimura, A; Minato, S; Tamaoki, H; Mizushima, H

    1976-06-01

    The activities of alcohol dehydrogease(ADH) in serum and in the subcellular fractions of rat liver were determined with n-amyl alcohol or ethanol as substrate and thionicotinamide-adenine dinucleotide as coenzyme. It was found that the enzyme's activity ratio on the amyl alcohol and ethanol(A/E value) of serum and on the particulate fractions of the liver were different, but the A/E value of the soluble fraction was similar to that of serum. The A/E value of the particulate fractions were higher than that of the soluble fraction. From the results of experimental liver damage in the rat, it seems that estimation of the A/E value of ADH activity in serum is a useful parameter for the diagnosis of active liver injury. Since the A/E values of patients' sera differed from those of the normal subjects, the estimation of the A/E value of serum may give diagnostic information on liver injury, especially in chronic liver injury. PMID:179739

  16. [The PQQ-dehydrogenases. A novel example of bacterial quinoproteins].

    PubMed

    Flores-Encarnación, Marcos; Sánchez-Cuevas, Mariano; Ortiz-Gutiérrez, Felipe

    2004-01-01

    The word "quinoprotein" describes four groups of different enzymes which have cofactors containing o-quinones. Pyrrolo-quinoline quinone (PQQ) is not covalently attached. PQQ is the cofactor of several quinoprotein bacterial dehydrogenases including glucose dehydrogenase (G-DH), alcohol dehydrogenase (A-DH) and aldehyde dehydrogenase (AL-DH). These dehydrogenases are located in the periplasm of Gram-negative bacteria. This report summarises the structural properties of quinoprotein dehydrogenases, such as the biological functions and biotechnological aspects more important.

  17. Alcohol

    MedlinePlus

    ... Got Homework? Here's Help White House Lunch Recipes Alcohol KidsHealth > For Kids > Alcohol Print A A A Text Size What's in ... What Is Alcoholism? Say No en español El alcohol Getting the Right Message "Hey, who wants a ...

  18. High efficiency preparation and characterization of intact poly(vinyl alcohol) dehydrogenase from Sphingopyxis sp.113P3 in Escherichia coli by inclusion bodies renaturation.

    PubMed

    Jia, Dongxu; Yang, Yu; Peng, Zhengcong; Zhang, Dongxu; Li, Jianghua; Liu, Long; Du, Guocheng; Chen, Jian

    2014-03-01

    Poly(vinyl alcohol) dehydrogenase (PVADH, EC 1.1.99.23) is an enzyme which has potential application in textile industry to degrade the poly(vinyl alcohol) (PVA) in waste water. Previously, a 1,965-bp fragment encoding a PVADH from Sphingopyxis sp. 113P3 was synthesized based on the replacement of the rare codons in Escherichia coli (E. coli). In this work, the deduced mature PVADH (mPVADH) gene of 1,887 bp was amplified by polymerase chain reaction (PCR) and inserted into the site between NcoI and HindIII in pET-32a(+). The constructed recombinant plasmid was transformed into E. coli Rosetta (DE3). In shake flask, the fusion protein of thioredoxin (Trx)-mPVADH was expressed precisely; however, Trx-mPVADH was found to accumulate mainly as inclusion bodies. After isolating, dissolving in buffer containing urea, purification, dialysis renaturation, and digesting with recombinant enterokinase/His (rEK/His), the bioactive mPVADH fragments were obtained with protein concentration of 0.56 g/L and enzymatic activity of 194 U/mL. The K m and V max values for PVA 1799 were 2.33 mg/mL and 15.7 nmol/(min·mg protein), respectively. (1)H-NMR and infrared (IR) spectrum demonstrated that its biological function was oxidizing hydroxyl groups of PVA 1799 to form diketone, and PVA 1799 could be degraded completely by successive treatment with mPVADH and oxidized PVA hydrolase (OPH).

  19. 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.

  20. Relevance of Frank's solvent classification as typically aqueous and typically non-aqueous to activities of firefly luciferase, alcohol dehydrogenase, and alpha-chymotrypsin in aqueous binaries.

    PubMed

    Fadnavis, Nitin Wasantrao; Seshadri, Ramanujam; Sheelu, Gurrala; Madhuri, Kallakunta Vasantha

    2005-01-15

    Effects of cosolvent concentration on activity of fire fly luciferase, alpha-chymotrypsin, and alcohol dehydrogenase from baker's yeast (Saccharomyces cerevisiae) have been studied for several solvents with varying hydrophobicities (logP from +1.0 to -1.65) and polarities (dielectric constant from 7.4 to 109). The inhibitory effect of the cosolvent is examined in light of Frank's classification of solvents into 'typically aqueous (TA)' and 'typically non-aqueous (TNA).' The solvent concentration at which the enzyme activity decreases to half, the C(50) values, for TA solvents such as 1-cyclohexyl-2-pyrrolidinone, 2-butoxyethanol, 1-methyl-2-pyrrolidinone, tetrahydrofuran, t-butanol, and ethanol correlate quite well with their critical hydrophobic interaction concentration, rather than logP, while those for TNA solvents such as acetonitrile, dimethyl formamide, formamide, and dimethyl sulfoxide correlate well with logP. The interactions of TA solvents with proteins appear to be governed mainly by hydrophobic interactions while both hydrophobic and hydrophilic interactions play important role in case of TNA solvents.

  1. The role of hydration in enzyme activity and stability: 2. Alcohol dehydrogenase activity and stability in a continuous gas phase reactor.

    PubMed

    Yang, F; Russell, A J

    1996-03-20

    The degree of enzyme hydration is the one of the most important factors which can affect enzyme activity and stability in water-limited environments. Alcohol dehydrogenase from baker's yeast (YADH) has been used as a model enzyme to study the effects of hydration on activity, stability, and cofactor stability with gas phase substrates. In all cases, the enzyme is essentially inactive until a temperature-independent degree of surface coverage by water molecules has been reached. The critical water content corresponds to 40-50% of a single monolayer. Careful control of the degree of hydration, by adjustments to gas humidity and temperature, enables the enzyme to be stabilized for periods exceeding 1 month, whereas in water the half-life of the enzyme is 30 min. The reaction with gas phase substrates follows a pseudo-first-order mechanism with an activation energy of 7.5 +/- kcal/mol, which is almost half of that in aqueous solution. (c) 1996 John Wiley & Sons, Inc.

  2. The role of hydration in enzyme activity and stability: 1. Water adsorption by alcohol dehydrogenase in a continuous gas phase reactor.

    PubMed

    Yang, F; Russell, A J

    1996-03-20

    The adsorption of water by alcohol dehydrogenase from baker's yeast (YADH) has been measured in a continuous-flow gas reactor at varying temperatures. Adsorption isotherms in the presence of gaseous organic substrates are compared to those from organic-free gas mixtures. Almost no effect of the hydrophobic molecule on total water adsorption was observed. A rarely mentioned multilayer isotherm model from the 1930s, the Huttig's isotherm, has been found to fit the experimental data with extremely good accuracy. The model enables the calculation of both the heat of adsorption of water to the enzyme and the total amount of water necessary for monolayer coverage. The heat of adsorption of water in the first layer is approximately -16 kcal/mol. This tight binding of water, which is much higher than the heat of condensation of pure water, helps to explain the kinetic properties of YADH-catalyzed reactions on vapor phase substrates. While the monolayer coverage is temperature independent, the enzyme demonstrates hysteresis when transitioning between adsorption and desorption. The hysteresis observed in water sorption studies may also explain previously reported properties of the enzyme. (c) 1996 John Wiley & Sons, Inc.

  3. Differing features of proteins in membranes may result in antioxidant or prooxidant action: opposite effects on lipid peroxidation of alcohol dehydrogenase and albumin in liposomal systems.

    PubMed

    Riedl, A; Shamsi, Z; Anderton, M; Goldfarb, P; Wiseman, A

    1996-02-01

    The influence of 3 thiol-containing compounds, bovine serum albumin (fatty acid free: BSA), glutathione (GSH) and yeast alcohol dehydrogenase (YADH) on lipid peroxidation in multilamellar liposomes, prepared from ox-brain phospholipid, was investigated. Thiol-compounds were added either before liposome formation, or after liposome formation; and their effects compared to a positive control. Bovine serum albumin (BSA), an acidic hydrophilic protein, displays a small, concentration dependent, antioxidant effect when added to preformed liposomes. A much larger antioxidant effect was observed when the BSA was entrapped inside the liposome, by adding BSA just prior to liposome preparation. In contrast, a Zn(2+) containing redox enzyme, YADH, a basic hydrophobic membrane-associating protein, displays a large pro-oxidant effect at much lower concentrations especially when entrapped inside the liposome. This was observed also with GSH; but per mole of -SH, YADH was about 18 times as powerful a pro-oxidant perhaps because of structural changes to the membrane. Oxidized glutathione and N-acetylcysteine were also pro-oxidant (cysteine and cystine showed little effect). Formation of thiyl radicals may occur in the presence of iron ions with these pro-oxidant sulphur-containing compounds. Partial protection against lipid peroxidation was observed with EDTA, desferrioxamine and protoporphyrin (IX), potent iron-chelating agents.

  4. Engineered ketol-acid reductoisomerase and alcohol dehydrogenase enable anaerobic 2-methylpropan-1-ol production at theoretical yield in Escherichia coli.

    PubMed

    Bastian, Sabine; Liu, Xiang; Meyerowitz, Joseph T; Snow, Christopher D; Chen, Mike M Y; Arnold, Frances H

    2011-05-01

    2-methylpropan-1-ol (isobutanol) is a leading candidate biofuel for the replacement or supplementation of current fossil fuels. Recent work has demonstrated glucose to isobutanol conversion through a modified amino acid pathway in a recombinant organism. Although anaerobic conditions are required for an economically competitive process, only aerobic isobutanol production has been feasible due to an imbalance in cofactor utilization. Two of the pathway enzymes, ketol-acid reductoisomerase and alcohol dehydrogenase, require nicotinamide dinucleotide phosphate (NADPH); glycolysis, however, produces only nicotinamide dinucleotide (NADH). Here, we compare two solutions to this imbalance problem: (1) over-expression of pyridine nucleotide transhydrogenase PntAB and (2) construction of an NADH-dependent pathway, using engineered enzymes. We demonstrate that an NADH-dependent pathway enables anaerobic isobutanol production at 100% theoretical yield and at higher titer and productivity than both the NADPH-dependent pathway and transhydrogenase over-expressing strain. Our results show how engineering cofactor dependence can overcome a critical obstacle to next-generation biofuel commercialization.

  5. Characterization of two novel alcohol short-chain dehydrogenases/reductases from Ralstonia eutropha H16 capable of stereoselective conversion of bulky substrates.

    PubMed

    Magomedova, Zalina; Grecu, Andreea; Sensen, Christoph W; Schwab, Helmut; Heidinger, Petra

    2016-03-10

    Biocatalysis has significant advantages over organic synthesis in the field of chiral molecule production and several types of stereoselective enzymes are already in use in industrial biotechnology. However, there is still a high demand for new enzymes capable of transforming bulky molecules with sufficient operability. In order to reveal novel high-potential biocatalysts, the complete genome of the β-proteobacterium Ralstonia eutropha H16 was screened for potential short-chain dehydrogenases/reductases (SDRs). We were able to identify two (S)-enantioselective SDRs named A5 and B3. These showed clear preference towards long-chain and aromatic secondary alcohols, aldehydes and ketones, with diaryl diketone benzil as one of the best substrates. In addition the phylogenetic analysis of all enzyme types, which are known to facilitate benzil reduction, revealed at least two separate evolutionary clusters. Our results indicate the biotechnological potential of SDRs A5 and B3 for the production of chiral compounds with potential commercial value.

  6. 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.

  7. 2-Butanol and Butanone Production in Saccharomyces cerevisiae through Combination of a B12 Dependent Dehydratase and a Secondary Alcohol Dehydrogenase Using a TEV-Based Expression System

    PubMed Central

    Ghiaci, Payam; Norbeck, Joakim; Larsson, Christer

    2014-01-01

    2-Butanol and its chemical precursor butanone (methyl ethyl ketone – MEK) are chemicals with potential uses as biofuels and biocommodity chemicals. In order to produce 2-butanol, we have demonstrated the utility of using a TEV-protease based expression system to achieve equimolar expression of the individual subunits of the two protein complexes involved in the B12-dependent dehydratase step (from the pdu-operon of Lactobacillus reuterii), which catalyze the conversion of meso-2,3-butanediol to butanone. We have furthermore identified a NADH dependent secondary alcohol dehydrogenase (Sadh from Gordonia sp.) able to catalyze the subsequent conversion of butanone to 2-butanol. A final concentration of 4±0.2 mg/L 2-butanol and 2±0.1 mg/L of butanone was found. A key factor for the production of 2-butanol was the availability of NADH, which was achieved by growing cells lacking the GPD1 and GPD2 isogenes under anaerobic conditions. PMID:25054226

  8. Deletion of a conserved regulatory element in the Drosophila Adh gene leads to increased alcohol dehydrogenase activity but also delays development.

    PubMed Central

    Parsch, J; Russell, J A; Beerman, I; Hartl, D L; Stephan, W

    2000-01-01

    In vivo levels of enzymatic activity may be increased through either structural or regulatory changes. Here we use Drosophila melanogaster alcohol dehydrogenase (ADH) in an experimental test for selective differences between these two mechanisms. The well-known ADH-Slow (S)/Fast (F) amino acid replacement leads to a twofold increase in activity by increasing the catalytic efficiency of the enzyme. Disruption of a highly conserved, negative regulatory element in the Adh 3' UTR also leads to a twofold increase in activity, although this is achieved by increasing in vivo Adh mRNA and protein concentrations. These two changes appear to be under different types of selection, with positive selection favoring the amino acid replacement and purifying selection maintaining the 3' UTR sequence. Using transgenic experiments we show that deletion of the conserved 3' UTR element increases adult and larval Adh expression in both the ADH-F and ADH-S genetic backgrounds. However, the 3' UTR deletion also leads to a significant increase in developmental time in both backgrounds. ADH allozyme type has no detectable effect on development. These results demonstrate a negative fitness effect associated with Adh overexpression. This provides a mechanism whereby natural selection can discriminate between alternative pathways of increasing enzymatic activity. PMID:10978287

  9. Experimentally Increased Codon Bias in the Drosophila Adh Gene Leads to an Increase in Larval, But Not Adult, Alcohol Dehydrogenase Activity

    PubMed Central

    Hense, Winfried; Anderson, Nathan; Hutter, Stephan; Stephan, Wolfgang; Parsch, John; Carlini, David B.

    2010-01-01

    Although most amino acids can be encoded by more than one codon, the synonymous codons are not used with equal frequency. This phenomenon is known as codon bias and appears to be a universal feature of genomes. The translational selection hypothesis posits that the use of optimal codons, which match the most abundant species of isoaccepting tRNAs, results in increased translational efficiency and accuracy. Previous work demonstrated that the experimental reduction of codon bias in the Drosophila alcohol dehydrogenase (Adh) gene led to a significant decrease in ADH protein expression. In this study we performed the converse experiment: we replaced seven suboptimal leucine codons that occur naturally in the Drosophila melanogaster Adh gene with the optimal codon. We then compared the in vivo ADH activities imparted by the wild-type and mutant alleles. The introduction of optimal leucine codons led to an increase in ADH activity in third-instar larvae. In adult flies, however, the introduction of optimal codons led to a decrease in ADH activity. There is no evidence that other selectively constrained features of the Adh gene, or its rate of transcription, were altered by the synonymous replacements. These results are consistent with translational selection for codon bias being stronger in the larval stage and suggest that there may be a selective conflict over optimal codon usage between different developmental stages. PMID:19966063

  10. Experimentally increased codon bias in the Drosophila Adh gene leads to an increase in larval, but not adult, alcohol dehydrogenase activity.

    PubMed

    Hense, Winfried; Anderson, Nathan; Hutter, Stephan; Stephan, Wolfgang; Parsch, John; Carlini, David B

    2010-02-01

    Although most amino acids can be encoded by more than one codon, the synonymous codons are not used with equal frequency. This phenomenon is known as codon bias and appears to be a universal feature of genomes. The translational selection hypothesis posits that the use of optimal codons, which match the most abundant species of isoaccepting tRNAs, results in increased translational efficiency and accuracy. Previous work demonstrated that the experimental reduction of codon bias in the Drosophila alcohol dehydrogenase (Adh) gene led to a significant decrease in ADH protein expression. In this study we performed the converse experiment: we replaced seven suboptimal leucine codons that occur naturally in the Drosophila melanogaster Adh gene with the optimal codon. We then compared the in vivo ADH activities imparted by the wild-type and mutant alleles. The introduction of optimal leucine codons led to an increase in ADH activity in third-instar larvae. In adult flies, however, the introduction of optimal codons led to a decrease in ADH activity. There is no evidence that other selectively constrained features of the Adh gene, or its rate of transcription, were altered by the synonymous replacements. These results are consistent with translational selection for codon bias being stronger in the larval stage and suggest that there may be a selective conflict over optimal codon usage between different developmental stages.

  11. Human liver alcohol dehydrogenase: amino acid substitution in the beta 2 beta 2 Oriental isozyme explains functional properties, establishes an active site structure, and parallels mutational exchanges in the yeast enzyme.

    PubMed Central

    Jörnvall, H; Hempel, J; Vallee, B L; Bosron, W F; Li, T K

    1984-01-01

    The homodimeric Oriental beta 2 beta 2 isozyme of human liver alcohol dehydrogenase, corresponding to an allelic variant at the ADH2 gene locus, was studied in order to define the amino acid exchange in relation to the beta 1 beta 1 isozyme, the predominant allelic form among Caucasians. Sequence analysis reveals that the amino acid substitution occurs at position 7 of the largest CNBr fragment, corresponding to position 47 of the whole protein chain. Here, the beta 2 form has a histidine residue, while, in common with other characterized mammalian liver alcohol dehydrogenases, the beta 1 form has an arginine residue. This exchange does not affect the adjacent cysteine-46 residue, which is a protein ligand to the active-site zinc atom, thus clarifying previously inconsistent results. The histidine/arginine-47 mutational replacement corresponds to a position that binds the pyrophosphate group of the coenzyme NAD(H); this explains the functional differences between the beta 1 beta 1 and beta 2 beta 2 isozymes, including both a lower pH optimum and higher turnover number of beta 2 beta 2, which is likely to be the mutant form. The exchange demonstrates the existence of parallel but separate mutations in the evolution of alcohol dehydrogenases because these mammalian enzymes differ at exactly the same position by the same type of substitution as is found between a mutant and the wild-type constitutive forms of the corresponding yeast enzyme. PMID:6374651

  12. 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.

  13. 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.; 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 componentmore » in making biofuels production from lignocellulosic biomass feasible.« less

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

    PubMed

    Breicha, Klaus; Müller, Marion; Hummel, Werner; Niefind, Karsten

    2010-07-01

    Gre2p [Genes de respuesta a estres (stress-response gene)] from Saccharomyces cerevisiae is a monomeric enzyme of 342 amino acids with a molecular weight of 38.1 kDa. The enzyme catalyses both the stereospecific reduction of keto compounds and the oxidation of various hydroxy compounds and alcohols by the simultaneous consumption of the cofactor NADPH and formation of NADP(+). Crystals of a Gre2p complex with NADP(+) were grown using PEG 8000 as a precipitant. They belong to the monoclinic space group P2(1). The current diffraction resolution is 3.2 A. In spite of the monomeric nature of Gre2p in solution, packing and self-rotation calculations revealed the existence of two Gre2p protomers per asymmetric unit related by a twofold noncrystallographic axis.

  15. 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 Central

    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

  16. 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

  17. Alcoholism

    PubMed Central

    Girard, Donald E.; Carlton, Bruce E.

    1978-01-01

    There are important measurements of alcoholism that are poorly understood by physicians. Professional attitudes toward alcoholic patients are often counterproductive. Americans spend about $30 billion on alcohol a year and most adults drink alcohol. Even though traditional criteria allow for recognition of the disease, diagnosis is often made late in the natural course, when intervention fails. Alcoholism is a major health problem and accounts for 10 percent of total health care costs. Still, this country's 10 million adult alcoholics come from a pool of heavy drinkers with well defined demographic characteristics. These social, cultural and familial traits, along with subtle signs of addiction, allow for earlier diagnosis. Although these factors alone do not establish a diagnosis of alcoholism, they should alert a physician that significant disease may be imminent. Focus must be directed to these aspects of alcoholism if containment of the problem is expected. PMID:685264

  18. Cofactor Specificity of the Bifunctional Alcohol and Aldehyde Dehydrogenase (AdhE) in Wild-Type and Mutant Clostridium thermocellum and Thermoanaerobacterium saccharolyticum

    PubMed Central

    Zheng, Tianyong; Olson, Daniel G.; Tian, Liang; Bomble, Yannick J.; Himmel, Michael E.; Lo, Jonathan; Hon, Shuen; Shaw, A. Joe; van Dijken, Johannes P.

    2015-01-01

    ABSTRACT Clostridium thermocellum and Thermoanaerobacterium saccharolyticum are thermophilic bacteria that have been engineered to produce ethanol from the cellulose and hemicellulose fractions of biomass, respectively. Although engineered strains of T. saccharolyticum produce ethanol with a yield of 90% of the theoretical maximum, engineered strains of C. thermocellum produce ethanol at lower yields (∼50% of the theoretical maximum). In the course of engineering these strains, a number of mutations have been discovered in their adhE genes, which encode both alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) enzymes. To understand the effects of these mutations, the adhE genes from six strains of C. thermocellum and T. saccharolyticum were cloned and expressed in Escherichia coli, the enzymes produced were purified by affinity chromatography, and enzyme activity was measured. In wild-type strains of both organisms, NADH was the preferred cofactor for both ALDH and ADH activities. In high-ethanol-producing (ethanologen) strains of T. saccharolyticum, both ALDH and ADH activities showed increased NADPH-linked activity. Interestingly, the AdhE protein of the ethanologenic strain of C. thermocellum has acquired high NADPH-linked ADH activity while maintaining NADH-linked ALDH and ADH activities at wild-type levels. When single amino acid mutations in AdhE that caused increased NADPH-linked ADH activity were introduced into C. thermocellum and T. saccharolyticum, ethanol production increased in both organisms. Structural analysis of the wild-type and mutant AdhE proteins was performed to provide explanations for the cofactor specificity change on a molecular level. IMPORTANCE This work describes the characterization of the AdhE enzyme from different strains of C. thermocellum and T. saccharolyticum. C. thermocellum and T. saccharolyticum are thermophilic anaerobes that have been engineered to make high yields of ethanol and can solubilize components of

  19. 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.

  20. Alcohol Dehydrogenase Protects against Endoplasmic Reticulum Stress-Induced Myocardial Contractile Dysfunction via Attenuation of Oxidative Stress and Autophagy: Role of PTEN-Akt-mTOR Signaling

    PubMed Central

    Pang, Jiaojiao; Fuller, Nathan D.; Hu, Nan; Barton, Linzi A.; Henion, Jeremy M.; Guo, Rui; Chen, Yuguo; Ren, Jun

    2016-01-01

    Background The endoplasmic reticulum (ER) plays an essential role in ensuring proper folding of the newly synthesized proteins. Aberrant ER homeostasis triggers ER stress and development of cardiovascular diseases. ADH is involved in catalyzing ethanol to acetaldehyde although its role in cardiovascular diseases other than ethanol metabolism still remains elusive. This study was designed to examine the impact of ADH on ER stress-induced cardiac anomalies and underlying mechanisms involved using cardiac-specific overexpression of alcohol dehydrogenase (ADH). Methods ADH and wild-type FVB mice were subjected to the ER stress inducer tunicamycin (1 mg/kg, i.p., for 48 hrs). Myocardial mechanical and intracellular Ca2+ properties, ER stress, autophagy and associated cell signaling molecules were evaluated. Results ER stress compromised cardiac contractile function (evidenced as reduced fractional shortening, peak shortening, maximal velocity of shortening/relengthening, prolonged relengthening duration and impaired intracellular Ca2+ homeostasis), oxidative stress and upregulated autophagy (increased LC3B, Atg5, Atg7 and p62), along with dephosphorylation of PTEN, Akt and mTOR, all of which were attenuated by ADH. In vitro study revealed that ER stress-induced cardiomyocyte anomaly was abrogated by ADH overexpression or autophagy inhibition using 3-MA. Interestingly, the beneficial effect of ADH was obliterated by autophagy induction, inhibition of Akt and mTOR. ER stress also promoted phosphorylation of the stress signaling ERK and JNK, the effect of which was unaffected by ADH transgene. Conclusions Taken together, these findings suggested that ADH protects against ER stress-induced cardiac anomalies possibly via attenuation of oxidative stress and PTEN/Akt/mTOR pathway-regulated autophagy. PMID:26807981

  1. 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

  2. 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.

  3. A Cladistic Analysis of Phenotypic Associations with Haplotypes Inferred from Restriction Endonuclease Mapping. I. Basic Theory and an Analysis of Alcohol Dehydrogenase Activity in Drosophila

    PubMed Central

    Templeton, Alan R.; Boerwinkle, Eric; Sing, Charles F.

    1987-01-01

    Because some genes have been cloned that have a known biochemical or physiological function, genetic variation can be measured in a population at loci that may directly influence a phenotype of interest. With this measured genotype approach, specific alleles or haplotypes in the probed DNA region can be assigned phenotypic effects. In this paper we address several problems encountered in implementing the measured genotype approach with restriction site data. A number of analytical problems arise in part as a consequence of the linkage disequilibrium that is commonly encountered when dealing with small DNA regions: 1) different restriction site polymorphisms are not statistically independent, 2) the sites being measured are not likely to be the direct cause of the associated phenotypic effects, 3) haplotype classes may be phenotypically heterogeneous, and 4) the sites that are most strongly associated with phenotypic effects are not necessarily the most closely linked to the actual genetic cause of the effects. When recombination and gene conversion are rare, the primary cause of linkage disequilibrium is history (mutational origin, genetic drift, hitchhiking, etc.). We deal with historical association directly by producing a cladogram that partially reconstructs the evolutionary history of the present-day haplotype variability. The cladogram defines a nested analysis of variance that simultaneously detects phenotypic effects, localizes the effects within the cladogram, and identifies haplotypes that are potentially heterogeneous in their phenotypic associations. The power of this approach is illustrated by an analysis of the associations between alcohol dehydrogenase (ADH) activity and restriction site variability in a 13-kb fragment surrounding the ADH locus in Drosophila melanogaster. PMID:2822535

  4. 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.

  5. 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

    Nixon, Julie E J; Wang, Amy; Field, Jessica; Morrison, Hilary G; McArthur, Andrew G; Sogin, Mitchell L; Loftus, Brendan J; Samuelson, John

    2002-04-01

    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.

  6. Hypoxic and Anoxic Induction of Alcohol Dehydrogenase in Roots and Shoots of Seedlings of Zea mays (Adh Transcripts and Enzyme Activity).

    PubMed Central

    Andrews, D. L.; Cobb, B. G.; Johnson, J. R.; Drew, M. C.

    1993-01-01

    Alcohol dehydrogenase (ADH) is one of a number of enzymes of glycolysis and fermentation known to be synthesized preferentially under low O2 conditions. We examined levels of Adh1 transcripts and of ADH activity in 5-mm root tips, root axes (the remainder of the seminal root), and shoots of maize (Zea mays L. cv TX 5855) seedlings. Seedlings with roots averaging about 60-mm long were transferred from fully aerobic conditions (solutions sparged with 40% [v/v] O2) to anaerobic (O2-free) conditions, or to an intermediate O2 concentration. There was no prior acclimation to low O2. In root tips, anoxia induced Adh1 transcripts and enzyme activity at 6 h, but this was followed by a rapid decline so that at 12 to 18 h neither were detectable and the root tips were dead. In contrast, higher levels of Adh1 transcripts and enzyme activity were maintained for at least 48 h in root axes and shoots. When induction at 6 h was measured over a wide range of O2 concentrations, a peak in ADH activity occurred in all tissues at 4% (v/v) O2. Maximum levels of transcripts, however, were in the range of 0 to 4% O2, depending on the tissue. The time course of hypoxic induction (at 4% O2) in root tips showed a peak in transcript levels at 6 h, whereas ADH activity continued to rise throughout the 24-h experiment. These results show that in root tips, ADH induction by anoxia was small and transient relative to induction by hypoxia. PMID:12231696

  7. 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. PMID:15726639

  8. Picosecond-resolved fluorescence studies of substrate and cofactor-binding domain mutants in a thermophilic alcohol dehydrogenase uncover an extended network of communication.

    PubMed

    Meadows, Corey W; Tsang, Jonathan E; Klinman, Judith P

    2014-10-22

    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

  9. Genomic clones of Aspergillus nidulans containing alcA, the structural gene for alcohol dehydrogenase and alcR, a regulatory gene for ethanol metabolism.

    PubMed

    Doy, C H; Pateman, J A; Olsen, J E; Kane, H J; Creaser, E H

    1985-04-01

    Our aim was to obtain from Aspergillus nidulans a genomic bank and then clone a region we expected from earlier genetic mapping to contain two closely linked genes, alcA, the structural gene for alcohol dehydrogenase (ADH) and alcR, a positive trans-acting regulatory gene for ethanol metabolism. The expression of alcA is repressed by carbon catabolites. A genomic restriction fragment characteristic of the alcA-alcR region was identified, cloned in pBR322, and used to select from a genomic bank in lambda EMBL3A three overlapping clones covering 24 kb of DNA. Southern genomic analysis of wild-type, alcA and alcR mutants showed that the mutants contained extra DNA at sites near the center of the cloned DNA and are close together, as expected for alcA and alcR. Transcription from the cloned DNA and hybridization with a clone carrying the Saccharomyces cerevisiae gene for ADHI (ADC1) are both confined to the alcA-alcR region. At least one of several species of mature mRNA is about 1 kb, the size required to code for ADH. For all species, carbon catabolite repression overrides control by induction. The overall characteristics of transcription, hybridization to ADC1 and earlier work suggest that alcA consists of a number of exons and/or that the alcA-alcR region represents a cluster of alcA-related genes or sequences.

  10. 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

  11. Picosecond-resolved fluorescent probes at functionally distinct tryptophans within a thermophilic alcohol dehydrogenase: relationship of temperature-dependent changes in fluorescence to catalysis.

    PubMed

    Meadows, Corey W; Ou, Ryan; Klinman, Judith P

    2014-06-12

    Two single-tryptophan variants were generated in a thermophilic alcohol dehydrogenase with the goal of correlating temperature-dependent changes in local fluorescence with the previously demonstrated catalytic break at ca. 30 °C (Kohen et al., Nature 1999, 399, 496). One tryptophan variant, W87in, resides at the active site within van der Waals contact of bound alcohol substrate; the other variant, W167in, is a remote-site surface reporter located >25 Å from the active site. Picosecond-resolved fluorescence measurements were used to analyze fluorescence lifetimes, time-dependent Stokes shifts, and the extent of collisional quenching at Trp87 and Trp167 as a function of temperature. A subnanosecond fluorescence decay rate constant has been detected for W87in that is ascribed to the proximity of the active site Zn(2+) and shows a break in behavior at 30 °C. For the remainder of the reported lifetime measurements, there is no detectable break between 10 and 50 °C, in contrast with previously reported hydrogen/deuterium exchange experiments that revealed a temperature-dependent break analogous to catalysis (Liang et al., Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 9556). We conclude that the motions that lead to the rigidification of ht-ADH below 30 °C are likely to be dominated by global processes slower than the picosecond to nanosecond motions measured herein. In the case of collisional quenching of fluorescence by acrylamide, W87in and W167in behave in a similar manner that resembles free tryptophan in water. Stokes shift measurements, by contrast, show distinctive behaviors in which the active-site tryptophan relaxation is highly temperature-dependent, whereas the solvent-exposed tryptophan's dynamics are temperature-independent. These data are concluded to reflect a significantly constrained environment surrounding the active site Trp87 that both increases the magnitude of the Stokes shift and its temperature-dependence. The results are discussed in the context

  12. Picosecond-Resolved Fluorescent Probes at Functionally Distinct Tryptophans within a Thermophilic Alcohol Dehydrogenase: Relationship of Temperature-Dependent Changes in Fluorescence to Catalysis

    PubMed Central

    2015-01-01

    Two single-tryptophan variants were generated in a thermophilic alcohol dehydrogenase with the goal of correlating temperature-dependent changes in local fluorescence with the previously demonstrated catalytic break at ca. 30 °C (Kohen et al., Nature1999, 399, 496). One tryptophan variant, W87in, resides at the active site within van der Waals contact of bound alcohol substrate; the other variant, W167in, is a remote-site surface reporter located >25 Å from the active site. Picosecond-resolved fluorescence measurements were used to analyze fluorescence lifetimes, time-dependent Stokes shifts, and the extent of collisional quenching at Trp87 and Trp167 as a function of temperature. A subnanosecond fluorescence decay rate constant has been detected for W87in that is ascribed to the proximity of the active site Zn2+ and shows a break in behavior at 30 °C. For the remainder of the reported lifetime measurements, there is no detectable break between 10 and 50 °C, in contrast with previously reported hydrogen/deuterium exchange experiments that revealed a temperature-dependent break analogous to catalysis (Liang et al., Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 9556). We conclude that the motions that lead to the rigidification of ht-ADH below 30 °C are likely to be dominated by global processes slower than the picosecond to nanosecond motions measured herein. In the case of collisional quenching of fluorescence by acrylamide, W87in and W167in behave in a similar manner that resembles free tryptophan in water. Stokes shift measurements, by contrast, show distinctive behaviors in which the active-site tryptophan relaxation is highly temperature-dependent, whereas the solvent-exposed tryptophan’s dynamics are temperature-independent. These data are concluded to reflect a significantly constrained environment surrounding the active site Trp87 that both increases the magnitude of the Stokes shift and its temperature-dependence. The results are discussed in the context

  13. 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

  14. 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.

  15. CAD/CAM/CNC.

    ERIC Educational Resources Information Center

    Domermuth, Dave; And Others

    1996-01-01

    Includes "Quick Start CNC (computer numerical control) with a Vacuum Filter and Laminated Plastic" (Domermuth); "School and Industry Cooperate for Mutual Benefit" (Buckler); and "CAD (computer-assisted drafting) Careers--What Professionals Have to Say" (Skinner). (JOW)

  16. 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)

  17. Isozyme multiplicity with anomalous dimer patterns in a class III alcohol dehydrogenase. Effects on the activity and quaternary structure of residue exchanges at "nonfunctional" sites in a native protein.

    PubMed

    Danielsson, O; Shafqat, J; Estonius, M; el-Ahmad, M; Jörnvall, H

    1996-11-19

    The isozymes of class III alcohol dehydrogenase/glutathione-dependent formaldehyde dehydrogenase from cod were characterized. They exhibited three unexpected properties of general interest. First, these dimeric isozymes, derived from two types of subunit (h and l, for high- and low-activity forms), were recovered from liver preparations in only the homodimeric ll and heterodimeric hl combinations. Dissociation and reassociation of the isolated hl form in vitro also resulted in lower yields of the hh than the ll homodimer, although class III subunits are usually freely associable over wide borders of divergence (human and Drosophila). The h and l primary structures show that both chain types are characteristic of class III enzymes, without large amino acid replacements at positions of known subunit interactions. Hence, the hh dimer partial restriction indicates nontraditional alterations at h-subunit interfaces. The structure provides a possible explanation, in the form of h-chain modifications that may influence the anchoring of a loop at positions of two potentially deamidative beta-aspartyl shifts at distant Asn-Gly structures. Second the ll and hl forms differ in enzymatic properties, having 5-fold different K(m) values for NAD+ at pH 8, different K(m) values for S-(hydroxymethyl)glutathione (10 versus 150 microM), and different specific activities (4.5 versus 41 units/mg), with ll resembling and hl deviating from human and other class III alcohol dehydrogenases. However, functional residues lining substrate and coenzyme pockets in the known conformations of homologous forms are largely identical in the two isozymes [only minor conservative exchanges of Val/Leu116, Val/Leu203, Ile/Val224, and Ile/Val269 (numbering system of the human class I enzyme)], again indicating effects from distantly positioned h-chain replacements. Third, the two isozymes differ a surprising amount in amino acid sequence (18%, the same as the piscine/ human difference), reflecting a

  18. A novel zinc-binding alcohol dehydrogenase 2 from Arachis diogoi, expressed in resistance responses against late leaf spot pathogen, induces cell death when transexpressed in tobacco.

    PubMed

    Kumar, Dilip; Rampuria, Sakshi; Singh, Naveen Kumar; Kirti, Pulugurtha B

    2016-03-01

    A novel zinc-binding alcohol dehydrogenase 2 (AdZADH2) was significantly upregulated in a wild peanut, Arachis diogoi treated with conidia of late leaf spot (LLS) pathogen, Phaeoisariopsis personata. This upregulation was not observed in a comparative analysis of cultivated peanut, which is highly susceptible to LLS. This zinc-binding alcohol dehydrogenase possessed a Rossmann fold containing NADB domain in addition to the MDR domain present in all previously characterized plant ADH genes/proteins. Transient over-expression of AdZADH2 under an estradiol inducible promoter (XVE) resulted in hypersensitive response (HR)-like cell death in tobacco leaf. However, the same level of cell death was not observed when the domains were transiently expressed individually. Cell death observed in tobacco was associated with overexpression of cell death related proteins, antioxidative enzymes such as SOD, CAT and APX and pathogenesis-related (PR) proteins. In A. diogoi, AdZADH2 expression was significantly upregulated in response to the plant signaling hormones salicylic acid, methyl jasmonate, and sodium nitroprusside. PMID:27047748

  19. Ergot alkaloid biosynthesis in Aspergillus fumigatus: conversion of chanoclavine-I to chanoclavine-I aldehyde catalyzed by a short-chain alcohol dehydrogenase FgaDH.

    PubMed

    Wallwey, Christiane; Matuschek, Marco; Li, Shu-Ming

    2010-02-01

    Ergot alkaloids are toxins and important pharmaceuticals which are produced biotechnologically on an industrial scale. A putative gene fgaDH has been identified in the biosynthetic gene cluster of fumigaclavine C, an ergot alkaloid of the clavine-type. The deduced gene product FgaDH comprises 261 amino acids with a molecular mass of about 27.8 kDa and contains the conserved motifs of classical short-chain dehydrogenases/reductases (SDRs), but shares no worth mentioning sequence similarity with SDRs and other known proteins. The coding region of fgaDH consisting of two exons was amplified by PCR from a cDNA library of Aspergillus fumigatus, cloned into pQE60 and overexpressed in E. coli. The soluble tetrameric His(6)-FgaDH was purified to apparent homogeneity and characterized biochemically. It has been shown that FgaDH catalyzes the oxidation of chanoclavine-I in the presence of NAD(+) resulting in the formation of chanoclavine-I aldehyde, which was unequivocally identified by NMR and MS analyzes. Therefore, FgaDH functions as a chanoclavine-I dehydrogenase and represents a new group of short-chain dehydrogenases. K (M) values for chanoclavine-I and NAD(+) were determined at 0.27 and 1.1 mM, respectively. The turnover number was 0.38 s(-1). PMID:20039019

  20. Crystallization and preliminary X-ray diffraction analysis of the trigonal crystal form of Saccharomyces cerevisiae alcohol dehydrogenase I: evidence for the existence of Zn ions in the crystal.

    PubMed

    Kim, Kyung Jin; Howard, Andrew J

    2002-08-01

    Saccharomyces cerevisiae alcohol dehydrogenase I crystallized as trigonal plates using 20% 2-propanol and 20% PEG 4000 in 0.1 M sodium citrate buffer pH 5.6 in the presence of 1 mM NAD(+). The crystals diffract to 3.0 A resolution and belong to the trigonal space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 146.3, c = 68.1 A, alpha = beta = 90, gamma = 120 degrees. X-ray data were collected from frozen crystals at the 17-ID beamline of the Advanced Photon Source. A Zn fluorescence scan of the crystal produced a peak at 9671.6 eV, suggesting the existence of Zn ions in the crystal.

  1. CAD/CAE Integration Enhanced by New CAD Services Standard

    NASA Technical Reports Server (NTRS)

    Claus, Russell W.

    2002-01-01

    A Government-industry team led by the NASA Glenn Research Center has developed a computer interface standard for accessing data from computer-aided design (CAD) systems. The Object Management Group, an international computer standards organization, has adopted this CAD services standard. The new standard allows software (e.g., computer-aided engineering (CAE) and computer-aided manufacturing software to access multiple CAD systems through one programming interface. The interface is built on top of a distributed computing system called the Common Object Request Broker Architecture (CORBA). CORBA allows the CAD services software to operate in a distributed, heterogeneous computing environment.

  2. CAD/CAM for optomechatronics

    NASA Astrophysics Data System (ADS)

    Zhou, Haiguang; Han, Min

    2003-10-01

    We focus at CAD/CAM for optomechatronics. We have developed a kind of CAD/CAM, which is not only for mechanics but also for optics and electronic. The software can be used for training and education. We introduce mechanical CAD, optical CAD and electrical CAD, we show how to draw a circuit diagram, mechanical diagram and luminous transmission diagram, from 2D drawing to 3D drawing. We introduce how to create 2D and 3D parts for optomechatronics, how to edit tool paths, how to select parameters for process, how to run the post processor, dynamic show the tool path and generate the CNC programming. We introduce the joint application of CAD&CAM. We aim at how to match the requirement of optical, mechanical and electronics.

  3. CAD for small hydro projects

    SciTech Connect

    Bishop, N.A. Jr. )

    1994-04-01

    Over the past decade, computer-aided design (CAD) has become a practical and economical design tool. Today, specifying CAD hardware and software is relatively easy once you know what the design requirements are. But finding experienced CAD professionals is often more difficult. Most CAD users have only two or three years of design experience; more experienced design personnel are frequently not CAD literate. However, effective use of CAD can be the key to lowering design costs and improving design quality--a quest familiar to every manager and designer. By emphasizing computer-aided design literacy at all levels of the firm, a Canadian joint-venture company that specializes in engineering small hydroelectric projects has cut costs, become more productive and improved design quality. This article describes how they did it.

  4. Computerized design of CAD

    NASA Astrophysics Data System (ADS)

    Paul, B. E.; Pham, T. A.

    1982-11-01

    A computerized ballistic design technique for CAD/PAD is described by which a set of ballistic design parameters are determined, all of which satisfy a particular performance requirement. In addition, the program yields the remaining performance predictions, so that only a very few computer runs of the design program can quickly bring the ballistic design within the specification limits prescribed. An example is presented for a small propulsion device, such as a remover or actuator, for which the input specifications define a maximum allowable thrust and minimum end-of-stroke velocity. The resulting output automatically satisfies the input requirements, and will always yield an acceptable ballistic design.

  5. Characterization of the Enantioselective Properties of the Quinohemoprotein Alcohol Dehydrogenase of Acetobacter pasteurianus LMG 1635. 1. Different Enantiomeric Ratios of Whole Cells and Purified Enzyme in the Kinetic Resolution of Racemic Glycidol.

    PubMed

    Machado, S S; Wandel, U; Jongejan, J A; Straathof, A J; Duine, J A

    1999-01-01

    Resting cells of Acetobacter pasteurianus LMG 1635 (ATCC 12874) show appreciable enantioselectivity (E=16-18) in the oxidative kinetic resolution of racemic 2,3-epoxy-1-propanol, glycidol. Distinctly lower values (E=7-9) are observed for the ferricyanide-coupled oxidation of glycidol by the isolated quinohemoprotein alcohol dehydrogenase, QH-ADH, which is responsible for the enantiospecific oxidation step in whole cells. The accuracy of E-values from conversion experiments could be verified using complementary methods for the measurement of enantiomeric ratios. Effects of pH, detergent, the use of artificial electron acceptors, and the presence of intermediate aldehydes, could be accounted for. Measurements of E-values at successive stages of the purification showed that the drop in enantioselectivity correlates with the separation of QH-ADH from the cytoplasmic membrane. It is argued that the native arrangement of QH-ADH in the membrane-associated complex favors the higher E-values. The consequences of these findings for the use of whole cells versus purified enzymes in biocatalytic kinetic resolutions of chiral alcohols are discussed.

  6. TRAD or CAD? A Comparison.

    ERIC Educational Resources Information Center

    Resetarits, Paul J.

    1989-01-01

    Studies whether traditional drafting equipment (TRAD) or computer aided drafting equipment (CAD) is more effective. Proposes that students using only CAD can learn principles of drafting as well as students using only TRAD. Reports no significant difference either on achievement or attitude. (MVL)

  7. CAD/CAM data management

    NASA Technical Reports Server (NTRS)

    Bray, O. H.

    1984-01-01

    The role of data base management in CAD/CAM, particularly for geometric data is described. First, long term and short term objectives for CAD/CAM data management are identified. Second, the benefits of the data base management approach are explained. Third, some of the additional work needed in the data base area is discussed.

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

    PubMed

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

    2015-07-01

    Klebsiella oxytoca KMS005 (∆adhE∆ackA-pta∆ldhA) was metabolically engineered to improve 2,3-butanediol (BDO) yield. Elimination of alcohol dehydrogenase E (adhE), acetate kinase A-phosphotransacetylase (ackA-pta), and lactate dehydrogenase A (ldhA) enzymes allowed BDO production as a primary pathway for NADH re-oxidation, and significantly reduced by-products. KMS005 was screened for the efficient glucose utilization by metabolic evolution. KMS005-73T improved BDO production at a concentration of 23.5±0.5 g/L with yield of 0.46±0.02 g/g in mineral salts medium containing 50 g/L glucose in a shake flask. KMS005-73T also exhibited BDO yields of about 0.40-0.42 g/g from sugarcane molasses, cassava starch, and maltodextrin. During fed-batch fermentation, KMS005-73T produced BDO at a concentration, yield, and overall and specific productivities of 117.4±4.5 g/L, 0.49±0.02 g/g, 1.20±0.05 g/Lh, and 27.2±1.1 g/gCDW, respectively. No acetoin, lactate, and formate were detected, and only trace amounts of acetate and ethanol were formed. The strain also produced the least by-products and the highest BDO yield among other Klebsiella strains previously developed. PMID:25895450

  9. Change in ATP-binding cassette B1/19, glutamine synthetase and alcohol dehydrogenase gene expression during root elongation in Betula pendula Roth and Alnus glutinosa L. Gaertn in response to leachate and leonardite humic substances.

    PubMed

    Tahiri, Abdelghani; Delporte, Fabienne; Muhovski, Yordan; Ongena, Marc; Thonart, Philippe; Druart, Philippe

    2016-01-01

    Humic substances (HS) are complex and heterogeneous compounds of humified organic matter resulting from the chemical and microbiological decomposition of organic residues. HS have a positive effect on plant growth and development by improving soil structure and fertility. They have long been recognized as plant growth-promoting substances, particularly with regard to influencing nutrient uptake, root growth and architecture. The biochemical and molecular mechanisms through which HS influence plant physiology are not well understood. This study evaluated the bioactivity of landfill leachate and leonardite HS on alder (Alnus glutinosa L. Gaertn) and birch (Betula pendula Roth) during root elongation in vitro. Changes in root development were studied in relation to auxin, carbon and nitrogen metabolisms, as well as to the stress adaptive response. The cDNA fragments of putative genes encoding two ATP-binding cassette (ABC) transporters (ABCB1 and ABCB19) belonging to the B subfamily of plant ABC auxin transporters were cloned and sequenced. Molecular data indicate that HS and their humic acid (HA) fractions induce root growth by influencing polar auxin transport (PAT), as illustrated by the modulation of the ABCB transporter transcript levels (ABCB1 and ABCB19). There were also changes in alcohol dehydrogenase (ADH) and glutamine synthetase (GS) gene transcript levels in response to HS exposure. These findings confirmed that humic matter affects plant growth and development through various metabolic pathways, including hormonal, carbon and nitrogen metabolisms and stress response or signalization.

  10. The active (ADHa) and inactive (ADHi) forms of the PQQ-alcohol dehydrogenase from Gluconacetobacter diazotrophicus differ in their respective oligomeric structures and redox state of their corresponding prosthetic groups.

    PubMed

    Gómez-Manzo, Saúl; González-Valdez, Alejandra Abigail; Oria-Hernández, Jesús; Reyes-Vivas, Horacio; Arreguín-Espinosa, Roberto; Kroneck, Peter M H; Sosa-Torres, Martha Elena; Escamilla, Jose E

    2012-03-01

    The membrane-bound alcohol dehydrogenase of Gluconacetobacter diazotrophicus contains one pyrroloquinoline quinone moiety (PQQ), one [2Fe-2S] cluster, and four c-type cytochromes. Here, we describe a novel and inactive enzyme. ADHi, similarly to ADHa, is a heterodimer of 72- and 44-kDa subunits and contains the expected prosthetic groups. However, ADHa showed a threefold molecular mass as compared to ADHi. Noteworthy, the PQQ, the [2Fe-2S] and most of the cytochromes in purified ADHi is in the oxidized form, contrasting with ADHa where the PQQ-semiquinone is detected and the [2Fe-2S] cluster as well as the cytochromes c remained fully reduced after purification. Reduction kinetics of the ferricyanide-oxidized enzymes showed that while ADHa was brought back by ethanol to its full reduction state, in ADHi, only one-quarter of the total heme c was reduced. The dithionite-reduced ADHi was largely oxidized by ubiquinone-2, thus indicating that intramolecular electron transfer is not impaired in ADHi. The acidic pH of the medium might be deleterious for the membrane-bound ADH by causing conformational changes leading to changes in the relative orientation of heme groups and shift of corresponding redox potential to higher values. This would hamper electron transfer resulting in the low activity observed in ADHi.

  11. Change in ATP-binding cassette B1/19, glutamine synthetase and alcohol dehydrogenase gene expression during root elongation in Betula pendula Roth and Alnus glutinosa L. Gaertn in response to leachate and leonardite humic substances.

    PubMed

    Tahiri, Abdelghani; Delporte, Fabienne; Muhovski, Yordan; Ongena, Marc; Thonart, Philippe; Druart, Philippe

    2016-01-01

    Humic substances (HS) are complex and heterogeneous compounds of humified organic matter resulting from the chemical and microbiological decomposition of organic residues. HS have a positive effect on plant growth and development by improving soil structure and fertility. They have long been recognized as plant growth-promoting substances, particularly with regard to influencing nutrient uptake, root growth and architecture. The biochemical and molecular mechanisms through which HS influence plant physiology are not well understood. This study evaluated the bioactivity of landfill leachate and leonardite HS on alder (Alnus glutinosa L. Gaertn) and birch (Betula pendula Roth) during root elongation in vitro. Changes in root development were studied in relation to auxin, carbon and nitrogen metabolisms, as well as to the stress adaptive response. The cDNA fragments of putative genes encoding two ATP-binding cassette (ABC) transporters (ABCB1 and ABCB19) belonging to the B subfamily of plant ABC auxin transporters were cloned and sequenced. Molecular data indicate that HS and their humic acid (HA) fractions induce root growth by influencing polar auxin transport (PAT), as illustrated by the modulation of the ABCB transporter transcript levels (ABCB1 and ABCB19). There were also changes in alcohol dehydrogenase (ADH) and glutamine synthetase (GS) gene transcript levels in response to HS exposure. These findings confirmed that humic matter affects plant growth and development through various metabolic pathways, including hormonal, carbon and nitrogen metabolisms and stress response or signalization. PMID:26595095

  12. Towards catalyst compartimentation in combined chemo- and biocatalytic processes: immobilization of alcohol dehydrogenases for the diastereoselective reduction of a β-hydroxy ketone obtained from an organocatalytic aldol reaction.

    PubMed

    Rulli, Giuseppe; Heidlindemann, Marcel; Berkessel, Albrecht; Hummel, Werner; Gröger, Harald

    2013-11-01

    The alcohol dehydrogenases (ADHs) from Lactobacillus kefir and Rhodococcus sp., which earlier turned out to be suitable for a chemoenzymatic one-pot synthesis with organocatalysts, were immobilized with their cofactors on a commercially available superabsorber based on a literature known protocol. The use of the immobilized ADH from L. kefir in the reduction of acetophenone as a model substrate led to high conversion (>95%) in the first reaction cycle, followed by a slight decrease of conversion in the second reaction cycle. A comparable result was obtained when no cofactor was added although a water rich reaction media was used. The immobilized ADHs also turned out to be suitable catalysts for the diastereoselective reduction of an organocatalytically prepared enantiomerically enriched aldol adduct, leading to high conversion, diastereomeric ratio and enantioselectivity for the resulting 1,3-diols. However, at a lower catalyst and cofactor amount being still sufficient for biotransformations with "free" enzymes the immobilized ADH only showed high conversion and >99% ee for the first reaction cycle whereas a strong decrease of conversion was observed already in the second reaction cycle, thus indicating a significant leaching effect of catalyst and/or cofactor.

  13. Evaluation of Five Microcomputer CAD Packages.

    ERIC Educational Resources Information Center

    Leach, James A.

    1987-01-01

    Discusses the similarities, differences, advanced features, applications and number of users of five microcomputer computer-aided design (CAD) packages. Included are: "AutoCAD (V.2.17)"; "CADKEY (V.2.0)"; "CADVANCE (V.1.0)"; "Super MicroCAD"; and "VersaCAD Advanced (V.4.00)." Describes the evaluation of the packages and makes recommendations for…

  14. Expression pattern, ethanol-metabolizing activities, and cellular localization of alcohol and aldehyde dehydrogenases in human large bowel: association of the functional polymorphisms of ADH and ALDH genes with hemorrhoids and colorectal cancer.

    PubMed

    Chiang, Chien-Ping; Jao, Shu-Wen; Lee, Shiao-Pieng; Chen, Pei-Chi; Chung, Chia-Chi; Lee, Shou-Lun; Nieh, Shin; Yin, Shih-Jiun

    2012-02-01

    Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) are principal enzymes responsible for metabolism of ethanol. Functional polymorphisms of ADH1B, ADH1C, and ALDH2 genes occur among racial populations. The goal of this study was to systematically determine the functional expressions and cellular localization of ADHs and ALDHs in human rectal mucosa, the lesions of adenocarcinoma and hemorrhoid, and the genetic association of allelic variations of ADH and ALDH with large bowel disorders. Twenty-one surgical specimens of rectal adenocarcinoma and the adjacent normal mucosa, including 16 paired tissues of rectal tumor, normal mucosae of rectum and sigmoid colon from the same individuals, and 18 surgical mixed hemorrhoid specimens and leukocyte DNA samples from 103 colorectal cancer patients, 67 hemorrhoid patients, and 545 control subjects recruited in previous study, were investigated. The isozyme/allozyme expression patterns of ADH and ALDH were identified by isoelectric focusing and the activities were assayed spectrophotometrically. The protein contents of ADH/ALDH isozymes were determined by immunoblotting using the corresponding purified class-specific antibodies; the cellular activity and protein localizations were detected by immunohistochemistry and histochemistry, respectively. Genotypes of ADH1B, ADH1C, and ALDH2 were determined by polymerase chain reaction-restriction fragment length polymorphisms. At 33mM ethanol, pH 7.5, the activity of ADH1C*1/1 phenotypes exhibited 87% higher than that of the ADH1C*1/*2 phenotypes in normal rectal mucosa. The activity of ALDH2-active phenotypes of rectal mucosa was 33% greater than ALDH2-inactive phenotypes at 200μM acetaldehyde. The protein contents in normal rectal mucosa were in the following order: ADH1>ALDH2>ADH3≈ALDH1A1, whereas those of ADH2, ADH4, and ALDH3A1 were fairly low. Both activity and content of ADH1 were significantly decreased in rectal tumors, whereas the ALDH activity remained

  15. Glucose-6-phosphate dehydrogenase

    MedlinePlus

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

  16. CAD Services: an Industry Standard Interface for Mechanical CAD Interoperability

    NASA Technical Reports Server (NTRS)

    Claus, Russell; Weitzer, Ilan

    2002-01-01

    Most organizations seek to design and develop new products in increasingly shorter time periods. At the same time, increased performance demands require a team-based multidisciplinary design process that may span several organizations. One approach to meet these demands is to use 'Geometry Centric' design. In this approach, design engineers team their efforts through one united representation of the design that is usually captured in a CAD system. Standards-based interfaces are critical to provide uniform, simple, distributed services that enable the 'Geometry Centric' design approach. This paper describes an industry-wide effort, under the Object Management Group's (OMG) Manufacturing Domain Task Force, to define interfaces that enable the interoperability of CAD, Computer Aided Manufacturing (CAM), and Computer Aided Engineering (CAE) tools. This critical link to enable 'Geometry Centric' design is called: Cad Services V1.0. This paper discusses the features of this standard and proposed application.

  17. The binding site of a steroid hormone receptor-like protein within the Drosophila Adh adult enhancer is required for high levels of tissue-specific alcohol dehydrogenase expression.

    PubMed Central

    Ayer, S; Benyajati, C

    1992-01-01

    Developmental and tissue-specific transcription from the Adh distal promoter is regulated in part by the Adh adult enhancer, located 450 to 600 bp upstream from the distal RNA start site. We have characterized four proteins (DEP1 to DEP4), present in Drosophila tissue culture cell nuclear extracts, which bind to this enhancer. DEP1 and DEP2 bind to a positive cis-acting element (-492 to -481) and share nucleotide contacts. A small linker replacement deletion mutation, which disrupts the overlapping DEP1- and DEP2-binding sites, reduces Adh distal transcription in an alcohol dehydrogenase (ADH)-expressing cultured cell line, in the adult fat body (the major tissue of ADH expression), as well as in some but not all adult tissues where ADH is normally expressed. This enhancer element contains an imperfect palindromic sequence similar to steroid hormone receptor superfamily response elements. Binding-site screening of a lambda gt11 expression library has identified the steroid receptor superfamily member fushi tarazu factor 1 (FTZ-F1) as a protein that binds to this site. Anti-FTZ-F1 antibodies have identified DEP1 as FTZ-F1. DEP2 also binds to the FTZ-F1 site from the fushi tarazu zebra element, suggesting that DEP2 may also be a steroid receptor superfamily member. Our results raise the possibility that Adh regulation in certain adult tissues involves a hormone-mediated pathway. Because DEP1 (FTZ-F1) and DEP2 contact some of the same nucleotides within the positive cis element, it is unlikely that they can bind simultaneously. Such alternative binding may play a role in the tissue-specific and developmental transcription of Adh. Images PMID:1732738

  18. CAD systems simplify engineering drawings

    SciTech Connect

    Holt, J.

    1986-10-01

    Computer assisted drafting systems, with today's technology, provide high-quality, timely drawings that can be justified by the lower costs for the final product. The author describes Exxon Pipeline Co.'s experience in deciding on hardware and software for a CAD system installation and the benefits effected by this procedure and equipment.

  19. CAD Skills Increased through Multicultural Design Project

    ERIC Educational Resources Information Center

    Clemons, Stephanie

    2006-01-01

    This article discusses how students in a college-entry-level CAD course researched four generations of their family histories and documented cultural and symbolic influences within their family backgrounds. AutoCAD software was then used to manipulate those cultural and symbolic images to create the design for a multicultural area rug. AutoCAD was…

  20. Cool-and Unusual-CAD Applications

    ERIC Educational Resources Information Center

    Calhoun, Ken

    2004-01-01

    This article describes several very useful applications of AutoCAD that may lie outside the normal scope of application. AutoCAD commands used in this article are based on AutoCAD 2000I. The author and his students used a Hewlett Packard 750C DesignJet plotter for plotting. (Contains 5 figures and 5 photos.)

  1. Temporal expression of the human alcohol dehydrogenase gene family during liver development correlates with differential promoter activation by hepatocyte nuclear factor 1, CCAAT/enhancer-binding protein alpha, liver activator protein, and D-element-binding protein.

    PubMed Central

    van Ooij, C; Snyder, R C; Paeper, B W; Duester, G

    1992-01-01

    The human class I alcohol dehydrogenase (ADH) gene family consists of ADH1, ADH2, and ADH3, which are sequentially activated in early fetal, late fetal, and postnatal liver, respectively. Analysis of ADH promoters revealed differential activation by several factors previously shown to control liver transcription. In cotransfection assays, the ADH1 promoter, but not the ADH2 or ADH3 promoter, was shown to respond to hepatocyte nuclear factor 1 (HNF-1), which has previously been shown to regulate transcription in early liver development. The ADH2 promoter, but not the ADH1 or ADH3 promoter, was shown to respond to CCAAT/enhancer-binding protein alpha (C/EBP alpha), a transcription factor particularly active during late fetal liver and early postnatal liver development. The ADH1, ADH2, and ADH3 promoters all responded to the liver transcription factors liver activator protein (LAP) and D-element-binding protein (DBP), which are most active in postnatal liver. For all three promoters, the activation by LAP or DBP was higher than that seen by HNF-1 or C/EBP alpha, and a significant synergism between C/EBP alpha and LAP was noticed for the ADH2 and ADH3 promoters when both factors were simultaneously cotransfected. A hierarchy of ADH promoter responsiveness to C/EBP alpha and LAP homo- and heterodimers is suggested. In all three ADH genes, LAP bound to the same four sites previously reported for C/EBP alpha (i.e., -160, -120, -40, and -20 bp), but DBP bound strongly only to the site located at -40 bp relative to the transcriptional start. Mutational analysis of ADH2 indicated that the -40 bp element accounts for most of the promoter regulation by the bZIP factors analyzed. These studies suggest that HNF-1 and C/EBP alpha help establish ADH gene family transcription in fetal liver and that LAP and DBP help maintain high-level ADH gene family transcription in postnatal liver. Images PMID:1620113

  2. Inhibition of alcohol dehydrogenases by thiol compounds.

    PubMed

    Cheng, L Y; Lek, L H

    1992-04-01

    2-Mercaptoethanol is a strong inhibitor of LADH. The inhibitory effect is likely due to the binding of the SH group to the enzymatic zinc ion. Various thiol compounds do not inhibit YADH and it is suggested that the zinc atoms involved in the catalytic mechanism of LADH and YADH may have different structural arrangements and that these zinc atoms in YADH may not be blocked by thiol compounds. Thiol compounds also quench the enhanced fluorescence of LADH-NADH in a pH-dependent manner. At pH 9.2, the binding of coenzyme to LADH is replaced by 2-mercaptoethanol, whilst at pH 7.3, it further quenches the fluorescence of NADH-LADH. This quenching of fluorescence is likely attributed to a conformational change and energy transfer upon binding of 2-mercaptoethanol to the LADH-NADH complex. Complete reversal of the inhibitory effect of thiol compounds on LADH can be obtained by dialysis.

  3. Alcohol dehydrogenase polymorphism in Apis mellifera.

    PubMed

    Martins, E; Mestriner, M A; Contel, E P

    1977-04-01

    A polymorphic system of ADH isozymes is described in the honeybee Apis mellifera. Three and six different electrophoretic patterns were found, respectively, in drone and worker pupae analysis. The data indicate that the ADH isozymes are controlled by three alleles, Adh-1(1), Adh-1(2), and Adh-1(3). The frequency of the Adh-1 alleles is different in two analyzed subspecies, Apis mellifera adansonii (African bees) and Apis mellifera ligustica (Italian bees). In the African bees, the frequencies are 0.256 and 0.697 for Adh-1(1) and Adh-1(2), respectively. In the Italian bees, these values are shown to be 0.902 and 0.098, respectively. The allele Adh-1(3) was not detected in the Italian bee population. The effect of NAD on the resolution of this system was investigated, and only one region of ADH activity was obtained in drone pupae analysis when NAD was used in the gels. However, two different regions of activity were observed in the same samples, in the absence of the coenzyme. ADH activity was not detected in young larvae, but it increased to a maximum in prepupal and white-eyed pupal phases. It then declined progressively to total absence in the emerging bees.

  4. Xanthine dehydrogenase and 2-furoyl-coenzyme A dehydrogenase from Pseudomonas putida Fu1: two molybdenum-containing dehydrogenases of novel structural composition.

    PubMed Central

    Koenig, K; Andreesen, J R

    1990-01-01

    The constitutive xanthine dehydrogenase and the inducible 2-furoyl-coenzyme A (CoA) dehydrogenase could be labeled with [185W]tungstate. This labeling was used as a reporter to purify both labile proteins. The radioactivity cochromatographed predominantly with the residual enzymatic activity of both enzymes during the first purification steps. Both radioactive proteins were separated and purified to homogeneity. Antibodies raised against the larger protein also exhibited cross-reactivity toward the second smaller protein and removed xanthine dehydrogenase and 2-furoyl-CoA dehydrogenase activity up to 80 and 60% from the supernatant of cell extracts, respectively. With use of cell extract, Western immunoblots showed only two bands which correlated exactly with the activity stains for both enzymes after native polyacrylamide gel electrophoresis. Molybdate was absolutely required for incorporation of 185W, formation of cross-reacting material, and enzymatic activity. The latter parameters showed a perfect correlation. This evidence proves that the radioactive proteins were actually xanthine dehydrogenase and 2-furoyl-CoA dehydrogenase. The apparent molecular weight of the native xanthine dehydrogenase was about 300,000, and that of 2-furoyl-CoA dehydrogenase was 150,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of both enzymes revealed two protein bands corresponding to molecular weights of 55,000 and 25,000. The xanthine dehydrogenase contained at least 1.6 mol of molybdenum, 0.9 ml of cytochrome b, 5.8 mol of iron, and 2.4 mol of labile sulfur per mol of enzyme. The composition of the 2-furoyl-CoA dehydrogenase seemed to be similar, although the stoichiometry was not determined. The oxidation of furfuryl alcohol to furfural and further to 2-furoic acid by Pseudomonas putida Fu1 was catalyzed by two different dehydrogenases. Images PMID:2170335

  5. Substrate specificity and stereoselectivity of horse liver alcohol dehydrogenase. Kinetic evaluation of binding and activation parameters controlling the catalytic cycles of unbranched, acyclic secondary alcohols and ketones as substrates of the native and active-site-specific Co(II)-substituted enzyme.

    PubMed

    Adolph, H W; Maurer, P; Schneider-Bernlöhr, H; Sartorius, C; Zeppezauer, M

    1991-11-01

    1. The steady-state parameters kcat and Km and the rate constants of hydride transfer for the substrates isopropanol/acetone; (S)-2-butanol, (R)-2-butanol/2-butanone; (S)-2-pentanol, (R)-2-pentanol/2-pentanone; 3-pentanol/3-pentanone; (S)-2-octanol and (R)-2-octanol have been determined for the native Zn(II)-containing horse-liver alcohol dehydrogenase (LADH) and the specific active-site-substituted Co(II)LADH. 2. A combined evaluation of steady-state kinetic data and rate constants obtained from stopped-flow measurements, allowed the determination of all rate constants of the following ordered bi-bi mechanism: E in equilibrium E.NAD in equilibrium E.NAD.R1R2 CHOH in equilibrium E.NADH.R1R2CO in equilibrium E.NADH in equilibrium E. 3. On the basis of the different substrate specificities of LADH and yeast alcohol dehydrogenase (YADH), a procedure has been developed to evaluate the enantiomeric product composition of ketone reductions. 2-Butanone and 2-pentanone reductions revealed (S)-2-butanol (86%) and (S)-2-pentanol (95%) as the major products. 4. The observed enantioselectivity implies the existence of two productive ternary complexes; E.NADH.(pro-S) 2-butanone and E.NADH.(pro-R) 2-butanone. All rate constants describing the kinetic pathways of the system (S)-2-butanol, (R)-2-butanol/2-butanone have been determined. These data have been used to estimate the expected enantiomer product composition of 2-butanone reductions using apparent kcat/Km values for the two different ternary-complex configurations of 2-butanone. Additionally, these data have been used for computer simulations of the corresponding reaction cycles. Calculated, simulated and experimental data were found to be in good agreement. Thus, the system (S)-2-butanol, (R)-2-butanol/2-butanone is the first example of a LADH-catalyzed reaction for which the stereochemical course could be described in terms of rate constants of the underlying mechanism. 5. The effects of Co(II) substitution on the

  6. Maintenance of homeostasis of endogenous ethanol as a method for the therapy of alcoholism.

    PubMed

    Nikolaenko, V N

    2001-03-01

    We propose a new method for the therapy of alcoholism based on maintenance of homeostasis of endogenous ethanol and inhibition of alcohol dehydrogenase with emetine. After the standard antialcohol therapy, activity of this enzyme remained high or even increased, and pathological alcohol addiction also increased. Emetine normalized activity of alcohol dehydrogenase and suppressed pathological alcohol addiction. After this therapy more than 50% patients achieved stable remissions from alcoholism over 1 year, which indicated high efficiency of the proposed method.

  7. Dynamic structures of horse liver alcohol dehydrogenase (HLADH): results of molecular dynamics simulations of HLADH-NAD(+)-PhCH(2)OH, HLADH-NAD(+)-PhCH(2)O(-), and HLADH-NADH-PhCHO.

    PubMed

    Luo, J; Bruice, T C

    2001-12-01

    Molecular dynamics simulations of the oxidation of benzyl alcohol by horse liver alcohol dehydrogenase (HLADH) have been carried out. The following three states have been studied: HLADH.PhCH(2)OH.NAD(+) (MD1), HLADH.PhCH(2)O(-).NAD(+) (MD2), and HLADH.PhCHO.NADH (MD3). MD1, MD2, and MD3 simulations were carried out on one of the subunits of the dimeric enzyme covered in a 32-A-radius sphere of TIP3P water centered on the active site. The proton produced on ionization of the alcohol when HLADH.PhCH(2)OH.NAD(+) --> HLADH.PhCH(2)O(-).NAD(+) is transferred from the active site to solvent water via a hydrogen bonding network consisting of serine48 hydroxyl, ribose 2'- and 3'-hydroxyl groups, and Hist51. Hydrogen bonding of the 3'OH of ribose to Ile269 carbonyl maintains this proton in position to be transferred to water. Molecular dynamic simulations have been employed to track water1287 from the TIP3 water pool to the active site, thus exhibiting the mode of entrance of water to the active site. With time the water1287 accumulates in two different positions in order to accept the proton from the ribose 3'-OH and from His51. There can be identified two structural substates for proton passage. In the first substate the imidazole Ne2 of His51 is adjacent to the nicotinamide ribose C2'-OH and hydrogen bonding distances for proton transfer through the hydrogen bonded relay series PhCH(2)OH...Ser48-OH...Ribose2'-OH...His51...OH(2) (path 1) average 2.0, 2.0, and 2.1 A and (for His51...OH(2)) minimal distances less or equal to 2.5 A. The structure for path 1 is present 20% of the time span. And in the second substate, there are two possible proton passages: path 1 as before and path 2. Path 2 involves the hydrogen-bonded relay series PhCH(2)OH...Ser48-OH...Ribose2'-OH...Ribose3'-OH...His51.OH(2) with the average bonding distances being 2.0, 2.0, 2.1, and 2.0 A and (for His51...OH(2)) minimal distances less or equal to 2.5 A (20% probability of the time span), respectively

  8. Viewing CAD Drawings on the Internet

    ERIC Educational Resources Information Center

    Schwendau, Mark

    2004-01-01

    Computer aided design (CAD) has been producing 3-D models for years. AutoCAD software is frequently used to create sophisticated 3-D models. These CAD files can be exported as 3DS files for import into Autodesk's 3-D Studio Viz. In this program, the user can render and modify the 3-D model before exporting it out as a WRL (world file hyperlinked)…

  9. Computing Mass Properties From AutoCAD

    NASA Technical Reports Server (NTRS)

    Jones, A.

    1990-01-01

    Mass properties of structures computed from data in drawings. AutoCAD to Mass Properties (ACTOMP) computer program developed to facilitate quick calculations of mass properties of structures containing many simple elements in such complex configurations as trusses or sheet-metal containers. Mathematically modeled in AutoCAD or compatible computer-aided design (CAD) system in minutes by use of three-dimensional elements. Written in Microsoft Quick-Basic (Version 2.0).

  10. Alcoholism and Alcohol Abuse

    MedlinePlus

    ... This means that their drinking causes distress and harm. It includes alcoholism and alcohol abuse. Alcoholism, or ... brain, and other organs. Drinking during pregnancy can harm your baby. Alcohol also increases the risk of ...

  11. Plant Formate Dehydrogenase

    SciTech Connect

    John Markwell

    2005-01-10

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

  12. Genetics of alcoholism.

    PubMed

    Edenberg, Howard J; Foroud, Tatiana

    2014-01-01

    Multiple lines of evidence strongly indicate that genetic factors contribute to the risk for alcohol use disorders (AUD). There is substantial heterogeneity in AUD, which complicates studies seeking to identify specific genetic factors. To identify these genetic effects, several different alcohol-related phenotypes have been analyzed, including diagnosis and quantitative measures related to AUDs. Study designs have used candidate gene analyses, genetic linkage studies, genomewide association studies (GWAS), and analyses of rare variants. Two genes that encode enzymes of alcohol metabolism have the strongest effect on AUD: aldehyde dehydrogenase 2 and alcohol dehydrogenase 1B each has strongly protective variants that reduce risk, with odds ratios approximately 0.2-0.4. A number of other genes important in AUD have been identified and replicated, including GABRA2 and alcohol dehydrogenases 1B and 4. GWAS have identified additional candidates. Rare variants are likely also to play a role; studies of these are just beginning. A multifaceted approach to gene identification, targeting both rare and common variations and assembling much larger datasets for meta-analyses, is critical for identifying the key genes and pathways important in AUD.

  13. The Challenging Academic Development (CAD) Collective

    ERIC Educational Resources Information Center

    Peseta, Tai

    2005-01-01

    This article discusses the Challenging Academic Development (CAD) Collective and describes how it came out of a symposium called "Liminality, identity, and hybridity: On the promise of new conceptual frameworks for theorising academic/faculty development." The CAD Collective is and represents a space where people can open up their contexts and…

  14. Informatics infrastructure of CAD system.

    PubMed

    Pietka, Ewa; Gertych, Arkadiusz; Witko, Krzysztof

    2005-01-01

    A computer aided diagnosis (CAD) system requires several components which influence its effectiveness. An image processing methodology is responsible for the analysis, database structure archives and distributes the patient demographics, clinical information, and image data. A graphical user interface is applied in order to enter the data and present it to the user. By designing dynamic Web pages a remote access to the entire is granted. The computer aided diagnosis system includes three layers, which might be installed on various platforms. Elements of the application software are designed independently. Integration of all components is another issue discussed in the presented paper. Implementation of a computer aided diagnosis system improves and accelerates the analysis by giving to the user objective measurement tools. It also standardizes the decision-making process and solves the problem of replicability. Finally, it permits a set of images and features to be collected and recognized as a medical standard and be applied in education and research. PMID:15755535

  15. Racial differences in alcohol sensitivity.

    PubMed

    Chan, A W

    1986-01-01

    The existence of racial differences in alcohol sensitivity between Oriental and Caucasian populations has been well documented. The primary manifestation is a highly visible facial flushing (47-85% in Orientals vs 3-29% in Caucasians) accompanied by other objective and subjective symptoms of discomfort. Even among different Oriental groups, subtle differences in the flushing response and alcohol consumption can exist. North and South American Indian populations differ in phenotypes for alcohol dehydrogenase and aldehyde dehydrogenase, but systematic studies comparing degree of flushing, alcohol elimination rates and blood acetaldehyde levels in these populations are lacking. Although flushing does not automatically 'immunize' an individual against alcohol use, those susceptible tend to consume less alcohol, at least in Orientals. However, the flushing phenomenon cannot be the sole explanation for differences in incidences of alcoholism among different racial groups. Socio-cultural, environmental and genetic factors also have to be considered. An increased incidence of flushing has been found to associate with a familial risk of development of future alcoholism in a Caucasian population. It remains to be determined whether the same is true in Orientals. Most biochemical investigations of the flushing phenomenon have focused on aspects of alcohol metabolism. Based on recent findings, a convincing mechanism is the higher accumulation of acetaldehyde in flushing subjects because they have an unusual, less-active liver aldehyde dehydrogenase isozyme (ALDHI). The possibility that an 'atypical' alcohol dehydrogenase, which is present in 85-90% of Oriental subjects, can contribute to increased blood acetaldehyde levels in flushing subjects cannot be ruled out. Based on results of a small number of pedigree studies which demonstrated familial resemblances in flushing, a pharmacogenetic defect in ALDHI has been proposed to be responsible for flushing. Other possible

  16. Flexible Concurrency Control for Legacy CAD to Construct Collaborative CAD Environment

    NASA Astrophysics Data System (ADS)

    Cai, Xiantao; Li, Xiaoxia; He, Fazhi; Han, Soonhung; Chen, Xiao

    Collaborative CAD (Co-CAD) systems can be constructed based on either 3D kernel or legacy stand-alone CAD systems, which are typically commercial CAD systems such as CATIA, Pro/E and so on. Most of synchronous Co-CAD systems, especially these based on legacy stand-alone CAD systems, adopt the lock mechanism or the floor control as concurrency controls which are very restrictive and stagnant. A flexible concurrency control method is proposed to support the flexible concurrency control in Co-CAD systems based on legacy stand-alone CAD systems. At first, a model of operation relationship is proposed with special consideration for the concurrency control of these kinds of Co-CAD system. Then two types of data structure, the Collaborative Feature Dependent Graph (Co-FDG) and the Collaborative Feature Operational List (Co-FOL), are presented as the cornerstone of flexible concurrency control. Next a Flexible Concurrency Control Algorithm (FCCA) is proposed. Finally a Selective Undo/Redo Algorithm is proposed which can improve the flexibility of Co-CAD furthermore.

  17. Role of Alcohol Metabolism in Non-Alcoholic Steatohepatitis

    PubMed Central

    Baker, Susan S.; Baker, Robert D.; Liu, Wensheng; Nowak, Norma J.; Zhu, Lixin

    2010-01-01

    Background Non-alcoholic steatohepatitis (NASH) is a serious form of non-alcoholic fatty liver disease (NAFLD), associated with obesity and insulin resistance. Previous studies suggested that intestinal bacteria produced more alcohol in obese mice than lean animals. Methodology/Principal Findings To investigate whether alcohol is involved in the pathogenesis of NASH, the expression of inflammation, fibrosis and alcohol metabolism related genes in the liver tissues of NASH patients and normal controls (NCs) were examined by microarray (NASH, n = 7; NC, n = 4) and quantitative real-time PCR (NASH, n = 6; NC, n = 6). Genes related to liver inflammation and fibrosis were found to be elevated in NASH livers compared to normal livers. The most striking finding is the increased gene transcription of alcohol dehydrogenase (ADH) genes, genes for catalase and cytochrome P450 2E1, and aldehyde dehydrogenase genes. Immunoblot analysis confirmed the increased expression of ADH1 and ADH4 in NASH livers (NASH, n = 9; NC, n = 4). Conclusions/Significance The augmented activity of all the available genes of the pathways for alcohol catabolism suggest that 1) alcohol concentration was elevated in the circulation of NASH patients; 2) there was a high priority for the NASH livers to scavenge alcohol from the circulation. Our data is the first human evidence that suggests alcohol may contribute to the development of NAFLD. PMID:20221393

  18. Advances in noninvasive detection of CAD

    SciTech Connect

    Kahn, J.K. )

    1991-04-01

    Advances in the noninvasive detection of myocardial ischemia are increasing our ability to diagnose coronary artery disease (CAD). Tomographic (SPECT) thallium imaging provides better identification of coronary arteries with atherosclerotic narrowing. Increased lung thallium uptake and transient ischemic dilatation of the heart are additional markers of severe CAD. Late thallium imaging, as well as reinjection imaging, provides more accurate identification of myocardial ischemia. Finally, new myocardial imaging agents, such as technetium Tc 99m sestamibi (Cardiolite), should improve detection of CAD by noninvasive methods.10 references.

  19. Train effectively for CAD/D

    SciTech Connect

    Not Available

    1983-04-01

    After failing with an unstructured computer-aided drafting/ design CAD/D program, Bechtel changed to a structured training program. Five considerations are presented here: teach CAD/D to engineers, not engineering to CAD/D experts; keep the program flexible enough to avoid rewriting due to fast technology evolution; pace information delivery; and rote learning of sequences only works if the students have a conceptual model first. On the job training is necessary, and better monitoring systems to test the OJT are needed. One such test is presented.

  20. Improving the radiologist-CAD interaction: designing for appropriate trust.

    PubMed

    Jorritsma, W; Cnossen, F; van Ooijen, P M A

    2015-02-01

    Computer-aided diagnosis (CAD) has great potential to improve radiologists' diagnostic performance. However, the reported performance of the radiologist-CAD team is lower than what might be expected based on the performance of the radiologist and the CAD system in isolation. This indicates that the interaction between radiologists and the CAD system is not optimal. An important factor in the interaction between humans and automated aids (such as CAD) is trust. Suboptimal performance of the human-automation team is often caused by an inappropriate level of trust in the automation. In this review, we examine the role of trust in the radiologist-CAD interaction and suggest ways to improve the output of the CAD system so that it allows radiologists to calibrate their trust in the CAD system more effectively. Observer studies of the CAD systems show that radiologists often have an inappropriate level of trust in the CAD system. They sometimes under-trust CAD, thereby reducing its potential benefits, and sometimes over-trust it, leading to diagnostic errors they would not have made without CAD. Based on the literature on trust in human-automation interaction and the results of CAD observer studies, we have identified four ways to improve the output of CAD so that it allows radiologists to form a more appropriate level of trust in CAD. Designing CAD systems for appropriate trust is important and can improve the performance of the radiologist-CAD team. Future CAD research and development should acknowledge the importance of the radiologist-CAD interaction, and specifically the role of trust therein, in order to create the perfect artificial partner for the radiologist. This review focuses on the role of trust in the radiologist-CAD interaction. The aim of the review is to encourage CAD developers to design for appropriate trust and thereby improve the performance of the radiologist-CAD team. PMID:25459198

  1. Analysis of rat cytosolic 9-cis-retinol dehydrogenase activity and enzymatic characterization of rat ADHII.

    PubMed

    Popescu, G; Napoli, J L

    2000-01-01

    We report the characterization of two enzymes that catalyze NAD(+)-dependent 9-cis-retinol dehydrogenase activity in rat liver cystol. Alcohol dehydrogenase class I (ADHI) contributes > 80% of the NA D+-dependent 9-cis-retinol dehydrogenase activity recovered, whereas alcohol dehydrogenase class II (ADHII), not identified previously at the protein level, nor characterized enzymatically in rat, accounts for approximately 2% of the activity. Rat ADHII exhibits properties different from those described for human ADHII. Moreover, rat ADHII-catalyzed rates of ethanol dehydrogenation are markedly lower than octanol or retinoid dehydrogenation rates. Neither ethanol nor 4-methylpyrazole inhibits the 9-cis-retinol dehydrogenase activity of rat ADHII. We propose that ADHII represents the previously observed additional retinoid oxidation activity of rat liver cytosol which occurred in the presence of either ethanol or 4-methylpyrazole. We also show that human and rat ADHII differ considerably in enzymatic properties. PMID:10606766

  2. CADS:Cantera Aerosol Dynamics Simulator.

    SciTech Connect

    Moffat, Harry K.

    2007-07-01

    This manual describes a library for aerosol kinetics and transport, called CADS (Cantera Aerosol Dynamics Simulator), which employs a section-based approach for describing the particle size distributions. CADS is based upon Cantera, a set of C++ libraries and applications that handles gas phase species transport and reactions. The method uses a discontinuous Galerkin formulation to represent the particle distributions within each section and to solve for changes to the aerosol particle distributions due to condensation, coagulation, and nucleation processes. CADS conserves particles, elements, and total enthalpy up to numerical round-off error, in all of its formulations. Both 0-D time dependent and 1-D steady state applications (an opposing-flow flame application) have been developed with CADS, with the initial emphasis on developing fundamental mechanisms for soot formation within fires. This report also describes the 0-D application, TDcads, which models a time-dependent perfectly stirred reactor.

  3. The CAD-EGS Project: Using CAD Geometrics in EGS4

    SciTech Connect

    Langeveld, Willy G.J.

    2002-03-28

    The objective of the CAD-EGS project is to provide a way to use a CAD system to create 3D geometries for use within EGS4. In this report, we describe an approach based on an intermediate file, written out by the CAD system, that is read by an EGS4 user code designed for the purpose. A prototype solution was implemented using a commonly used CAD system and the Virtual Reality Modeling Language (VRML) as an intermediate file format. We report results from the prototype, and discuss various problems arising from both the approach and the particular choices made.

  4. NADH electrochemical sensor coupled with dehydrogenase enzymes

    SciTech Connect

    Yamanaka, Hideko; Mascini, Marco )

    1992-06-01

    A graphite electrode assembled in a flow cell has shown to be a good detector for NADH. Current is linearly dependent on concentration in the range 10{sup {minus}7}-10{sup {minus}3} M without any mediator at the potential applied of 300 mV vs Ag/AgCl. Lactate and alcohol dehydrogenases were immobilized near to the electrode surface or in a reactor to obtain an NADH-based biosensor for lactate or ethanol. With lactate the authors succeeded to obtain a response only if the reactor was used and for alcohol a current proportional to the concentration was obtained either if the enzyme was immobilized in a membrane and placed near the electrode surface or when the enzyme was immobilized in a reactor form. By FIA procedures fast responses and recoveries were obtained, but with a short linear range.

  5. AutoCAD-To-NASTRAN Translator Program

    NASA Technical Reports Server (NTRS)

    Jones, A.

    1989-01-01

    Program facilitates creation of finite-element mathematical models from geometric entities. AutoCAD to NASTRAN translator (ACTON) computer program developed to facilitate quick generation of small finite-element mathematical models for use with NASTRAN finite-element modeling program. Reads geometric data of drawing from Data Exchange File (DXF) used in AutoCAD and other PC-based drafting programs. Written in Microsoft Quick-Basic (Version 2.0).

  6. A CAD System for Hemorrhagic Stroke.

    PubMed

    Nowinski, Wieslaw L; Qian, Guoyu; Hanley, Daniel F

    2014-09-01

    Computer-aided detection/diagnosis (CAD) is a key component of routine clinical practice, increasingly used for detection, interpretation, quantification and decision support. Despite a critical need, there is no clinically accepted CAD system for stroke yet. Here we introduce a CAD system for hemorrhagic stroke. This CAD system segments, quantifies, and displays hematoma in 2D/3D, and supports evacuation of hemorrhage by thrombolytic treatment monitoring progression and quantifying clot removal. It supports seven-step workflow: select patient, add a new study, process patient's scans, show segmentation results, plot hematoma volumes, show 3D synchronized time series hematomas, and generate report. The system architecture contains four components: library, tools, application with user interface, and hematoma segmentation algorithm. The tools include a contour editor, 3D surface modeler, 3D volume measure, histogramming, hematoma volume plot, and 3D synchronized time-series hematoma display. The CAD system has been designed and implemented in C++. It has also been employed in the CLEAR and MISTIE phase-III, multicenter clinical trials. This stroke CAD system is potentially useful in research and clinical applications, particularly for clinical trials.

  7. An application protocol for CAD to CAD transfer of electronic information

    NASA Technical Reports Server (NTRS)

    Azu, Charles C., Jr.

    1993-01-01

    The exchange of Computer Aided Design (CAD) information between dissimilar CAD systems is a problem. This is especially true for transferring electronics CAD information such as multi-chip module (MCM), hybrid microcircuit assembly (HMA), and printed circuit board (PCB) designs. Currently, there exists several neutral data formats for transferring electronics CAD information. These include IGES, EDIF, and DXF formats. All these formats have limitations for use in exchanging electronic data. In an attempt to overcome these limitations, the Navy's MicroCIM program implemented a project to transfer hybrid microcircuit design information between dissimilar CAD systems. The IGES (Initial Graphics Exchange Specification) format is used since it is well established within the CAD industry. The goal of the project is to have a complete transfer of microelectronic CAD information, using IGES, without any data loss. An Application Protocol (AP) is being developed to specify how hybrid microcircuit CAD information will be represented by IGES entity constructs. The AP defines which IGES data items are appropriate for describing HMA geometry, connectivity, and processing as well as HMA material characteristics.

  8. Project CAD as of July 1978: CAD support project, situation in July 1978

    NASA Technical Reports Server (NTRS)

    Boesch, L.; Lang-Lendorff, G.; Rothenberg, R.; Stelzer, V.

    1979-01-01

    The structure of Computer Aided Design (CAD) and the requirements for program developments in past and future are described. The actual standard and the future aims of CAD programs are presented. The developed programs in: (1) civil engineering; (2) mechanical engineering; (3) chemical engineering/shipbuilding; (4) electrical engineering; and (5) general programs are discussed.

  9. Next Generation CAD/CAM/CAE Systems

    NASA Technical Reports Server (NTRS)

    Noor, Ahmed K. (Compiler); Malone, John B. (Compiler)

    1997-01-01

    This document contains presentations from the joint UVA/NASA Workshop on Next Generation CAD/CAM/CAE Systems held at NASA Langley Research Center in Hampton, Virginia on March 18-19, 1997. The presentations focused on current capabilities and future directions of CAD/CAM/CAE systems, aerospace industry projects, and university activities related to simulation-based design. Workshop attendees represented NASA, commercial software developers, the aerospace industry, government labs, and academia. The workshop objectives were to assess the potential of emerging CAD/CAM/CAE technology for use in intelligent simulation-based design and to provide guidelines for focused future research leading to effective use of CAE systems for simulating the entire life cycle of aerospace systems.

  10. cadDX Operon of Streptococcus salivarius 57.I▿

    PubMed Central

    Chen, Yi-Ywan M.; Feng, C. W.; Chiu, C. F.; Burne, Robert A.

    2008-01-01

    A CadDX system that confers resistance to Cd2+ and Zn2+ was identified in Streptococcus salivarius 57.I. Unlike with other CadDX systems, the expression of the cad promoter was negatively regulated by CadX, and the repression was inducible by Cd2+ and Zn2+, similar to what was found for CadCA systems. The lower G+C content of the S. salivarius cadDX genes suggests acquisition by horizontal gene transfer. PMID:18165364

  11. cadDX operon of Streptococcus salivarius 57.I.

    PubMed

    Chen, Yi-Ywan M; Feng, C W; Chiu, C F; Burne, Robert A

    2008-03-01

    A CadDX system that confers resistance to Cd(2+) and Zn(2+) was identified in Streptococcus salivarius 57.I. Unlike with other CadDX systems, the expression of the cad promoter was negatively regulated by CadX, and the repression was inducible by Cd(2+) and Zn(2+), similar to what was found for CadCA systems. The lower G+C content of the S. salivarius cadDX genes suggests acquisition by horizontal gene transfer. PMID:18165364

  12. Alcoholism, Alcohol, and Drugs

    ERIC Educational Resources Information Center

    Rubin, Emanuel; Lieber, Charles S.

    1971-01-01

    Describes research on synergistic effects of alcohol and other drugs, particularly barbiturates. Proposes biochemical mechanisms to explain alcoholics' tolerance of other drugs when sober, and increased sensitivity when drunk. (AL)

  13. Cyanobacterial NADPH dehydrogenase complexes

    SciTech Connect

    Ogawa, Teruo; Mi, Hualing

    2007-07-01

    Cyanobacteria possess functionally distinct multiple NADPH dehydrogenase (NDH-1) complexes that are essential to CO2 uptake, photosystem-1 cyclic electron transport and respiration. The unique nature of cyanobacterial NDH-1 complexes is the presence of subunits involved in CO2 uptake. Other than CO2 uptake, chloroplastic NDH-1 complex has similar role as cyanobacterial NDH-1 complexes in photosystem-1 cyclic electron transport and respiration (chlororespiration). In this mini-review we focus on the structure and function of cyanobacterial NDH-1 complexes and their phylogeny. The function of chloroplastic NDH-1 complex and characteristics of plants defective in NDH-1 are also described forcomparison.

  14. Quantifying alcohol-related emergency admissions in a UK tertiary referral hospital: a cross-sectional study of chronic alcohol dependency and acute alcohol intoxication

    PubMed Central

    Vardy, J; Keliher, T; Fisher, J; Ritchie, F; Bell, C; Chekroud, M; Clarey, F; Blackwood, L; Barry, L; Paton, E; Clark, A; Connelly, R

    2016-01-01

    Objectives Alcohol is responsible for a proportion of emergency admissions to hospital, with acute alcohol intoxication and chronic alcohol dependency (CAD) implicated. This study aims to quantify the proportion of hospital admissions through our emergency department (ED) which were thought by the admitting doctor to be (largely or partially) a result of alcohol consumption. Setting ED of a UK tertiary referral hospital. Participants All ED admissions occurring over 14 weeks from 1 September to 8 December 2012. Data obtained for 5497 of 5746 admissions (95.67%). Primary outcome measures Proportion of emergency admissions related to alcohol as defined by the admitting ED clinician. Secondary outcome measures Proportion of emergency admissions due to alcohol diagnosed with acute alcohol intoxication or CAD according to ICD-10 criteria. Results 1152 (21.0%, 95% CI 19.9% to 22.0%) of emergency admissions were thought to be due to alcohol. 74.6% of patients admitted due to alcohol had CAD, and significantly greater than the 26.4% with ‘Severe’ or ‘Very Severe’ acute alcohol intoxication (p<0.001). Admissions due to alcohol differed to admissions not due to alcohol being on average younger (45 vs 56 years, p<0.001) more often male (73.4% vs 45.1% males, p<0.001) and more likely to have a diagnosis synonymous with alcohol or related to recreational drug use, pancreatitis, deliberate self-harm, head injury, gastritis, suicidal ideation, upper gastrointestinal bleeds or seizures (p<0.001). An increase in admissions due to alcohol on Saturdays reflects a surge in admissions with acute alcohol intoxication above the weekly average (p=0.003). Conclusions Alcohol was thought to be implicated in 21% of emergency admissions in this cohort. CAD is responsible for a significantly greater proportion of admissions due to alcohol than acute intoxication. Interventions designed to reduce alcohol-related admissions must incorporate measures to tackle CAD. PMID:27324707

  15. The polymorphism in acetaldehyde dehydrogenase 2 gene, causing a substitution of Glu > Lys(504), is not associated with coronary atherosclerosis severity in Han Chinese.

    PubMed

    Xu, Feng; Chen, Yu-Guo; Geng, Yong-Jian; Zhang, He; Jiang, Chun-Xiao; Sun, Yi; Li, Rui-Jian; Sagar, Madi Bidya; Xue, Li; Zhang, Yun

    2007-11-01

    Alcohol consumption has an important effect on coronary atherosclerotic heart disease (CAD). Acetaldehyde dehydrogenase 2 (ALDH2) is a key enzyme in alcohol metabolism. A G-to-A missense mutation of ALDH2 gene, which causes a Glu > Lys(504) substitution, was recently shown to be associated with carotid atherosclerosis; however, its relationship with coronary atherosclerosis has not been well studied. We, therefore, investigated this relationship in Han Chinese. There are two ALDH2 alleles (1 and 2) and their combination: 1/1 (GG, typical homozygote), 1/2 (GA, heterozygote) and 2/2 (AA, atypical homozygote) in the population. Successive Han Chinese, including 89 with myocardial infarction (MI) and 142 with unstable angina, were recruited, and underwent coronary angiography and gene sequencing. Coronary atherosclerosis severity was expressed by the number of lesioned coronary arteries (>or=50% diameter stenosis) and Gensini score, calculated based on the luminal narrowing degree and its geographic importance, as assessed by angiography. Based on their ALDH2 genotypes, the 231 patients were divided into wild-type (1/1, n = 145) and mutation groups (1/2 and 2/2, n = 86). There were no significant differences in basic clinical data between the two groups; however, the mutation group had significantly higher rates of diabetes mellitus and MI, and lower prevalence of alcohol consumption than wild-type group. Yet, the two groups were not significantly different in coronary atherosclerosis severity. Multiple regression analysis has shown that the ALDH2 genotype 1/2 or 2/2 is an independent risk factor for MI, but is not associated with coronary atherosclerosis severity in Han Chinese. PMID:17984618

  16. Genetics Home Reference: pyruvate dehydrogenase deficiency

    MedlinePlus

    ... control the activity of the complex: pyruvate dehydrogenase phosphatase turns on (activates) the complex, while pyruvate dehydrogenase ... binding protein (the PDHX gene), and pyruvate dehydrogenase phosphatase (the PDP1 gene) have been identified in people ...

  17. Pharmacogenetics of alcohol response and alcoholism: the interplay of genes and environmental factors in thresholds for alcoholism.

    PubMed

    Radel, M; Goldman, D

    2001-04-01

    Recent advances in neuroscience and genetics have enabled a better understanding of genetically influenced differences in ethanol ("alcohol")-related responses and differential vulnerability to alcohol dependence at the cellular and molecular levels. Heritability studies reveal that the role of genetic factors in alcoholism is largely substance-specific, with the exception of nicotine. One focus of genetic research in alcoholism is the study of functional polymorphisms influencing alcohol metabolism, such as the aldehyde dehydrogenase type 2 Glu487Lys and alcohol dehydrogenase type 2 His47Arg polymorphisms, which affect vulnerability to alcoholism via pharmacokinetic mechanisms, and cross-population studies have begun to reveal important gene-environment interactions. The other focus is on functional genetic variants of proteins involved in the neuronal response to alcohol, including alcohol sensitivity, reward, tolerance, and withdrawal. Studies on the roles of GABA(A) alpha6-amino acid substitutions in rodents in alcohol and benzodiazepine sensitivity, and potential roles in human alcohol and benzodiazepine sensitivity are reviewed. These studies, together with recently developed knowledge on a GABA(A) receptor gene cluster at a quantitative trait loci for alcohol withdrawal on mouse chromosome 11, indicate that research investigation of variation at GABA(A) neurotransmission is a promising area in the pharmacodynamics of alcohol and in differential susceptibility to alcoholism. Genes for proteins involved in alcohol-mediated reward include genes for transporters and receptors for dopamine, serotonin, opioids, and GABA. These genes and their functional variants also represent important targets for understanding alcohol's effects in humans. Identification of genes for alcoholism vulnerability is important in the near future, not only for prevention, but also for development and targeting treatments.

  18. Some Workplace Effects of CAD and CAM.

    ERIC Educational Resources Information Center

    Ebel, Karl-H.; Ulrich, Erhard

    1987-01-01

    Examines the impact of computer-aided design (CAD) and computer-aided manufacturing (CAM) on employment, work organization, working conditions, job content, training, and industrial relations in several countries. Finds little evidence of negative employment effects since productivity gains are offset by various compensatory factors. (Author/CH)

  19. Pipe Drafting with CAD. Teacher Edition.

    ERIC Educational Resources Information Center

    Smithson, Buddy

    This teacher's guide contains nine units of instruction for a course on computer-assisted pipe drafting. The course covers the following topics: introduction to pipe drafting with CAD (computer-assisted design); flow diagrams; pipe and pipe components; valves; piping plans and elevations; isometrics; equipment fabrication drawings; piping design…

  20. A Case Study in CAD Design Automation

    ERIC Educational Resources Information Center

    Lowe, Andrew G.; Hartman, Nathan W.

    2011-01-01

    Computer-aided design (CAD) software and other product life-cycle management (PLM) tools have become ubiquitous in industry during the past 20 years. Over this time they have continuously evolved, becoming programs with enormous capabilities, but the companies that use them have not evolved their design practices at the same rate. Due to the…

  1. Mechanical Drafting with CAD. Teacher Edition.

    ERIC Educational Resources Information Center

    McClain, Gerald R.

    This instructor's manual contains 13 units of instruction for a course on mechanical drafting with options for using computer-aided drafting (CAD). Each unit includes some or all of the following basic components of a unit of instruction: objective sheet, suggested activities for the teacher, assignment sheets and answers to assignment sheets,…

  2. Joining Astrobiology to Medicine, Resurrecting Ancient Alcohol Metabolism

    NASA Astrophysics Data System (ADS)

    Carrigan, M. A.; Uryasev, O.; Davis, R. W.; Chamberlin, S. G.; Benner, S. A.

    2010-04-01

    We apply an astrobiological approach to understand how primates responded to the emergence of ethanol in their environment by resurrecting two enzymes involved in the degradation of ethanol, alcohol dehydrogenase and aldehyde dehydrgenase.

  3. PC Board Layout and Electronic Drafting with CAD. Teacher Edition.

    ERIC Educational Resources Information Center

    Bryson, Jimmy

    This teacher's guide contains 11 units of instruction for a course on computer electronics and computer-assisted drafting (CAD) using a personal computer (PC). The course covers the following topics: introduction to electronic drafting with CAD; CAD system and software; basic electronic theory; component identification; basic integrated circuit…

  4. CAD/CAM. High-Technology Training Module.

    ERIC Educational Resources Information Center

    Zuleger, Robert

    This high technology training module is an advanced course on computer-assisted design/computer-assisted manufacturing (CAD/CAM) for grades 11 and 12. This unit, to be used with students in advanced drafting courses, introduces the concept of CAD/CAM. The content outline includes the following seven sections: (1) CAD/CAM software; (2) computer…

  5. Education and Training Packages for CAD/CAM.

    ERIC Educational Resources Information Center

    Wright, I. C.

    1986-01-01

    Discusses educational efforts in the fields of Computer Assisted Design and Manufacturing (CAD/CAM). Describes two educational training initiatives underway in the United Kingdom, one of which is a resource materials package for teachers of CAD/CAM at the undergraduate level, and the other a training course for managers of CAD/CAM systems. (TW)

  6. A CAD (Classroom Assessment Design) of a Computer Programming Course

    ERIC Educational Resources Information Center

    Hawi, Nazir S.

    2012-01-01

    This paper presents a CAD (classroom assessment design) of an entry-level undergraduate computer programming course "Computer Programming I". CAD has been the product of a long experience in teaching computer programming courses including teaching "Computer Programming I" 22 times. Each semester, CAD is evaluated and modified for the subsequent…

  7. Genes contributing to the development of alcoholism: an overview.

    PubMed

    Edenberg, Howard J

    2012-01-01

    Genetic factors (i.e., variations in specific genes) account for a substantial portion of the risk for alcoholism. However, identifying those genes and the specific variations involved is challenging. Researchers have used both case-control and family studies to identify genes related to alcoholism risk. In addition, different strategies such as candidate gene analyses and genome-wide association studies have been used. The strongest effects have been found for specific variants of genes that encode two enzymes involved in alcohol metabolism-alcohol dehydrogenase and aldehyde dehydrogenase. Accumulating evidence indicates that variations in numerous other genes have smaller but measurable effects.

  8. Alcohol Alert

    MedlinePlus

    ... Us You are here Home » Alcohol Alert Alcohol Alert The NIAAA Alcohol Alert is a quarterly bulletin that disseminates important research ... text. To order single copies of select Alcohol Alerts, see ordering Information . To view publications in PDF ...

  9. Alcoholism - resources

    MedlinePlus

    Resources - alcoholism ... The following organizations are good resources for information on alcoholism : Alcoholics Anonymous -- www.aa.org Al-Anon/Alateen -- www.al-anon.org/home National Institute on Alcohol ...

  10. Alcoholic neuropathy

    MedlinePlus

    Neuropathy - alcoholic; Alcoholic polyneuropathy ... The exact cause of alcoholic neuropathy is unknown. It likely includes both a direct poisoning of the nerve by the alcohol and the effect of poor nutrition ...

  11. Alcohol Facts

    MedlinePlus

    ... raquo Alcohol Facts Alcohol Facts Listen Drinks like beer, malt liquor, wine, and hard liquor contain alcohol. Alcohol is the ingredient that gets you drunk. Hard liquor—such as whiskey, rum, or gin—has more ...

  12. Genetic differences in response to alcohol.

    PubMed

    Matsushita, Sachio; Higuchi, Susumu

    2014-01-01

    The level of response to alcohol, which reflects individual differences in sensitivity to the pharmacologic effects of alcohol, is considered to be an important endophenotype of alcohol use disorder (AUD). By comparing monozygotic and dizygotic twins, the heritability of the level of response to alcohol has been estimated to be 60%. Many genes have been implicated as potential contributors toward heavy drinking, alcohol-related problems, and AUD through a low level of response to alcohol, each with a small effect. Identified are genes for gamma-aminobutyric acid (GABA) receptors, serotonin transporter, opioid receptor, and nicotinic acetylcholine receptor, but the most well-characterized genes that have a strong impact on the level of response to alcohol are those for alcohol-metabolizing enzymes. Although two genetic variations in alcohol and aldehyde dehydrogenases, which have been the most intensively studied, exist almost exclusively in Asian populations, studies on the effect of genetic variations in alcohol-metabolizing enzymes on the response to alcohol are gradually expanding in non-Asian populations. In this chapter, we focus on genetic studies in humans. After analyzing the overall influence of genetic factors on the response to alcohol, we explore individual genes that may influence the response to alcohol. Lastly, we review studies examining the effects of genetic variations in alcohol-metabolizing enzymes on the level of response to alcohol.

  13. Alcohol Alert: Genetics of Alcoholism

    MedlinePlus

    ... and Reports » Alcohol Alert » Alcohol Alert Number 84 Alcohol Alert Number 84 Print Version The Genetics of ... immune defense system. Genes Encoding Enzymes Involved in Alcohol Breakdown Some of the first genes linked to ...

  14. ProperCAD: A portable object-oriented parallel environment for VLSI CAD

    NASA Technical Reports Server (NTRS)

    Ramkumar, Balkrishna; Banerjee, Prithviraj

    1993-01-01

    Most parallel algorithms for VLSI CAD proposed to date have one important drawback: they work efficiently only on machines that they were designed for. As a result, algorithms designed to date are dependent on the architecture for which they are developed and do not port easily to other parallel architectures. A new project under way to address this problem is described. A Portable object-oriented parallel environment for CAD algorithms (ProperCAD) is being developed. The objectives of this research are (1) to develop new parallel algorithms that run in a portable object-oriented environment (CAD algorithms using a general purpose platform for portable parallel programming called CARM is being developed and a C++ environment that is truly object-oriented and specialized for CAD applications is also being developed); and (2) to design the parallel algorithms around a good sequential algorithm with a well-defined parallel-sequential interface (permitting the parallel algorithm to benefit from future developments in sequential algorithms). One CAD application that has been implemented as part of the ProperCAD project, flat VLSI circuit extraction, is described. The algorithm, its implementation, and its performance on a range of parallel machines are discussed in detail. It currently runs on an Encore Multimax, a Sequent Symmetry, Intel iPSC/2 and i860 hypercubes, a NCUBE 2 hypercube, and a network of Sun Sparc workstations. Performance data for other applications that were developed are provided: namely test pattern generation for sequential circuits, parallel logic synthesis, and standard cell placement.

  15. Cost reduction advantages of CAD/CAM

    NASA Astrophysics Data System (ADS)

    Parsons, G. T.

    1983-05-01

    Features of the CAD/CAM system implemented at the General Dynamics Convair division are summarized. CAD/CAM was initiated in 1976 to enhance engineering, manufacturing and quality assurance and thereby the company's competitive bidding position. Numerical models are substituted for hardware models wherever possible and numerical criteria are defined in design for guiding computer-controlled parts manufacturing machines. The system comprises multiple terminals, a data base, digitizer, printers, disk and tape drives, and graphics displays. The applications include the design and manufacture of parts and components for avionics, structures, scientific investigations, and aircraft structural components. Interfaces with other computers allow structural analyses by finite element codes. Although time savings have not been gained compared to manual drafting, components of greater complexity than could have been designed by hand have been designed and manufactured.

  16. AutoCAD-To-GIFTS Translator Program

    NASA Technical Reports Server (NTRS)

    Jones, Andrew

    1989-01-01

    AutoCAD-to-GIFTS translator program, ACTOG, developed to facilitate quick generation of small finite-element models using CASA/GIFTS finite-element modeling program. Reads geometric data of drawing from Data Exchange File (DXF) used in AutoCAD and other PC-based drafting programs. Geometric entities recognized by ACTOG include points, lines, arcs, solids, three-dimensional lines, and three-dimensional faces. From this information, ACTOG creates GIFTS SRC file, which then reads into GIFTS preprocessor BULKM or modified and reads into EDITM to create finite-element model. SRC file used as is or edited for any number of uses. Written in Microsoft Quick-Basic (Version 2.0).

  17. DFE workbench: a CAD integrated DFE tool

    NASA Astrophysics Data System (ADS)

    Man, Elena; Diez-Campo, Juan E.; Roche, Thomas

    2002-02-01

    Because of the emergent legislation (e.g. WEEE and EOLV), environmental standards (e.g. ISO 14000) and a shift in consumer opinion toward environmentally superior products, there is an increased need for CAD integrated Design for Environment tools to assist the designer in the development of environmentally superior products (ESP). Implementing Design for the Environment practices is an extremely effective strategy, as it is widely believed that 95% of development costs are determined at this stage. Many methodologies and tools have been developed to perform environmental analysis; however, many existing methodologies are inadequately integrated in the design process. This paper addresses these problems and presents a CAD integrated DFE tool that has been under development in the authors' institutes for the last number of years.

  18. Generating Composite Overlapping Grids on CAD Geometries

    SciTech Connect

    Henshaw, W.D.

    2002-02-07

    We describe some algorithms and tools that have been developed to generate composite overlapping grids on geometries that have been defined with computer aided design (CAD) programs. This process consists of five main steps. Starting from a description of the surfaces defining the computational domain we (1) correct errors in the CAD representation, (2) determine topology of the patched-surface, (3) build a global triangulation of the surface, (4) construct structured surface and volume grids using hyperbolic grid generation, and (5) generate the overlapping grid by determining the holes and the interpolation points. The overlapping grid generator which is used for the final step also supports the rapid generation of grids for block-structured adaptive mesh refinement and for moving grids. These algorithms have been implemented as part of the Overture object-oriented framework.

  19. Activity of select dehydrogenases with Sepharose-immobilized N6-carboxymethyl-NAD

    PubMed Central

    Beauchamp, Justin; Vieille, Claire

    2015-01-01

    N6-carboxymethyl-NAD (N6-CM-NAD) can be used to immobilize NAD onto a substrate containing terminal primary amines. We previously immobilized N6-CM-NAD onto sepharose beads and showed that Thermotoga maritima glycerol dehydrogenase could use the immobilized cofactor with cofactor recycling. We now show that Saccharomyces cerevisiae alcohol dehydrogenase, rabbit muscle L-lactate dehydrogenase (type XI), bovine liver L-glutamic dehydrogenase (type III), Leuconostoc mesenteroides glucose-6-phosphate dehydro-genase, and Thermotoga maritima mannitol dehydrogenase are active with soluble N6-CM-NAD. The products of all enzymes but 6-phospho-D-glucono-1,5-lactone were formed when sepharose-immobilized N6-CM-NAD was recycled by T. maritima glycerol dehydrogenase, indicating that N6-immobilized NAD is suitable for use by a variety of different dehydrogenases. Observations of the enzyme active sites suggest that steric hindrance plays a greater role in limiting or allowing activity with the modified cofactor than do polarity and charge of the residues surrounding the N6-amine group on NAD. PMID:25611453

  20. Activity of select dehydrogenases with sepharose-immobilized N(6)-carboxymethyl-NAD.

    PubMed

    Beauchamp, Justin; Vieille, Claire

    2015-01-01

    N(6)-carboxymethyl-NAD (N(6)-CM-NAD) can be used to immobilize NAD onto a substrate containing terminal primary amines. We previously immobilized N(6)-CM-NAD onto sepharose beads and showed that Thermotoga maritima glycerol dehydrogenase could use the immobilized cofactor with cofactor recycling. We now show that Saccharomyces cerevisiae alcohol dehydrogenase, rabbit muscle L-lactate dehydrogenase (type XI), bovine liver L-glutamic dehydrogenase (type III), Leuconostoc mesenteroides glucose-6-phosphate dehydro-genase, and Thermotoga maritima mannitol dehydrogenase are active with soluble N(6)-CM-NAD. The products of all enzymes but 6-phospho-D-glucono-1,5-lactone were formed when sepharose-immobilized N(6)-CM-NAD was recycled by T. maritima glycerol dehydrogenase, indicating that N(6)-immobilized NAD is suitable for use by a variety of different dehydrogenases. Observations of the enzyme active sites suggest that steric hindrance plays a greater role in limiting or allowing activity with the modified cofactor than do polarity and charge of the residues surrounding the N(6)-amine group on NAD.

  1. Computer-aided-diagnosis (CAD) for colposcopy

    NASA Astrophysics Data System (ADS)

    Lange, Holger; Ferris, Daron G.

    2005-04-01

    Uterine cervical cancer is the second most common cancer among women worldwide. Colposcopy is a diagnostic method, whereby a physician (colposcopist) visually inspects the lower genital tract (cervix, vulva and vagina), with special emphasis on the subjective appearance of metaplastic epithelium comprising the transformation zone on the cervix. Cervical cancer precursor lesions and invasive cancer exhibit certain distinctly abnormal morphologic features. Lesion characteristics such as margin; color or opacity; blood vessel caliber, intercapillary spacing and distribution; and contour are considered by colposcopists to derive a clinical diagnosis. Clinicians and academia have suggested and shown proof of concept that automated image analysis of cervical imagery can be used for cervical cancer screening and diagnosis, having the potential to have a direct impact on improving women"s health care and reducing associated costs. STI Medical Systems is developing a Computer-Aided-Diagnosis (CAD) system for colposcopy -- ColpoCAD. At the heart of ColpoCAD is a complex multi-sensor, multi-data and multi-feature image analysis system. A functional description is presented of the envisioned ColpoCAD system, broken down into: Modality Data Management System, Image Enhancement, Feature Extraction, Reference Database, and Diagnosis and directed Biopsies. The system design and development process of the image analysis system is outlined. The system design provides a modular and open architecture built on feature based processing. The core feature set includes the visual features used by colposcopists. This feature set can be extended to include new features introduced by new instrument technologies, like fluorescence and impedance, and any other plausible feature that can be extracted from the cervical data. Preliminary results of our research on detecting the three most important features: blood vessel structures, acetowhite regions and lesion margins are shown. As this is a new

  2. CAD Integration : new optical design possibilities

    NASA Astrophysics Data System (ADS)

    Haumonte, Jean-Baptiste; Venturino, Jean-Claude

    2005-09-01

    The development of optical design and analysis tools in a CAD software can help to optimise the design, size and performance of tomorrow's consumer products. While optics was still held back by software limitations, CAD programs were moving forward in leaps and bounds, improving manufacturing technologies and making it possible to design and produce highly innovative and sophisticated products. The problem was that in the past, 'traditional' optical design programs were only able to simulate spherical and aspherical lenses, meaning that the optical designers were limited to designing systems which were a series of imperfect lenses, each one correcting the last. That is why OPTIS has created the first optical design program to be fully integrated into a CAD program. The technology is available from OPTIS in an integrated SOLIDWORKS or CATIA V5 version. Users of this software can reduce the number of lenses needed in a system. Designers will now have access to complex surfaces such as NURBS meaning they will now be able to define free shape progressive lenses and even improve on optical performances using fewer lenses. This revolutionary technology will allow mechanical designers to work on optical systems and to share information with optical designers for the first time. Previously not possible in a CAD program you may now determine all the optical performances of any optical system, providing first order and third order performances, sequential and non-sequential ray-tracing, wavefront surfaces, point spread function, MTF, spot-diagram, using real optical surfaces and guaranteeing the mechanical precision necessary for an optical system.

  3. Computer-aided diagnosis (CAD) for colonoscopy

    NASA Astrophysics Data System (ADS)

    Gu, Jia; Poirson, Allen

    2007-03-01

    Colorectal cancer is the second leading cause of cancer deaths, and ranks third for new cancer cases and cancer mortality for both men and women. However, its death rate can be dramatically reduced by appropriate treatment when early detection is available. The purpose of colonoscopy is to identify and assess the severity of lesions, which may be flat or protruding. Due to the subjective nature of the examination, colonoscopic proficiency is highly variable and dependent upon the colonoscopist's knowledge and experience. An automated image processing system providing an objective, rapid, and inexpensive analysis of video from a standard colonoscope could provide a valuable tool for screening and diagnosis. In this paper, we present the design, functionality and preliminary results of its Computer-Aided-Diagnosis (CAD) system for colonoscopy - ColonoCAD TM. ColonoCAD is a complex multi-sensor, multi-data and multi-algorithm image processing system, incorporating data management and visualization, video quality assessment and enhancement, calibration, multiple view based reconstruction, feature extraction and classification. As this is a new field in medical image processing, our hope is that this paper will provide the framework to encourage and facilitate collaboration and discussion between industry, academia, and medical practitioners.

  4. Measuring Negative Consequences of College Student Substance Use: A Psychometric Evaluation of the Core Alcohol and Drug Survey

    ERIC Educational Resources Information Center

    Martens, Matthew P.; Brown, Natashia T.; Donovan, Brooke M.; Dude, Kim

    2005-01-01

    A commonly used instrument to assess negative consequences of substance use among college students is the Core Alcohol and Drug Survey (CADS; C. A. Presley, P. W. Meilman, & J. S. Leichliter, 1998; C. A. Presley, P. W. Meilman, & R. Lyerla, 1993). Results from 2 studies suggest that a subset of CADS negative consequences items can be explained by…

  5. CAD in the processing plant environment or managing the CAD revolution

    SciTech Connect

    Woolbert, M.A.; Bennett, R.S.; Haring, W.I.

    1985-10-01

    The author presents a case report on the use of a Computer Aided Design/Computer Aided Drafting (CAD) system. Illustrated is a four-work station system, in addition to which there are two 70 megabyte disk drives, a check plotter and a 24-inch wide electrostatic plotter, a 300 megabyte disk for on-line storage, a tape drive for archive and backup, and a 1-megabyte network process server. It is a distributed logic system. The author states that the CAD system both inexpensive enough and powerful enough for the plant environment is relatively new on the market, made possible by the advent of super microcomputers. Also discussed is the impact the CAD system has had on productivity.

  6. Functional Analysis of a Mosquito Short Chain Dehydrogenase Cluster

    PubMed Central

    Mayoral, Jaime G.; Leonard, Kate T.; Defelipe, Lucas A.; Turjansksi, Adrian G.; Nouzova, Marcela; Noriegal, Fernando G.

    2013-01-01

    The short chain dehydrogenases (SDR) constitute one the oldest and largest families of enzymes with over 46,000 members in sequence databases. About 25% of all known dehydrogenases belong to the SDR family. SDR enzymes have critical roles in lipid, amino acid, carbohydrate, hormone and xenobiotic metabolism as well as in redox sensor mechanisms. This family is present in archaea, bacteria, and eukaryota, emphasizing their versatility and fundamental importance for metabolic processes. We identified a cluster of eight SDRs in the mosquito Aedes aegypti (AaSDRs). Members of the cluster differ in tissue specificity and developmental expression. Heterologous expression produced recombinant proteins that had diverse substrate specificities, but distinct from the conventional insect alcohol (ethanol) dehydrogenases. They are all NADP+-dependent and they have S-enantioselectivity and preference for secondary alcohols with 8–15 carbons. Homology modeling was used to build the structure of AaSDR1 and two additional cluster members. The computational study helped explain the selectivity towards the (10S)-isomers as well as the reduced activity of AaSDR4 and AaSDR9 for longer isoprenoid substrates. Similar clusters of SDRs are present in other species of insects, suggesting similar selection mechanisms causing duplication and diversification of this family of enzymes. PMID:23238893

  7. False positive reduction for lung nodule CAD

    NASA Astrophysics Data System (ADS)

    Zhao, Luyin; Boroczky, Lilla; Drysdale, Jeremy; Agnihotri, Lalitha; Lee, Michael C.

    2007-03-01

    Computer-aided detection (CAD) algorithms 'automatically' identify lung nodules on thoracic multi-slice CT scans (MSCT) thereby providing physicians with a computer-generated 'second opinion'. While CAD systems can achieve high sensitivity, their limited specificity has hindered clinical acceptance. To overcome this problem, we propose a false positive reduction (FPR) system based on image processing and machine learning to reduce the number of false positive lung nodules identified by CAD algorithms and thereby improve system specificity. To discriminate between true and false nodules, twenty-three 3D features were calculated from each candidate nodule's volume of interest (VOI). A genetic algorithm (GA) and support vector machine (SVM) were then used to select an optimal subset of features from this pool of candidate features. Using this feature subset, we trained an SVM classifier to eliminate as many false positives as possible while retaining all the true nodules. To overcome the imbalanced nature of typical datasets (significantly more false positives than true positives), an intelligent data selection algorithm was designed and integrated into the machine learning framework, thus further improving the FPR rate. Three independent datasets were used to train and validate the system. Using two datasets for training and the third for validation, we achieved a 59.4% FPR rate while removing one true nodule on the validation datasets. In a second experiment, 75% of the cases were randomly selected from each of the three datasets and the remaining cases were used for validation. A similar FPR rate and true positive retention rate was achieved. Additional experiments showed that the GA feature selection process integrated with the proposed data selection algorithm outperforms the one without it by 5%-10% FPR rate. The methods proposed can be also applied to other application areas, such as computer-aided diagnosis of lung nodules.

  8. Myths about drinking alcohol

    MedlinePlus

    ... to. I spend a lot of time getting alcohol, drinking alcohol, or recovering from the effects of alcohol. ... Institute on Alcohol Abuse and Alcoholism. Overview of Alcohol Consumption. www.niaaa.nih.gov/alcohol-health/overview-alcohol- ...

  9. Mammogram CAD, hybrid registration and iconic analysis

    NASA Astrophysics Data System (ADS)

    Boucher, A.; Cloppet, F.; Vincent, N.

    2013-03-01

    This paper aims to develop a computer aided diagnosis (CAD) based on a two-step methodology to register and analyze pairs of temporal mammograms. The concept of "medical file", including all the previous medical information on a patient, enables joint analysis of different acquisitions taken at different times, and the detection of significant modifications. The developed registration method aims to superimpose at best the different anatomical structures of the breast. The registration is designed in order to get rid of deformation undergone by the acquisition process while preserving those due to breast changes indicative of malignancy. In order to reach this goal, a referent image is computed from control points based on anatomical features that are extracted automatically. Then the second image of the couple is realigned on the referent image, using a coarse-to-fine approach according to expert knowledge that allows both rigid and non-rigid transforms. The joint analysis detects the evolution between two images representing the same scene. In order to achieve this, it is important to know the registration error limits in order to adapt the observation scale. The approach used in this paper is based on an image sparse representation. Decomposed in regular patterns, the images are analyzed under a new angle. The evolution detection problem has many practical applications, especially in medical images. The CAD is evaluated using recall and precision of differences in mammograms.

  10. Genetics Home Reference: lactate dehydrogenase deficiency

    MedlinePlus

    ... dehydrogenase-B pieces (subunits) of the lactate dehydrogenase enzyme. This enzyme is found throughout the body and is important ... cells. There are five different forms of this enzyme, each made up of four protein subunits. Various ...

  11. An animal model of human aldehyde dehydrogenase deficiency

    SciTech Connect

    Chang, C.; Mann, J.; Yoshida, A.

    1994-09-01

    The genetic deficiency of ALDH2, a major mitochondrial aldehyde dehydrogenase, is intimately related to alcohol sensitivity and the degree of predisposition to alcoholic diseases in humans. The ultimate biological role of ALDH2 can be exposed by knocking out the ALDH2 gene in an animal model. As the first step for this line of studies, we cloned and characterized the ALDH2 gene from mouse C57/6J strain which is associated with a high alcohol preference. The gene spans 26 kbp and is composed of 13 exons. Embryonic stem cells were transfected with a replacement vector which contains a partially deleted exon3, a positive selection cassette (pPgk Neo), exon 4 with an artificial stop codon, exons 5, 6, 7, and a negative selection cassette (pMCI-Tk). Genomic DNAs prepared from drug resistant clones were analyzed by polymerase chain reaction and by Southern blot analysis to distinguish random integration from homologous recombination. Out of 132 clones examined, 8 had undergone homologous recombination at one of the ALDH2 alleles. The cloned transformed embryonic stem cells with a disrupted ALDH2 allele were injected into blastocysts. Transplantation of the blastocysts into surrogate mother mice yielded chimeric mice. The role of ALDH2 in alcohol preference, alcohol sensitivity and other biological and behavioral characteristics can be elucidated by examining the heterozygous and homozygous mutant strains produced by breeding of chimeric mice.

  12. An Evaluation of Internet-Based CAD Collaboration Tools

    ERIC Educational Resources Information Center

    Smith, Shana Shiang-Fong

    2004-01-01

    Due to the now widespread use of the Internet, most companies now require computer aided design (CAD) tools that support distributed collaborative design on the Internet. Such CAD tools should enable designers to share product models, as well as related data, from geographically distant locations. However, integrated collaborative design…

  13. Preparing Students for Computer Aided Drafting (CAD). A Conceptual Approach.

    ERIC Educational Resources Information Center

    Putnam, A. R.; Duelm, Brian

    This presentation outlines guidelines for developing and implementing an introductory course in computer-aided drafting (CAD) that is geared toward secondary-level students. The first section of the paper, which deals with content identification and selection, includes lists of mechanical drawing and CAD competencies and a list of rationales for…

  14. Teach CAD and Measuring Skills through Reverse Engineering

    ERIC Educational Resources Information Center

    Board, Keith

    2012-01-01

    This article describes a reverse engineering activity that gives students hands-on, minds-on experience with measuring tools, machine parts, and CAD. The author developed this activity to give students an abundance of practical experience with measuring tools. Equally important, it provides a good interface between the virtual world of CAD 3D…

  15. An Instructional Method for the AutoCAD Modeling Environment.

    ERIC Educational Resources Information Center

    Mohler, James L.

    1997-01-01

    Presents a command organizer for AutoCAD to aid new uses in operating within the 3-D modeling environment. Addresses analyzing the problem, visualization skills, nonlinear tools, a static view of a dynamic model, the AutoCAD organizer, environment attributes, and control of the environment. Contains 11 references. (JRH)

  16. CAD/CAM: Practical and Persuasive in Canadian Schools

    ERIC Educational Resources Information Center

    Willms, Ed

    2007-01-01

    Chances are that many high school students would not know how to use drafting instruments, but some might want to gain competence in computer-assisted design (CAD) and possibly computer-assisted manufacturing (CAM). These students are often attracted to tech courses by the availability of CAD/CAM instructions, and many go on to impress employers…

  17. Evaluating the Learning Process of Mechanical CAD Students

    ERIC Educational Resources Information Center

    Hamade, R. F.; Artail, H. A.; Jaber, M. Y.

    2007-01-01

    There is little theoretical or experimental research on how beginner-level trainees learn CAD skills in formal training sessions. This work presents findings on how trainees develop their skills in utilizing a solid mechanical CAD tool (Pro/Engineer version 2000i[squared] and later version Wildfire). Exercises at the beginner and intermediate…

  18. Making a Case for CAD in the Curriculum.

    ERIC Educational Resources Information Center

    Threlfall, K. Denise

    1995-01-01

    Computer-assisted design (CAD) technology is transforming the apparel industry. Students of fashion merchandising and clothing design must be prepared on state-of-the-art equipment. ApparelCAD software is one example of courseware for instruction in pattern design and production. (SK)

  19. Alcohol and Alcoholism.

    ERIC Educational Resources Information Center

    National Inst. of Mental Health (DHEW), Chevy Chase, MD. National Clearinghouse for Mental Health Information.

    This concise survey presents some of the highlights of modern research on drinking and alcoholism, as based on technical articles published in the scientific literature and the views expressed by leading authorities in the field. Contents include discussions about: (1) the nature and scope of the problem; (2) the chemical composition of alcoholic…

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

    PubMed

    Barry, S; O'Carra, P

    1973-12-01

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

  1. Training a CAD classifier with correlated data

    NASA Astrophysics Data System (ADS)

    Dundar, Murat; Krishnapuram, Balaji; Wolf, Matthias; Lakare, Sarang; Bogoni, Luca; Bi, Jinbo; Rao, R. Bharat

    2007-03-01

    Most methods for classifier design assume that the training samples are drawn independently and identically from an unknown data generating distribution (i.i.d.), although this assumption is violated in several real life problems. Relaxing this i.i.d. assumption, we develop training algorithms for the more realistic situation where batches or sub-groups of training samples may have internal correlations, although the samples from different batches may be considered to be uncorrelated; we also consider the extension to cases with hierarchical--i.e. higher order--correlation structure between batches of training samples. After describing efficient algorithms that scale well to large datasets, we provide some theoretical analysis to establish their validity. Experimental results from real-life Computer Aided Detection (CAD) problems indicate that relaxing the i.i.d. assumption leads to statistically significant improvements in the accuracy of the learned classifier.

  2. Convergent evolution of Trichomonas vaginalis lactate dehydrogenase from malate dehydrogenase

    PubMed Central

    Wu, Gang; Fiser, András; ter Kuile, Benno; Šali, Andrej; Müller, Miklós

    1999-01-01

    Lactate dehydrogenase (LDH) is present in the amitochondriate parasitic protist Trichomonas vaginalis and some but not all other trichomonad species. The derived amino acid sequence of T. vaginalis LDH (TvLDH) was found to be more closely related to the cytosolic malate dehydrogenase (MDH) of the same species than to any other LDH. A key difference between the two T. vaginalis sequences was that Arg91 of MDH, known to be important in coordinating the C-4 carboxyl of oxalacetate/malate, was replaced by Leu91 in LDH. The change Leu91Arg by site-directed mutagenesis converted TvLDH into an MDH. The reverse single amino acid change Arg91Leu in TvMDH, however, gave a product with no measurable LDH activity. Phylogenetic reconstructions indicate that TvLDH arose from an MDH relatively recently. PMID:10339579

  3. SAXS fingerprints of aldehyde dehydrogenase oligomers

    PubMed Central

    Tanner, John J.

    2015-01-01

    Enzymes of the aldehyde dehydrogenase (ALDH) superfamily catalyze the nicotinamide adenine dinucleotide-dependent oxidation of aldehydes to carboxylic acids. ALDHs are important in detoxification of aldehydes, amino acid metabolism, embryogenesis and development, neurotransmission, oxidative stress, and cancer. Mutations in genes encoding ALDHs cause metabolic disorders, including alcohol flush reaction (ALDH2), Sjögren–Larsson syndrome (ALDH3A2), hyperprolinemia type II (ALDH4A1), γ-hydroxybutyric aciduria (ALDH5A1), methylmalonic aciduria (ALDH6A1), pyridoxine dependent epilepsy (ALDH7A1), and hyperammonemia (ALDH18A1). We previously reported crystal structures and small-angle X-ray scattering (SAXS) analyses of ALDHs exhibiting dimeric, tetrameric, and hexameric oligomeric states (Luo et al., Biochemistry 54 (2015) 5513–5522; Luo et al., J. Mol. Biol. 425 (2013) 3106–3120). Herein I provide the SAXS curves, radii of gyration, and distance distribution functions for the three types of ALDH oligomer. The SAXS curves and associated analysis provide diagnostic fingerprints that allow rapid identification of the type of ALDH oligomer that is present in solution. The data sets provided here serve as a benchmark for characterizing oligomerization of ALDHs. PMID:26693506

  4. SAXS fingerprints of aldehyde dehydrogenase oligomers.

    PubMed

    Tanner, John J

    2015-12-01

    Enzymes of the aldehyde dehydrogenase (ALDH) superfamily catalyze the nicotinamide adenine dinucleotide-dependent oxidation of aldehydes to carboxylic acids. ALDHs are important in detoxification of aldehydes, amino acid metabolism, embryogenesis and development, neurotransmission, oxidative stress, and cancer. Mutations in genes encoding ALDHs cause metabolic disorders, including alcohol flush reaction (ALDH2), Sjögren-Larsson syndrome (ALDH3A2), hyperprolinemia type II (ALDH4A1), γ-hydroxybutyric aciduria (ALDH5A1), methylmalonic aciduria (ALDH6A1), pyridoxine dependent epilepsy (ALDH7A1), and hyperammonemia (ALDH18A1). We previously reported crystal structures and small-angle X-ray scattering (SAXS) analyses of ALDHs exhibiting dimeric, tetrameric, and hexameric oligomeric states (Luo et al., Biochemistry 54 (2015) 5513-5522; Luo et al., J. Mol. Biol. 425 (2013) 3106-3120). Herein I provide the SAXS curves, radii of gyration, and distance distribution functions for the three types of ALDH oligomer. The SAXS curves and associated analysis provide diagnostic fingerprints that allow rapid identification of the type of ALDH oligomer that is present in solution. The data sets provided here serve as a benchmark for characterizing oligomerization of ALDHs. PMID:26693506

  5. Alcohol use disorder

    MedlinePlus

    ... Alcohol abuse; Problem drinking; Drinking problem; Alcohol addiction; Alcoholism - alcohol use; Substance use - alcohol ... The National Institute on Alcohol Abuse and Alcoholism ... 1 drink per day Men should not drink more than 2 drinks per day

  6. CAD-centric Computation Management System for a Virtual TBM

    SciTech Connect

    Ramakanth Munipalli; K.Y. Szema; P.Y. Huang; C.M. Rowell; A.Ying; M. Abdou

    2011-05-03

    HyPerComp Inc. in research collaboration with TEXCEL has set out to build a Virtual Test Blanket Module (VTBM) computational system to address the need in contemporary fusion research for simulating the integrated behavior of the blanket, divertor and plasma facing components in a fusion environment. Physical phenomena to be considered in a VTBM will include fluid flow, heat transfer, mass transfer, neutronics, structural mechanics and electromagnetics. We seek to integrate well established (third-party) simulation software in various disciplines mentioned above. The integrated modeling process will enable user groups to interoperate using a common modeling platform at various stages of the analysis. Since CAD is at the core of the simulation (as opposed to computational meshes which are different for each problem,) VTBM will have a well developed CAD interface, governing CAD model editing, cleanup, parameter extraction, model deformation (based on simulation,) CAD-based data interpolation. In Phase-I, we built the CAD-hub of the proposed VTBM and demonstrated its use in modeling a liquid breeder blanket module with coupled MHD and structural mechanics using HIMAG and ANSYS. A complete graphical user interface of the VTBM was created, which will form the foundation of any future development. Conservative data interpolation via CAD (as opposed to mesh-based transfer), the regeneration of CAD models based upon computed deflections, are among the other highlights of phase-I activity.

  7. Investigation of IGES for CAD/CAE data transfer

    NASA Technical Reports Server (NTRS)

    Zobrist, George W.

    1989-01-01

    In a CAD/CAE facility there is always the possibility that one may want to transfer the design graphics database from the native system to a non-native system. This may occur because of dissimilar systems within an organization or a new CAD/CAE system is to be purchased. The Initial Graphics Exchange Specification (IGES) was developed in an attempt to solve this scenario. IGES is a neutral database format into which the CAD/CAE native database format can be translated to and from. Translating the native design database format to IGES requires a pre-processor and transling from IGES to the native database format requires a post-processor. IGES is an artifice to represent CAD/CAE product data in a neutral environment to allow interfacing applications, archive the database, interchange of product data between dissimilar CAD/CAE systems, and other applications. The intent here is to present test data on translating design product data from a CAD/CAE system to itself and to translate data initially prepared in IGES format to various native design formats. This information can be utilized in planning potential procurement and developing a design discipline within the CAD/CAE community.

  8. A new CAD approach for improving efficacy of cancer screening

    NASA Astrophysics Data System (ADS)

    Zheng, Bin; Qian, Wei; Li, Lihua; Pu, Jiantao; Kang, Yan; Lure, Fleming; Tan, Maxine; Qiu, Yuchen

    2015-03-01

    Since performance and clinical utility of current computer-aided detection (CAD) schemes of detecting and classifying soft tissue lesions (e.g., breast masses and lung nodules) is not satisfactory, many researchers in CAD field call for new CAD research ideas and approaches. The purpose of presenting this opinion paper is to share our vision and stimulate more discussions of how to overcome or compensate the limitation of current lesion-detection based CAD schemes in the CAD research community. Since based on our observation that analyzing global image information plays an important role in radiologists' decision making, we hypothesized that using the targeted quantitative image features computed from global images could also provide highly discriminatory power, which are supplementary to the lesion-based information. To test our hypothesis, we recently performed a number of independent studies. Based on our published preliminary study results, we demonstrated that global mammographic image features and background parenchymal enhancement of breast MR images carried useful information to (1) predict near-term breast cancer risk based on negative screening mammograms, (2) distinguish between true- and false-positive recalls in mammography screening examinations, and (3) classify between malignant and benign breast MR examinations. The global case-based CAD scheme only warns a risk level of the cases without cueing a large number of false-positive lesions. It can also be applied to guide lesion-based CAD cueing to reduce false-positives but enhance clinically relevant true-positive cueing. However, before such a new CAD approach is clinically acceptable, more work is needed to optimize not only the scheme performance but also how to integrate with lesion-based CAD schemes in the clinical practice.

  9. The Protective Effects of Buzui on Acute Alcoholism in Mice

    PubMed Central

    Wen, Da-Chao; Gao, Shu-di; Hu, Xiao-yu; Yi, Cheng

    2016-01-01

    This study was designed to investigate the role of a traditional buzui recipe in anti-inebriation treatment. Buzui consists of Fructus Schisandrae Chinensis, Fructus Chebulae, Fructus Mume, Fructus Crataegi, Endothelium Corneum Gigeriae Galli, and Excrementum Bombycis. The buzui mixture was delivered by gavage, and ethanol was delivered subsequent to the final treatment. The effects of buzui on the righting reflex, inebriation rates, and the survival curve are depicted. Blood alcohol concentrations, alanine aminotransferase (ALT) levels, aspartate aminotransferase (AST) levels, and alkaline phosphatase (ALP) levels were recorded. The activities of alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH), and superoxide dismutase (SOD), as well as malonaldehyde (MDA) levels, were also measured. Our results demonstrated that a traditional buzui recipe showed significant effects on promoting wakefulness and the prevention of acute alcohol intoxication, accelerating the metabolism of alcohol in the liver and reducing the oxidative damage caused by acute alcoholism. PMID:26884793

  10. Turnkey CAD/CAM systems' integration with IPAD systems

    NASA Technical Reports Server (NTRS)

    Blauth, R. E.

    1980-01-01

    Today's commercially available turnkey CAD/CAM systems provide a highly interactive environment, and support many specialized application functions for the design/drafting/manufacturing process. This paper presents an overview of several aerospace companies which have successfully integrated turnkey CAD/CAM systems with their own company wide engineering and manufacturing systems. It also includes a vendor's view of the benefits as well as the disadvantages of such integration efforts. Specific emphasis is placed upon the selection of standards for representing geometric engineering data and for communicating such information between different CAD/CAM systems.

  11. Antidotes for poisoning by alcohols that form toxic metabolites.

    PubMed

    McMartin, Kenneth; Jacobsen, Dag; Hovda, Knut Erik

    2016-03-01

    The alcohols, methanol, ethylene glycol and diethylene glycol, have many features in common, the most important of which is the fact that the compounds themselves are relatively non-toxic but are metabolized, initially by alcohol dehydrogenase, to various toxic intermediates. These compounds are readily available worldwide in commercial products as well as in homemade alcoholic beverages, both of which lead to most of the poisoning cases, from either unintentional or intentional ingestion. Although relatively infrequent in overall occurrence, poisonings by metabolically-toxic alcohols do unfortunately occur in outbreaks and can result in severe morbidity and mortality. These poisonings have traditionally been treated with ethanol since it competes for the active site of alcohol dehydrogenase and decreases the formation of toxic metabolites. Although ethanol can be effective in these poisonings, there are substantial practical problems with its use and so fomepizole, a potent competitive inhibitor of alcohol dehydrogenase, was developed for a hopefully better treatment for metabolically-toxic alcohol poisonings. Fomepizole has few side effects and is easy to use in practice and it may obviate the need for haemodialysis in some, but not all, patients. Hence, fomepizole has largely replaced ethanol as the toxic alcohol antidote in many countries. Nevertheless, ethanol remains an important alternative because access to fomepizole can be limited, the cost may appear excessive, or the physician may prefer ethanol due to experience.

  12. OXIDATION OF SECONDARY ALCOHOLS BY EXTRACTS OF A CORYNEBACTERIUM.

    PubMed

    ROSENBERG, E; HOLMES, P

    1965-05-01

    Rosenberg, Eugene (University of California, Los Angeles), and Paul Holmes. Oxidation of secondary alcohols by extracts of a Corynebacterium. J. Bacteriol. 89:1212-1216. 1965.-A Corynebacterium was isolated from soil by use of the enrichment culture technique. This Corynebacterium oxidized and grew on the chemically synthesized secondary alcohol, 3-tetrahydrofuranol. Extracts of this organism contained at least two different nicotinamide adenine dinucleotide-requiring soluble secondary alcohol dehydrogenases. These enzymes were distinguished by the relative rates at which they oxidized thymidylic acid and 3-tetrahydrofuranol. In addition to their substrate specificity, the two enzymes differed in pH optima and thermal stability. Also, the 3-tetrahydrofuranol dehydrogenase was induced by 3-tetrahydrofuranol, whereas the thymidylic acid dehydrogenase was constitutive.

  13. Computer-aided diagnosis of alcoholism-related EEG signals.

    PubMed

    Acharya, U Rajendra; S, Vidya; Bhat, Shreya; Adeli, Hojjat; Adeli, Amir

    2014-12-01

    Alcoholism is a severe disorder that affects the functionality of neurons in the central nervous system (CNS) and alters the behavior of the affected person. Electroencephalogram (EEG) signals can be used as a diagnostic tool in the evaluation of subjects with alcoholism. The neurophysiological interpretation of EEG signals in persons with alcoholism (PWA) is based on observation and interpretation of the frequency and power in their EEGs compared to EEG signals from persons without alcoholism. This paper presents a review of the known features of EEGs obtained from PWA and proposes that the impact of alcoholism on the brain can be determined by computer-aided analysis of EEGs through extracting the minute variations in the EEG signals that can differentiate the EEGs of PWA from those of nonaffected persons. The authors advance the idea of automated computer-aided diagnosis (CAD) of alcoholism by employing the EEG signals. This is achieved through judicious combination of signal processing techniques such as wavelet, nonlinear dynamics, and chaos theory and pattern recognition and classification techniques. A CAD system is cost-effective and efficient and can be used as a decision support system by physicians in the diagnosis and treatment of alcoholism especially those who do not specialize in alcoholism or neurophysiology. It can also be of great value to rehabilitation centers to assess PWA over time and to monitor the impact of treatment aimed at minimizing or reversing the effects of the disease on the brain. A CAD system can be used to determine the extent of alcoholism-related changes in EEG signals (low, medium, high) and the effectiveness of therapeutic plans.

  14. CAD-CAE in Electrical Machines and Drives Teaching.

    ERIC Educational Resources Information Center

    Belmans, R.; Geysen, W.

    1988-01-01

    Describes the use of computer-aided design (CAD) techniques in teaching the design of electrical motors. Approaches described include three technical viewpoints, such as electromagnetics, thermal, and mechanical aspects. Provides three diagrams, a table, and conclusions. (YP)